Preprint of "Complete biodiverse lignocellulosic biomass fractionation process using the green solvent γ-valerolactone"

RSC Sustainability Complete biodiverse lignocellulosic biomass fractionation process using the green solvent γvalerolactone Moritz Schweiger, Thomas Lang, Didier Touraud, Eva Müller and Werner Kunz Vojtěch Jeřábek, Magdalena Bendová, Karel Řehák, Jan Heyda, Martin Klajmon Biomass pretreatment processes using organic solvents have historically been investigated for the separation of lignin from lignocellulosic biomass. This research explores how the pretreatment process can be expanded to fractionate all biomass components from various feedstock and waste streams and transform them into valuable bio-based molecules using green solvent mixtures and mild conditions. Already in the past, the bio-based solvent γ-valerolactone (GVL) has been identified as highly efficient for lignin separation and the dissolution of residual lignocellulosic components. A computational investigation employing the quantum-mechanicsaided COSMO-SAC thermodynamic model and molecular dynamics simulations provided more detailed insights into the efficiency of GVL/water mixtures in maximizing solubilization of lignin, and into the molecular mechanism of lignin dissolution. Using GVL for this process instead of hazardous solvents like 1,4-dioxane or N-methyl-2-pyrrolidone achieves a green process for full lignin removal from lignocellulosic biomass (92-99 wt% yield, 93-99 % purity, depending on biomass source). Additionally, in the frame of this work we could show that GVL also effectively solubilizes residual hemicellulose, facilitating its degradation into sugar monomers at high yield (90 wt%) and purity (100 %), which can easily be separated from lignin through non-polar lignin precipitation. Moreover, in the course of this work, we could recover mostly lignin-free cellulose with high purity (> 99 %) from lignocellulosic biomass. GVL emerges as a significantly promising solvent for the complete fractionation of all components of lignocellulosic biomass, regardless of the source material. The fractionation process proved to be similarly efficient in every step for different biomasses and waste-stream biomasses. GVL could be recovered and reused in every separation step and even be prepared from hemicellulose monomers, enabling a sustainable biomass dissolution cycle. This cycle stands as a basis for producing valuable biopolymer molecules, from biodiverse origin, that can be further converted into quality products for the chemical industry. 1. Introduction The primary goal of the chemical industry today is its transition towards renewable and sustainable resources. Currently, there is an enormous demand for fossil-based chemicals and their derived products, a demand that is projected to double over the next 25 years1,2. However, with the continuous depletion of fossil resources, future production will be unable to meet this rising demand. Therefore, it is crucial to identify and develop alternative feedstocks that can replace fossil-based materials in the chemical industry. One such promising alternative is lignocellulosic biomass, with an estimated global annual production of 181.5 billion tons. This feedstock holds great potential due to its renewability, economic viability, and carbon neutrality3. Lignocellulosic biomass encompasses renewable resources such as wood, plants, agricultural residues, grasses, and plant-based industrial waste streams like nutshells4. Despite its vast availability, only 8.2 billion tons are currently utilized5, primarily for livestock feed (61 %), food (15 %), and energy production (16 %)2. Traditionally, biomass has been used in the paper industry to produce cellulose fibers3. However, the concept of biorefineries, which seek to fully exploit the potential of lignocellulosic biomass, is gaining attraction due to the versatility of the usable rawmaterials. These components could serve as precursors for producing sustainable energy6, biofuels7, bioplastics8, and various biochemicals9, such as amino acids10, platform chemicals like furfural11, or organic solvents like γ-valerolactone (GVL)12, thereby offering a viable alternative to fossil-based chemical production. Nevertheless, several challenges must be addressed to fully realize the biorefinery concept, including reducing processing costs, improving process efficiency, ensuring the complete valorization of all biomass components, scaling up to industrial levels, and competing with the established fossil-based market13. This study highlights the improvements to a complete separation of all components of lignocellulosic biomass using green solvents. With the green solvent GVL, complete lignocellulosic biomass fractionation can be achieved through a simple, mild, and optimized process. Additionally, the production of lignocellulose-based monomers can be subsequently done to receive valuable chemicals. This can lead to application-based development of specific biomass-based molecules in future studies. One such project is the creation of bio-based resin monomers replacing the toxic bisphenol A, which will be further addressed in our future work. Lignocellulosic biomass consists mainly of cellulose (44 %), hemicellulose (28 %), and lignin (20 %), with smaller fractions of proteins, other extractives (6 %), and ash (2 %). The overall structures of these three main biopolymers are depicted in figure (1). These proportions vary by approximately ± 10 %, depending on the biomass source14. Cellulose, the most abundant component, is a linear polysaccharide made up of β-(1,4)-D-glucopyranose units linked by glycosidic bonds. The degree of polymerization (DP) of cellulose ranges from 300 to 10,000, depending on its biomass origin15. This article shows the efficient separation of impurity-free cellulose from lignocellulosic biomass using GVL as a solvent. An issue is the dissolution of cellulose, which will be addressed in another publication. Figure 1: (a) Structural excerpt of lignin. (b) Structural excerpt of cellulose chains with intermolecular and intramolecular hydrogen bonding. (c) Structural excerpt of hemicellulose. (d) Possible monomeric sugar molecules that can be part of the substituted hemicellulose backbone in place of R. Hemicellulose, the second most abundant component of lignocellulosic biomass, is a heterogeneous polysaccharide primarily composed of xylose and glucose backbones, with varying pentose or hexose substituents depending on the biomass type16,17. Its structure is much less regular than cellulose, making it easier to dissolve and break down. Hemicellulose has significant potential for applications in the renewable chemical industry, historically being used primarily for biofuel production18. More recent applications include biodegradable films19, medical coatings for cancer therapy and wound dressings20,21. In this study, the degradation products of hemicellulose, particularly xylose, are being dissolved and separated from other biomass extractives. Xylose can act as a precursor for important platform chemicals in the future22. This aligns with the growing interest in replacing different fossil-based chemicals with renewable alternatives. However, a key challenge is to efficiently extract and isolate hemicellulose from biomass in a sustainable manner. Using GVL as the main solvent, this study aims to optimize a process that extracts all key biopolymers. This study intends to maximize the valorization of the entire biomass within a single energy efficient and ecofriendly process that is studied in more detail over future articles. Lignin is a complex, three-dimensional heteropolymer. It is composed mainly of the aromatic monomers sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol, which are interconnected by ether and carbon-carbon linkages. These monomers form the corresponding subunits syringyl (S), guaiacyl (G), and hydroxyphenyl (H). The monomers can bond through carbon-carbon linkages, including β-β´, β-1´, and 5-5´, or through carbon-oxygen bonds, such as β-O-4´, α-O-4´, and 4-O-5´ linkages. Additionally, combinations of carbon-carbon and carbon-oxygen bonds result in structures like phenyl coumaran and resinol within the lignin polymer network. The precise aromatic monomer and linkage composition of lignin varies significantly depending on the type of biomass23-39. Lignin in biomass is typically present in lignin-carbohydrate complexes with hemicellulose, enabling their co-extraction23. Due to its polyaromatic structure, lignin is one of the most promising bio-based materials for replacing fossil-derived products in the chemical industry, positioning lignocellulosic biomass as a promising renewable and sustainable feedstock. While lignin is currently mostly burned to recover process energy in the paper industry3, it has potential for a variety of applications. For instance, its high UV absorptivity (250–300 nm) and antimicrobial properties make it suitable for biodegradable UV-protective films in food packaging or skincare30-32. Additionally, lignin's antioxidant properties, which are linked to its hydroxy group content on benzyl rings, allow it to be used as an