Enhanced Enzymatic Hydrolysis and Lignin Extraction of Wheat Straw by Triethylbenzyl Ammonium Chloride/Lactic Acid-Based Deep Eutectic Solvent Pretreatment.

Efficient and feasible pretreatment of lignocellulosic biomass waste is an important prerequisite step to promote subsequent enzymatic hydrolysis and enhance the economics of biofuels production. This study focuses on the pretreatment of wheat straw (WS) with triethylbenzyl ammonium chloride/lactic acid (TEBAC/LA)-based deep eutectic solvents to enhance biomass fractionation and lignin extraction. Effects of pretreatment time, temperature, and TEBAC/LA molar ratio on pretreatment were evaluated systematically. Results suggested that 89.06 ± 1.05% of cellulose and 71.00 ± 1.03% of xylan were hydrolyzed with enzyme loadings of 35 FPU cellulase and 82 CBU β-glucosidase (per gram of dry biomass) after pretreatment by TEBAC/LA (1:9) at 373 K for 10 h. A total monosaccharide yield of 0.550 g/g WS (91.27% of the theoretical yield) was achieved with 79.73 ± 0.93% of lignin removal. Furthermore, the 1H-13C two-dimensional heteronuclear single quantum correlation (2D-HSQC) NMR spectroscopy showed that the regenerated lignin (75.69 ± 1.32% purity) was mainly composed of the syringyl units and the guaiacyl units. Overall, the results in this study provide an effective and facile pretreatment method for lignocellulosic biomass waste to enhance enzymatic hydrolysis saccharification.

Introduction

Lignocellulosic biomass is a renewable carbon resource that stores solar energy in the form of chemical energy through photosynthesis of CO2 and H2O.[1] With the depletion of fossil resources and the demand for energy, combined with the requirement of emission reduction and environmental protection, the urgent need for renewable alternative carbon resources has arisen in the market.[2−5] Approximately 700 million tons of straw agricultural wastes, including rice, sorghum, wheat, barley, and corn, is produced every year in China.[6]

Wheat straw (WS) agricultural residues contain a significant amount of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose can be used as feedstocks for sustainable production of valuable platform chemical and biofuels.[7−11] Lignin is also regarded as a versatile feedstock in adhesive and resin industries due to its renewable nature and special aromatic structure.[12] However, lignin is a cross-linked polymer that contains aromatic precursors, which play a key role in highly recalcitrant to chemical and biological degradation.[13,14] Therefore, efficient pretreatment of lignocellulosic biomass waste is a key step to remove lignin to improve the enzymatic hydrolysis performance of cellulose components.[14−17]

Various pretreatment techniques have been investigated and used to remove lignin from WS, such as hydrothermal and microwave,[18] steam expansion,[19] acids (sulfuric acid, phosphoric acid, and atmospheric acetic acid, etc.),[20−22] bases (sodium hydroxide, potassium hydroxide, sodium sulfide, and sodium borohydride, etc.),[23−25] organic solvents (methanol, ethanol, butanol, and glycerol),[26−28] and ionic liquids (1-butyl-3-methy-limidazolium acetate, 1-ethyl-3-methylimidazolium acetate, N,N-dimethylethanolammonium formate, [Emim] [OAC], cholinium taurinate, cholinium glycinate, cholinium levulinate, and cholinium pentanoate, etc.).[29−32] Each method possesses its respective advantages and disadvantage in terms of pretreatment cost, energy requirement, operational simplicity, reaction condition, and difficulty of industrial applications.[33] For example, the cellulose and xylan conversions of WS pretreated by steam explosion integrated with 2% (w/v) sodium hydroxide were 87.8 and 43.8%, respectively. However, these pretreatment methods usually require a high pretreatment temperature (198–200 °C) and pressure (1.03–13 bar), resulting in the total loss of xylan.[19,23] Although pretreatment of WS using ionic liquids (ILs) overcomes some of these disadvantages of traditional techniques in terms of equipment corrosion, high temperature, and pressure, their use in industrial applications was restricted by high synthesis cost and complicated synthesis process.[33] In recent years, deep eutectic solvents (DESs), generally formed by complexion of hydrogen-bond acceptors (HBAs) with hydrogen-bond donors (HBDs),[34−36] have emerged as potential solvents in lignocellulosic biomass pretreatment due to its high efficiency on the depolymerization of lignin, low cost, low viscosity, low vapor pressure, biocompatibility, and environmental friendliness, gaining great attention from academic and industrial fields.[37−43] Most of the reported studies on DESs for lignocellulosic biomass pretreatment focus on choline chloride (ChCl) as an HBA, mainly due to the fact that ChCl can be mixed with various HBDs, such as amino acid, p-toluenesulfonic acid, carboxylic acid, and alcohol, to prepare DESs with certain solubility to lignin.[44−47] However, ChCl belongs to a non-aromatic ring quaternary ammonium salt, and its phase transfer catalytic ability is not as good as an aromatic ring quaternary ammonium salt, which makes the solubility of lignin in these DESs limited. There are few DESs reported that utilize aromatic ring quaternary alkyl ammonium salts;[14] however, their application to WS pretreatment is not well reported.

