A Green DES for the fractionation of lignocellulosic biomass

doi:10.21203/rs.3.rs-2241104/v1

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A Green DES for the fractionation of lignocellulosic biomass

https://doi.org/10.21203/rs.3.rs-2241104/v1

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In this study, a DES system composed of benzoic acid (BA) and choline chloride (ChCl) was fabricated, and successfully applied to the lignocellulose fractionation. The DES has low toxicity and little pollution. In this system, 67.8% of lignin and 91.2% of hemicellulose were removed, leaving 95.8% of cellulose intact as solid residue. Due to the removal of the amorphous components, crystallinity of CRS substantially increased from 55.6–68.6%, and CRS was used as feedstock for nanocrystalline cellulose preparation with excellent properties. The results showed that the obtained lignin had similar properties to MWL by GPC, FT-IR, 2D-NMR and TGA. A high-purity lignin rich in G units was recovered with a well-preserved structure, which has β-O-4 linkage content up to 53.01%, low molecular weight, low polydispersity (1.99). This study demonstrated that BA is suitable for DES design with excellent properties on lignin extraction, and this promising DES enable efficient pretreatment for economically feasible biomass conversion.

Deep eutectic solvent

Fractionation

Characterization

Lignin

The excessive consumption on fossil reserves in the modern society has incessantly provoked ponderous pressure on resources and environmental protection. Correspondingly, the sustainable healthy development of human society has been bothered by environmental pollution, resources depletion and high energy demand (Haldar & Purkait, 2021; Lynam, Kumar, & Wong, 2017) .In recent years, exploring practicable substitutes for fossil resource have been undertaking both in academic and industrial communities. Among them, lignocellulosic biomass has been extensively studied due to its abundance, renewability, sustainability, biodegradability, and carbon neutral, which showed huge potential application (Vu et al., 2020). Structurally, lignocellulose is mainly composed of cellulose (40–50%), hemicellulose (25–35%) and lignin (10–30%) (Qaseem & Wu, 2020; Satlewal, Agrawal, Bhagia, Sangoro, & Ragauskas, 2018; Shrotri, Kobayashi, & Fukuoka, 2018). These components orderly assembled into compact and robust structure, which posed recalcitrance of biomass. Correspondingly, efficient and cost-effective biorefinery, analogous to the traditional petroleum-based refinery was inhibited by this structural recalcitrance. Moreover, the physical and chemical properties of each component are extremely disparate. Lignin is a complex water-insoluble aromatic polymer and mainly composed of various phenylpropanoid units interlinked by C-C and ether linkages, which form a highly heterogeneous branched three-dimensional network. The semicrystalline cellulose is linear macromolecule with anhydro-D-glucose unit interconnected by β-1,4-glycosidic bond, and the orderly crystalline zone of cellulose has inferior accessibility and reactivity (Rinaldi et al., 2016). And the amorphous hemicellulose is a kind of short chain heteropolysaccharide with branched structure, which has a relatively high reactivity.

Considering the recalcitrance and different components of biomass, fractionation is one of the important techniques in biorefinery (Schutyser et al., 2018; Shen & Sun, 2021). Thus, maximum valorization of each component in biomass could be achieved, and chemicals, fuels and functional materials could be produced. For example, pulp and paper were fractionated from wood chips by kraft cooking at industrial scale for more than a century, which were extensively used in every aspect of manufacture and daily life (Galkin & Samec, 2016). Numerous fractionation techniques involved treating the lignocellulosic biomass with acid or alkali in water, supercritical fluid (SCF), hot liquid water (HLW) and various organic solvents. Especially, discovery of innovative green solvents, including ionic liquids (ILs) and deep eutectic solvents (DESs) have opened new pathway for biorefinery. Many unique results in biorefinery could be achieved using these solvents, such as biomass fractionation, biomacromolecule dissolution, catalytic transformation, chemical derivation, and preparation of functional material (Bungay, 1982; van Osch et al., 2017).

However, the industrial application of ILs was hampered by its high cost, poor biodegradability, and questionable toxicity. Comparatively, DESs have advantages of low-cost, environmental-friendly, tunable properties and simple synthesis, and are promising in the future application. DES is a mixture of two or more chemical substances at an appropriate ratio, one of which is a hydrogen bond donor (HBD) and the other is a hydrogen bond acceptor (HBA) (Guo et al., 2019). Choline chloride (ChCl) is the most popular substance acting as HBA in DES preparation for its availability and biodegradability. Correspondingly, the prepared DES exhibited a melting point below that of its individual component. Essentially, DES provided a mild catalytic mechanism that caused the controlled cleavage of unstable ether bonds between phenylpropane units, and separation of lignin from biomass was obtained with reduced condensation reaction (Alvarez-Vasco et al., 2016). Therefore, DES could be used as an ideal medium for selective and efficient fractionation of biomass. DES involving p-toluenesulfonicacid (p-TsOH)/ChCl was used to treat wheat straw and miscanthus, dissolved most lignin and hemicellulose (W. Wang et al., 2020). Due to the cellulose nanocrystal production needed mild processing conditions, acidic DESs can be the hydrolytic media. The less ordered amorphous region of cellulose was selectively hydrolyzed (Sirvio, Visanko, & Liimatainen, 2016).

