Lignin-cellulose mixtures (LCMs) generated as intermediates in wood biorefineries are commonly separated into lignin and cellulose. However, using ultrasound (US) to pro-cess these mixtures could create novel, valuable materials not possible with conven-tional methods. This study looked at how lignin affects the US modification of these mixtures. Crude and partially delignified LCMs were successfully prepared using aqueous solutions of EtOH, THF and dilute NaOH and then subjected to short, high-power US treatment. The resulting materials were characterised using FT-IR spectroscopy, particle size analysis, water retention value analysis, SEM and XRD. Sonication rapidly reduced the mean particle size, generating cellulose nanofibril-like structures in all samples according to SEM. The response depended strongly on lignin content, with samples containing lower amounts of lignin exhibiting substantially higher hydration capacity and stronger US responsiveness. At the molecular level, lig-nin removal exposes cellulose surfaces and enhances hydrophilic interface formation, increasing water uptake and suspension stability. Thus, results show that lignin limits accessible hydrophilic cellulose surface area rather than preventing fragmentation by sonication. US is therefore a chemical-lean strategy to tune the physicochemical prop-erties of partly delignified LCMs and expand the product portfolio of integrated wood biorefineries towards novel advanced lignocellulosic materials.

Background: The utilization of Pseudomonas putida was explored in this study as a promising approach for lignin valorization. To this end, dry hydrolysis lignin was used as a feedstock for the first time. Hydrolysis lignin is a product of the enzymatic hydrolysis and separation of cellulose and hemicellulose from the lignin backbone in diverse lignocellulosic sources. Various fractionation techniques were applied to obtain lignin monomers and multimers in solution for use as a growth medium for P. putida , whose tolerance of inhibitory phenolic compounds distinguishes it from most bacteria. Results: Physiological evaluations revealed that Pseudomonas putida strains KT2440 and PaW85 exhibited broad pH tolerance ranges, with robust growth observed at elevated pH levels. Batch fermentations using hydrolysis lignin (HL) solutions showed complete consumption of sugars within 24 hours, demonstrating the viability of fractionated HL as a substrate for P. putida cultivation. HPLC analysis of HL monomer concentrations during simulated fed-batch fermentation revealed rapid catabolism of catechol and increased CCMA concentration, followed by stabilization, indicating that CCMA is synthesized more quickly than degraded when the initial catechol concentration is high. Filtered alkaline HL fractionations yielded more than twice as much catechol as unfiltered fractionations. Screening of indigenous bacterial strains isolated from various soil and water samples (CELMS Collection, website http://eemb.ut.ee ) identified five new candidate strains for CCMA production, two for PCA production, and three for vanillic acid production. Conclusions: The novel use of fractionated hydrolysis lignin as a growth medium shows potential for lignin valorization and chemical production. Filtered alkaline fractionation yields more catechol and is superior for cis,cis -muconic acid production; however, unfiltered fractionations may be more suitable for other compounds and upscaling. Further investigation of screened strains could reveal more efficient enzymes, which could be optimized and transformed into P. putida in future research.

Lignin–cellulose mixtures (LCMs) generated as intermediates in wood biorefineries are commonly separated into lignin and cellulose. However, using ultrasound (US) to process these mixtures could create novel, valuable materials not possible with conventional methods. This study looked at how lignin affects the US modification of these mixtures. Crude and partially delignified LCMs were successfully prepared using aqueous solutions of EtOH, THF and dilute NaOH and then subjected to short, high-power US treatment. The resulting materials were characterised using FT-IR spectroscopy, particle size analysis, water retention value analysis, SEM and XRD. Sonication rapidly reduced the mean particle size, generating cellulose nanofibril-like structures in all samples according to SEM. The response depended strongly on lignin content, with samples containing lower amounts of lignin exhibiting substantially higher hydration capacity and stronger US responsiveness. At the molecular level, lignin removal exposes cellulose surfaces and enhances hydrophilic interface formation, increasing water uptake and suspension stability. Thus, results show that lignin limits accessible hydrophilic cellulose surface area rather than preventing fragmentation by sonication. US is therefore a chemical-lean strategy to tune the physicochemical properties of partly delignified LCMs and expand the product portfolio of integrated wood biorefineries towards novel advanced lignocellulosic materials.

Recent rapid developments in forest biomass valorisation have highlighted lignin as a key value driver for the economic sustainability of modern biorefining. Sugar-based biorefinery hydrolysis lignin (HL) from wood biomass has an immense potential as an alternative for fossil-based aromatic feedstock. However, impurities, heterogeneous structures and limited solubility in common solvents are the main challenges associated with HL functionalisation. Aqueous organic solvent fractionation represents a promising approach to improve on HL properties and subsequently increase its valorisation options. In this study, for the first time, biorefinery HL derived from birch was purified and fractionated with water-EtOH and -THF mixtures across the whole co-solvent concentration range at ambient temperature. The highest lignin solubility was achieved with 70 wt% THF mixture, which dissolved a remarkable 81 wt% of lignin available in HL. Moreover, fractionated lignin purity was further increased to 95 wt% when employing an extra water-washing step. In general, fractions soluble in THF mixtures had high dispersity, syringyl-to-guaiacyl (S/G) subunit ratio and aliphatic OH-groups, whereas EtOH fractions had narrower molecular weight distributions, lower S/G subunit ratio and higher concentration of phenolic OH-groups. A brief description of the solvent effects at the molecular level was given using molecular dynamics simulations which revealed specific solvation between the dissolved lignin and solvent mixture. Protocol presented in this paper enables to purify HL and control the physicochemical properties of the soluble lignin fractions by simply altering the composition of the solvent mixture, and therefore, expanding the possibilities in valorisation of HL.