It is therefore the objective of the present study to test triethylbenzyl ammonium chloride (TEBAC)/lactic acid (LA)-based DESs for the pretreatment of WS. The lignin removal, xylan loss, and the cellulose reservation from WS by TEBAC/LA-based DESs were systematically investigated. The effects of different pretreatment times (2, 4, 6, 8, 10, and 12 h), reaction temperatures (353, 363, 373, and 383 K), and TEBAC/LA molar ratios (1:5, 1:7, 1:9, 1:11, and 1:13) on lignin removal from WS were systematically investigated. Moreover, the cellulose-enriched residues were systemically investigated for the enzymatic saccharification to produce sugars. Furthermore, scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectrometry, X-ray diffraction (XRD), and 1H–13C two-dimensional heteronuclear single quantum correlation (2D-HSQC) NMR spectroscopy were used to characterize the cellulose-enriched residues and extracted lignin. Overall, a systematic understanding of this pretreatment process will be beneficial for separation and valorization of three biomass components (cellulose, hemicellulose, and lignin) from WS in future large-scale biorefinery.

Results and Discussion

Effect of Pretreatment Conditions on the Enzymatic Hydrolysis

Pretreatment Time

The effects of different pretreatment times on the WS pretreated by a TEBAC/LA (1:9)-based DES and enzymatic hydrolysis efficiency of cellulose-enriched residues were investigated at various pretreatment times of 2, 4, 6, 8, 10, and 12 h. The ratio of solid to liquid was 1:15, and the pretreatment temperature was kept at 373 K. The recovery of cellulose-enriched residues and the compositions of cellulose, xylan, and lignin are displayed in Figure A. It can be observed from Figure A that the recovery rate of the cellulose-enriched residues gradually decreases with the increase in the pretreatment time, reducing from 100% (untreated WS) to 48.7 ± 2.54% at a pretreatment time of 12 h. Figure B presents the related parameters in terms of lignin removal, xylan loss, and cellulose maintenance. Clearly, as the treated reaction time prolonged, the removal rate of lignin gradually increased from 48.76 ± 1.12% (2 h) to 73.71 ± 1.19% (12 h). Meanwhile, the rate of xylan loss gradually increased from 43.44 ± 1.61% (2 h) to 78.37 ± 1.98% (12 h). However, the cellulose reservation was slightly reduced from 83.14 ± 2.58% (2 h) to 76.23 ± 1.32% (12 h). Furthermore, the effect of pretreatment time on the cellulose-enriched residues enzymatic hydrolysis after TEBAC/LA-based DES pretreatment is listed in Table . It can be seen from Table that the untreated WS presented low cellulose and xylan digestibilities of 25.82 ± 0.94 and 9.69 ± 1.08%, respectively. This is mainly due to the presence of the lignin polymer and coating effect of xylan in the cell wall, which severely hindered enzymatic hydrolysis.[48] In contrast, the cellulose and xylan digestibilities of cellulose-enriched residues after TEBAC/LA-based DES pretreatment (10 h) were dramatically improved to 89.06 ± 1.05 and 71.00 ± 1.03%, respectively, which are 3.45- and 7.33-fold higher than that of untreated WS. As we all know, shorter pretreatment time usually leads to higher production efficiency in industrialized production; therefore, it is preferable to select the pretreatment reaction time to 10 h, and the highest yields of glucose and xylose reached 66.32 ± 1.58 and 20.09 ± 1.39%, respectively.

Figure 1

Effects of different pretreatment reaction times on three major component removal from WS. The molar ratio of TEBAC/LA was 1:9, the ratio of solid to liquid was 1:15, and the pretreatment temperature was kept at 373 K. (A) Recovery of residues and the composition of cellulose, xylan, and lignin. (B) Lignin removal, xylan loss, and cellulose reservation.