In addition, the previous studies demonstrated that many DESs have high lignin solubility and negligible cellulose solubility, and performance of DESs could be readily tuned by adjusting the HBDs and HBAs (Škulcová et al., 2016). Considering its versatile designability, many DESs with excellent properties could be potentially prepared and novel strategy in biorefinery could be fabricated. In regard of lignin extraction from lignocellulosic matrix, two pathways are typically required, including the extensive breakage of linkage among lignin units and high affinity between lignin and the solvent. Therein, many aromatic acids can hydrolyze ether-linkage among lignin, and its molecular interaction with lignin is strong for their abundant π-electron cloud. Therefore, benzoic acid (BA) with balanced acidity and affinity with lignin is an excellent option as HBD for DES design. In this study, we fabricated a green DES with BA and ChCl for efficient fractionation of lignocellulose. The properties of fractionation and the lignin structure were analyzed. And the solid residual rich in cellulose was favorable for preparation of cellulose nano crystal (CNC). This novel DES opened an new avenue for lignin separation and efficient utilization of lignocellulose, and design of task-specific DESs for lignocellulose treatment could be potentially optimized.

2.1 Materials

The poplar was sampled from Linqing, Shandong Province, China. The air-dried sample was ground with Willy mill (IKA MF10 Basic, Germany). A series of American standard sieves was used to fractionate the wood powder, and wood powder with size of 40–60 mesh was collected for future use. The raw poplar wood contains 45.7% glucan, 17.4% xylan, 25.5% lignin, 2.82% mannan, 1.67% galactose, 2.65% ash and 3.12% benzo-ethanol extractives. BA (purity ≥ 99.5%) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; ChCl (purity of 98%) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; anhydrous ethanol (purity ≥ 99.7%) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; 1,4-dioxane was purchased from Tianjin Guangcheng Chemical Reagent Co., Ltd.; dimethyl sulfoxide (DMSO) was obtained from isotope laboratory, Cambridge. Cellulase (Cellic® CTec2) was supplied by Novozymes (Beijing, China). All other reagents used in the experiment are analytical grade and used without further purification.

2.2 ChCl-BA DES synthesis

DES was synthesized by simply stoichiometrically mixing BA and ChCl under magnetic stirring (300 rpm) at 75°C, until a homogeneous colorless liquid formed. The liquid was cooled to room temperature and stored in a desiccator for future use.

2.3 Fractionation of poplar using DES treatment

DES treatment was performed in a 250 mL three-neck round bottom flask, which was placed in an oil bath under magnetic stirring (300 rpm). The flask was equipped with a condenser to avoid solvent evaporation during the treatment and a thermometer to monitor the temperature of the reaction. The poplar sample (4 g) and DES (24 mL) were added into the flask and treated at the specified conditions. After treatment, the reaction mixture was cooled down to room temperature, then diluted with anhydrous ethanol and stirred for 3–5 min. Afterwards, the solution was filtered on a Buchner funnel. The filtrate was collected and dialyzed for a week to neutral, where the dissolved lignin (EL) was obtained. And the undissolved portion was thoroughly washed with deionized water to remove dissolved substances and chemicals, and then freeze-dried for 12 h. The yield (Y) of the undissolved portion was calculated according to Eq. (1).

$$\text{Y}\text{ (\%)}\text{=}\frac{{\text{m}}_{\text{s}}}{{\text{m}}_{\text{r}}}\text{×100}$$

Where ${\text{m}}_{\text{r}}$is the weight of the raw material added into the flask, g; ${\text{m}}_{\text{s}}$ is the weight of the undissolved solid portion after treatment, g.

2.4 Determination of biomass components

The structural carbohydrates and lignin, including Klason lignin and acid-soluble lignin (ASL) of raw poplar and the DES treated poplar were determined using National Renewable Energy Laboratory analytical procedure (NREL/TP-51042618). ASL was determined by using an ultraviolet-visible (UV-Vis) spectrophotometer (Thermo Scientific Genesys 10S) at 205 nm using an extinction coefficient of 110 L⋅g^− 1⋅cm^− 1. In addition, the purity of the separated lignin samples was also determined according to this protocol.

Ion chromatography (ICS-5000 + system, Dionex) was used to determine monosaccharides, and integrated amperometric detector, CarboPac PA20 column and guard column were equipped. The column temperature was 30°C and the flow rate was 0.4 mL/min. The degradation products of the filtrate were analyzed using a high-performance liquid chromatography (HPLC) system (Agilent 1260, USA), and a Bio-Rad Aminex HPX-87H Column (300 ×7.8 mm, USA) column was used with a differential refractive index detector (Jiang et al. al., 2020), and 5 mmol/L sulfuric acid as the mobile phase (0.6 mL/min) at a column temperature of 60°C.

