Ionic Liquid Pretreatment Research Articles

Coupling of hydrothermal and ionic liquid pretreatments for sequential biorefinery of Tamarix austromongolica

Fractional pretreatment of lignocellulose is compelling to synergistically enhance its carbohydrate and lignin outputs for the extension of renewable energy sources. A two-stage approach using hydrothermal and ionic liquid (1-butyl-3-methylimidazoliumacesulfamate, [bmim]Ace) pretreatments was evaluated for hierarchically extracting hemicelluloses and lignin from Tamarix austromongolica, while providing a cellulose-rich fraction that could be readily hydrolyzed by cellulase for the recovery of fermentable glucose and residual lignin. Autohydrolysate-derived xylooligosaccharides (XOS), high-quality lignin, and accessible cellulose were successively acquired at the end of the process. The molecular weights, structural characteristics, functional groups and transitional fate of hemicelluloses and lignin were thoroughly explored towards a waste-free concept. This integrated biorefinery removed the amorphous portions, disrupted the rigid structure and altered cellulose I to II, thereby improving the enzymatic digestibility from 11.6 to 90.2%. Results confirmed that 60.98 mg/g XOS (DP 2–6), 352 mg/g glucose and 178.2 mg/g lignin were harvested via this coupled process. In-depth HSQC and 31P NMR techniques signified that the lignins contained large amounts of β-aryl ether units and hydroxyl groups, rendering them essentially suitable for depolymerizing into aromatic chemicals from biomass to energy. It was reasonable to diversify the energy-oriented product portfolio as a function of this sequential process, such as high-purity XOS, fermentable glucose and lignin, which can remarkably boost the feasibility of bioenergy.

Scale-up of biomass conversion using 1-ethyl-3-methylimidazolium acetate as the solvent

This scale-up study demonstrated the feasibility of an ionic liquid (IL) pretreatment process at 40 kg scale, using the IL 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) as the solvent. The pretreatment was followed by enzymatic hydrolysis through which the process efficiency for biomass conversion to monomeric sugars was determined. The results show that 43 wt% of switchgrass was dissolved in IL after 2 h of pretreatment at 160 °C with 15 wt% solid loading. A 120 h enzymatic hydrolysis of the pretreated switchgrass results in 96% glucan and 98% xylan conversion. [C2C1Im][OAc] pretreatment has been successfully scaled up to 40 kg with improved sugar titers and yields relative to bench scale (6 kg). The mass flow of the overall process was established and the major scale-up challenges of the process were identified.

Evaluation of hardwood and softwood fractionation using autohydrolysis and ionic liquid microwave pretreatment

Differences in hardwood and softwood, elucidates their behaviour against pretreatments varies. In this work, microwave ionic liquid (IL) and autohydrolysis (AH) pretreatments were applied to Eucalyptus globulus (as a model of hardwood) and Pinus radiata (as a model of softwood). The comparison between hardwood and softwood of microwave ionic liquid (IL) and autohydrolysis (AH) were evaluated in terms of chemical composition of pretreated solids, liquid by streams composition (hemicellulose and lignin extraction) and, substrates enzymatic digestibility. Furthermore, micrographs using scanning electron microscopy (SEM) and confocal fluorescence microscopy supported results obtained. In this study, it has been demonstrated that autohydrolysis pretreatment effectiveness, through maximizing enzymatic digestibility, is opposite in hardwood (73 g glucan/100 g glucan introduced at severe conditions) and softwood (10 g/100 g glucan). IL pretreatment has been especially effective in softwood with higher digestibilities (78 g glucan/100 g glucan introduced) than those obtained in hardwood (68 g glucan/100 g glucan introduced). Confocal fluorescence microscopy images, together with SEM images have resulted to be a clarifying technique to explain enzymatic digestibility results. Final sugars yields after the whole process have shown that low solid yields recoveries obtained in AH treatments have considerably worsened final glucose production, mainly in softwood. IL microwave pretreatment have resulted in higher glucose yields in softwood than in hardwood.

Extraction of lignin, structural characterization and bioconversion of sugarcane bagasse after ionic liquid assisted pretreatment.