Table 1

Effect of Different Pretreatment Time on Wheat Straw Pretreatmenta

		digestibility (%)		sugar yield (%)
pretreatment reaction time (h)	lignin purity (%) (PL%)	cellulose (DC%)	xylan (DL%)	glucose (YGlucose)	xylose (YXylose)
untreated		25.82 ± 0.94	9.69 ± 1.08	25.82 ± 2.67	10.66 ± 0.58
2	81.68 ± 2.38	55.76 ± 1.17	20.28 ± 1.27	44.43 ± 1.36	11.11 ± 2.29
4	82.77 ± 1.87	67.96 ± 1.42	55.64 ± 1.35	44.84 ± 0.98	21.52 ± 1.28
6	77.50 ± 1.23	64.00 ± 3.22	65.60 ± 2.39	51.93 ± 1.83	22.04 ± 1.11
8	75.69 ± 1.86	73.36 ± 1.31	73.97 ± 3.18	56.29 ± 2.47	23.36 ± 1.35
10	75.69 ± 1.32	89.06 ± 1.05	71.00 ± 1.03	66.32 ± 1.58	20.09 ± 1.39
12	68.90 ± 2.37	86.73 ± 2.84	68.27 ± 2.75	60.12 ± 1.93	15.09 ± 1.87

The molar ratio of TEBAC/LA was 1:9, the ratio of solid to liquid was 1:15, and the pretreatment temperature was kept at 373 K.

Pretreatment Temperature

Figure displays the effect of pretreatment temperature ranging from 353 to 383 K on the WS pretreated by a TEBAC/LA (1:9)-based DES and enzymatic hydrolysis efficiency of cellulose-enriched residues. The ratio of solid to liquid was 1:15, and the pretreatment time was 10 h. It can be observed clearly from Figure A that, with the reaction temperature ranging from 353 to 383 K, the recovery rate of cellulose-enriched residues correspondingly decreased from 67.45 ± 3.34% (353 K) to 45.76 ± 2.58% (383 K), while the cellulose content increased gradually from 45.02 ± 2.70 to 60.39 ± 3.60%, and the xylan content decreased from 18.87 ± 0.64 to 9.19 ± 1.02% as the pretreatment temperature increase from 353 to 383 K. This may be due to the increase in temperature, which is beneficial to not only the removal of lignin but also the removal of xylan. Besides, as the temperature increased, the lignin content in the residue after DES pretreatment was decreased from 18.12 ± 0.60 to 6.10 ± 0.58%, and the lignin removal rate also significantly increased from 51.27 ± 1.76 to 84.00 ± 1.03% (see Figure B), indicating that the high temperature could promote the DES to break the chemical bond between lignin, cellulose, and xylan and result in the removal of lignin.[49]

Figure 2

Effects of different pretreatment reaction temperatures on three major component removal from WS. The molar ratio of TEBAC/LA was 1:9, the ratio of solid to liquid was 1:15, and the pretreatment time was 10 h. (A) Recovery of residues and the composition of cellulose, xylan, and lignin. (B) Lignin removal, xylan loss, and cellulose reservation.

The enzymatic hydrolysis of cellulose-enriched residues at different pretreatment temperatures is listed in Table . By comparison, it can be found that the enzymatic digestion rate of cellulose and xylan can be greatly improved after DES treatment. Among them, the cellulose digestibility sharply increased to 91.23 ± 2.19% (pretreated at 383 K) and was approximately 3.53-fold higher than the untreated WS. The digestibility of xylan was also increased from 9.69 ± 0.58% (untreated WS) to 77.59 ± 2.18% (pretreated at 383 K). This phenomenon may be due to the increase in temperature, which facilitates the removal of lignin and xylan, thereby increasing the accessibility of the enzyme and accelerating the enzymatic hydrolysis of cellulose.[37,50] However, when the pretreatment temperature was 373 K, the cellulose digestibility reached 89.06 ± 1.05%, and when the pretreatment reaction temperature rises to 383 K, the cellulose conversion rate was 91.23 ± 2.19%, which only increased slightly by around 2.44%. Although higher cellulose and xylan digestibility were achieved at a high reaction temperature, the recovery rate of cellulose-enriched residues was low under the high temperature. The total sugar yield depended on the residue recovery and polysaccharide digestibility. Furthermore, considering that a high temperature requires larger energy consumption, the optimal pretreatment reaction temperature for WS was selected as 373 K. A total monosaccharide yield of 0.550 g/g WS (0.471 g of glucose and 0.079 g of xylose) was achieved, which is 91.27% of the theoretical yield (0.603 g/g).