antioxidant33,34. Other potential applications include its use in flavor or fragrance monomers35 and medical treatments for conditions such as diabetes and cancer36. Currently, vanillin is the only product industrially derived from lignin, accounting for 1% of global vanillin production37,38. In this work, lignin extraction was carried out using a well-established extraction method involving an acidic mixture of water and the green organic solvent GVL39,40. These results were compared with lignin extraction and characterization from a similar process using the same solvent under the addition of certain aldehydes to prohibit extensive lignin condensation reactions, as first described by Lan et al. (2018)41. The default extraction process made the condensation of lignin molecules at their diol-sites towards carbon-carbon linked oligomers relatively common. As these oligomers come with very inconsistent properties, which are unfavorable for further using extracted lignin in specific ways that demand certain structural elements, the aldehyde-assisted process was employed to increase consistency and largely reduce condensation in extracted lignin oligomers. With an extraction-based condensation rate of close to zero, the extraction efficiency was predicted to increase significantly compared to the extraction of condensed lignin oligomers. A central focus of this study was the use of the green solvent GVL. The aim was to partly explore GVL's potential for complete fractionation of lignocellulosic biomass from various sources. GVL is considered a green solvent because it poses minimal risks to human health and the environment, while also adhering to most of the twelve principles of green chemistry42,43. It offers numerous advantageous chemical and physical properties: GVL is a colorless, low-viscosity liquid with a high boiling point of 207 °C and a low melting point of -31 °C. It is an intermediately polar, aprotic solvent, miscible with water and many organic solvents, and has a low vapor pressure of 32 Pa at 20 °C, reducing its flammability risk. GVL only begins to decompose at temperatures above 150 °C under acidic conditions, or under basic conditions, undergoing a reversible ring-opening reaction to form 4-hydroxyvaleric acid. Strong acids can also catalyze this reaction at lower temperatures, but the equilibrium concentration of degradation products remains at only 4%44. GVL can be produced from biomass through various pathways, including the selective hydrogenation of biomass-derived levulinic acid45. Some very interesting prospectives are the selective catalytic conversion of xylose and subsequently furfural to GVL46-48, or the currently ongoing industrialization of fructose to GVL49. Many of these biomass-originating platform chemicals can therefore be used to efficiently produce GVL, facilitating its use as a solvent in its own production process and thereby contributing to an eco-responsible lignocellulosic biomass extraction cycle50. Given GVL's ability to dissolve lignin51 and hemicellulose52 in substantial quantities, and its prospect to improve cellulose dissolution53, it was considered an ideal solvent for this study to achieve complete biomass fractionation, replacing more hazardous solvents from methods such as the aldehyde-extraction process. Additionally, GVL can be easily recycled through simple distillation techniques54 or CO2 extraction55. The fractionation process of lignocellulosic biomass is primarily built on the efficient and mild separation of lignin. This is due to the linkages between lignin and the cellulose and hemicellulose, binding the biomass together23. With an effective separation of lignin in maximally natural constitution with its native ether content comes the fractionation of all biopolymers in high yields and quality. Since GVL/water mixtures showed good lignin solubility already in the past39,53, we investigated the performance of GVL/water solvent system by means of advanced computational techniques, namely, the quantum-mechanics-aided COSMO-SAC model and atomistic molecular simulations, to provide further theoretical insights into the behavior of the proposed fractionation media. Hereby, we provide mechanistical arguments to explain why a mixture of GVL and water can be the optimal green solvent for lignin separation by comparison with other common lignin solvents and investigation of its interaction with lignin molecules. Green biomass fractionation processes have been developed in the past to improve the sustainability and efficiency of biomass fractionation. A common approach is the use of ionic liquids (IL´s) to dissolve biomass components and fractionate them. IL´s are mainly organic salts with melting points < 100 °C. They provide advantages such as low volatility, high thermal stability, and high adaptability to certain processes due to the large number of possible combinations of cations and anions. IL´s mainly disrupt the intermolecular hydrogen bonds between biopolymers, facilitating their fractionation and dissolution56. Examples for IL´s being developed for biomass fractionation are especially imidazolium-based IL´s like 1-ethyl-3-methylimidazolium acetate57 or 1-butyl-3-methylimidazolium acetate58. These IL´s have high cost and toxicity, and do not show complete fractionation ability of all biopolymers57,58. Protic IL´s such as triethylammonium hydrogen sulfate dissolved up to 85 % lignin and 100 % hemicellulose from Miscanthus x giganteus with low production cost and high recovery. Still, the purity and quality of the extracted biopolymers was not yet efficient enough for industrial application59. Other general problems of using IL´s for biomass fractionation are their high costs, potential toxicity, and difficult recovery56,60. A better alternative to IL´s are deep eutectic solvents (DES). DES are mainly formed by mixing a hydrogen bond accepting and a hydrogen bond donating component, resulting in a mixture with a melting point lower than that of the individual components. DES are generally considered more biodegradable, less toxic, and more cost-efficient than IL´s59,61. Several DES formulations have been developed to fractionate biomass. A choline chloride/oxalic acid DES showed medium delignification capabilities from wheat straw of 57.9 % lignin removal. This process allows a breakdown of the lignin structure, but struggles with further processing cellulose into valuable products, while fully removing lignin and hemicellulose from the biomass62. A DES based on choline chloride/levulinic acid could effectively fractionate cellulose (3 % lignin left), lignin, and hemicellulose from acacia wood at high temperatures of 160 °C, still being very energy-intensive and the lignin being of very poor quality, meaning high condensation and low purity63. Other DES, or natural deep eutectic solvents (NADES), based on lactic acid64, or three components DES, with additional protection agents like ethylene glycol, that ensure an uncondensed state of lignin65 also yielded great results for biomass fractionation, yet still struggle with DES recovery, high temperatures up to 160 °C, recovery of the hemicellulose component, biodiversity, and long reaction times up to 24 hours. DES provide clear advantages compared to IL´s in lignin separation and subsequent complete biomass fractionation but are not yet effective enough for industrial application56,61,65. A last possibility is the use of organosolv processes to fractionate biomass. This method has been highly developed, with the use of different organic solvents like ethanol, methanol or acetone in combination with water and other catalysts/additives to solubilize lignin and hemicellulose, leaving behind a pure cellulose residue. Core mechanisms are the disruption of lignin-hemicellulose complexes due to slightly acidic conditions, and the dissolution of lignin and hemicellulose fragments through the organic solvents. While pathways to efficiently separate lignin, hemicellulose, and cellulose have been found, problems like solvent toxicity, energy-intensive and solvent-intensive process conditions, lack of biodiversity, or unsatisfactory quality of biopolymer products still reside66,67. While simple mixtures of acidic organic solvent/water mostly extract lignin in high quantities, elevated temperatures are necessary due to the condensation of lignin during the process, leading to lower value products. Additionally, many organic solvents pose the risk of flammability or other safety/environmental risks due to their high volatility6870. GVL has proven to be an excellent option for organosolv processes due to its advantageous properties67, with its large-scale availability increasing significantly over the next few years. Due to additives that can reduce lignin condensation and thus fractionate all biopolymers at relatively low temperatures below 100 °C, GVL could be industrially used in the future. The challenge of designing a process using the advantages of GVL combined with simple solvent recovery, low process temperatures, low solvent quantities, high biopolymer yields and purities, high lignin quality, low extraction times, and especially biodiversity of the process has been developed in the frame of this work. In summary, this study proposes a comprehensive process for the separation of all components of lignocellulosic biomass from various sources using GVL as the main solvent. The overarching goal of this research was to complete a process for the full and biodiverse fractionation of all components of lignocellulosic biomass using green solvent mixtures at mild conditions. Cellulose, lignin and hemicellulose were extracted from biomass using mild GVL extraction processes. The differences between a default extraction process with GVL and an aldehyde-assisted GVL extraction process were examined qualitatively. The extractives from all three biopolymers can be considered for further applications, as pathways for specific applications were developed, which will also be specifically addressed in future research. 