The yield of components in solid residual after DES treatment was calculated using Eq. (2).

$${\text{Y}}_{\text{i}}\text{=}\frac{{\text{C}}_{\text{ti}}\text{×}\text{Y}}{{\text{C}}_{\text{ri}}}\text{×}\text{100}$$

Where ${\text{Y}}_{\text{i}}$ is the yield of the individual component after DES treatment, %; ${\text{C}}_{\text{ri}}$ is the content of the corresponding component in the raw material, %; ${\text{C}}_{\text{ti}}$ is the content of the corresponding component in the DES treated solid material, %; $\text{Y}$ is the yield of DES treatment, %.

2.5 X-ray diffraction (XRD) analysis

A D8-advanced X-ray diffractometer (Bruker, Germany) was used to scan the samples under a Co radiation source at 40 kW and 35 mA. Prior to analysis, the samples were pressed into flat sheets on a sample holder. The scanning speed was 2.0 °/min with step size 0.02°. The spectra were normalized, and the crystallinity index (CrI) of the of the samples was calculated according to empirical Eq. (3).

$$\text{CrI}\text{ }\text{(\%)}\text{=}\frac{{\text{I}}_{\text{200}}\text{-}{\text{I}}_{\text{am}}}{{\text{I}}_{\text{200}}}\text{×100}$$

Where I₂₀₀ is the maximum intensity of diffraction from the main crystal plane (200) at about 22.8°, and I_am is the maximum intensity of the amorphous cellulose region at about 18.0°.

2.6 Lignin samples preparation

The lignin samples were prepared according to our previous report (P. Li et al., 2020). Briefly, the samples were sequentially treated by ball milling, enzymatic hydrolysis and dioxane extraction to obtain milled wood lignin (MWL), as poplar natural lignin, denoted as NL-P and residual lignin samples (RL). The filtrate was diluted with water to precipitate the lignin, then the sample was dialyzed against water to remove impurities. Finally, the dialyzed samples were freeze-dried to obtain dissolved lignin samples (EL).

2.7 Fourier transform infrared spectroscopy (FTIR) analysis

FTIR spectra of the lignin samples were recorded using an Alpha infrared spectrometer (Bruker, Germany). The samples for FTIR analysis were obtained by KBr pellet technique. The lignin samples were ground and mixed with KBr, then pressed in an evacuated die under 17 MPa for 30 s. The scanning range is in wavelength from 400 cm^− 1 to 4000 cm^− 1 for 16 times at resolution of 2.0 cm^− 1.

2.8 Thermal properties analysis

A thermogravimetric analysis (TGA) system (TGA Q50, TA Instruments) was used to analyze the thermal properties of the lignin samples. The measurement was performed under a flowing nitrogen atmosphere (20 mL/min), and the sample was heated from 50°C to 800°C at a heating rate of 10°C/min.

2.9 Molecular weight and distribution analysis of the lignin samples

Gel permeation chromatography (GPC, Omnisec, Waters Co., USA) was used to determine the weight average molecular weight (M_w), number average molecular weight (M_n) and polydispersity (M_w/M_n) of the lignin samples. The freeze-dried lignin (50 mg) was added into pyridine/acetic anhydride solution (1:1, v/v, 4 mL) and stirred at room temperature for 24 h in the dark. After acetylation, the lignin solution was diluted with ethanol and stirred for 30 min, then the solvents were removed using a rotary evaporator. Acidic water (pH < 2) was added to induce precipitation of lignin, and the acetylated lignin was collected by freeze-drying. The acetylated lignin was dissolved in tetrahydrofuran (THF) (2.0 mg/mL), and the solution was filtered through a 0.22 µm filter. The samples were analyzed by a chromatographic column (Styragel HR 4 THF, 7.8×300 mm, Ireland) with an injection volume of 20 µl and a column temperature of 35°C. THF was used as the mobile phase, with flow rate of 0.6 mL/min and ultra-violent detector at 280 nm. The polystyrene samples with molecular weight 1930, 2900 and 31400 was used for calibration.

2.10 2D- heteronuclear single quantum coherence (HSQC) NMR analysis of lignin samples

The samples were prepared by mixing the lignin samples (40 mg) with DMSO-d₆ (0.6 mL) solution. The measurement was performed on Bruker 600 MHz (Avance III, Bruker, Germany) operating at 400.13 and 100.61 MHz for ¹H and ¹³C, respectively. The chemical shifts (δ) and coupling constants (J values) were denoted as parts per million and hertz, respectively. The central DMSO-d₆ peak was designated as internal reference (δ ¹³C 39.5 ppm; δ ¹H 2.49 ppm). The 2D-HSQC spectra were acquired with spectral widths of 12 ppm for ¹H and 220 ppm for ¹³C using a 2-s relaxation delay, 1 K data points, 256 t1 increments, and 64 transients. In the obtained 2D spectrum, the X and Y axis are H and C spectrum, respectively.

