The influence of the putative lignin-carbohydrate complexes (LCCs) on enzymatic saccharification was elucidated for the first time by examining two groups of transgenic black cottonwood (P. trichocarpa) comprised of the lignin down-regulated and the xylan down-regulated lines. Any adventitious contaminants that could affect the characterization of LCCs and the enzymatic saccharification were removed by performing a thorough extraction on the samples. The crude milled wood lignin was subsequently isolated from which the phenyl glycoside, benzyl ether and c-ester linkages representative of the LCCs were identified and quantified with the combination of C-13 and H-1-C-13 Heteronuclear Single Quantum Coherence (HSQC) NMR. The result indicated that the samples showed different levels of the three LCC linkages, depending on the xylan and/or lignin content. The correlation between the LCCs and enzymatic saccharification nearly conclusively demonstrated that the LCCs accounting for the recalcitrance of lignocellulosic biodegradation. (C) 2013 Elsevier Ltd. All rights reserved.