The primary focus of this work was to recover lignin and investigate the structural changes in sugarcane bagasse after pretreatment with ionic liquid 1-ethyl-3-methylimidazolium acetate ([EMIM]oAc). 90% lignin recovery was achieved while bagasse was treated with [EMIM]oAc at 140°C, 120min reaction time and 1:20 bagasse to the ionic liquid ratio (w/w). The impact of ionic liquid pretreatment on bagasse was confirmed by qualitative analysis of untreated and pretreated bagasse. Scanning electron microscopy analysis exhibited the porous and irregular structure of bagasse after pretreatment. X-ray powder diffraction analysis verified a decrease in crystallinity as a result of the pretreatment process by showing a 14.7% reduction of Crystallinity index after ionic liquid treatment. The efficacy of [EMIM]oAc on bagasse treatment was also examined by enzymatic hydrolysis which manifested an increase in reducing sugar yield as a result of pretreatment. Maximum yield of 54.3% reducing sugar was obtained after 72h enzymatic hydrolysis of pretreated bagasse. Recovered lignin was analyzed qualitatively. 1D NMR spectroscopy of lignin revealed the presence of essential functional groups whereas 2D NMR spectroscopy showed the dominance of etherified syringyl unit. Further ionic liquid recovery and reuse were substantiated by Gel permeation chromatography analysis of lignin. Weight average molecular weight (Mw) of lignin extracted by fresh [EMIM]oAc was obtained as 1769g/mol (in the previous study) while lignin recovered by recycled [EMIM]oAc showed almost equal Mw 1765g/mol in this study. Thus, the current investigation corroborated satisfactory performance of [EMIM]oAc in lignocellulose processing which further enhanced enzymatic hydrolysis in the subsequent step.

Application of ionic liquid and alkali pretreatment for enhancing saccharification of sunflower stalk biomass for potential biofuel-ethanol production

Biorefining of lignocellulosic biomass to fuels/chemicals has recently gained immense research momentum. Current study reports sequential pretreatment of sunflower stalk (SFS) biomass in a combinatorial regime involving alkali (NaOH) and ionic liquid 1-butyl-3-methyl imidazolium chloride. The pretreatment enhanced the enzymatic digestibility, and resulted in increased sugar yield (163.42 mg/g biomass) as compared to standalone pretreatment using alkali (97.38 mg/g biomass) or ionic liquid (79.6 mg/g biomass). Ultrastructural and morphological analysis (FTIR and SEM) of pretreated biomass showed that the combined ionic liquid and alkali pretreatment causes more drastic alterations in the biomass ultrastructure as compared to alone ionic liquid or alkali pretreatment. Thus, combined pretreatment led to ease of enzymatic saccharification and consequent increased sugar yield, and this observation was corroborated by physicochemical analysis of the pretreated biomass. The pretreated SFS biomass was subjected to consolidated bioprocessing for its direct conversion to bioethanol in a single vessel.

Three Way Comparison of Hydrophilic Ionic Liquid, Hydrophobic Ionic Liquid, and Dilute Acid for the Pretreatment of Herbaceous and Woody Biomass