2. Materials and Methods 2.1 Chemicals and materials Sulfuric acid (95-98 %, CAS 7664-93-9), L-cysteine (98 %, CAS 52-90-4), DMSO-d6 (99.8 %, CAS 2206-27-1), acetaldehyde (for synthesis, CAS 75-07-0), acetone (>99.5 %, CAS 6764-1), dimethyl sulfoxide (DMSO) (for analysis, CAS 67-68-5), 1,4-dioxane (for analysis, CAS 123-91-1), ethanol (for analysis, CAS 64-17-5), ethyl acetate (for analysis, CAS 141-78-6), para-formaldehyde (37 % in water, CAS 50-00-0), hydrochloric acid (37 %, CAS 7647-01-0), methanol (for analysis, CAS 67-56-1), toluene (for analysis, CAS 108-88-3), sodium hydroxide (for analysis, CAS 1310-73-2), propionic aldehyde (> 98 %, CAS 123-38-6), were purchased from Merck KGaA (Darmstadt, Germany). γ-Valerolactone (GVL) (anal. Grade, CAS 108-29-2) was provided by KVT-Technology/Glaconchemie (Graz, Austria). n-Hexane (> 99 %, CAS 110-54-3), n-heptane (> 99 %, CAS 142-82-5), methyl-tetrahydrofuran (> 99 %, CAS 96-47-9) were purchased from Fisher Scientific GmbH (Schwerte, Germany). Sodium hydrogen carbonate (> 99.5 %, CAS 144-55-8) was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Millipore distilled water was cleaned in a Millipore purification system (electrical conductivity p > 18 MΩcm). Peanut shells and skin were removed from store bought peanuts; pistachio shells were removed from store bought pistachios; hazelnut shells were removed from store bought hazelnuts. Walnut shells were removed from home grown walnuts. Coffee silverskin was provided by Rheorik Rösterei & Feinkost GmbH (Regensburg, Germany). Birch and Beech wood were provided by Schreinerei Heitzner (Traitsching, Germany). Cashew shell powder was provided by Orpia-Innovation (Paris, France). Almond shells were provided by Otto A. Müller Recycling GmbH (Ahrensburg, Germany). Spruce sawdust was provided by HCR Holz Centrum Regensburg GmbH (Regensburg, Germany). 2.2 Lignin and Hemicellulose extraction process The default lignin extraction follows a method established by Cheng et al.39. Using GVL/water mixtures as extraction solvent, the optimum extraction parameters were determined through variation in temperature, solid loading, extraction time, solvent composition and pH value. Biomass samples were dried at 75 °C for 48 h to remove water residues, then milled into powder with a diameter of 2 mm. An amount of 9 g of the milled biomass powder was transferred into a 250 mL round flask. To the flask, 90 mL of a GVL/water (9/1 wt/wt) mixture were added. The mixture was subjected to ultrasonic treatment for 30 min at 60 °C to achieve optimal swelling of the lignocellulose structure, enabling a good solvent penetration for the extraction. A concentration of 0.075 mol/L sulfuric acid was set in the mixture. The round flask was equipped with a magnetic stirring bar and subjected into an oil bath at 120 °C for 2 h under continuous stirring. These optimum conditions for the GVL-based organosolv process were determined by variation of solid loading, acid concentration, solvent composition, extraction time, and reaction temperature to maximize yield and purity of the extracted lignin (Details see SI chapter 1). After the extraction finished, the round flask was air-cooled to room temperature before being put in an ice bath for 20 min to prohibit intense condensation reactions. Residual biomass residue, mostly cellulose, was removed through a Buechner filter from the lignin/hemicellulose/GVL/water solution. The filter cake was washed with 15 mL of GVL and 15 mL of water three times each. The filtrate was collected in an appropriate beaker and subjected to 10 times its volume of water to precipitate the lignin. The lignin was collected through centrifugation at 4000 rpm for 15 min. The residual solution was subjected to vacuum distillation at 60 °C and 0.001 bar to completely remove the solvent mixture and collect the hemicellulose residues. The solid lignin was washed with distilled water multiple times to remove GVL and hemicellulose residues (which were in the end added to the hemicellulose fraction) and then dried at 75 °C for 48 h in a drying oven, then for 24 h in a vacuum desiccator equipped with silica gel. Finally, light brown colored lignin powder was obtained. The color varied for different biomass sources. Each biomass sample underwent three separate extraction steps to ensure nearly complete lignin extraction. The lignin yield was calculated through equation (1). 𝑌𝑖𝑒𝑙𝑑 = 𝑚𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑙𝑖𝑔𝑛𝑖𝑛 𝑚𝑏𝑖𝑜𝑚𝑎𝑠𝑠 ∙ 𝑙𝑖𝑔𝑛𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 ∙ 100 (1) Following this, the purity of the extracted lignin was assessed using a modified version of the CASA method, see section 2.6.2. 2.3 Aldehyde assisted extraction process The modified extraction method of lignin and hemicellulose is based on the aldehyde-assisted fractionation established by Lan et al. (2018)41. The method was optimized for our extraction process using GVL by varying temperature, extraction time, acid content, water content, and neutralization method (for details see SI chapter 2). 5 g of biomass, 27.5 mL of GVL, 5.3 mL of propionaldehyde, and 0.94 mL of 37% HCl solution (instead of sulfuric acid, details see SI) were added into a 100 mL glass flask containing a 20 mm PTFE-coated stir bar. After connecting the flask to a reflux condenser, the mixture was heated to 85 °C for 3 h (heat up time included). To ensure ideal stirring during the reaction, the stirrer was turned on 15 min into the reaction and was set to approx. 400 rpm. After completion of the extraction, the flask was set aside until it cooled down to room temperature. The mixture was filtered using a Buechner funnel and washed with 5 x 5 mL GVL and 2 x 10 mL methanol (important for neutralization, see SI chapter 2). The collected cellulose was set aside. The filtrate was neutralized by adding 1.7 g of NaHCO3 and stirred for approx. 1 h at room temperature. After neutralization residual NaHCO3 and resulting NaCl were filtered using a Buechner funnel. Neutralization was necessary to avoid deprotection and recondensation during lignin recovery. GVL from the neutralized filtrate was evaporated by using a vacuum distillation setup (1 x 10-3 bar, 60 °C). The resulting slurry was redissolved in 10 mL ethyl acetate until it was not viscous anymore. Lignin was precipitated by slowly pipetting one part (volume) of the prepared solution to 10 parts of vigorously stirring heptane. The stirring was stopped after the residue was disaggregated, and the yellow-colored supernatant was decanted into an Erlenmeyer flask. The remaining lignin-rich residue was solidified by adding 20 mL of distilled water to the gel-like residue. The residue was filtered using a Buechner funnel and washed with 2 x 10 mL distilled water. The recovered lignin was dried overnight using a desiccator. The resulting lignin powder was purified using the following procedure: 10 mL diethyl ether (see SI, chapter 2) were added to the lignin powder and placed into an ultrasonic bath. The yellow-colored supernatant liquid was decanted through a fritted filter funnel (Pore size: G3) and added to the hemicellulose fraction. The purification process was repeated one more time. The resulting lignin was dried at 40 °C for 1 h and stored in a closed container. The depolymerized hemicellulose from the heptane/ethyl acetate filtrate and the washing step was received by distillation using a rotary evaporator. The heptane/ethyl acetate solvent mixture was used instead of water for precipitation of the lignin to reduce the overall solvent consumption from a total of 322.5 mL of solvents in the default extraction process to 197.5 mL of solvents in the aldehyde assisted extraction process. Moreover, the high enthalpy of vaporization for water could be avoided, and easily vaporizable n-heptane could reduce the distillation energy needed. Still, aqueous precipitation can be used in this process if a complete set of green solvents is necessary. The theoretical lignin extraction yield was calculated by using equation 1 to further calculate the actual lignin extraction yield, as the introduction of protecting groups altered the molecular weight of lignin structures. This was suggested by lan et al. (2019)71. 