2.11 Preparation and characterization of CNC

First, the CIS samples (2.0 g) were treated in NaClO₂ solution for 3 h at 80°C to remove the residual lignin (Feng et al., 2015). After filtering and washing 2–3 times, TEMPO-mediated oxidation was conducted for surface modification for 2 h (B. Li et al., 2015). Then, after ultrasonic treatment for 1 h, an equal volume of absolute ethanol was added and centrifuged (8000 rmp, 10 min), and then washed 3–5 times.

The properties of as-prepared CNC were analyzed by scanning electron microscopy (SEM), XRD, TGA, FTIR and dynamic light scattering (DLS). Scanning electron microscopy (Regulus 8220, Hitachi, Japan) images were obtained by scanning sample at an accelerating voltage of 5 KV. Gold sputtering of the samples was performed, then the samples were observed and pictures were captured. DLS data were acquired from Zetasizer Nano (Malvern, United Kingdom)

3.1 DES preparation and effect of DES treatment on poplar fractionation

ChCl-BA at molar ratio of 1:0.8 can form DES. The melting point of choline chloride benzoic acid is 302°C and 122°C, respectively. ChCl and BA can form transparent liquid at 75°C (Fig. 1), however, recrystallization occurred, and DES turned into solid at room temperature.

Fractionation of the poplar samples was investigated using ChCl-BA DES treatment, and the change on chemical compositions of the raw and DES treated poplar was analyzed (Table 1, Fig. 2). The poplar samples after DES treatment were fractionated into cellulose-rich water-insoluble solid (CIS) and a spent stream mainly containing dissolved lignin and hemicellulose. With increase on treatment intensity, yield of CIS gradually lowered due to degradation and dissolution of the components. Especially, effect of temperature, time, and solid-liquid ratio on performance of fractionation varied significantly. In contrast to lignin and cellulose, dissolution of hemicellulose smoothly proceeded, and removal ratio of hemicellulose increased significantly with temperature, time, and solid-to-liquid ratio. For example, the removal ratio of hemicellulose increased from 37.2–97.0% when temperature increased from 120°C to 150°C. The glycosidic bonds of hemicellulose with short-branched chain and amorphous structure are easy to be cleaved, leading to its rapid degradation and dissolution.

Lignin dissolution is another scenario other than hemicellulose, and removal ratio of lignin firstly increased then decreased with temperature. The removal ratio of lignin increased from 17.9% (120°C) to 46.5% (140°C), then dropped to 43.0% (150°C) and 36.4% (160°C). This phenomenon is similar to that p-TsOH-ChCl DES treatment (Zhou et al., 2021). The decrease on lignin removal at high temperature may be due to the adsorption and recondensation of the dissolved lignin units. The 2D-HSQC results showed that the relative decrease of β-O-4 linkages and increase of β-β and β-5 linkages, which can support this hypothesis. The dissolved lignin was adsorbed and redeposited on the solid surface. In addition, the DES treated residuals were darkened at high temperatures (Table S1), which may be due to humin formation by carbonization reactions. Correspondingly, further conversion of carbohydrates into value-added products was impaired, for example, inhibited enzymatic hydrolysis. Besides, lignin dissolution could be enhanced by increasing time and solid-liquid ratio.

Table 1

Chemical compositions of poplar samples treated in DES under different conditions
Sample	Pretreatment	Solid yield (%)	Glucan (%)	Xylan (%)	Lignin (%)
P	Paplor	100	45.7 (100)	17.4 (100)	25.5 (100)
L1	T120t3R1/6	92.7	47.4 (96.2)	11.8 (62.8)	22.6 (82.1)
L2	T130t3R1/6	79.0	54.4 (94.0)	6.6 (30.0)	22.5 (69.9)
L3	T140t3R1/6	65.2	64.5 (92.1)	3.1 (11.7)	20.9 (53.5)
L4	T150t3R1/6	64.8	62.9 (89.2)	0.8 (3.0)	22.4 (57.0)
L5	T160t3R1/6	63.4	62.4 (86.5)	0.8 (2.8)	25.6 (63.6)
L6	T140t2R1/6	72.8	58.0 (92.4)	6.7 (28.1)	20.2 (57.6)
L7	T140t4R1/6	59.8	65.2 (85.4)	3.1 (10.8)	15.5 (36.4)
L8	T140t5R1/6	54.7	77.8 (93.1)	3.7 (11.7)	15.2 (32.6)
L9	T140t3R1/4	76.2	59.8 (99.8)	3.1 (13.4)	21.0 (63.0)
L10	T140t3R1/8	64.2	69.3 (97.3)	3.6 (13.2)	18.0 (45.3)
L11	T140t3R1/10	60.8	72.7 (96.8)	2.6 (9.2)	17.3 (41.4)
L12	T140t3R1/12	59.2	72.3 (93.6)	1.8 (6.2)	16.5 (38.3)
L13	T140t3R1/14	56.6	77.3 (95.8)	2.7 (8.8)	14.5 (32.2)

Note:The data in parentheses are component yields based on its counterparts in the raw poplar.