This paper examines the efficacy of ionic liquid (IL) pretreatment on seven different commercially harvested biomass types: corn stover, miscanthus, pine, sorghum, sugarcane bagasse, switchgrass, and wheat straw in an effort to improve the production of renewable fuels and chemicals from biomass derived sugars. Initial experiments screened the pretreatment of lodgepole pine, a particularly recalcitrant biomass feedstock, with nine different imidazolium based ionic liquids. After screening, one hydrophilic and one hydrophobic ionic liquid was selected for pretreatment tests on six commercially harvested biomasses. Ultimately, the hydrophilic ionic liquid functioned better for biomass pretreatment than the hydrophobic ionic liquid. These results were then compared to a traditional dilute acid pretreatment to examine the relative effectiveness of ionic liquid pretreatment across a variety of biomass and ionic liquid types. Total theoretical sugar yields after IL pretreatment varied widely by IL and biomass type and ranged from 4.9 to 90.2%. Dilute acid pretreatment showed consistent sugar yields for herbaceous material (from 71.4 to 80.8%) but low yield for lodgepole pine (22.8%). Overall, ILs showed the potential to reach slightly higher sugar yields than dilute acid and were particularly effective for woody feedstocks. More importantly, the sugar release kinetics for IL pretreatment were three times faster than dilute acid and gave maximum sugar yields after about 24 hours. Additional characterization of IL treated materials included scanning electron microscopy (SEM), x-ray diffraction (XRD), and compositional analysis. SEM and XRD showed qualitative and quantitative reductions in cellulose crystallinity (respectively) that correlated well to improved sugar release during enzymatic hydrolysis for hydrophilic ionic liquids. However, reductions in crystallinity associated with hydrophobic ionic liquids resulted in lower sugar release during enzymatic hydrolysis. Compositional analysis generally showed increased sugars content for hydrophilic ILs and increased lignin content for hydrophobic ILs.

Ionic Liquid and Sulfuric Acid-Based Pretreatment of Bamboo: Biomass Delignification and Enzymatic Hydrolysis for the Production of Reducing Sugars

The present work investigates the efficiency of two pretreatment pathways of biomass, namely ionic liquid (IL) and dilute sulfuric acid (H2SO4) hydrolysis. Both processes are compared in terms of their composition and enzymatic saccharification efficacy. For the IL process, bamboo was dissolved in 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) at different temperatures (90, 110, 130, and 150 °C) for 3 h. These pretreated bamboo samples were then characterized by thermogravimetric analysis and X-ray diffraction to evaluate the biomass crystallinity and thermal decomposition temperature. The crystallinity index, thermal decomposition temperature, hemicellulose, and lignin content of bamboo were found to decrease after [Emim][OAc] pretreatment. Further, the IL pretreated biomass significantly enhanced the enzymatic saccharification of cellulose component of bamboo. The enzymatic hydrolysis rate for IL pretreated biomass was 4.7 times higher than that of the acid pretreated biomass. This was primarily attr...

Obtaining Multiple Coproducts from Red Grape Pomace via Anthocyanin Extraction and Biogas Production.

Redgrape pomace (RGP), a byproduct of redwine production, is an abundant foodprocessingwaste stream in California, rich in both anthocyanins, a class of red-blue pigments, and lignocellulose. Extraction of anthocyanins and biofuel production from RGP have been investigated independently, but no research has examined employing both strategies together for maximal valorization. In this study, anthocyanins were most effectively extracted from RGP at 80 °C. Convection- and vacuum-oven drying of the pomace were found to decrease anthocyanin yield, whereas lyophilization did not significantly affect yield. Fermentable sugars were successfully separated from the crude extract via solid-phase extraction. Ionicliquid pretreatment of RGP was determined to be a nonviable option for application to anaerobic digestion. Extraction reduced biomethane output, but supplementation with the aqueous fraction of the extract mitigated much of this difference, indicating sequential extraction and fractionation of anthocyanins from RGP can minimize the impact on biofuel yields.

Evaluation of the chemical composition of a mixture of sugarcane bagasse and straw after different pretreatments and their effects on commercial enzyme combinations for the production of fermentable sugars

Replacement of fossil fuels with renewable sources has been studied as a strategy to mitigate the environmental impacts of energy production. These concerns have encouraged the use of alternative sources of cost-competitive and sustainable biofuels, such as lignocellulosic biomass, for second-generation ethanol production. As the enzymes required for biomass hydrolysis are costly, the development of less expensive enzyme mixtures for the hydrolysis of sugarcane biomass is important. The efficient conversion of biomass to fermentable sugars requires a critical pretreatment step to increase enzyme accessibility to the substrates during hydrolysis. Ionic liquids (ILs) have recently emerged as interesting solvents for biomass pretreatment. Extensive research has been conducted, showing that IL pretreatment can increase fermentable sugar productivity and biomass hydrolysis yields in comparison with conventional pretreatments, such as dilute sulfuric acid (DSA). The objective of this work was to evaluate changes in the chemical composition of a sugarcane biomass mixture composed of straw and bagasse in a 1:1 (w/w) ratio after pretreatment with the IL 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]) or with diluted sulfuric acid and determine the effect of the pretreatments on the interaction between commercial enzymes for the production of fermentable sugars by enzymatic hydrolysis. Pretreatments with IL were more efficient in the removal of lignin than pretreatments with DSA. Cellulase and endo-1,4-xylanase had a higher contribution to the release of glucose and xylose for both pretreated samples. The highest values of glucose and xylose yields, 99.96% and 86.52%, respectively, were achieved with the enzymatic hydrolysis of the biomass pretreated with IL.