𝑌𝑖𝑒𝑙𝑑 =𝑚𝑏𝑖𝑜𝑚𝑎𝑠𝑠 ∗𝐿𝐶∗[(236 196∗%𝐺+266 226∗%𝑆)∗𝐸𝐶+(1−𝐸𝐶)] (2) In equation (2), m means weight of biomass subjected to extraction, LC means lignin content in the biomass, %G/%S means percentage of guaiacyl/syringyl units present in lignin, and EC means the ether content. S/G-ratio and ether content are determined by 2D HSQC NMR spectroscopy. The molecular weights of the β–O–4 bonded monomeric units in native lignin are 196 g/mol for a G unit and 226 g/mol for an S unit. Following protection with propionaldehyde, these molecular weights increase to 236 g/mol for G and 266 g/mol for S. 2.4 Solvent recovery process Solvents from all mixtures containing GVL could be recovered for solvent reusability. GVL was recovered through simple vacuum distillation at 60 °C and 10-3 bar. Water can be separated from GVL through rotary vacuum evaporation at 40 °C and 0.1 bar. Similarly, ethyl acetate (50 °C, 0.150 bar) and heptane (50 °C, 0.07 bar) can be separated and reused for lignin precipitation after rotary vacuum evaporation. 2.5 NMR spectroscopy NMR experiments were conducted using an Avance III HD 400 spectrometer (400.13 MHz Proton, 5 mm BBO 400 SB BB-H-D sample head with Z-gradient). Lignin samples were dissolved in DMSO-d6 at 60 mg/mL and transferred to NMR glass tubes after complete dissolution. For all samples, 1H-NMR and 2D-HSQC-NMR spectra were measured to evaluate the S/G-ratio and the ether content of lignin from different biomass sources to accurately calculate lignin yields through equation (1). For lignin from the aldehyde extraction process, the protection rate can also be calculated through 2D-HSQC-NMR. SpinWorks was used to analyze the NMR spectra. The detailed NMR assignment method can be found in SI chapter 3. 2.6 UV/Vis spectroscopy 2.6.1 Spectroscopy UV/Vis spectroscopy experiments were conducted on a double-beam UV/Vis spectrophotometer from Perkin Elmer Lambda 19 UV/Vis/NIR (Dodgau, Germany). Examined samples were measured in micro-UV cuvettes with an optical path length (L) of 1 cm from brand GmbH & Co.KG (Wertheim, Germany) against a reference sample at 25 °C in a wavelength range from 200 nm to 400 nm. 2.6.2 CASA lignin content and purity determination In all biomass samples, the lignin content was determined through the Cysteine Assisted Sulfuric Acid (CASA) method, which was first described by Lu et al. (2021)72. A stock solution of 0.1 g/mL L-cysteine in 72 % sulfuric acid was prepared. In 1 mL of stock solution, 20 mg of a ground biomass sample (m) were dissolved under stirring at room temperature, before being diluted to 100 mL (V) with distilled water. The UV/Vis absorbance (𝐴283𝑏𝑖𝑜𝑚𝑎𝑠𝑠) was measured at 283 nm to determine the overall lignin content using equation (3) with a molar absorption coefficient ε of 17.25 L/g cm. The lignin content can be overestimated due to other aromatic extractives from proteins, but this is generally negligible due to their very low content. 𝐿𝑖𝑔𝑛𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 [%]=𝐴283𝑏𝑖𝑜𝑚𝑎𝑠𝑠 ∙𝑉 𝜀∙𝐿∙𝑚 ∙ 100 (3) Additionally, the lignin purity could be determined by using a modification of the CASA method, which was validated internally. The loss of lignin in a representative biomass sample modeled with extracted lignin was measured, which correlated to the purity of the extracted lignin sample. To this end, instead of biomass, an equivalent amount of extracted lignin to the lignin content in the respective biomass (e.g. 1 g biomass, 20 % lignin content → 0.2 g extracted lignin) was dissolved in 1 mL stock solution and diluted to 100 mL with distilled water. Equation (4) was used to calculate the lignin purity by dividing the absorbance of the extracted lignin sample (𝐴283) by the absorbance of the respective biomass sample (𝐴283𝑏𝑖𝑜𝑚𝑎𝑠𝑠). Since aromatic parts from proteins are also removed during extraction, the lignin purity can be underestimated. can be drawn on how to efficiently extract lignin from lignocellulosic biomass without wasting the potential of the residual biopolymers. The lignin isolated from biomass through the default GVL-based extraction process was qualitatively identified through 1H-NMR spectroscopy to confirm the extraction products. Table 1: Analysis of the lignin content for different types of biomasses, including wood and different waste streams, using the CASA method described in chapter 2.7.2. The lignin was extracted through the GVL-assisted organosolv process described in chapter 2.2. The extractions were conducted in triplicates, meaning each biomass sample was used as a substrate three subsequent times to remove most of the lignin from the biomass. The yield is given as a percentage of the determined lignin content in the respective type of biomass. Additionally, the purity of the extracted lignin was determined through the CASA method, as certain extractives (degraded sugars, proteins, ash) might be co-extracted and reside in the lignin samples. The different biomass sources showed very different overall lignin contents, which is due to their different structural properties. Woods like beech, birch and spruce show lignin contents from 22-23 wt%. Generally, nutshells show a higher lignin content (27-33 wt%), which can be attributed to their overall more rigid structure with higher mechanical strength and reduced water absorptivity, making them more durable and protected95. Nutshells also have specialized cells called sclereids, that are heavily lignified, contributing to the overall higher lignin content and to the hardness of the shell96. These cells only occur in the part of the biomass that is the shell, meaning that typical nut skins show lower lignin contents, which can be seen in the lignin contents of coffee silverskin and peanut skin (21-26 wt%). Overall, all biomasses enabled high triplicate lignin extraction yields from 89-97 % with high purity values from 83-96 % depending on the biomass source. GVL/water (9/1 wt/wt) proved to be an effective solvent for lignin extraction for not only wood residues but also different lignocellulosic waste streams like nutshells or nut skins. This underlines the biodiversity of using the process based on GVL for lignocellulosic biomass extraction. The efficiency of the process was still improvable, as high yields of lignin were only achieved after three subsequent extraction steps at high temperatures of 120 °C. The extraction yield decreased with higher Type of biomass Lignin content [wt%] Step 1 extraction yield [% of lignin content] Step 1 lignin purity [%] Step 2 extraction yield [% of lignin content] Step 2 lignin purity [%] Step 3 extraction yield [% of lignin content] Step 3 lignin purity [%] Triplicate extraction yield [% of lignin content] Purity of extracted lignin [%] spruce sawdust 22 57 ± 2 94 ± 1 28 ± 1 93 ± 1 12 ± 1 93 ± 1 97 ± 3 93 ± 1 coffee silverskin 21 61 ± 2 96 ± 1 21 ± 2 96 ± 1 5 ± 1 96 ± 1 87 ± 6 96 ± 1 walnut shell 28 53 ± 1 94 ± 1 27 ± 1 93 ± 1 10 ± 0 93 ± 1 90 ± 2 93 ± 1 hazelnut shell 29 51 ± 1 95 ± 1 28 ± 0 93 ± 1 13 ± 0 93 ± 1 92 ± 1 93 ± 1 peanut skin 26 46 ± 1 94 ± 1 34 ± 0 90 ± 1 15 ± 0 90 ± 1 95 ± 1 90 ± 1 peanut shell 33 41 ± 1 87 ± 1 34 ± 0 83 ± 1 14 ± 1 83 ± 2 89 ± 2 83 ± 2 pistachio shell 15 43 ± 1 91 ± 1 33 ± 1 89 ± 3 14 ± 0 89 ± 3 90 ± 2 89 ± 3 almond shell 29 50 ± 2 91 ± 1 30 ± 1 90 ± 1 12 ± 0 90 ± 1 92 ± 3 90 ± 1 cashew shell 27 45 ± 4 95 ± 2 32 ± 1 93 ± 3 15 ± 1 96 ± 4 92 ± 6 96 ± 4 Beech sawdust 22 51 ± 1 94 ± 1 28 ± 1 94 ± 1 15 ± 0 93 ± 1 94 ± 2 93 ± 1 Birch sawdust 23 53 ± 1 94 ± 1 27 ± 0 94 ± 1 15 ± 0 93 ± 1 95 ± 1 93 ± 1 extraction steps, as less lignin is present in the fiber structure. Residual lignin also showed higher condensation when examining the fractions through 2D-HSQC-NMR spectroscopy, which could be seen in an increasingly overlapping signal for the aromatic subunits G and S for lignin fractions from higher extraction steps (figure 4 (b)). The condensation mechanism of lignin structures is shown in figure 4 (a). The first extraction steps yielded 41-61 % lignin, second extraction steps yielded 21-34 % lignin, and third extraction steps yielded 5-15 % lignin, depending on the biomass origin. The purity of the lignin from different extraction steps did not show any significant changes, which is probably due to no change in extraction conditions. Another reason for the decrease in lignin extraction efficiency with the number of extraction steps is the increase in condensation of lignin structures. This is shown in figure 4 (b). Lignin, extracted from the guaiacyl (G) rich hardwood biomass spruce, after the first extraction step already shows medium condensation. Signals for G6 and G5 overlap, indicating condensation. With an increasing number of extraction steps, the overlap of G signals increases. After extraction step 3, the extracted lignin shows large condensation, as G2, G5, and G6 signals all overlap. For the exact assignment of the signals, see SI chapter 3. The resulting condensed lignin structures become more complex and less consistent. With the decrease in hydroxy groups and the increase in C-C bonds, the lignin structures become more thermally and chemically stable. This also comes with a loss in polarity and an increase in structural rigidity97, making its solvent penetration and solubilization with the intermediately polar solvent GVL more challenging. For the extraction process, GVL is