Integrated Multi-analytical Framework for Comprehensive Characterization of Lignocellulosic Hydrolysates

ABSTRACT Lignocellulosic hydrolysates are promising second-generation feedstocks for sustainable biomanufacturing. However, biomass compositional complexities and a lack of analytical framework, particularly for lignin fraction (LF), have limited holistic characterization and impeded downstream valorization. In this study, we present an integrated multi-analytical framework for in-depth compositional profiling of spruce sawdust hydrolysate (SSH). Combining high-performance anion-exchange chromatography and liquid chromatography, sugars and inhibitors were quantified, with monomeric arabinose (78.8±0.6 g/L) and acetic acid (14.9±0.1 g/L) as predominant components, respectively. Additionally, we developed a workflow to quantify and evaluate lignin structures and interlinkages, determining factors in biomanufacturing using a unique spectroscopic-chromatographic approach enabling a comprehensive understanding of the LF. UV-Vis spectroscopy quantified a total aromatic content of 58.2±0.1g/L, while quantitative 31 P NMR clarified the hydroxyl group distribution in the LF. 1 H- 13 C HSQC NMR provided structural identification of lignin units and interconnecting linkages, and gel permeation chromatography showed the molecular weight distribution. Gas chromatography-mass spectrometry identified and quantified 38 lignin-derived monomers and extractives, with benzoic acid (2.7±0.2 mg/L) as the most abundant. This integrated approach provides holistic biomass compositional insights and cross-validation across methods, overcomes the limitations of a single technique, and delivers a robust workflow to evaluate the biomass suitability for bio-valorization. ABSTRACT GRAPHIC SYNOPSIS This study develops an integrated multi-analytical workflow to comprehensively profile biomass hydrolysate for downstream valorization.