RSM based optimization of PEG assisted ionic liquid pretreatment of sugarcane bagasse for enhanced bioethanol production: Effect of process parameters

Production of bioethanol from lignocelluloses is highly dependent on pretreatment process. Therefore, a better understanding of this process is an essential prerequisite to consider the whole bioethanol production from lignocelluloses in a cost and energy efficient way. The first step in performing the pretreatment is to discover the effects of different process parameters on various chemical features of the lignocelluloses. In order to achieve this aim, response surface methodology (RSM) was chosen to assess the exact impact of variables including temperature, polyethylene glycol (PEG) concentration and time on glucan, xylan and acid insoluble lignin contents as responses, also to optimize the pretreatment process to finally achieve higher bioethanol production yields. 1-butyl-3-methylimidazolium chloride (BMIMCl) and PEG in the concentration range of 1–5 (%w/w) were used as pretreatment agents. Temperature and time levels of 100–160 °C and 60–120 min were considered for experimental design, respectively. For further understanding of the effects of pretreatment on lignocelluloses, structural features examined by FT-IR, SEM and XRD analysis, in addition, enzymatic hydrolysis and fermentation were implemented on the samples pretreated in optimum conditions. The optimum pretreatment condition is 154.6 °C, 60 min and 5 (%w/w) of PEG concentration which resulted to the high enzymatic conversion and bioethanol production.

Effect of ionic liquid/inorganic salt/water pretreatment on the composition, structure and enzymatic hydrolysis of rice straw

Albeit ionic liquid (IL) pretreatment has been demonstrated to effectively enhance the saccharification of lignocellulosic biomass, extremely high costs of ILs still restrict its scale-up. If the ILs at high water content can be used as pretreatment solvents for lignocellulosic biomass, the chemical costs will be significantly reduced. However, the dissolving capacity of the ILs was notably decreased when water contents exceeded within limits. In this work, inorganic salts (K3PO4 and K2CO3) were added as additives of imidazolium-based IL/water systems to improve tolerance of ILs in the presence of water. The pretreatment with 50% 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl) + 49.5% water + 0.5% K2CO3 at 95 °C for 3 h improved lignin removal to 69.32%, and also increased the surface areas as compared to untreated rice straw. The reducing sugar yield of the regenerated materials was increased to 82.45% after 72 h enzymatic hydrolysis.

Recalcitrance Assessment of the Agro-industrial Residues from Five Agave Species: Ionic Liquid Pretreatment, Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.

Efficient pretreatment of bagasse at high loading in an ionic liquid

A high biomass loading in ionic liquid (IL) pretreatments that reduces the total cost of the process is potentially a key technique in biorefining. Previously, we demonstrated that, with microcrystalline cellulose, a higher cellulose loading could lead to improved accessibility and consequently higher hydrolytic reactivity, when catalyzed by an acid. In this study, we expand this concept to the lignocellulosic biomass such as bagasse. The pretreatment of bagasse was performed with a representative IL,1-ethyl-3-methylimidazolium acetate, over a biomass loading range of 5–50 wt% and was subsequently subjected to crystallinity measurements and acid hydrolysis. The results indicated a criterion of the pretreatment condition, which showed a higher pretreatment efficiency at a higher biomass loading. At the optimum pretreatment condition, the hydrolytic reactivity for 33 wt% biomass loading was 1.4 times higher than that at the conventional loading of 5 wt%. This concurred with the results that demonstrated a high biomass loading condition produces a lower crystallinity.