essential due to its ability to dissolve the lignin very efficiently. GVL acts as the main solvent for the fractionation of all biopolymers in lignocellulosic biomass, creating a green solvent mixture for the process. Due to its size and intermediate polar properties, GVL cannot only swell the biopolymer structure, but then disrupt hydrogen bonding and hydrophobic interactions between cellulose and lignin-hemicellulose-complexes. This leads to lignin being isolated from the main cellulose fiber much easier than with other green solvents98-100. Additionally, acidic conditions are necessary to break apart the large lignin structures into smaller oligomers, enabling their dissolution in the solvent system. Many applications can be considered for isolated lignin from various waste streams, depending on its composition. Condensed lignin structures might form stronger interactions with the cellulose and hemicellulose components in the biomass, coming from increased hydrophobic interactions between the now more hydrophobic lignin and hydrophobic parts of the other biopolymers101, and the reduced flexibility of the lignin structures102. Another reason for this can be the increased hydrogen bonding strength between lignin hydroxy groups, now surrounded by larger high electron density aromatic systems103, and cellulose/hemicellulose hydroxy groups. Additionally, π-π stacking interactions between large aromatic domains of lignin molecules might increase, which also adds to the decreased extraction efficiency97. To efficiently extract lignin with GVL from biomass, the extraction method had to be altered to preserve native lignin structures (these only include native C-C linkages). This should increase the extraction efficiency by preserving an intermediately polar lignin structure, while creating lignin extraction products with consistent structural properties. This will be addressed in the following chapter. Figure 4: (a) Lignin condensation process, starting from a guaiacol subunit with β-1,4-diol function and ether linkage to another guaiacol subunit, representing the guaiacol-rich hardwood lignin of spruce sawdust. The condensation reaction is an acid-catalyzed nucleophilic aromatic substitution, with another lignin fragment forming a C-C bond with the starting lignin molecule. The product, condensed lignin, can further undergo condensation, forming a highly complex and random lignin structure [76, 77]. (b) 2D-HSQC-NMR spectrum excerpts of extracted lignin from spruce sawdust biomass after different numbers of extraction steps (1, 2, and 3 extraction steps). The excerpts show the aromatic region of the 2D-HSQC-NMR spectra, ranging from 6.4 to 7.3 ppm on the 1H-scale (x-axis), and from 110 to 120 ppm on the 13C-scale (y-axis), respectively. Due to its hardwood nature, spruce sawdust lignin consists mainly of guaiacol subunits. The signals for G2, G5, and G6 are labelled inside the spectra. An overlap of multiple guaiacol subunit signals in the 2D-HSQC-NMR indicates condensation of lignin structures. Before any lignin extraction, some biomasses can provide valuable extractives that can be separated from the fiber part of the biomass before. These extractives are low molecular weight compounds extrinsic to the plant cell walls to protect them from degradation and growth alteration agents like bacteria or fungi. They can be divided into terpenoids, polyphenols, waxes and fats, salts of organic acids, proteins, and alkaloids. The exact extractive composition depends largely on the type and nature variance of a certain lignocellulosic biomass104. The simple removal of valuable extractives before insoluble fiber fractionation was conducted for coffee silverskin, which is very rich in caffeine and certain polyphenols. These were extracted with water in a multicycle process, which was studied in a separate work with Chemat et al. (2024)105. By removing these extractives, the CASA lignin content and the purity did not change, which verifies the insignificance of partly aromatic extractives in the employed lignin purity determination method. This concludes that by a simple cascade process, different extractives can be separated from certain biomasses with green solvents before fractionation of the insoluble fibers, creating more valuable products. This can be very interesting in terms of valorization of wastebiomass, like coffee silverskin or other waste products. 3.2.2 Aldehyde-assisted lignin extraction For the modified lignin and hemicellulose extraction process from lignocellulosic biomass, the method of aldehyde-assisted fractionation, first established by Lan et al. (2018)41, was adapted and optimized for our process based on GVL. This method uses aldehydes as protecting agents for the β-1,4-diol functions in lignin polymers, forming acetal-functionalities in the process. While this process does not use GVL/water mixtures from the start, the aqueous solution of HCl provides enough water to establish the synergistic solvent effect that was found with the COSMO-SAC and MD simulations. The reaction of the acetal-protection process is depicted in figure 5 (a). These acetals cannot be broken down in the lightly acidic environment of extraction, thus protecting the lignin structures from condensation during the extraction process. This was expected to yield more consistent lignin structures with higher ether-contents and an overall more consistent structure. These lignin structures were also expected to be more suitable for subsequent hydrogenolysis towards lignin monomers, as higher overall ether-contents and less condensed aromatic structures lead to more cleavable sites between lignin subunits. This process was previously conducted with 1,4-dioxane but was now performed with GVL under mild conditions. The optimization of extraction parameters has shown that 13 % solid loading of dried and ground biomass, 84/16 wt/wt of GVL/aldehyde (in this case, propionic aldehyde was used) mixture composition, and 0.34 mol/L of hydrochloric acid at a temperature of 85 °C for an extraction time of 3h were the optimal extraction conditions (more details for process optimization see SI, chapter 2). The lignin yield (calculated through comparison with the theoretical yield in equation (1) due to alteration of molecular weight of lignin after acetal functionalization) and purity for each biomass using this improved method, as well as the lignin protection rate analyzed through 2D-HSQC-NMR (details see SI chapter 3) is shown in table 2. Table 2: Lignin quantitative analysis for the modified extraction process using an acidic mixture of GVL/propionic-aldehyde, including the overall lignin content for each biomass determined through CASA UV/Vis spectroscopy, the extraction yield calculated through equation (1), the purity of extracted lignin determined through CASA UV/Vis spectroscopy, and the lignin protection rate analyzed through 2D-HSQC-NMR spectroscopy. Type of biomass Lignin content [wt%] Extraction yield [% of lignin content] Purity of extracted lignin [%] Lignin protection rate [%] spruce sawdust 22 99 ± 1 99 ± 1 100 coffee silverskin 21 96 ± 2 97 ± 1 100 walnut shell 28 95 ± 2 96 ± 1 100 hazelnut shell 29 95 ± 1 96 ± 1 100 peanut skin 26 95 ± 1 97 ± 1 100 peanut shell 33 93 ± 2 93 ± 2 100 pistachio shell 15 92 ± 2 94 ± 2 100 almond shell 29 95 ± 3 97 ± 1 100 cashew shell 27 93 ± 6 97 ± 1 100 Beech sawdust 22 99 ± 1 98 ± 1 100 Birch sawdust 23 97 ± 1 99 ± 1 100 Overall, the modified process achieves higher lignin yields with higher purities after only one extraction step than the default extraction process achieves after triplicate extraction steps on the same biomass. In contrast to the dioxane-based aldehyde assisted extraction process, the partly mentioned advantages of the GVL-based aldehyde assisted extraction process are manifold: zero solvent toxicity, no solvent flammability, no formation of explosive hydroperoxides, solvent recovery without degradation, green synthesis, or biodegradability. However, all these advantages are only relevant if the performance of the GVL process is comparable. The optimized process conditions (with non-polar precipitation) were used with both solvents to obtain a performance comparison of both methods. Spruce and birch were used as the extracted biomass. Both isolated lignins behaved and appeared very similar, with highly similar yields, and both sharing zero extraction-based condensation. In summary, GVL offers a highly similar performance as a solvent in biomass fractionation compared to dioxane. Differences in the quality of the isolated lignins are not significant. These are highly promising results for the future use of GVL as the solvent in AAF biorefineries or for developing new biomass fractionation procedures. The protection of the β-1,4-diol groups was very successful, leading to protection rates of 100 % for lignin from every examined biomass source. The 2D-HSQC-NMR signals for the protection group hydrogen atoms are shown in figure 5 (d), and the signals for the lignin subunit compositions are shown in figure 5 (c) (both for spruce sawdust lignin). Figure 5: (a) Aldehyde induced protection reaction of β-1,4-diol functions in lignin structures. The