In respect of cellulose, removal ratio of cellulose changed negligibly, and the obtained CIS was cellulose rich. Efficient removal of hemicellulose (97%) occurred at 150°C and 160°C, whereas lignin dissolution was relatively inferior. The treatment time and solid-liquid ratio were adjusted to 140°C and 1:14, respectively. Indeed, increasing time and solid-liquid ratio ensured both dissolution of hemicellulose and lignin. Removal of 91% hemicellulose and 67.8% lignin could be obtained at 140°C for 3 h, and cellulose removal was less than 5%. For example (Table S2), poplar fractionation using benzoic acid and p-hydroxybenzoic acid forming DES had similar capability on lignin and hemicellulose dissolution (Y. Wang et al., 2020), however, cellulose loss was extremely high (20.3%). In contrast, ChCl-BA forming DES can retain high portion of cellulose.

The filtrates after treatment were analyzed (Table 2). The filtrate comprised of oligosaccharides, monosaccharides, 5-HMF, furfural humus, and other degradation products. The sugar concentration is high under low severities of treatment (L3). The sugar content significantly reduced with increasing severities of treatment, and the degradation products, such as HMF and furfural, increased accordingly (L8).

Table 2

Chemical composition of filtrate after DES treatment
Sample	Glucan (%)	Xylan (%)	HMF (%)	Furfural (%)
L3	1.89	8.28	0	0.33
L8	0.65	2.29	0.45	1.80
L13	0.78	2.16	0.14	0.53

Note: The numbers are component yields based on components in the untreated poplar

3.2 CrI and the functional groups of CIS

The crystalline structure of the raw material and the CIS samples was analyzed by XRD technique (Fig. 3). All the samples showed the typical characteristics of type I cellulose (peaks at 2θ = 16.7° and 22.2°), indicating that the crystalline cellulose was well retained. The CrI of the raw poplar was the lowest (55.64%). After DES treatment, the CrI significantly increased. For example, the CrI of the sample L8, L3 and L13 is 63.9%, 66.8% and 68.6%, respectively. which may be due to the gradual removal of amorphous constituents, such as lignin, hemicellulose and the cellulose in the non-crystalline zone. The sample L13 dissolved 91.2% of hemicellulose and 67.8% of lignin, and the high CrI was conducive for the preparation of CNC with excellent properties.

The functional groups of the samples were analyzed by FTIR (Fig. S1). The bands around 900 cm^− 1 and 1100 cm^− 1 are the C-H deformation of cellulose and the C-O stretching vibration of the crystalline cellulose, respectively. The intensity change of peak at 900 cm^− 1 of the treated samples is consistent with cellulose content. The peak at 1103 cm^− 1 after pretreatment confirmed the increase in CrI, which was in agreement with XRD result. After treatment, the peaks at 990 and 1164 cm^− 1 (C-O valence vibration and C-O-C asymmetrical stretching of cellulose, respectively) confirmed the enrichment of cellulose after treatment. The peak at 1240 cm^− 1 (C-O vibration of lignin) indicated the presence of lignin and hemicellulose. Due to the efficient removal of hemicellulose, the intensity of the carbonyl group at 1738 cm^− 1 corresponding to the hemicellulose decreased with its content (Z. Chen & Wan, 2018).

3.3 Lignin characterization

3.3.1 Molecular weight distribution and purity of lignin

The weight average molecular weight (M_w), number average molecular weight (M_n) and PDI of the natural lignin and treated lignin samples were analyzed using GPC technique for further unraveling the solubilization and depolymerization of lignin during DES treatment (Table 3). The results showed that M_w and M_n of the samples after treatment were significantly reduced compared to that of the natural lignin. In case of the sample treated at 140°C for 3 h, the molecular weight decreased due to the breakage of inter-unit linkage. The depolymerization of lignin is the dominant reaction during DES treatment, and the M_w of lignin reduced from 5105 g·mol^− 1 to 1860 g·mol^− 1, and the M_n also reduced from 2516 g·mol^− 1 to 912 g·mol^− 1. The reaction time and solid-liquid ratio had limited effects on molecular weight and polydispersity. Therefore, from the perspective of lignin dissolution, extending the treatment time to greater than 3 h and increase the solid-liquid ratio could not facilitate lignin dissolution.

