Understanding the structural changes and depolymerization of Eucalyptus lignin under mild conditions in aqueous AlCl₃

Abstract

Lignin is a unique renewable source of phenolic products for the potential replacement of fossil fuels. In this study, swollen residual enzyme lignin (SREL) was prepared and then depolymerized in aqueous AlCl₃ to produce phenolic compounds. The residual lignin after depolymerization was further characterized by FT-IR, advanced NMR (2D-HSQC, ¹³C, and ³¹P NMR spectra) and GPC techniques, and the degradation products were identified and quantified by GC-MS. The results showed that the depolymerization reaction became the leading reaction under these conditions. Meanwhile, the depolymerization process also resulted in a large amount of a volatile fraction, which was composed of phenols, guaiacols, and syringols. Among the volatile fraction, syringaldehyde accounted for about 31.70% of the bio-oil under optimum conditions (at 160 °C for 1 h). Furthermore, the mechanism for lignin depolymerization in aqueous AlCl₃ was discussed and an enhanced understanding of the effects of aqueous AlCl₃ pretreatment on lignin chemistry was provided, which will improve the pretreatment methodology to reduce biomass recalcitrance.

---

1. Introduction

Facing the impending depletion of fossil resources and environmental problems, utilization of lignocellulosic biomass as an alternative for fossil carbon sources is one of the greatest challenges in the current world,^1,2 which can be converted into a variety of products, such as chemicals, materials and energy. Lignocellulosic biomass is mainly composed of three components: cellulose, hemicelluloses and lignin with unique chemical, physical, and structural properties that offer specific opportunities to produce a variety of valuable chemicals. In particular, lignin is one of the most interesting natural polymers due to its high content of aromatic units and many kinds of functional groups, which makes it a promising source of commercial chemicals and renewable products.

In fact, annually exceeding 50 million tons of industrial lignin (Kraft lignin, lignosulfonate, and alkali lignin) is produced worldwide from multitudinous pulping plants. However, the vast majority of lignin is simply disposed as low-cost fuel to burn out and only a small amount of lignin was used for commercial purposes.³ Moreover, the burgeoning biorefinery industry also produce different lignin stream with special composition and chemical structures.⁴ Therefore, it is necessary and important to exploit the technologies and processes for lignin valorization.

Lignin is second most abundant biopolymer on the earth followed cellulose and produced in large quantities by pulping and recent biorefinery industries. In general, lignin is consisted of randomly cross-linked phenylpropanoid unit: coniferyl alcohol (G), and sinapyl alcohol (S), 4-hydroxycinnamyl alcohol (H).⁵ The basic monomers are linked together by ether inter-units (e.g., β-O-4 and α-O-4) and carbon–carbon bonds (e.g., β–β, β-5, β-1, and 5–5) via free radical coupling reaction.⁶ In the plant cell wall, lignin is also covalently linked to polysaccharides to form lignin-carbohydrate complexes (LCCs), which restrict the pretreatment and subsequent enzymatic hydrolysis. Recently, the effects of aluminum chloride-catalyzed hydrothermal pretreatment on the residual lignin in the substrate and enzymatic hydrolysis have been investigated and it was found that most of β-O-4 linkages in the residual lignin were cleaved at higher temperature during the pretreatment.^7,8 However, due to the accompanying polysaccharides, the effects of aluminum chloride-catalyzed hydrothermal pretreatment on the structural changes and depolymerization of lignin have not been thoroughly investigated.