reaction takes place in a lightly acidic environment within the solvent GVL. For this work, propionic aldehyde, acetaldehyde, and formaldehyde were used. Full acetals are formed, protecting lignin structures from condensation. (b) Nomenclature of protected lignin structures after aldehyde protection reaction for this work. (c) 2D-HSQC-NMR spectrum excerpt from spruce sawdust lignin after aldehyde-assisted extraction. For the guaiacol-rich spruce lignin, the signals in the aromatic region for G2, G5, and G6 are distinctly visible in the excerpt. The x-axis shows the 1H-scale, and the y-axis shows the 13C-scale for the 2D-HSQC-NMR spectrum excerpt. Distinctly visible signals for each aromatic proton linked to its carbon instead of signal overlap show the successful prevention of lignin condensation. Instead, the structure of native lignin was preserved during the extraction. (d) 2D-HSQCNMR spectrum excerpt from spruce sawdust lignin after aldehyde-assisted extraction. The excerpt shows the aliphatic region from 3.2-5.2 ppm on the 1H-x-axis and 50-100 ppm on the 13C-y-axis. The signals for the acetal-protected lignin linkage structures A1, Aα, Aβ, and Aγ, as well as signals for methoxy groups, and residual native β-β´ and β-5´ linkages are assigned respectively. The intensity of native C-C linkage signals and thus their compositional percentage depends entirely on the source of biomass used for lignin extraction. The β-1,4-diol structures were converted to acetal groups, which can be examined through the signal assignment for the respective A1, Aα, Aβ, and Aγ signals. Signals for native β-O-4´ linkages were not retained, but signals for native C-C linkages (β-β´ and β-5´) were still identified. The β-O-4´ linkages were quantitatively replaced by acetal functions, with some β-O-4´ linkages also being broken down during extraction. Thus, the lignin received after aldehyde-assisted extraction from biomass retained its native structure besides cleavage at random ether linkages due to the acidic environment. The extraction-based condensation is effectively zero, as signals for G2, G5, and G6 are distinctly visible and show no overlap. Using only one extraction step with largely milder conditions, compared to the default extraction process, preserves a large part of the lignin structure while interrupting intermolecular interactions to separate lignin from the carbohydrate fibers and eventually dissolve it in the GVL-based solvent. Comparing the extraction of lignin from spruce sawdust with aldehyde protecting agents at 85 °C (yield: 99 %) to the extraction without aldehyde protecting agents (default extraction process) at 85 °C (yield: 5 %), both after one extraction step, there is a clear gap in extraction yield. Reducing extractionbased condensation to effectively zero, the lignin can be extracted in its native form under milder conditions. Also, precipitation-based condensation was eliminated by switching the precipitation system from water towards a n-heptane/ethyl acetate based system. Using water led to lower protection and higher condensation even with the aldehyde extraction process (for more details, see SI chapter 2). With the protection of the diol groups, the polarity of the lignin is only slightly decreased compared to unprotected lignin. Instead of condensed, largely hydrophobic lignin, the now extracted lignin structures stay intermediately polar106, retaining the ability to be readily dissolved by GVL at high percentages. For condensed lignin, hydroxy groups at the γ-position can form stronger hydrogen bonds with cellulose and hemicellulose due to the increased electron density through the enlarged aromatic surrounding103. When using the aldehyde-protection process, lignin hydroxy groups are fully converted to acetals. This completely prevents hydrogen bonding between lignin β-O-4´ linkage domains and cellulose/hemicellulose fibers, enabling an efficient separation and subsequent dissolution in GVL. Still, hydrogen bonding can occur due to the presence of e.g. free phenolic hydroxy groups or ether linkages in the lignin structure. The swelling process of the biomass also plays a big role in efficient dissolution of lignin. Due to the flexibility and intermediate polarity of the acetalprotected lignin structures, solvent molecules have a much easier way of migrating into the biopolymer structure, swelling it and enabling dissolution by interrupting intermolecular hydrophobic interactions and hydrogen bonding interactions between lignin and cellulose/hemicellulose. The residual native C-C linkages that are present in lignin structures after extraction are only of small concentrations in most woods (beech, birch, spruce). Carbon linkages are more prevalent in lignin of more rigid biomasses, as they provide rigidity and less flexibility to the lignin and thus to the overall fiber structure. A correlation can be seen in the achieved lignin yields for different biomasses after aldehyde-assisted extraction. Lignin from biomasses such as nutshells achieve slightly lower yields compared to woods. In lignin from these biomasses, the content of native C-C linkages is also higher than in lignin from woods107. This native condensation of aromatic units provides stability and rigidity, but also slightly decreases the potential for efficient extraction of lignin. This again confirms the correlation between condensation and extraction efficiency for this GVL-based extraction process. In summary, the aldehyde-assisted GVL-based lignin extraction process leads to effectively no condensation between lignin structures during extraction, which leads to almost complete lignin removal after one extraction step at milder conditions than with the default extraction process. This is influenced by the different properties of the acetal-protected lignin compared to the condensed lignin. Native lignin condensation only slightly influences the extraction efficiency, but greatly influences the rigidity of the biomass source. 3.2.3 Hemicellulose extraction After the extraction of lignin and separation through either heptane or aqueous precipitation, the hemicellulose residues dissolved in the liquid fraction can be recovered through reduced pressure evaporation of the solvent. For the default extraction process using an acidic mixture of GVL/water, the hemicellulose residues are a mixture of monomeric sugars like xylose and glucose, which can be separated and further processed towards sugar-based products (see upcoming article). For the modified extraction process using an acidic mixture of GVL/protecting agent, these monomeric sugars are obtained in their acetal protected form. These acetal-protected sugar molecules show great perspectives for valorization, as the protection groups can be varied and even modified to yield different functionalities attached to the sugar molecules. Their qualitative assessment through 2D-HSQC-NMR spectroscopy and their structures, extracted from beech wood sawdust, are shown in figure 6. Since beech wood has a xyloglucan backbone and thus a high xylose and glucose content, the yield (see table 3, for all biomasses) of hemicellulose decomposition products and their composition (45 mol% of protected xylose, 40 mol% of protected glucose, 15 mol% of different other protected hexoses or pentoses, determined through quantitative 1H-NMR, see SI chapter 3) were determined. Using the GVL/protecting agent modified extraction process, monomeric protected sugars can be extracted in high yields and purities from all types of biomasses. The purity here describes the presence of lignin or cellulose residues in the hemicellulose solution, which is 0 % for all biomass sources due to the insolubility of lignin and cellulose in the hemicellulose solvent. Since lignin fully precipitates in the precipitation medium, and cellulose fully precipitates in the extraction solvent, the hemicellulose residue was completely ligninand cellulose-free. From the default extraction process, lignin-hemicellulose complexes are more intact during and after extraction, making it more difficult to separate hemicellulose from the other biopolymers. This leads to lower purities for both cellulose and lignin in the default extraction process due to hemicellulose residues present in their structure. The aldehyde-assisted extraction process in a GVL-based medium provides higher one-step yields of hemicellulose residues at milder conditions, which however now are di-acetalized. These protected sugar monomers can be tuned and used for various applications in the future. Their possible valorization will be addressed over the next articles. Table 3: Hemicellulose content for different biomass sources determined in through the carbohydrate determination method. The hemicellulose yields for the default extraction process and the aldehyde-assisted extraction process are given in respect to the overall hemicellulose content. Type of biomass Hemicellulose content [wt%] Hemicellulose yield default [%] Hemicellulose yield prot. [%] spruce sawdust 25 80 ± 4 98 ± 1 Figure 6: 2D-HSQC-NMR spectrum of the residual hemicellulose fraction from beech wood sawdust. Hemicellulose was decomposed into protected sugar