Table 2

Effect of Different Pretreatment Temperatures on Wheat Straw Pretreatmenta

		digestibility (%)		sugar yield (%)
pretreatment reaction temperature (K)	lignin purity (%) (PL%)	cellulose (DC%)	xylan (DL%)	glucose (YGlucose)	xylose (YXylose)
untreated		25.82 ± 0.94	9.69 ± 1.08	25.82 ± 2.67	9.69 ± 0.58
353	56.57 ± 1.18	61.67 ± 2.01	21.61 ± 0.43	55.48 ± 2.06	14.25 ± 0.49
363	82.68 ± 2.74	76.21 ± 2.93	54.12 ± 1.41	59.49 ± 1.32	21.07 ± 1.77
373	75.69 ± 1.32	89.06 ± 1.05	71.00 ± 1.03	66.32 ± 1.58	20.09 ± 1.39
383	65.71 ± 2.42	91.23 ± 2.19	77.59 ± 2.18	66.49 ± 1.83	17.97 ± 2.11

The molar ratio of TEBAC/LA was 1:9, the ratio of solid to liquid was 1:15, and the pretreatment time was 10 h.

Molar Ratio

The effect of different molar ratios of TEBAC/LA-based DESs on the WS pretreatment is presented in Figure . The pretreatment reaction temperature was kept at 373 K, the reaction time was 10 h, and the solid–liquid ratio is 1:15. It could be seen from Figure A that the recovery rate of residues decreases first and then increases as the amount of LA molar increases, which was due to the fact that the different molar ratios of TEBAC/LA have various pretreatment effects on the WS. Furthermore, the differences in cellulose and xylan contents obtained by different molar ratios of TEBAC/LA were also obvious. It is worth noting that the lowest cellulose reservation (77.79 ± 0.70%) was obtained using the TEBAC/LA (1:9)-based DES. This may be attributed to the better removal of lignin (73.96 ± 1.26%; see Figure B) from this type of DES, resulting in more cellulose exposure and loss. It also can be seen from Table that after TEBAC/LA (1:9)-based DES pretreatment, the enzymatic digestion rate could be significantly increased, and the digestibility of cellulose was improved from 25.82 ± 0.94% (untreated WS) to 89.06 ± 1.05%, increasing by 3.45-fold, which was higher than the pretreatment by a TEBAC/LA (1:7)-based DES (77.27 ± 1.37%). With no doubt, a high glucose yield (66.32 ± 1.58%) was achieved with more lignin removal. Based on the abovementioned results, the 1:9 molar ratio of TEBAC/LA was selected for the optimum ratio.

Figure 3

Effects of different molar ratios of TEBAC/LA on three major component removal from WS. The ratio of solid to liquid was 1:15, the pretreatment temperature was kept at 373 K, and the pretreatment time was 10 h. (A) Recovery of residues and the composition of cellulose, xylan, and lignin. (B) Lignin removal, xylan loss, and cellulose reservation.

Table 3

Effect of Different Molar Ratios of TEBAC/LA on Wheat Straw Pretreatmenta

		digestibility (%)		sugar yield (%)
pretreatment reaction time (h)	lignin purity (%) (PL%)	cellulose (DC%)	xylan (DL%)	glucose (YGlucose)	xylose (YXylose)
untreated		25.82 ± 0.94	9.69 ± 1.08	25.82 ± 2.67	9.69 ± 0.58
1:5	60.67 ± 1.88	65.19 ± 2.51	75.33 ± 2.17	56.76 ± 1.24	28.52 ± 1.47
1:7	63.61 ± 2.23	77.27 ± 1.37	73.50 ± 1.62	63.81 ± 1.26	27.81 ± 1.65
1:9	75.69 ± 1.32	89.06 ± 1.05	71.00 ± 1.03	66.32 ± 1.58	20.09 ± 1.39
1:11	77.08 ± 1.64	74.89 ± 2.01	77.90 ± 1.96	63.53 ± 3.01	26.61 ± 1.54
1:13	68.68 ± 1.79	70.81 ± 1.33	74.27 ± 2.58	56.23 ± 1.77	23.16 ± 1.08

The ratio of solid to liquid was 1:15, the pretreatment temperature was kept at 373 K, and the pretreatment time was 10 h.