Understanding Lignin Fractionation and Characterization from Engineered Switchgrass Treated by an Aqueous Ionic Liquid

Aqueous ionic liquids (ILs) have received increasing interest because of their high efficacy in fractionating and pretreating lignocellulosic biomass while at the same time mitigating several challenges associated with IL pretreatment such as IL viscosity, gel formation during pretreatment, and the energy consumption and costs associated with IL recycling. This study investigated the fate of lignin, its structural and compositional changes, and the impact of lignin modification on the deconstruction of cell wall compounds during aqueous IL (10% w/w cholinium lysinate) pretreatment of wild-type and engineered switchgrass. The 4CL genotype resulting from silencing of 4-coumarate:coenzyme A ligase gene (Pv4CL1) had a lower lignin content, relatively higher amount of hydroxycinnamates, and higher S/G ratio and appeared to be less recalcitrant to IL pretreatment likely due to the lower degree of lignin branching and more readily lignin solubilization. The results further demonstrated over 80% of lignin dissolu...

Pretreatment of Guinea grass (Panicum maximum) with the ionic liquid 1-ethyl-3-methyl imidazolium acetate for efficient hydrolysis and bioethanol production

In the present study, we pretreated the plant Guinea grass (Panicum maximum) with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [EMIM][OAc] to expose its cellulose fibers. For this, we first determined the composition of Guinea grass, with 6.87 ± 1.21% of ash, 14.33 ± 1.18% of lignin, 70.55 ± 1.15% of holocellulose, 29.9 ± 1.20% of cellulose, and 40.65 ± 1.20% of hemicellulose. After characterizing the material, we determined the IL pretreatment conditions that maximized the enzymatic and acid hydrolysis stages [namely: temperature = 157 °C, reaction time = 30 min, and biomass concentration (relative to total mass) = 14%]. Under these conditions, the enzymatic hydrolysis conversions of glucose and total reducing sugars were respectively 69.8 and 54.2%, determined by enzymatic and DNS methods. The acid hydrolysis was also carried out, and conversions of 57.4 and 27.2% were verified by the enzymatic and DNS methods, respectively. Finally, the hydrolysate obtained after the enzymatic hydrolysis was fermented by the yeast Saccharomyces cerevisiae, and the ethanol yield (81.9%) was determined through gas chromatography.

Optimization of Ionic Liquid Pretreatment of Mixed Softwood by Response Surface Methodology and Reutilization of Ionic Liquid from Hydrolysate

We investigated the feasibility of producing bioethanol from mixed softwood pretreated with the ionic liquid 1-butyl-3-methylimidazolium acetate ([Bmim]Ac). The optimal pretreatment conditions were determined by response surface methodology to be 100°C for 15 h, and the fermentable sugar yield was estimated to be 92.5%. Efficient pretreatment of softwood was maintained even after reutilizing [Bmim]Ac up to four times. Through the enzymatic saccharification and subsequent fermentation, bioethanol was produced with 0.42 g/g of yield and 0.24 g/L/h of productivity, which clearly suggests that efficient and economical bioethanol production can be achieved by optimizing pretreatment processes and reutilizing ionic liquid.

Towards a multi-scale understanding of dilute hydrochloric acid and mild 1-ethyl-3-methylimidazolium acetate pretreatment for improving enzymatic hydrolysis of poplar wood

Sole pretreatment methods always have limitations in improving enzymatic hydrolysis of wood due to the multi-scale biomass recalcitrance. Herein, dilute acid pretreatment (DAP) with 2% hydrochloric acid at 160 °C and mild ionic liquid pretreatment (MILP) with 1-ethyl-3-methylimidazolium acetate at 90 °C were combined to increase cellulose accessibility in poplar wood. Additionally, the compositional, structural, and topochemical changes of wood during pretreatment and their effect on cellulose digestibility were comprehensively investigated and well understood. The sequential DAP and then MILP led to the highest yield of fermentable sugar (87.15%) after 72 h enzymatic hydrolysis when compared with the sole pretreatments and the sequential MILP and then DAP (21.31–73.64%). We suggested that the significant hemicellulose removal and cell wall deconstruction during first-step DAP exposed more cellulose fibrils to ionic liquid, thereby improving the efficacy of subsequent MILP to delignify wood and change cellulose crystal structure. The considerable removal of both hemicellulose and lignin, the damage of wood structure, and the crystal transformation from cellulose I to cellulose II dramatically improved the enzymatic hydrolysis of the DA + MIL sample. This study provides a better understanding of decomposition mechanism of cell walls during DAP and MILP, which can offer valuable insights into the development of promising pretreatment strategies.