units; in this case the protection agent was propionic aldehyde. Protected xylose and glucose are depicted in the spectrum with their respective signal assignments to specific protons and carbon atoms. coffee silverskin 32 82 ± 3 91 ± 1 walnut shell 22 81 ± 6 96 ± 3 hazelnut shell 22 81 ± 4 95 ± 3 peanut skin 19 75 ± 2 93 ± 1 peanut shell 16 77 ± 2 93 ± 1 pistachio shell 22 81 ± 4 96 ± 1 almond shell 23 84 ± 6 95 ± 4 cashew shell 11 68 ± 3 86 ± 1 Beech sawdust 31 87 ± 1 97 ± 1 Birch sawdust 32 85 ± 2 97 ± 1 3.2.4 Cellulose fractionation From the improved lignin and hemicellulose extraction process using an acidic mixture of GVL/water/protecting agent at 85 °C, cellulose was recovered through simple filtration, washing and mild drying processes. To analyze the yield and purity of cellulose from each extracted biomass sample, the cellulose content was determined using simple acid hydrolysis, and the cellulose purity after lignin and hemicellulose extraction was determined using the CASA method, of which the results are shown in table 4. An important note is that the lignin-free cellulose was received after one single lignin/hemicellulose extraction step with the aldehyde-assisted GVL extraction process (~ 0.6 wt% residual lignin for spruce sawdust measured with CASA), compared to the default extraction process using only an acidic mixture of GVL/water at 120 °C, which led to less pure cellulose (~ 3.2 wt% residual lignin for spruce sawdust measured with CASA) even after 3 subsequent extraction steps. To dissolve the cellulose, it is important to first completely remove the lignin and hemicellulose, as it interacts with the cellulose chains, forming lignin-hemicellulose-complexes around the chains and acting as a sort of fiber-glue, preventing efficient dissolution108. The aldehyde-assisted GVL extraction process removes more lignin and hemicellulose from the cellulose chains under milder conditions and less extraction steps (92-99 %) compared to the default extraction process (87-97 %). This also leads to cellulose being less degraded due to a lower possibility of polysaccharide hydrolysis. Additionally, only very small amounts of natively condensed lignin structures stick with the cellulose chains leading to higher cellulose purity for the aldehyde-assisted process (> 99 %) compared to the default process (> 89 %). This enables the cellulose from the aldehyde-assisted extraction process for further valorization. One particular way of cellulose valorization is connected to its efficient dissolution and regeneration to produce fibers. Current processes are either not green or not efficient enough109, which is why the problem of dissolving cellulose in a biodiverse dissolution process will be addressed in future work. Table 4: Cellulose content determined through acid hydrolysis for every biomass. The cellulose yield (determined gravimetrically) and purity (determined through CASA method) after a default extraction using an acidic mixture of GVL/water are given. Additionally, the cellulose yield and purity after the modified extraction using an acidic mixture of GVL/protecting agent are given. Type of biomass Cellulose content [%] Cellulose yield default [%] Cellulose purity default [%] Cellulose yield prot. [%] Cellulose purity prot. [%] spruce sawdust 43 91 ± 2 97 ± 1 97 ± 2 99 ± 1 coffee silverskin 22 87 ± 5 93 ± 1 98 ± 1 99 ± 1 walnut shell 40 92 ± 3 95 ± 3 97 ± 2 99 ± 1 hazelnut shell 41 92 ± 1 96 ± 2 97 ± 2 99 ± 1 peanut skin 35 90 ± 4 94 ± 1 98 ± 2 99 ± 1 peanut shell 41 90 ± 3 94 ± 1 98 ± 2 99 ± 1 pistachio shell 56 89 ± 3 92 ± 3 96 ± 1 99 ± 1 almond shell 39 93 ± 1 96 ± 2 97 ± 1 99 ± 1 cashew shell 37 81 ± 5 89 ± 7 93 ± 4 99 ± 1 Beech sawdust 41 92 ± 3 97 ± 1 98 ± 2 99 ± 1 Birch sawdust 40 93 ± 2 96 ± 2 98 ± 1 99 ± 1 3.3 Overview of the process for complete green lignocellulosic biomass dissolution The overarching goal of this research was to complete a process for the full and biodiverse fractionation of all components of lignocellulosic biomass using green solvent mixtures at mild conditions. GVL in an acidic mixture of GVL/water/lignin-protecting-agent at 85 °C proved to be effective in separating lignin and hemicellulose in an uncondensed way from lignocellulosic biomass, leaving behind pure cellulose. Hemicellulose is degraded to sugar units, mostly xylose and glucose, during the extraction process. The protecting agent that is responsible for extracting uncondensed lignin structures also protects the sugar molecules over acetal functions, Figure 7: Visualization of the complete lignocellulosic biomass separation, dissolution, and degradation using the green solvent GVL. Zero waste is left from the initial biomass. This process can be used for every lignocellulosic biomass, be it wood, nutshells, or different lignocellulosic waste-streams. leading to stable protected sugar molecules that can be isolated. Figure 7 shows a simple visualization for the full process studied in this work, including all process products. The lignin extraction was compared between an acidic GVL/water mixture and an acidic GVL/water/aldehyde mixture. Using aldehydes to protect the β-1,4-diol groups in lignin structures from condensation can be efficiently done in GVL to achieve full protection. This leads to higher lignin yields, higher lignin purity, and effectively no extraction-based condensation. The condensation of lignin structures influences their polarity, flexibility, and thus their solubilization and swelling behavior for specific solvents. Using aldehydes as protecting agents provides native lignin structures without altered properties, that can be deprotected and further broken down towards valuable monomers, which will be addressed in future work. The protected monomeric sugar units were also received from the extraction process and analyzed qualitatively and quantitatively. Using different biomasses leads to different yields of xylose, glucose, and smaller amounts of different hexoses and pentoses, as hemicellulose composition varies depending on the origin biomass. Depending on the need for certain applications, other protected sugars can be isolated for biomasses that have a low xylose content and higher contents of other sugar monomers. For protected xylose and glucose, applications can be found by varying the protecting agent and performing modification reactions, introducing functional groups into the xylose/glucose structure that can, e.g., induce curing reactions in resin formulations. Additionally, xylose can always be deprotected in a slightly acidic environment and used to produce the solvent GVL110. The isolated cellulose was analyzed on its yield and purity, which was compared for a default extraction process using only an acidic mixture of GVL/water, and a modified extraction process introducing a protecting agent for lignin and hemicellulose extraction. The latter yielded a purer cellulose after one extraction step at milder conditions compared to three extraction steps at harsher conditions for the default extraction process. The purer cellulose can be used for further valorization, which involves finding a green and efficient solvent for cellulose dissolution. Lowering the dissolution temperature and increasing the soluble amount of cellulose while maintaining the fast dissolution times and low viscosities are all challenges on cellulose solubilization that will be addressed in future work. The detailed, [56] Van Osch DJGP, Kollau LJBM, Van den Bruinhorst A, Asikainen S, Rocha MAA, Kroon MC. Ionic liquids and deep eutectic solvents for lignocellulosic biomass fractionation. Phys Chem Chem Phys. 2017;19:2636-55. [57] Da Costa Lopes AM, João KG, Morais AR, Bogel-Lukasik E, Bogel-Lukasik R. Ionic liquids as a tool for lignocellulosic biomass fractionation. Sus Chem Proc. 2013;1:3. [58] Smuga-Kogut M, Szymanowska-Powalowska D, Markiewicz R, Piskier T, Kogut T. Ionic liquid pretreatment of stinging nettle stems and giant miscanthus for bioethanol production. Sci Rep. 2021;11:184565. [59] Brandt-Talbot A, Gschwend FJV, Fennell PS, Lammens TM, Tan B, Weale J, et al. An economically viable ionic liquid for the fractionation of lignocellulosic biomass. Green Chem. 2017;19:3078-3102. [60] Husanu E, Mero A, Rivera JG, Mezzetta A, Ruiz JC, D’Andrea F, et al. Exploiting deep eutectic solvents and ionic liquids for the valorization of chestnut shell waste. ACS Sustain Chem Eng. 2020;8:18386-99. [61] Gholami M. Regeneration of deep eutectic solvent post biomass delignification [dissertation]. University of Twente; 2024. [62] Jablonsky M, Skulcova A, Kamenska L, Vrska M, Sima J. Deep eutectic solvents: fractionation of wheat straw. BioRes. 2015;10(4):8039-47. [63] Magalhaes S, Moreira A, Almeida R, Cruz PF, Alves L, Costa C, et al. Acacia wood fractionation using deep eutectic solvents: extraction, recovery, and characterization of the different fractions. ACS Omega. 2022;7:26005-14. [64] Mero A, Moody NR, Husanu E, Mezzetta A, D’Andrea F, Pomelli CS, et al. Challenging DES and ILs in the valorization of food waste: a case study. Front Chem. 2023;11:1270221. [65] Liu Y, Deak N, Wang Z, Yu H, Harneleers L, Jurak E, et al. Tunable and functional deep eutectic solvents for lignocellulose valorization. Nat Commun. 