Comparison of Different Methods for WS Pretreatment

Compared to the chloride (ChCl)/LA (1:2)-based DES treatment of WS,[46] low temperature and shorter time and a similar lignin removal can be obtained by using the TEBAC/LA (1:9)-based DES in the current study. For example, 73.96 ± 1.26% of lignin can be removed from WS after the TEBAC/LA (1:9)-based DES treatment at 373 K for 10 h, whereas ChCl/LA (1:2) pretreatment of WS at 393 K for 12 h led to 78.6 ± 0.7% lignin removal. This is mainly because TEBAC is an aromatic ring quaternary ammonium salt with strong phase transfer ability, which can dissolve in both water and organic phases. The positive and negative ions in the quaternary ammonium salt form an ion pair in the water phase, which can transfer negative ions from the water phase to the organic phase, while in the organic phase, negative ions have no solventization role. Furthermore, because the positive ions are large, the distance between the positive and negative ions is also large, and the role between each other is weak. Also, negative ions can be regarded as bare, so the reaction activity is greatly improved. Furthermore, compared to the TEBAC/LA (1:9)-based DES pretreatment of corncobs, the sugar yield was increased to 94.0% under optimal conditions at 413 K for 2 h.[14] The main reason is that the fiber cell wall ultrastructure, vascular tissue structure, and lignin characteristics between corncobs and WS are different.[46]

Characterization of Solid Fraction

Scanning Electron Microscopy

Figure S1 displays the morphologies of the untreated and pretreated WS with the TEBAC/LA (1:9)-based DES samples as characterized by SEM. As shown in Figure S1A,B, the surface of untreated WS is smooth without obvious damage. However, some wrinkles and holes appeared on the surface of the pretreated WS after TEBAC/LA (1:9)-based DES pretreatment (see Figure S1C,D). This is due to the fact that DESs removed the lignin and hemicellulose that wrapped and exposed the cellulose.[33] The loose structure of pretreated WS is conducive to penetration and hydrolysis by enzymes, thus providing favorable conditions for the enzymatic treatment of cellulose in the later stage and significantly improves the enzyme digestion efficiency. Furthermore, the surface of obtained solid residues is not as smooth as expected because the residues after pretreatment by DES still contain some non-fiber components.

FT-IR Spectra

The FT-IR spectra of untreated WS and pretreated WS using the TEBAC/LA (1:9)-based DES are presented in Figure S2. The absorption peaks at 3396 cm–1 denote O–H stretching vibration, and the absorption peaks at 2924 cm–1 represent C–H stretching vibration, while the absorption bands at 1727 cm–1 are the stretching vibration peaks of C=O, which are the characteristic absorption peak of xylan. The peaks at 1631 cm–1 are the skeleton vibration of lignin benzene ring with weak C=O vibration, and those at 1511 cm–1 are the skeleton vibration of aromatic ring.[32] The absorption peaks at 1380 cm–1 are the C–O or OH bending vibration of lignin benzene ring, and the absorption bands at 1233 cm–1 denote the ether linkages between hemicellulose and lignin.[33] The absorption bands at 1066 cm–1 are the C–O stretching vibration on C–O–C, and those at 890 cm–1 are a β-(1,4) glycosidic bond.[14] Clearly, comparing with untreated WS, the characteristic absorption peaks of 1511, 1380, and 1233 cm–1 were weakened or even disappeared in the DES pretreated WS, indicating that the lignin and hemicellulose were depolymerized during the pretreatment. Furthermore, the absorption bands at 1066 cm–1 after pretreatment using TEBAC/LA were stronger than untreated WS, demonstrating that the cellulose was not damaged.

X-ray Diffraction (XRD) Analysis of WS

XRD analyses of the WS before and after pretreatment by different DESs were performed to monitor the changes in the crystal structure. Figure S3 shows the X-ray diffraction spectra of WS before and after pretreatment using the TEBAC/LA (1:9)-based DES. The crystallization index (CrI) was calculated using the following equation[51,52]where I002 is the maximum diffraction peak intensity of the 002 crystal plane (2θ ≈ 22.5°) and Iam is the intensity of the amorphous cellulose, hemicellulose, and lignin at 2θ ≈ 18.2°.