In principle, the abundant aryl ether bonds in lignin make it possible that lignin can be converted into valuable aromatic chemicals due to low bond dissociation energy of aryl ether bonds.⁹ To obtain fuels and chemicals from lignin, multiple pretreatment, depolymerization and upgrading strategies have been emerged, such as, homogeneous and heterogeneous catalysis.^10,11 Wide variety of degradation products could be obtained from lignin depolymerization, such as gases (e.g. low-molecule gas), liquid (e.g. phenols and benzene, aromatic aldehydes) and solid (e.g. activated carbon). However, many kinds of degraded components and expensive procedures are the primary barriers to achieve effective degradation.¹² To overcome this shortcoming, hot compressed water has been used for lignin depolymerization because of its low-cost and eco-friendly. For instance, some researchers have investigated the depolymerization of lignin into value-added aromatic monomers from 215 to 270 °C in the hot compressed water with the assist of solid acid catalyst, in which lignin could be converted into liquid products with 60% yields.¹³ In fact, both catalyst and solvents are vital for lignin depolymerization. Recently, the enhancement effects of heterogeneous catalyst (Pd/C, Pd/C, Raney-Ni, Ni-HTC, Ru/TiO₂, Pt–Re/ZrO₂, and Ni₇Au₃ nanoparticles) on the phenolic product of lignin depolymerization have been evidenced.^14–20 However, due to high cost and complication in the preparation of noble metal catalyst, it is difficult to use noble metal catalyst to depolymerize lignin in industry. Different from heterogeneous catalyst, homogeneous catalyst could be more reactive in conversion of lignin due to the adequately mixing. In recent years, some researchers have investigated the degradation of alkali lignin using metal chlorides as catalysts. In the metal chlorides solution, the ether linkages (e.g., β-O-4 and α-O-4) could be weakened by the rich electronegativity group Cl⁻ and cleaved more easily under the acid center of MCl_x.⁷ It has been found that about 80% β-O-4 bonds of guaiacylglycerol-β-guaiacylether (GG) were hydrolyzed in the presence of AlCl₃.²¹ Besides, Zhang et al. have reported that the guaiacol in volatile was maximum (3.41%) through catalytic depolymerization of alkali lignin with AlCl₃ than those with other metal chlorides.²² Although the depolymerization of lignin in metal chlorides has been studied, only the degradation products in liquid fraction were identified and determinated. In addition, the structural characteristics of starting lignin and residual lignin after depolymerization were not illuminated, which is vital for understanding the whole depolymerization process of lignin.

In this study, swollen residual enzyme lignin (SREL) was chosen as the starting feedstock²³ since SREL was a representative lignin (different from the industrial lignins, such as alkali lignin and organosolv lignin) to understand the structural transformations of lignin during AlCl₃-catalyzed depolymerization processes. To investigate the changes in molecular weight, chemical bonds, and functional groups of lignin during AlCl₃-catalyzed depolymerization process, the SREL pretreated under varying AlCl₃ catalyzed conditions (i.e. 140–180 °C, 1 h) was characterized by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) techniques. In addition, gas chromatography mass-spectrometry (GC-MS) was used to detect the aromatic compounds in the hydrolysate. In short, it is hoped that the results will provide useful information to better understand the structural changes of the lignin during the depolymerization process, which plays an important role in reducing the recalcitrance of biomass and depolymerizing of lignin into aromatic compounds.

2. Material and method

2.1. Material

The raw biomass, Eucalyptus camaldulensis, was obtained from Guangxi province, China. It was cut into pieces, milled and screened in a mill (DFT-200A, Shanghai) to obtain a 40–60 mesh fraction. Eucalyptus sawdust was extracted with toluene–ethanol (2 : 1, v/v). The chemical composition of Eucalyptus sawdust used was 43.5% glucan, 15.7% xylan, 0.6% galactan, 0.8% mannan, 26.3% Klason lignin and 3.94% acetyl groups in terms of dry weight, which was analyzed according to the literature.²⁴ The extractive-free Eucalyptus sawdust was ball-milled (5 h, 450 rpm) in a Fritsch planetary ball mill according to a previous publication.²⁴ All chemicals were analytical grade and purchased from Sigma-Aldrich without further purification.

2.2. Preparation of swollen residual enzyme lignin (SREL)

SREL was prepared according to the recent method with minor modification.²⁵ The ball-milled Eucalyptus wood powder was directly dissolved in 2% NaOH (equivalent to 1 : 25, solid : liquid ratio) for 24 h under stirring at ambient temperature (RT, 25 °C). After stirring, the pH of the resulting mixture was adjusted to 4.8 with acetic acid. Then the mixture was subjected to enzymatic hydrolysis (50 FPU cellulase per g substrate and 50 IU β-glucosidase per g substrate) at 50 °C in a rotary shaker (200 rpm) for 72 h. After enzymatic hydrolysis, the residue (SREL) was obtained by centrifugation and thoroughly washed with hot water (pH = 2.0) to remove the residual enzymes and hydrolyzed carbohydrates, then freeze-dried.