2021;12:5424. [66] Espinoza-Acosta JL, Ramirez-Wong B, Carvajal-Millan E, Torres-Chaves PI, Montano-Leyva B, BelloPerez LA. Ionic liquids and organic solvents for recovering lignin from lignocellulosic biomass. BioRes. 2014;9(2):3660-87. [67] Nair LG, Agrawal K, Verma P. Organosolv pretreatment: an in-depth purview of mechanics of the system. Bioresour Bioprocess. 2023;10:50. [68] Chin DWK, Lim S, Pang YL, Lam MK. Fundamental review of organosolv pretreatment and its challenges in emerging consolidated bioprocessing. Biofuels Bioprod Bioref. 2020;14:808-29. [69] Chen M, Malaret F, Firth AEJ, Verdia P, Abouelela AR, Chen Y, et al. Design of a combined ionosolvorganosolv biomass fractionation process for biofuel production and high value-added lignin valorization. Green Chem. 2020;22:5161-80. [70] Chin DWK, Lim S, Pang YL, Lim CH, Shuit SH, Lee KM, et al. Effects of organic solvents on the organosolv pretreatment of degraded empty fruit bunch for fractionation and lignin removal. Sustainability. 2021;13:6757. [71] Lan W, de Bueren JB, Luterbacher JS. Highly selective oxidation and depolymerization of α,γ-diol-protected lignin. Angew Chem Int Ed. 2019;58:2649-54. [72] Lu F, Wang C, Chen M, Yue F, Ralph J. A facile spectroscopic method for measuring lignin content in lignocellulosic biomass. Green Chem. 2021;23:5106-12. [73] Chu Y, He X. MoDoop: an automated computational approach for COSMO-RS prediction of biopolymers in ionic liquids. ACS Omega. 2019;4(1):1-12. [74] Casas A, Omar S, Palomar J, Oliet M, Alonso MV, Rodriguez F. Relation between differential solubility of cellulose and lignin in ionic liquids and activity coefficients. RSC Adv. 2013;3(10):3453-60. [75] Lin S-T, Sandler SI. A priori phase equilibrium prediction from a segment contribution solvation model. Ind Eng Chem Res. 2002;41(5):899-913. [76] Hsieh C-M, Sandler SI, Lin S-T. Improvements of COSMO-SAC for vapor-liquid and liquid-liquid equilibrium predictions. Fluid Phase Equilib. 2010;297(1):90-7. [77] Bell IH, Mickoleit E, Hsieh C-M, Lin S-T, Vrabec J, Breitkopf C, et al. J Chem Theory Comput. 2020;16(4):2635-46. [78] Mohan M, Simmons BA, Sale KL, Singh S. Multiscale molecular simulations for the solvation of lignin in ionic liquids. Sci Rep. 2023;13:271. [79] Antolovic I, Vrabec J, Klajmon M. COSMOPharm: Drug-polymer compatibility of pharmaceutical amorphous solid dispersions from COSMO-SAC. Mol Pharmaceutics. 2024;21(9):4395-415. [80] Klajmon M. Purely predicting the pharmaceutical solubility: what to expect from PC-SAFT and COSMORS? Mol Pharmaceutics. 2022;19(11):4212-32. [81] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 16 (Revision C.01), Gaussian, Inc., Pittsburgh, PA, 2016. [82] Cossi M, Rega N, Scalmani G, Barone V. Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model. J Comput Chem. 2003;24(6):669-81. [83] Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;12:19-25. [84] Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. J Comput Chem. 2004;25(9):1157-74. [85] Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J Am Chem Soc. 1995;117(19):5179-97. [86] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 09 (Revision A.02), Gaussian, Inc., Pittsburgh, PA, 2016. [87] Abascal JLF, Vega C. A general purpose model for the condensed phases of water: TIP4P/2005. J Chem Phys. 2005;123(23):234505. [88] Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126(1):014101. [89] Bernetti M, Bussi G. Pressure control using stochastic cell rescaling. J Chem Phys. 2020;153(11):114107. [90] Hess B. P-LINCS: A parallel linear constraint solver for molecular simulation. J Chem Theory Comput. 2008;4(1):116-22. [91] Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103(19):8577-93. [92] Martinez L, Andrade R, Birgin EG, Martinez JM. PACKMOL: A package for building initial configurations for molecular dynamics simulations. J Comput Chem. 2009;30(13):2157-64. [93] Polák J, Ondo D, Heyda J. Thermodynamics of N-Isopropylacrylamide in water: insights from experiments, simulations, and Kirkwood-Buff analysis teamwork. J Phys Chem B. 2020;124(12):2495-504. [94] Lé HQ, Zaitseva A, Pokki J-P, Stahl M, Alopaeus V, Sixta H. Solubility of organosolv lignin in γvalerolactone/water binary mixtures. ChemSusChem. 2016;9(20):2939-47. [95] Barbu MC, Sepperer T, Tudor EM, Petutschnigg A. Walnut and hazelnut shells: untapped industrial resources and their suitability in lignocellulosic composites. Appl Sci. 2020;10:6340. [96] Zhao S, Nia J, Yun L, Liu K, Wang S, Wen J, et al. The relationship among the structural, cellular, and physical properties of walnut shells. Hortscience. 2019;54(2):275-81. [97] Sannigrahi P, Ragauskas AJ, Miller SJ. Lignin structural modifications resulting from ethanol organosolv treatment of loblolly pine. Energy Fuels. 2010;24:683-9. [98] Luterbacher JS, Rand JM, Alonso DM, Han J, Youngquist JT, Maravelias CT, et al. Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone. Science. 2014;343(6168):277-80. [99] Alonso DM, Hakim SH, Zhou S, Won W, Hosseinaei O, Tao J, et al. Increasing the revenue from lignocellulosic biomass: maximizing feedstock utilization. Sci Adv. 2017;3:e1603301. [100] Horvath IT, Mehdi H, Fabos V, Boda L, Mika LT. γ-Valerolactone – a sustainable liquid for energy and carbon-based chemicals. Green Chem. 2008;10:238-42. [101] Zhang Y-HP, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng. 2004;88(7):797-824. [102] Pu Y, Hu F, Davison BH, Ragauskas AJ. Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnol Biofuels. 2013;6:15. [103] Carey FA, Sundberg RJ. Advanced Organic Chemistry, Part A: Structure and Mechanisms. 5th ed. Springer; 2007. [104] N’Guessan JLL, Niamké BF, Yao NJC, Amusant N. Wood extractives: Main families, functional properties, fields of application and interest of wood waste. For Prod J. 2023;73(3):194-208. [105] Chemat A, Schweiger M, Touraud D, Müller R, Lajoie L, Mazzitelli JB, et al. Cascade extractions of coffee silverskin: towards zero solid waste valorization of a byproduct. Sus Chem Pharm. 2024;42:101779. [106] Dehne L, Babarro CV, Saake B, Schwarz KU. Influence of lignin source and esterification on properties of lignin-polyethylene blends. Ind Crops Prod. 2016;86:320-8. [107] Nishide RN, Truong JH, Abu-Omar MM. Organosolv fractionation of walnut shell biomass to isolate lignocellulosic components for chemical upgrading of lignin to aromatics. ACS Omega. 2021;6:8142-50. [108] Protz R, Lehmann A, Ganster J, Pink H. Solubility and spinnability of cellulose-lignin blends in aqueous NMMO. Carbohydr Polym. 2021;251:117027. [109] Nawaz H, He A, Wu Z, Wang X, Jiang Y, Ullah A, et al. Revisiting various mechanistic approaches for cellulose dissolution in different solvent systems: A comprehensive review. Int J Biol Macromol. 2024;273:133012. [110] Li X, Yuan X, Xia G, Liang J, Liu C, Wang Z, et al. Catalytic production of γ-valerolactone from xylose over delaminated Zr-Al-SCM-1 zeolite via a cascade process. J Catal. 2020;392:175-85. [111] Xiao L, Liu W, Huang J, Lou H, Qiu X. Study on the antioxidant activity of lignin and its application performance in SBS elastomer. Ind Eng Chem Res. 2020;60(1):1-12. [112] Wang B, Wang S-F, Lam SS, Sonne C, Yuan T-Q, Song G-Y, et al. A review on production of lignin-based flocculants: sustainable feedstock and low carbon footprint applications. Renew Sustain Energy Rev. 2020;134:110384. [113] Foyer G, Chenfi B-H, Boutevin B, Caillol S, David G. New method for the synthesis of formaldehydefree phenolic resins from lignin-based aldehyde precursors. Eur Polym J. 2016;74:296-309. [114] Meng Y, Cheng Y, Lu J, Wang H. A review on lignin-based phenolic resin adhesive. Macromol Chem Phys. 2022;223:2100434. [115] Ren T, Zhang Z, You S, Qi W, Su R, He Z. Isolation and purification of 4-propylguaiacol and 4-propylsyringol by extraction and crystallization from the products of reductive catalytic fractionation processes. Green Chem. 2022;24(19):7355-61. [116] Li Y, Zhu J, Zhang Z, Qu Y. Preparation of syringaldehyde from lignin by catalytic oxidation of perovskite-type oxides. ACS Omega. 2020;5(5):2107-13. [117] Li K, Zhong W, Li P, Ren J, Jiang K, Wu W. Recent advances in lignin antioxidant: antioxidant mechanism, evaluation methods, influence factors and various applications. Int J Biol Macromol. 2023;251:125992. [118] Heinze T, Liebert T. 10.05 – Cellulose and Polyoses/Hemicelluloses. In: Polymer Science: A Comprehensive Reference. Vol 10. 2012. p. 83-152. [119] Heinze T, Koschella A. Solvents applied in the field of cellulose chemistry: a mini review. Polimeros. 2005;15(2):84-90. [120] Zhang S, Chen C, Duan C, Hu H, Li H, Li J, et al. Regenerated cellulose by the Lyocell process, a brief review of the process and properties. BioRes. 2018;13(2):4577-92. [121] Michels C, Kosan B. Lyocell process - material and technological restrictions. Chem Fibers Int. 2000;50:556-61. [122] Rosenau T, French AD. N-Methylmorpholine-N-oxide (NMMO): hazards in practice and pitfalls in theory. Cellulose. 2021;28:5985-90. [123] Hytönen E, Sorsamäki L, Kolehmainen E, Sturm M, Weymarn NV. Lyocell fibre production using NMMO – a simulation-based techno-economic analysis. BioRes. 2023;18(3):6384-411.