Table 3

Molecular weight distribution of the lignin samples
Sample	Mw	Mn	PDI
MWL-P	5105	2516	2.03
EL3	1860	912	2.04
EL8	1640	855	1.92
EL13	1818	915	1.99
RL3	1137	664	1.71
RL8	1094	655	1.67
RL13	1173	676	1.74

For understanding on efficiency of lignin extraction using DES treatment, the yield and purity of the extracted lignin were determined (Table 4). The dissolved lignin in filtrate was precipitated by adding water then dialyzed against water, thus the dissolved lignin was recovered. The lignin yield increased with residence time and biomass-liquid ratio. The lignin yield was 67.8% for the sample treated at 140°C for 3 h with a biomass-liquid ratio of 1:14. The purity of the lignin samples was analyzed. All lignin samples contained a small amount of carbohydrates. The glucan and xylan contents in the lignin sample (L13) treated at 140°C for 3 h were 0.46% and 0.94%, respectively. The total sugar content in the lignin samples decreased with growing residence time and biomass-liquid ratio. Therefore, LCC structure could be effectively cleaved under this DES treatment. Herein, the lignin purity of sample L13 is 88.76%, which is relatively high among the studied conditions.

The purity of lignin is crucial for its application value. The lignin extracted in this study ranged between 87–89% after precipitation and dialysis. The lignin content by soda pulping and sulfite pulping is 48–49% (Gellerstedt, Tomani, Axegård, & Backlund, 2012), and the purity of lignin obtained by kraft pulping method reached about 88% after purification. However these pulping processes are complicated with severe pollution concerns (wang & Liu, 2014). Generally, lignin by DES fractionation method has high purity, for example, ChCl-lactic acid treated D. fir lignin has purity of 87.1%(Alvarez-Vasco et al., 2016), and ChCl-ethylene glycol treatment switchgrass lignin has purity of 86.14% (Zhu Chen, Bai, A, & Wan, 2018). Therefore, DES treatment has potential application on lignin purification.

Table 4

Yield and purity of dissolved lignin
Sample	Lignin yield (%)	Lignin purity
Sample	Lignin yield (%)	AKL (%)	Glu (%)	Xyl (%)	Ash (%)	ASL (%)
EL3	46.4	87.61	0.57	1.56	0.13	0.06
EL8	67.3	87.99	0.50	1.30	0.13	0.06
EL13	67.8	88.69	0.46	0.94	0.06	0.07

3.3.2 FTIR analysis of the lignin structure

The FTIR spectra of the dissolved and residual lignin samples were analyzed (Fig. 4). Apparently, the distinctive functional groups of the original lignin were relatively intact, and the contours of the spectra after treatment slightly changed. Particularly, FTIR spectra of the dissolved lignin were significantly affected by treatment intensity, especially biomass-liquid ratio. All the obtained lignin samples had a strong absorption at 3434 cm^− 1, which is assigned to the stretching vibration of hydroxyl in aromatics and aliphatic sidechains. The absorption peaks at the -OH at 2930 cm^− 1 and 2845 cm^− 1 are attributed to the methylene vibration of holocellulose and the C-H stretching vibration of the methoxy group in the lignin, respectively. In addition, other characteristic absorption peaks of lignin are the stretching vibration of carboxyl and ketone C = O at 1710 cm^− 1, and the benzene ring at 1597 cm^− 1, 1506 cm^− 1 and 1418 cm^− 1. Skeletal vibration and asymmetric bending vibrations of -CH₃- and -CH₂- appeared at 1457 cm^− 1. And syringyl and guaiacyl CO stretching vibrations appeared at 1326 cm^− 1 and 1266 cm^− 1, respectively. These peaks of the dissolved lignin samples were weakened, indicating the removal or damage of these groups. The phenolic hydroxyl CO stretching vibration at 1226 cm^− 1, the guaiacyl CH stretching vibration at 1121 cm^− 1 and the deformation vibration of aromatic C-H plane at 1033 cm^− 1 are also detected. With the emergence of aromatic C-H plane deformation and vibration, these peaks in lignin were weakened to slight extent.

3.3.3 Effect of DES treatment on the thermal stability of the lignin samples

TGA was used to analyze the thermal properties of the obtained lignin samples (Fig. 5). The thermal stability and volatility of lignin are important properties for its subsequent valorization. High stability is conducive to biochar preparation, while high volatility can be used to synthesize fuels and chemicals (Zhu Chen, Bai, A, Zhang, & Wan, 2020). The negligible weight loss of all the samples below 150°C was observed due to the trace amount of moisture. The initial stage of decomposition of lignin mainly attributed to the breakage of weak C-O and aryl ether linkages (β-O-4, α-O-4, and 4-0-5). At this stage, the thermal stability of the dissolved lignin was significantly superior to that of the natural lignin. The partial cleavage of the ether linkages possibly occurred during DES treatment, leaving the more stable bonds in the dissolved lignin. When the temperature rose above 400°C, the aromatic ring and the C-C bond began to crack. It is worth noting that the weight loss of sample EL13 was significant, indicating that the condensation of the dissolved lignin structure under the condition of high solid-to-liquid ratio.