2.3. AlCl₃-catalyzed depolymerization of lignin

All the reactions were carried out in a 100 mL stirred E100 batch reactor (Beijing Century Sen Long Instruments Company, Beijing, China). In a typical run, 0.5 g of SREL and 0.121 g AlCl₃·6H₂O (0.5 mmol) were added into the reactor with 20 mL deionized water. Then the autoclave was heated to setting temperature (140, 150, 160, 170 and 180 °C) under stirring at 1000 rpm, and the reaction was maintained at this temperature for 1 h. After completion of the reaction, the reactor was rapidly cooled to room temperature by built-in cooling coils inside the reactor. After releasing pressure, the pH of the mixed reaction slurry was measured by pH meter, and then adjusted to 2 by 1 M HCl. The liquid and solid fractions (residual SREL) were separated by filtration. The liquid fraction was extracted with CH₂Cl₂ (15 mL × 3). The CH₂Cl₂ soluble phase (bio-oil) was dried at 60 °C and stored for further analysis. After drying at 105 °C, the residual SREL was weighed and stored for further analysis. The residual SRELs were labeled as S₁₄₀, S₁₅₀, S₁₆₀, S₁₇₀ and S₁₈₀, respectively, corresponding to the residue pretreated at various temperatures. S₀ represents for the original SREL.

2.4. Analysis methods

FT-IR spectra of SRELs were obtained using a Thermo Scientific Nicolet iN10 FT-IR microscope as previously.²⁴ The weight average molecular weight (M_w) and number average molecular weight (M_n) of the SRELs were performed with GPC after acetylation. About 30 mg of dry lignin was dissolved in 3 mL of a solution of dimethyl sulfoxide: 1-methylimidazole (2 : 1, v/v) and stirred without direct light at room temperature for 12 h. Acetic anhydride (1.0 mL) was added to the reaction mixture and continued reacting for 2 h. The reaction mixture was dropped slowly into 100 mL acid water (pH = 2) to induce precipitation followed by centrifugation. The acetylated lignin was dissolved in tetrahydrofuran (THF) (2 mg mL⁻¹), and the solution was filtered through a 0.22 μm filter. The filtered solution (20.0 μL) was injected into the HPLC system and detected using an UV detector set at 280 nm. THF was used as the mobile phase and the flow rate was 0.5 mL min⁻¹. Standard PL polystyrene samples were used for calibration. Thermal stability of the SRELs was performed using thermogravimetric analysis (TGA) (DTG-60, Shimadzu, Japan) according to a previous publication.^25,26 The SREL was heated in an aluminum crucible to 800 °C at a heating rate of 10 °C min⁻¹. NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer at 25 °C. Solid state CP/MAS ¹³C-NMR spectra of the SRELs were obtained at 100.6 MHz and 2D-HSQC spectra of the SRELs were performed according to a previous literature.^26,31 P NMR spectra were acquired after the reaction of lignin with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) according to the literatures.^26–28 The compounds in bio-oil were identified by GC/MS (Agilent 7890/5978, USA) using a 30 m × 0.25 mm × 0.25 μm capillary column (HP-5MS). Gas chromatography was carried out at 35 °C for 5 min before the temperature was increased to 280 °C at 2 °C min⁻¹. The injection volume was 1 μL and the injector temperature was 300 °C with a split ratio of 5 : 1. The compounds were identified by comparison with library spectra supplied by the NIST database.

3. Results and discussion

The effective lignin preparation method “swollen residual enzyme lignin” (SREL) facilitates development of more effective depolymerization strategies in the current biorefinery and catalytic conversion process. In this study, SREL was subjected to aqueous AlCl₃ at 140–180 °C for 1.0 h, and the residual lignin and liquids were separated. Liquid yield, pH values of the liquids and molecular weight of the residual lignin were listed in Table 1. As shown in Table 1, the pH of the initial solution without pretreatment was 3.55, while it sharply reduced to 1.86 in the pretreated liquid when the pretreatment temperature reached to 180 °C. Accordingly, the liquid yield increased from 23.34 to 35.76%. However, the liquid yield of lignin in the present study is lower than those reported in the recent publications.^22,29,30 By contrast, the yield of residual lignin is still 64.24% under the optimal condition. The reason for the low liquid yield of lignin is probably ascribed to poor solubilization of lignin and corresponding depolymerized products in aqueous phase during depolymerization, which makes the generated intermediates instable and tends to form condensed structures, thus decreasing the liquid yield.³¹ To investigate the detailed structural changes of the residual lignin after the homogeneous depolymerization, the molecular weights, chemical bonds, functional groups, and thermal properties of the lignin fractions were all investigated as compared to the control SREL.