Obviously, the crystalline structure is classified as cellulose I crystal type as suggested by the peaks at 16.2° (110 planes), 21.8° (200 planes), and 34.8° (004 planes). Moreover, the TEBAC/LA (1:9)-based DES pretreated WS did not undergo the change in the cellulose I crystal form, suggesting that the TEBAC/LA (1:9)-based DES used in the present work does not disrupt the crystal structure of WS. The crystallinity of WS after pretreatment by the TEBAC/LA (1:9)-based DES (47.27%) is higher than that of untreated WS (38.17%), indicating the amorphous components in terms of partial cellulose, hemicellulose, and lignin removal.[53,54] Furthermore, more xylan and lignin removal results in higher CrI values, which is similar to the results reported by other researchers.[33,55]

Regenerated Lignin Structural Characterization

2D-HSQC NMR Analysis

The 2D-HSQC NMR spectroscopy analysis was carried out to demonstrate the structural characteristics of regenerated lignin and is shown in Figure . Generally, the 2D-HSQC NMR spectroscopy could be divided into three regions of 13C–1H correlation corresponding to aliphatic (about δC/δH 0–50/0–2.5 ppm), oxygenated aliphatic (approximately δC/δH 50–100/2.5–6.5 ppm), and aromatic (around δC/δH 100–160/5.5–9.0 ppm) regions.[56] In the oxygenated aliphatic region of 2D-HSQC NMR spectra, the relative abundances of interunit linkages were estimated from intensities of CR–HR, while lignin acylation was estimated from Cγ–Hγ correlations in acylated and nonacylated side chains.[57,58] In the aromatic region, the C2,6–H2,6 correlations from S units and the sum of C2–H2 and C6–H6 correlations from G units were used to estimate the S/G ratio.[57,58] The main interunit linkages of ether or the C–C bond are displayed in Figure , and their proportions were estimated and are listed in Table . Clearly, it can be seen from Figure that the δC/δH = 52.55/3.13 denotes the Cβ–Hβ in β-β′ resinol structures, δC/δH = 55.33/3.46 is the β–5′ in phenylcoumaran substructures, δC/δH = 56.55/3.78 is the C–H in −OCH3 structures, and δC/δH = 66.76/3.87 and 4.19 represents the Cγ–Hγ in β-β′ resinol structures. Interestingly, the structural signal peak of Hibbert ketone (HK) appeared at δC/δH = 69.22/4.93, while there is no Cβ–Hβ signal in the β-O-4′ structure in the side-chain regions, which may be because TEBAC/LA breaks the β-O-4′ bond in the lignin structure and leads to no signal or weak signal here. Furthermore, the β-O-4′ bond breaks lead to the formation of the HK structure, suggesting that the connection structure between lignin extracted from DES is mainly the C–C bond. This is consistent with the similar results of the β-O-4′ fracture in the DES-extracted lignin reported by a previous study.[59] Noticeably, in aromatic (approximately δC/δH 100–160/5.5–9.0 ppm) regions, the signal peaks of the syringyl units (S), guaiacyl units (G), and p-hydroxybenzene (H) units can be detected. It shows that these three basic units exist in the lignin structure. Among them, the signal peaks of the syringyl units (S) and the guaiacyl units (G) are relatively strong, indicating that the extracted lignin is mainly composed of the syringyl units (S) and the guaiacyl units (G).

Figure 4

(A) Side-chain regions in the 2D-HSQC NMR spectra of the regenerated lignin. (B) Aromatic regions in the 2D-HSQC NMR spectra of the regenerated lignin.

Figure 5

Main structures identified in regenerated WS lignin: (A) resinol structures formed by β-β′, α-O-γ′, and γ-O-α′ linkages, (B) phenylcoumaran structures formed by β-5′ and α-O-4′ linkages, (HK) Hibbert ketone, (C) p-coumaroylated substructures formed by Cα′–Hα′, (G) guaiacyl unit, (S) syringyl unit, and (S′ and S″) oxidized syringyl units with a Cα ketone or a Cα carboxyl group.

Table 4

Assignment of the Main Lignin 13C–1H Cross Signals in the 2D-HSQC Spectra of Regenerated WS Lignin

type	δc/δH (ppm)	assignment
Cα′	145.7/7.35	Cα′–Hα′ in p-coumaroylated substructures (C)
C2′,6′	130.7/7.47	C2′,6′–H2′,6′ in p-coumaroylated substructures (C)
H2,6	128.5/7.18	C2,6–H2,6 in p-hydroxybenzene
G6	119.34/6.77	C2,6–H2,6 in guaiacyl units (G)
G5	115.8/6.52116.7/6.77	C5–H5 in guaiacyl units (G)
C3′,5′	116.05/6.85	C3′,5′–H3′,5′ in p-coumaroylated substructures (C)
G2	111.5/6.95	C2–H2 in guaiacyl units (G)
S′2,6	105.1/7.29	C2,6–H2,6 in oxidized (Cα=O) syringyl units (S′)
S″2,6	104.8/7.03	C2,6–H2,6 in oxidized (CαOOH) syringyl units (S″)
S2,6	104.5/6.67	C2,6–H2,6 in syringyl units (S)
HKα	69.22/4.93	Cα=O in Hibbert ketone (HK)
Aγ	66.76/3.87 and 4.19	Cγ–Hγ in β-β′ resinol structures (A)
-OMe	56.55/3.78	C–H in −OCH3 structures
Bβ	56.33/3.46	β–5′ in phenylcoumaran substructures (B)
Aβ	52.55/3.13	Cβ–Hβ in β-β′ resinol structures (A)