Chen et al. found that the initial depolymerization of lignin can form active free radicals, and the formation of free radical intermediates is an important step in lignin depolymerization (Zhu Chen et al., 2018). Due to the lack of hydrogen in lignin, active free radicals cannot be effectively stabilized and mutually coupled, leading to the formation of various C-O and C-C bonds that require high dissociation energy. Therefore, lignin extracted at high temperature or acid concentration has higher thermal stability and lower volatility. In this experiment, the pretreatment with lower solid content may cause serious depolymerization of lignin, thus the secondary reaction in the L13 pretreatment process will be more obvious. In contrast, aryl ether bonds and some other weak bonds were well preserved in L3. Finally, when the pyrolysis reached 800°C, natural lignin has the highest volatility with the lowest amount of solid residue (Fig. 5). The abundant aromatic ether linkages in natural lignin are easily thermally decomposed, resulting in low thermal stability and high volatility. In contrast, the dissolved lignin samples exhibited higher thermal stability and lower volatility because of the thermally resistant structure. The contours of the residual lignin under the various conditions are consistent in the full range. Compared to the natural lignin, the difference was slight, indicating that the aryl ether linkage and other weak bonds were well preserved. Furthermore, the M_n and M_w of the residual lignin were higher than of the natural lignin. Therefore, the large molecular weight portion was retained in the residual lignin, while the small molecular weight portion was dissolved out.

3.3.4 NMR analysis of lignin structure

The structure of the lignin samples was analyzed using 2D-HSQC NMR, and milled wood lignin (MWL) in the raw poplar was used as the natural lignin for comparison. The main cross signals of lignin were assigned according to the previous literature (Table S3). In case of the lignin samples after DES treatment at 140°C, polysaccharide signal was not observed in the polysaccharide ectopic region (δ_C/δ_H 90–105/3.9–5.4), which further proved the high purity of the dissolved lignin. All lignin samples were detected in the aromatic zone (δ_C/δ_H 90–160/5.5-8.0 ppm) to be associated with cross peaks of guaiacol (G), syringyl (S) and p-hydroxybenzoate (PB) units (Fig. 6), and distribution and linkage of the subunits were obtained (Table 5). Compared to that of the natural lignin (2.09), S/G ratio of the dissolved and residual lignin reduced at varying degrees after treatment, indicating more S units were removed and degradation occurred. And the dissolved lignin was rich in G units, resulting in a decrease in the S/G ratio. This phenomenon may be due to that guaiacyl unit is more stable than syringyl unit during DES treatment.

In the side chain region (δ_H/δ_C 2.5 5.8/50 90), methoxy (-OCH₃) structure can be observed. In the natural lignin, β-O-4 linkage content is the highest, followed by β-5 and β-β (Table 5). The content of β-O-4 linkage in dissolved lignin decreased after DES treatment, indicating that the ether bond was partially cleaved, whereas the content of β-β and β-5 linkages increased due to condensation of the dissolved lignin units. The results showed that β-O-4 linkage was readily to be cleaved and dissolved out during the DES treatment, while this linkage is basically intact in the residual lignin. C-C bonds, the increase in the content of β-5 and β-β bonds in the 2D-HSQC NMR results also explains the above TGA profile.

Table 5

Information of lignin samples in HSQC spectra
Sample	β-O-4 (%)	β-5 (%)	β-β (%)	S (%)	S_condensed (%)	G (%)	S/G Ratio
NL-P	79.52	14.22	6.26	68.03	0	31.97	2.09
EL3	64.00	33.71	2.29	48.11	10.26	41.64	1.16
EL8	56.17	28.71	15.12	47.18	9.25	43.56	1.08
EL13	53.01	36.01	10.97	46.14	10.87	42.98	1.07
RL3	80.18	9.83	10.00	59.79	1.77	38.44	1.55
RL8	79.29	17.67	3.04	58.08	4.03	37.89	1.53
RL13	75.53	22.55	1.92	52.38	5.16	42.46	1.23

DES treatment can cleave various ether bonds in lignin without effective breakage of C-C bond, and phenolic compounds containing glycosidic bonds were formed simultaneously. Two possible pathways can account for breakage of the ether bonds. In Pathway 1, free protons directly attack the ether bond, followed by protonation of hydroxyl group at α-carbon, dehydration at a-carbon and deprotonation at β-carbon, formation of carbocation and/or enol-ether intermediate and hydroxylation at β -carbon, finally β-O-4 ether bond cleavage occurred (Xu, Kong, Peng, Wang, & Li, 2021). Supposedly, the extraction efficiency of lignin can be significantly improved in the presence of sufficient free protons. Entry L13 and L8 treatment at wood-liquor ration of 6 achieved close lignin extraction. Therefore, the adequate number of protons in the system is the crucial. In case of Pathway 2, chloride ion directly cleaves the ether bond, which includes protonation of hydroxyl group, nucleophile attack α-carbon by halide anion and release of oxonium ion (dehydration), and dehydrochlorination and formation of enol-ether occurred (da Costa Lopes, Gomes, Coutinho, & Silvestre, 2020).