Table 1 Liquid yield, pH and molecular weight of the SREL before and after depolymerization process

Entry	Temperature (°C)	pH	Liquid yield^a (%)	Mw (g mol^−1)	Mn (g mol^−1)	Mw/Mn
a ; Mdry, dried raw lignin (g); MSR, solid residue (g).
1	Control	3.55	—	4210	1250	3.38
2	140	2.24	23.34	2770	1880	1.48
3	150	2.21	33.18	2400	1740	1.38
4	160	2.55	33.76	2630	1910	1.38
5	170	2.13	35.50	3050	2110	1.45
6	180	1.86	35.76	3690	2410	1.53

---

3.1. Molecular weights analysis of the residual SREL

Changes in molecular weights of residual lignin can provide important insights into depolymerization and recondensation of lignin during the AlCl₃-catalyzed depolymerization process. To confirm the depolymerization of lignin, molecular weights and polydispersity index were determined by GPC. Results listed in Table 1 demonstrated clearly that the average molecular weight (M_w) of the lignin first decreased significantly (140–160 °C) and then increased with the increase of reaction temperature (170–180 °C). The depolymerization and recondensation of lignin were competing reactions during the AlCl₃-catalyzed depolymerization process, which was observed by 2D-HSQC analysis. The depolymerization reaction became the leading reaction with the increased temperatures, thus leading to the reduced M_w of lignin under mild conditions. As the temperature was elevated to 160–180 °C, recondensation reaction probably became the dominant reaction. Additionally, polydispersity index (PDI) was slightly increased with the rise of the temperature (160–180 °C), which could be attributed to the condensation phenomena of several phenolic oligomers (repolymerization).³²

3.2. FT-IR analysis

FT-IR spectroscopy is frequently used to investigate the structural changes of the SREL during the depolymerization process. Fig. 1 shows the FT-IR spectra of the SREL samples before and after depolymerization, and peaks were assigned according to the literatures.^33,34 The spectrum of the SREL shows a wide absorption band at 3400 cm⁻¹, which is attributed to phenolic or aliphatic OH groups, followed by the bands at 2939 cm⁻¹ and 2844 cm⁻¹ originated from the C–H asymmetric and symmetrical vibrations of the methyl and methylene groups, respectively. The peak at 1658 cm⁻¹ is related to the C=O stretching vibrations in conjugated carboxylic acid and ketone groups. Simultaneously, the signals at 1592 and 1504 cm⁻¹ correspond to aromatic skeletal vibrations and the C–H deformation vibrations, respectively. The band of the C–H deformations asymmetric in –CH₃ of methoxy groups is at 1459 cm⁻¹ and 1420 cm⁻¹. The bands at 1326, 1266, 1119 cm⁻¹, corresponding to breathing vibration of syringyl and condensed guaiacyl, guaiacyl ring breathing with C=O stretching vibrations, are unmistakable signals of a typical G–S lignin. In addition, the peaks of 1223, 1031 and 838 cm⁻¹, which are attributed to the C–C or C=O stretching vibrations, aromatic C–H in-plane deformation vibrations and the C–H out-of plane stretching vibrations, could be distinctly observed in the Fig. 1. The similarity of these spectra implied that the “core” of the lignin structure didn't observably change during different depolymerization temperatures. However, some changes of the lignin structure occurred with the increase of depolymerization temperature. For example, the intensity band at 1326 and 1266 cm⁻¹ (syringyl, condensed guaiacyl and guaiacyl C=O units) was gradually reduced, which was in agreement with the analysis of the following CP-MAS ¹³C NMR spectra of the SRELs before and after depolymerization process (Fig. 2), suggesting that the SREL was clearly depolymerized. Meanwhile, the peak intensity of the C–H deformations asymmetric in –CH₃ of methoxy groups was also decreased, suggesting that demethoxylation reaction of lignin probably occurred during this process.

