Abstract
Eucalypt feedstock was pretreated with Myceliophthora thermophila laccase and methyl syringate, as mediator, in a multistage sequence consisting of successive enzymatic and alkaline peroxide stages, directly on the ground wood. Lignin modification was studied by two-dimensional (2D) nuclear magnetic resonance (NMR) of wood (at the gel state) after each stage of the sequence. Decrease of guaiacyl and syringyl lignin units (with preferential removal of the former) and aliphatic (mainly β-O-4′-linked) side chains was observed along the sequence, without a substantial change in polysaccharide cross-signals along the sequence, was observed. However, the most noticeable modification was the formation of Cα-oxidized syringyl units in the enzymatic stages, which were partially removed in the alkaline extractions. Further insight was attained by 2D NMR of the cellulolytic-enzyme lignins, isolated from the pretreated samples, and the filtrates from the different stages. Additionally, a comparison of eucalypt delignification with the high redox potential laccase from Pycnoporus cinnabarinus in the presence of the synthetic mediator 1-hydroxybenzotriazole was carried out. Wood delignification already occurred at the first cycle of the pretreatment with P. cinnabarinus laccase and 1-hydroxybenzotriazole, which directly correlated with increases in glucose (~10 %) yields after enzymatic hydrolysis. In contrast, the first cycle of pretreatment with M. thermophila laccase and methyl syringate did not produce any delignification or saccharification improvement. However, at the end of the sequence, similar delignification (~50 %) of eucalypt feedstock and increases (~30 %) in glucose yields were attained in both laccase-mediator pretreatments. Finally, the influence of lignin content and the ratio of its syringyl and guaiacyl units on recalcitrance of eucalypt feedstock to enzymatic saccharification is discussed.
Similar content being viewed by others
References
Papa G, Varanasi P, Sun L, Cheng G, Stavila V, Holmes B, Simmons BA, Adani F, Singh S (2012) Exploring the effect of different plant lignin content and composition on ionic liquid pretreatment efficiency and enzymatic saccharification of Eucalyptus globulus L. mutants. Bioresource Technol 117:352–359
Studer MH, DeMartini JD, Davis MF, Sykes RW, Davison B, Keller M, Tuskan GA, Wyman CE (2011) Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci U S A 108:6300–6305
Li X, Ximenes E, Kim Y, Slininger M, Meilan R, Ladisch M, Chapple C (2010) Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment. Biotechnol Biofuels 3
Martín-Sampedro R, Rahikainen JL, Johansson LS, Marjamaa K, Laine J, Kruus K, Rojas OJ (2013) Preferential adsorption and activity of monocomponent cellulases on lignocellulose thin films with varying lignin content. Biomacromolecules 14:1231–1239
Pareek N, Gillgren T, Jönsson LJ (2013) Adsorption of proteins involved in hydrolysis of lignocellulose on lignins and hemicelluloses. Bioresource Technol 148:70–77
Rahikainen JL, Martin-Sampedro R, Heikkinen H, Rovio S, Marjamaa K, Tamminen T, Rojas OJ, Kruus K (2013) Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Bioresource Technol 133:270–278
Nakagame S, Chandra RP, Saddler JN (2010) The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis. Biotechnol Bioeng 105:871–879
Martínez AT, Ruiz-Dueñas FJ, Martínez MJ, del Río JC, Gutiérrez A (2009) Enzymatic delignification of plant cell wall: from nature to mill. Curr Opin Biotechnol 20:348–357
Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett 267:99–102
Call H-P (1994) Verfahren zur Veränderung, Abbau oder Bleichen von Lignin, ligninhaltigen Materialien oder ähnlichen Stoffen. Patent (International) WO 94/29510
Poppius-Levlin K, Wang W, Tamminen T, Hortling B, Viikari L, Niku-Paavola M-L (1999) Effects of laccase/HBT treatment on pulp and lignin structures. J Pulp Paper Sci 25:90–94
Camarero S, García O, Vidal T, Colom J, del Río JC, Gutiérrez A, Gras JM, Monje R, Martínez MJ, Martínez AT (2004) Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system. Enzyme Microb Technol 35:113–120
Gutiérrez A, del Río JC, Martínez AT (2009) Microbial and enzymatic control of pitch in the pulp and paper industry. Appl Microbiol Biotechnol 82:1005–1018
Prasetyo EN, Kudanga T, Ostergaard L, Rencoret J, Gutiérrez A, del Río JC, Santos JI, Nieto L, Jimenez-Barbero J, Martínez AT et al (2010) Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresource Technol 101:5054–5062
Widsten P, Kandelbauer A (2008) Laccase applications in the forest products industry: a review. Enzyme Microb Technol 42:293–307
