Structural Heterogeneity and Functional Group Distribution in Spruce and Beech Cellulolytic Enzyme Lignins

This study aimed to analyze the structure of near-native lignin isolated from spruce and beech and to evaluate the relationship between their molar mass and functional group content. In addition, the work assessed how well experimentally isolated cellulolytic enzyme lignin (CEL) fractions represent close-to-native lignin structures and whether existing lignin models are sufficient to describe and predict their functional group contents. CELs were isolated by sequential extraction with 96% and 60% dioxane. The CELs were then dissolved in ammonium hydroxide and fractionated by preparative SEC. The crude lignins and selected fractions were characterized by acidic methanolysis, HSQC NMR, quantitative 31P-NMR, and DMSO/LiBr SEC. Acidic methanolysis showed residual carbohydrate contents of 11–13% in spruce CEL-96 and 15% in beech CEL-96, while CEL-60 fractions contained higher sugar contents of 16–22%. Analysis by DMSO/LiBr SEC showed a good preparative separation for beech CEL, but not an ideal separation for spruce CEL. Beech fractions decreased in Mw from 146 to 6.5 kDa, while spruce fractions remained broad and overlapping. HSQC NMR showed that spruce fractions were structurally similar, with β–O–4 contents remaining at 32–34 per 100 Ar, whereas beech fractions showed clearer structural shifts, including a decrease in β–O–4 content from 62–64 to 52–54 per 100 Ar from high to lower molar mass fractions. The experimentally determined OH profiles only partly agreed with the literature lignin models: aliphatic OH contents were lower than predicted by the Ralph models, while phenolic OH contents were closer to the Ralph models than to the highly phenolic Balakshin model. These results indicate that current literature lignin models insufficiently represent the experimentally observed contents of aliphatic and phenolic OH groups in near-native CEL fractions.