Fig. 1 FT-IR spectra of the lignin samples before and after depolymerization.

Fig. 2 ¹³C CP-MAS NMR spectra of the lignin samples before and after depolymerization.

3.3. CP-MAS ¹³C NMR spectral analysis

To further investigate the chemical and structural changes of SREL during the depolymerization process, CP-MAS ¹³C NMR spectra of the SRELs are shown in Fig. 2. The detailed assignments of the SREL could be obtained by a previous publication.³⁵ It was found that the ¹³C NMR spectra of the SRELs were similar except for different intensities of these signals. The obvious signals are listed as follows, such as, 152.3 ppm (etherified S_3/5 unit, mainly β-O-4), 148.0–147.1 ppm (nonetherified S_3,5 unit), 135.0–134.4 ppm (etherified G₁ or S₁), and 103.1 ppm (S_2,6), respectively. Meanwhile, the C_β and C_α of β-O-4 linkages as well as methoxy group in the SREL fractions could be distinguished by 83.9, 71.8–71.4 and 54.8 ppm, respectively. In addition, the signals between 171.3 and 201.5 ppm are attributed to carbonyl group (C=O) in the SREL, the peaks appeared at 201.5, 196.8, 185.6–184.7 and 171.0 ppm represent for ketone, aldehyde, carboxylic acid and ester of the SREL, respectively.³⁵ Moreover, the signals of etherified S_3,5 unit and β-O-4 linkages were significantly decreased, while the signals of nonetherified S_3,5 unit were increased as the temperature rose. This fact indicated that S-type units in the SREL were more susceptible to be degraded than G-unit under the AlCl₃-catalyzed depolymerization, in accordance with the FT-IR analyses aforementioned. Interestingly, it was found that the signals of the ketone, carboxylic acid and ester in the SREL were significantly decreased, while the signal of aldehyde was increased with the temperature increased. Therefore, a possible speculation is that cleavage of the C_α–C_β bond of side chain in the lignin led to the fact that C=O of ketone in the C_α position has been transformed into C=O of aldehyde in the C_α position.

3.4. 2D-HSQC NMR spectral analysis

2D-HSQC is an efficient method to further understand the compositional and structural changes of the SREL during different depolymerization temperatures. The side-chain region (δ_C/δ_H 50.0–90.0/2.50–6.00) and aromatic region (δ_C/δ_H 100.0–150.0/5.50–8.50) of the SREL are shown in Fig. 3, the detailed assignments of HSQC spectra of SREL are listed in Table S1† and the major substructures are depicted in Fig. 4 based on recent publications.^33,35

Fig. 3 Side-chain (a) and aromatic region (b) in the 2D-HSQC NMR spectra of the lignins. Lignin correlations are labeled with color-coded structures as given in Fig. 4.

Fig. 4 Main classical substructures, involving different side-chain linkages, and aromatic units identified by 2D NMR of Eucalyptus lignin: (A) β-O-4 aryl ether linkages with a free –OH at the γ-carbon (blue); (B) resinol substructures formed by β–β, α-O-γ, and γ-O-α linkages; (C) phenylcoumarane substructures formed by β-5 and α-O-4 linkages; (G) guaiacyl units; (S) syringyl units; (S′) oxidized syringyl units with a C_α ketone.

In the aliphatic region, the cross-peaks of methoxy groups in side chains and the inter-unit linkages in lignin, such as β-O-4 aryl ether linkages (A) and resinol (β–β, B) were the most predominant substructures. Obviously, the signal of methoxyl groups could be observed at δ_C/δ_H 56.4/3.70. The C_α–H_α correlations in classical β-O-4 substructures were observed at δ_C/δ_H 71.6/4.83, while the C_β–H_β correlations in β-O-4 substructures linked to G unit and S unit could be distinguished at δ_C/δ_H 83.9/4.30 and 85.9/4.11, respectively. The C_γ–H_γ correlations in β-O-4 substructures were observed at δ_C/δ_H 59.5/3.69. Besides, signals for resinol substructures (β–β, B) were observed in the spectra, with their C_α–H_α, C_β–H_β and the double C_γ–H_γ correlations at δ_C/δ_H 85.3/4.70, 54.0/3.10, and 71.4/3.85 and 4.22, respectively. As compared with the raw SREL, the spectra of SRELs after depolymerization have only faint signals of β-O-4 linkages. This phenomenon is attributable to the cleavage of β-O-4 aryl ether linkages in SREL. As shown in Fig. 3a, the signals of β-O-4 linkages were almost disappeared at 160 °C in residual SREL. The previous studies on lignin/lignin model compounds have proven that the β-ether and α-ether bonds in the β-O-4 and α-O-4 linkages in lignin are readily cleaved during high temperatures, while the 5–5 (biphenyl)-type and aromatic rings structures are relatively stable.^36–39 Therefore, the raw lignin could not be completely degraded into oligomer and monomer under the depolymerization in the presence of AlCl₃. However, the obvious cleavage of β-O-4 linkages would facilitate the degradation of SREL because β-O-4 aryl ether bonds are the main linkages in lignin.