Dimethyl Sulfoxide Assisted Ionic Liquid Pretreatment of Switchgrass for Isoprenol Production

The production cost and viscosity of certain ionic liquids (ILs) are among the major factors preventing the establishment of economically viable IL-based biomass pretreatment technologies. Recently, mixtures of an IL with an organic solvent have been proposed for cellulose processing and biomass pretreatment. Dimethyl sulfoxide (DMSO) is an inexpensive organic solvent that is industrially produced from lignin, a byproduct of the pulping process. We carry out a mechanistic study of DMSO-assisted IL pretreatment of switchgrass. The physical structures of biomass samples are studied by X-ray diffraction (XRD), N2 adsorption analysis, and small angle neutron scattering (SANS). Both dry and aqueous suspensions of biomass samples are measured by SANS which provides unique information on biomass pretreatment. A mixture of 42 wt % [C2C1Im][OAc] and 58 wt % DMSO is proposed as the optimal pretreatment solution, and the recycling and reuse of the mixture of solvents are also studied. The fermentability of the hydrolysates generated after pretreatment is evaluated using an E. coli strain engineered to produce isoprenol. This study suggests an avenue for developing more efficient and cost-effective IL-based processes for the production of lignocellulosic biofuels and bioproducts.

Comparison of ultrasound-assisted ionic liquid and alkaline pretreatment of Eucalyptus for enhancing enzymatic saccharification

Two ultrasound-assisted pretreatment technologies, ultrasound-assisted alkaline and ultrasound-assisted aqueous ionic liquid tetrabutylammonium hydroxide ([TBA][OH]), are compared systematically in regard to enzymatic saccharification. Pretreated Eucalyptus samples were characterized by powder X-ray diffraction, 13C cross polarization/magic-angle spinning solid state NMR spectroscopy, Fourier transform infrared spectroscopy, Scanning electron microscope (SEM) and chemistry composition analysis. These results not only explain the enzymatic saccharification difference between samples from the microstructure level, but also provide helpful information for relevant pretreatment research. Ultrasound-assisted [TBA][OH] pretreatment acquired a significant enhancement in the initial enzymatic rate of cellulose (79.39 mg/g/h), and a reducing sugar yield of 426.6 mg/g at 48 h. The pretreatment combining inexpensive aqueous ionic liquid and ultrasound may provide a promising strategy in the field of bio-refinery because of its unique advantages.

Characterization of Lignin Streams during Bionic Liquid-Based Pretreatment from Grass, Hardwood, and Softwood

Delignification as a function of ionic liquid (IL) pretreatment has potential in terms of recovering and converting the fractionated lignin streams to renewable products. Renewable biogenic ionic liquids, or bionic liquids (e.g., cholinium lysinate, ([Ch][Lys])), provide opportunities in terms of effective, economic, and sustainable lignocellulosic biomass pretreatment. We have evaluated [Ch][Lys] pretreatment in terms of sugar and lignin yields for three different feedstocks: switchgrass, eucalyptus, and pine. Four lignin streams isolated during [Ch][Lys] pretreatment and enzymatic hydrolysis were comprehensively analyzed, tracking their changes in physical–chemical structures. We observed changes in major lignin linkages and lignin aromatics units (p-hydroxyphenyl (H), guaiacyl (G), and syringil (S)) that occurred during pretreatment. A compositional analysis of the different process streams and a comprehensive mass balance in conjunction with multiple analytical techniques (nuclear magnetic resonance (...