Gutiérrez A, Rencoret J, Cadena EM, Rico A, Barth D, del Río JC, Martínez AT (2012) Demonstration of laccase-mediator removal of lignin from wood and non-wood plant feedstocks. Bioresource Technol 119:114–122
Palonen H, Viikari L (2004) Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood. Biotechnol Bioeng 86:550–557
Chen Q, Marshall MN, Geib SM, Tien M, Richard TL (2012) Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover. Bioresource Technol 117:186–192
Rico A, Rencoret J, del Río JC, Martínez AT, Gutiérrez A (2014) Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol Biofuels 7:6
Babot ED, Rico A, Rencoret J, Kalum L, Lund H, Romero J, del Río JC, Martínez AT, Gutiérrez A (2011) Towards industrially feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator. Bioresource Technol 102:6717–6722
Tappi: 2006–2007 TAPPI test methods. Norcoss, GA 30092, USA: TAPPI Press
Selvendran RR, March JF, Ring SG (1979) Determination of aldoses and uronic-acid content of vegetable fiber. Anal Biochem 96:282–292
Rencoret J, Marques G, Gutiérrez A, Nieto L, Santos I, Jiménez-Barbero J, Martínez AT, del Río JC (2009) HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state. Holzforschung 63:691–698
Kim H, Ralph J, Akiyama T (2008) Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6. Bioenerg Res 1:56–66
Rencoret J, Gutiérrez A, del Río JC (2007) Lipid and lignin composition of woods from different eucalypt species. Holzforschung 61:165–174
Rencoret J, Marques G, Gutiérrez A, Ibarra D, Li J, Gellerstedt G, Santos JI, Jiménez-Barbero J, Martínez AT, del Río JC (2008) Structural characterization of milled wood lignin from different eucalypt species. Holzforschung 62:514–526
Rencoret J, Gutiérrez A, Nieto L, Jiménez-Barbero J, Faulds CB, Kim H, Ralph J, Martínez AT, del Río JC (2001) Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiol 155:667–682
Kishimoto T, Chiba W, Saito K, Fukushima K, Uraki Y, Ubukata M (2010) Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins. J Agric Food Chem 58:895–901
Santos RB, Treasure T, Gonzalez R, Phillips R, Lee JM, Jameel H, Chang HM (2012) Impact of hardwood species on production cost of second generation ethanol. Bioresource Technol 117:193–200
Martínez-Íñigo MJ, Kurek B (1997) Oxidative degradation of alkali wheat straw lignin by fungal lignin peroxidase, manganese peroxidase and laccase: a comparative study. Holzforschung 51:543–548
Du XY, Li J, Gellerstedt G, Rencoret J, del Río JC, Martínez AT, Gutiérrez A (2013) Understanding pulp delignification by laccase-mediator systems through isolation and characterization of lignin-carbohydrate complexes. Biomacromolecules 14:3073–3080
Calcaterra A, Galli C, Gentili P (2008) Phenolic compounds as likely natural mediators of laccase: a mechanistic assessment. J Mol Catal B-Enzym 51:118–120
Lahtinen M, Kruus K, Heinonen P, Sipila J (2009) On the reactions of two fungal laccases differing in their redox potential with lignin model compounds: products and their rate of formation. J Agric Food Chem 57:8357–8365
Zhang YHP, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 88:797–824
Yu ZY, Jameel H, Chang HM, Park S (2011) The effect of delignification of forest biomass on enzymatic hydrolysis. Bioresource Technol 102:9083–9089
Santos RB, Lee JM, Jameel H, Chang HM, Lucia LA (2012) Effects of hardwood structural and chemical characteristics on enzymatic hydrolysis for biofuel production. Bioresource Technol 110:232–238
Chen F, Dixon R (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25:759–761
Acknowledgments
This study was funded by the INDOX (KBBE-2013-7-613549) EU-project and the LIGNOCELL (AGL2011-25379) and HIPOP (BIO2011-2669) Spanish MICINN projects (co-financed by FEDER funds). J. Rencoret acknowledges the CSIC JAE-Doc contract of the program “Junta para la Ampliación de Estudios” co-financed by the European Social Fund (ESF). H. Lund and L. Kalum from Novozymes (Bagsvaerd, Denmark) are acknowledged for the cellulases (Celluclast and Novozym 188) and the M. thermophila laccase, Beldem (Andenne, Belgium) for the P. cinnabarinus laccase and J. Romero (ENCE, Spain) for the eucalypt wood samples. Manuel Angulo (CITIUS, University of Seville) is acknowledged for providing technical assistance in the NMR analyses.
Author information
Authors and Affiliations
Corresponding author
Additional information
Alejandro Rico and Jorge Rencoret contributed equally to this work.
Rights and permissions
About this article
Cite this article
Rico, A., Rencoret, J., del Río, J.C. et al. In-Depth 2D NMR Study of Lignin Modification During Pretreatment of Eucalyptus Wood with Laccase and Mediators. Bioenerg. Res. 8, 211–230 (2015). https://doi.org/10.1007/s12155-014-9505-x
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12155-014-9505-x