In the aromatic region, the signals of S and G units can be clearly observed in the spectra. The normal S unit showed a remarkable signal for the C_2,6–H_2,6 (S_2,6) at δ_C/δ_H 104.0/6.72. In addition, the gradually increasing signal at δ_C/δ_H 106.3/7.26 was corresponds to C_2,6–H_2,6 in oxidized S unit (C=O in α position of S′_2,6).³⁵ By contrast, the signals at δ_C/δ_H 111.0/6.99 (C₂–H₂), 119.0/6.80 (C₆–H₆), 114.8/6.68 (C₅–H₅) and 114.8/6.98 (C₅–H₅) exhibited the presence of G unit. The double signals of C₅–H₅ showed some heterogeneity among the G unit, which especially affects the C₅–H₅ correlation, probably due to different substituents at C₄ (etherified and no-etherified C-4 in aromatic ring).⁴⁰ As can be seen, with the increasing temperature, the normal G-type and S-type in lignin were gradually decreased, especially the evanescent signal for G₆, while the increasing signal of condensed G and S-type lignin structure appeared, suggesting the condensed G and S units in lignin were likely formed in a relatively high temperature.³²

Quantification of the SRELs samples by 2D-HSQC NMR method can provide explicit structural evolution at different depolymerization temperatures. According to the computing method of the literatures, the different linkages could be expressed by a comparative mode.^35,41 Fig. 3b shows the S/G ratios of SREL and relative abundance of the inter-unit linkages involved in the main substructures in the lignin. As shown in Fig. 3b, the relative content of β-O-4 linkage in the untreated lignin is 46.4/100Ar, while the signal of β-O-4 gradually disappeared as the temperature increased to 180 °C, implying that AlCl₃-catalyzed depolymerization enhanced the degradation of lignin. The data herein indicated that the cleavage of β-O-4 aryl ether was the main reaction during the AlCl₃ depolymerization at high temperatures. In addition, S/G ratios of the SREL were another prominent structural alterations observed after depolymerization. The S/G ratio increased as the depolymerization temperature rose and reached a maximum ratio (S/G = 6.02) in S₁₅₀ suggesting that the G-type lignin was more easily degraded at relatively low temperature. However, as the depolymerization temperature further increased from 160 to 180 °C, the S/G ratio gradually decreased to 2.62 in S₁₈₀, suggesting that both G-type and S-type lignin were degraded at a relatively high temperature.

3.5. ³¹P-NMR analysis

To further investigate the fundamental chemistry of the SREL obtained after AlCl₃-catalyzed depolymerization, the SREL samples were analyzed using the ³¹P NMR technique.⁴² Table 2 shows the quantitative data on the distribution of the different OH groups in the SREL. The ³¹P NMR data confirmed that SREL belonged to the guaiacyl–syringyl type, as evidenced by the presence of syringyl- and guaiacyl-type phenolic structures in the ³¹P NMR spectra of all SRELs.