FT-IR Analysis

Furthermore, Figure S4 shows the FT-IR spectra of regenerated lignin extracted by the TEBAC/LA-based DES. It can be found that these typical lignin characteristic peaks of 1600, 1511, 1456, and 1427 cm–1 exist in the regenerated lignin extracted by the TEBAC/LA-based DES. Among them, 1600 cm–1 is lignin aromatic ring vibration accompanied by weak C=O vibration, 1511 cm–1 is aromatic ring skeleton vibration, 1456 cm–1 is C–H asymmetric deformation signal peak, and 1427 cm–1 is aromatic ring skeleton vibration surface. Other peaks were due to the aromatic ring breathing of syringyl units (1300 cm–1) and syringyl units (1265 cm–1) and out-of-plane C–H bending of syringyl units (835 cm–1), from which it can be inferred that the extracted lignin structure is dominated by the syringyl units (S) and the guaiacyl units (G) structures.[60]

Conclusions

The present study comprehensively demonstrated the effects of pretreatment time, temperature, and molar ratio of TEBAC/LA on enhanced enzymatic digestibility and lignin valorization. The total sugar concentration was significantly increased to 0.550 g/g WS (0.471 g of glucose and 0.079 g of xylose) under optimal pretreatment conditions with the TEBAC/LA (1:9) system at 373 K for 10 h, which reached 91.27% of the theoretical yield (0.603 g/g). Furthermore, the regenerated lignin (75.69 ± 1.32% purity) was mainly composed of the syringyl units and the guaiacyl units. Thus, the TEBAC/LA (1:9)-based DES provides a promising pretreatment method for selective delignification of WS to enhance enzymatic hydrolysis.

Experimental Section

Materials

The WS milled using a lapping machine and sieved to 60 meshes was collected from Lianyungang (Jiangsu Province, China). Before use, the WS was washed with deionized water and dried to constant weight at 105 °C. The composition of WS was 34.94 ± 1.2% of cellulose, 18.87 ± 0.64% of xylan, and 18.12 ± 0.6% of lignin in terms of dry weight. Triethylbenzyl ammonium chloride (TEBAC; 98%) and lactic acid (LA; AR, ≥85% purity) were purchased from Shanghai Macklin Biochemical Co., Ltd. All chemicals were used without any further purification.

Preparation of DES Mixtures

TEBAC and LA were mixed at specific molar ratios of 1:5, 1:7, 1:9, 1:11, and 1:13. The solutions were added into the three-neck bottles and stirred at 80 °C for 1 h until a homogeneous colorless liquid was obtained. Then, the prepared DESs were stored in a desiccator before use.

General Process for Pretreatment of WS Using DESs

Figure displays a general flow diagram of the WS pretreatment process using the TEBAC/LA-based DES. Typically, the WS pretreatment was carried out in a three-neck bottle with 2.0 g of WS and 30.0 g of DES. Then, the three-neck bottle was placed in a constant temperature oil bath and reacted at different temperatures (353, 363, 373, and 383 K) for a certain time (2, 4, 6, 8, 10, and 12 h) at an agitation speed of 200 rpm. After the reaction, the mixtures were cooled to room temperature, washed with anhydrous ethanol, centrifuged at 10,000 rpm for 5 min, and then transferred the sediment from the lower layer to the vacuum drying chamber to dry the residue. Ethanol was recovered by rotary evaporation from the upper liquid at 323 K to remove the ethanol. Deionized water was added to the concentrated liquid about three times. After a period, the stratified mixture was centrifuged under 10,000 rpm for 5 min. The sediment was washed repeatedly with deionized water, and regenerated lignin was obtained after drying. The upper liquid was removed by rotary evaporation to recover DESs. After the pretreatment step, recovered WS was characterized with regard to its composition, crystallinity, and enzymatic digestibility.