3.4 Mass balance analysis of DES treatment

Effective fractionation and conversion of poplar components could be achieved by this DES treatment, and the mass balance analysis was performed in the case of DES treatment with wood-liquid ratio of 1:6 at 140°C for 5 h (Fig. 7). All calculations are based on the experimental results of 100 g poplar wood as starting material, and the mass recovery percentage of the obtained CIS was 54.7%, and its main components were cellulose (42.55 g), lignin (8.31 g) and hemicellulose (2.05 g) (The part in bold italics). After DES treatment, the total solid content in the filtrate (mainly reducing sugars and polysaccharides) was 21.97 g, theoretically 16.69 g lignin fraction was obtained with purity of 88.77%. A small amount of degradation products and humus were formed. This DES treatment can selectively isolate hemicellulose and lignin, and a cellulose-rich solid could be obtained for further utilization.

3.5 Preparation and Characterization of CNC

The CNC using CIS as feedstock was prepared by TEMPO-mediated oxidation method. By observing the images after natural settlement for different time (Fig. 8A), the CNC had high stability. The rod-like products were obtained (Fig. 8B), and the average particle size of CNC by DLS measurement is 278.4 nm. The absolute value of zeta potential (-23.6 mV) is also high, which may account for its higher dispersibility. Figure 4C showed that the crystallinity of the CNC sample is 61.2%, which is somewhat lower than that of the L13 sample (68.6%). Figure 8D showed the TGA profile of the sample, CNC has experienced a slight weight loss due to water at low temperature range (Kaffashsaie et al., 2021). The initial temperature of CNC decomposition is 225°C, and the thermal stability is slightly reduced.

In respect of FTIR result (Fig. 8E), peak at 1055 cm^− 1 is C-O-C asymmetrical stretching of cellulose and hemicellulose, and peak at 1425 cm^− 1 is C-H in-plane deformation of lignin (wood). Peak at 2910 cm^− 1 is C-H stretching (related to methyl/methylene group of cellulose), and peak at 3348 cm^− 1 attributed to strong O-H stretching of cellulose (Kumar, Mago, Balan, & Wyman, 2009).The peak at 1610 cm^− 1 is assigned to the stretching vibration in the sodium carboxylate group (COONa) (Lin, Bruzzese, & Dufresne, 2012). Based on FTIR results, cellulose was successfully oxidized.

This study demonstrated effective biomass fractionation using ChCl-BA DES treatment, and the products were comprehensively characterized. The lignin was degraded and comprehensively extracted from lignocellulosic matrix. The residual cellulose in the solid residual changed slightly under different conditions using DES solution (ChCl:BA = 1:0.8). The retention rate of cellulose was 95.8% at 140°C for 3 h, more than 91% of hemicellulose was dissolved. Delignification rate reached 67.8%, and recovered lignin had a purity of 88%. Compared with the residual lignin, the extracted lignin has a well-preserved structure and low molecular weight, which can be potentially further catalytic upgraded. Therefore, this aromatic acid as HBD for DES design is applicable for lignin extraction. In addition, the CIS could be converted into CNC by the TEMPO-mediated oxidization method. This novel ChCl-BA DES offered a novel pathway for biomass fractionation and utilization.

Ethics approval and consent to participate

No ethics approval was required in this work.

Consent for publication

All authors agree to publish the data in this work.

Availability of data and materials

The authors confrm that the data supporting the fndings of this study are available within the article.

Competing interests

The authors declare that they have no conﬂict of interest.

Funding

The authors are grateful for the financial support from International Cooperation Funding of Qilu University of Technology (TGJHZ2018030) and the QUTJBZ Program (No. 2022JBZ01-05)

Authors' contributions -

Pengfei Li: Resources, Methodology, Validation, Writing - Original Draft, Writing: Review & Editing.

Cuihua Dong:Formal analysis, Data Curation, Writing - Original Draft

Zhiqiang Pang: Methodology, Project administration, Funding acquisition; Writing: Review & Editing.

Acknowledgements

Not applicable.

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FigS1.jpg
FTIR spectra the raw polplar and DES treated CRS samples

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A Green DES for the fractionation of lignocellulosic biomass

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Abstract

Figures

1. Introduction

2 Materials And Methods

2.1 Materials

2.2 ChCl-BA DES synthesis

2.3 Fractionation of poplar using DES treatment

2.4 Determination of biomass components

2.5 X-ray diffraction (XRD) analysis

2.6 Lignin samples preparation

2.7 Fourier transform infrared spectroscopy (FTIR) analysis

2.8 Thermal properties analysis

2.9 Molecular weight and distribution analysis of the lignin samples

2.10 2D- heteronuclear single quantum coherence (HSQC) NMR analysis of lignin samples

3 Result And Discussion

3.1 DES preparation and effect of DES treatment on poplar fractionation

3.2 CrI and the functional groups of CIS

3.3 Lignin characterization

3.3.1 Molecular weight distribution and purity of lignin

3.3.2 FTIR analysis of the lignin structure

3.3.3 Effect of DES treatment on the thermal stability of the lignin samples

3.3.4 NMR analysis of lignin structure

3.4 Mass balance analysis of DES treatment

3.5 Preparation and Characterization of CNC

4 Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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