Table 2 Quantification of the SREL by quantitative ³¹P NMR (mmol g⁻¹)

Lignins	Aliphatic OH	Syringyl OH		Guaiacyl OH		Carboxylic group	Total phenolic OH
		C^a	NC^b	C	NC
a C, condensed.b NC, non-condensed.
S0	4.04	0.07	0.15	0.04	0.18	0.09	0.44
S140	1.00	0.27	0.41	0.13	0.17	0.05	0.98
S150	1.23	0.26	0.56	0.19	0.25	0.07	1.26
S160	1.01	0.28	0.65	0.16	0.25	0.07	1.34
S170	2.72	0.37	0.78	0.20	0.30	0.08	1.65
S180	0.54	0.51	1.12	0.31	0.44	0.08	2.38

---

As shown in Table 2, the contents of S- and G-type phenolic OH groups were greatly increased from S₀ to S₁₈₀, which was attributable to the cleavage of the β-O-4 aryl ether linkages. During the cleavage of the β-O-4 linakges of the lignin macromolecule, small phenolic compounds were simultaneously released, resulting in the appearance of lignin-degraded products, which will be discussed in the subsequent section. Additionally, the content of S-type phenolic OH was higher than that of G-type phenolic OH in SREL and the increasing rate of S-type lignin was faster than that of the corresponding G-type lignin, suggesting that most of the β-O-4 linkages cleaved are mainly composed of S unit. Moreover, the gradually increased condensed S and G phenolic OH implied that increasing depolymerization temperature also resulted in condensation reactions during this process, which is in agreement with the 2D HSQC NMR aforementioned. The content of aliphatic OH was reduced (4.04–0.54 mmol g⁻¹), while all the SREL showed similar content of COOH (0.05–0.09 mmol g⁻¹), suggesting that a part of aliphatic OH were oxidized to carbonyl groups in air atmosphere during the pretreatment, as revealed by the aforementioned CP-MAS NMR and 2D-HSQC characterization of the residural lignin. Similarly, the chemical conversion of aliphatic OH into carbonyl groups has been mentioned in some recent publications.^43,44

3.6. Thermogravimetric analysis

The thermal stability of lignin is significantly important for its utilization in thermochemical transformation into chemicals and energy, particularly, thermolysis.⁴⁵ TG curves reveal the relationship between the weight losses of lignin and depolymerization temperature, while DTG curve represents the corresponding rates of weight loss. The thermolysis of lignin was closely related to its specific structures. The rate of lignin thermo-decomposition is mainly influenced by its inherent structure and the various functional groups of lignin.⁴⁶ To investigate the relationship between structural features and thermal properties, both TGA and DTG curves of the SREL before and after depolymerization are shown in Fig. 5.

Fig. 5 TG/DTG curves of the lignin samples before and after depolymerization.

As shown in Fig. 5, the thermo-decomposition rate of the SREL (S₀) was faster than those of the degraded SREL (S₁₄₀, S₁₅₀, S₁₆₀, S₁₇₀ and S₁₈₀) at the beginning of decomposition, which was attributable to the fact that the initial reaction involved deformation of the weaker C–O bond in the β-O-4 linkages during the SREL thermal decomposition.⁴⁷ Majority of the aryl ether bond linkages would be cleaved at the stage of 200–350 °C, which is shown by the high content of β-O-4 in S₀ (Fig. 3a). When decomposition temperature was around 350–400 °C, the increased decomposition rate was probably due to the lignin side chain oxidation (i.e. carbonylation/carboxylation of aliphatic hydroxyl group, side chain dehydrogenation).⁴⁸ Afterward, the increased decomposition rate above 400 °C was attributed to cleavage of 5–5 (biphenyl) linkages within the SREL. It has been reported that the aromatic ring, C–C bond cleavage of lignin as well as the release of H₂O, CO₂ and CO also begin above 400 °C under nitrogen.⁴⁹ Finally, typical weight losses in TG curves flatten out above 500 °C, with a slow release of the final products before final char formation. The “char residues” at 700 °C were 34.1% for S₀, 38.6% for S₁₄₀, 40.5% for S₁₅₀, 40.6% for S₁₆₀, 45.6% for S₁₇₀ and 43.6% for S₁₈₀ in this work, implying that more condensed lignin structures were found at the high depolymerization temperature, as revealed in the aforementioned NMR spectra. In addition, the T_M of the SREL shifted to higher temperatures with the raised temperature, suggesting that more stable lignin structures (e.g. condensed lignin structures) were formed as the temperature rose, which correlates well to the aforementioned the NMR results (Fig. 3).

