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bioRxiv · 10.1101/2025.09.12.675780

Integrated Multi-analytical Framework for Comprehensive Characterization of Lignocellulosic Hydrolysates

Kumar, S.·Nousiainen, P.·Kamravamanesh, D.·Mangayil, R.
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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 31P NMR clarified the hydroxyl group distribution in the LF. 1H-13C 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/675780v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@db6eceorg.highwire.dtl.DTLVardef@47b9b3org.highwire.dtl.DTLVardef@f0a85aorg.highwire.dtl.DTLVardef@1bcb66c_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISThis study develops an integrated multi-analytical workflow to comprehensively profile biomass hydrolysate for downstream valorization.

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Kumar, S., Nousiainen, P., Kamravamanesh, D., Mangayil, R.. 2025-09-17. Integrated Multi-analytical Framework for Comprehensive Characterization of Lignocellulosic Hydrolysates. https://doi.org/10.1101/2025.09.12.675780

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