Figure 6

Flow chart of DES pretreatment of wheat straw.

The residue recovery (R%) of WS was calculated by the following equationwhere mR and MWS represent the masses of the residue and raw WS (g), respectively.

The lignin removal (RL%), xylan loss (LXyl%), and cellulose reservation (RC%) of WS are measured based on the content of each component in residues, as displayed in the following equationswhere mRL, mRXyl, and mRC are the masses of lignin, xylan, and cellulose in the residues (g), respectively. MWSL, MWSXyl, and MWSC are the masses of lignin, xylan, and cellulose in the WS (g), respectively.

The purity of lignin (PL%) in the isolated lignin was calculated as followswhere mrL is the mass of lignin in regenerated products (g). Mr is the mass of regenerated products (g).

The morphological analysis of solid fraction coated with gold was observed using a scanning electron microscope (SU70, Japan). FT-IR spectroscopy was determined by a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Germany) using potassium bromide method. Each spectrum was collected from 4000 to 400 cm–1 with a resolution of 4 cm–1. X-ray diffraction (XRD) was confirmed on a Panalytical X’pert Pro diffractometer with Cu Kα irradiation (40 kV and 40 mA) and zero background. Each diffraction pattern was collected in the angles (2θ) from 5° to 50° using a rotation speed of 80 rpm.

1H–13C two-dimensional heteronuclear single quantum correlation (2D-HSQC) NMR spectroscopy of regenerated lignin was conducted on an AVANCE III HD 400 MHz spectrometer and recorded in the gradient HSQC adiabatic version. The sample was dissolved with 80 mg of regenerated lignin in 0.75 mL of deuterated dimethyl sulfoxide (DMSO-d6). The spectral widths of 1H and 13C were 8333 and 22,347 Hz, respectively. The number of collected complex points was 1024 for the 1H dimension with a recycle delay of 1.5 s, the number of transients was 128, and 256 time increments were recorded in the 13C dimension.

Enzymatic Hydrolysis Saccharification

Enzymatic hydrolysis saccharification was carried out in an Erlenmeyer flask (50 mL) containing 0.4 g of the WS samples (pretreated or untreated) and 20 mL of 50 mM citrate buffer (pH 4.8). The dosage of cellulase (cellulase 1.5 L, Novozymes) and β-glucosidase (AMG300L, Novozymes) used were 35 filter paper units (FPU)/g biomass and 82 CBU/g biomass, respectively. The reaction was performed in a constant temperature incubator shaker (ZQZY-80BS, Shanghai Zhichu Instrument Co., Ltd., Shanghai, China) with a speed of 200 rpm at 323 K. Then, 1.00 mL of supernatants was withdrawn and terminated after inactivation in boiling water for 5 min at a time of 60 h. The supernatant was then centrifuged (10,000 rpm for 5 min) and diluted twice by the mobile phase and then filtered by a 0.22 μm membrane to measure the concentrations of sugars using high-performance liquid chromatography (HPLC). The digestibilities of cellulose and xylan were calculated as followswhere Cglucose, Ccellobiose, and Cxylan are the concentrations of glucose cellobiose and xylose in enzymatic hydrolysis system (g/L), respectively. 20/19 is the multiplication factor that converts cellobiose to equivalent glucose. Csubstrate is the substrate loading (g/L), and Cellulose_content and Xylan_content are the content of cellulose and xylan in the substrate (g), respectively. Values 0.90 and 0.88 represent the conversion factors used to convert cellulose and xylan into glucose and xylose, respectively.

The yields of glucose and xylose were calculated using the following equationswhere MHydrolysis-glucose and MHydrolysis-xylose are the mass of glucose and xylose released from enzymatic hydrolysis residues (g), respectively. MWS-glucose and MWS-xylose are the theoretical mass of glucose and xylose in untreated WS (g), respectively.

Analysis Method

The cellulose, xylan, and lignin contents of the untreated and pretreated WS were determined using the National Renewable Energy Laboratory (NREL) analytical method.[61] All the experiments were carried out in duplicate to show the reproducibility of the experiments. The concentrations of sugar were determined by HPLC (Waters 2685 systems, Waters Corp., USA) using an Aminex HPX-87H anion exchange column (300 mm × 7.8 mm, Bio-Rad Corp., USA) equipped with a refractive index detector (Waters 2414). The column temperature was maintained at 328 K, and the flow rate of the mobile phase (5 mM sulfuric acid) was 0.5 mL/min.[62] The quantity of the sample was 10 μL. External standards were established for calibration.