3.7. GC-MS characterization of degradation product in bio-oils

To further understand the lignin depolymerization reactions, the obtained bio-oils were analyzed by GC-MS. The chemical composition and retain time of degradation product in bio-oils are shown in Table 3 and Fig. 6. The bio-oils were composed of a series of complex organic compounds mixtures. As shown in Table S3,† aldehydes, ketones, carboxylic acids, esters, phenolics, hydrocarbons, furan derivatives, benzene derivatives can be detected and identified due to their detectable amount. The peak area percentages of the identified compounds in each temperature are listed in Table 3. It was observed that the main chemical compounds of the bio-oils after depolymerization of lignin were phenols (0.13–0.6%), guaiacols (5.31–10.41%) and syringols (23.28–38.04%) (Table S2†), such as vanillin, 4-hydroxy-3-methoxy-benzoic acid, 2,6-dimethoxy-phenol, 4-hydroxy-3,5-dimethoxy-benzaldehyde, 1-(4-hydroxy-3,5-dimethoxyphenyl)-ethanone, 4-hydroxy-3,5-dimethoxy-benzoic acid; the amount of these compounds were in the range of 85–93% of bio-oils depending on different reaction conditions. Meanwhile, the aromatic aldehyde was major product and the yield was up to 39.96%, including 8.26% vanillin and 31.7% syringaldehyde, implying that the ether bonds (e.g., α-O-4, 5-O-4, and β-O-4) and C–C bonds (e.g., β-5) were cleaved, and small molecular organic compounds were simultaneously formed. The result was in agreement with a previous report.⁵⁰ As shown in Table 3, the yield of total aromatic aldehyde firstly increased from 27.70% to 39.96% at 160 °C, while decreased to 25.35% at 180 °C, implying that the generation of aromatic compounds was main reaction under the low temperatures (140–160 °C), while these aromatic compounds further repolymerized with the high temperatures (170–180 °C). Moreover, small amounts of furan derivatives and levulinic acid were also detected and confirmed by GC-MS, implying that these compounds were produced by the transformation of cellulosic residues in the lignin.^51,52

Table 3 Main monomeric products from SREL depolymerization

Degradation product	Area percentage^a (%)
	140 °C	150 °C	160 °C	170 °C	180 °C
a The matching degree of all the compounds here were more than 80%, and the compounds whose matching degree less than 80% were not listed.b Not detected.
Vanillin	4.42	5.26	8.26	7.21	5.55
Benzoic acid, 4-hydroxy-3-methoxy-	0.71	0.87	1.36	1.27	1.11
2,4′-Dihydroxy-3′-methoxyacetophenone	—^b	—	0.5	0.19	—
Phenol, 2,6-dimethoxy-	0.33	0.27	0.54	1.08	1.61
Syringaldehyde	19.07	23.21	31.7	24.47	19.77
Ethanone, 1-(4-hydroxy-3,5-dimethoxyphenyl)-	1.23	1.5	1.97	1.81	1.59
Benzoic acid, 4-hydroxy-3,5-dimethoxy-	1.81	2.62	3.57	3.38	2.78

---

Fig. 6 GC-MS for the bio-oil obtained from lignin depolymerization (T = 160 °C).

3.8. Proposed degradation mechanism

As discussed above, the AlCl₃-catalyzed hydrothermal depolymerization mainly results in the release of vanillin and syringaldehyde from lignin macromolecule. Therefore, its depolymerization mechanism was proposed based on the detailed structural analysis of the residual SREL, the phenolic monomer and other volatile products with AlCl₃ catalysts (Fig. 6). It was also reported that AlCl₃ was a high-effective catalyst for cellulose conversion to furfural and the conversion mechanism demonstrated that Al³⁺ could coordinate with the electron-rich oxygen to form a complex, which can further degrade into HMF.^53,54 Based on the existing knowledge about the processes and the results obtained from this work, a possible reaction pathway for the aromatic aldehyde generation was proposed. In this pathway (Fig. 7), (1) primary hydroxyl group at the C_γ position of the lignin was first oxidized to aldehyde group; (2) underwent keto–enol tautomerism to form intermediate; (3) [AlCl₄]⁻ was conjuncted with O atom and the electron-rich benzene ring of lignin is to form stable intermediate; (4). Simultaneously, the electron of the β-O-4 ether linkage transfers to oxygen atom, resulting in the weakening of C–O bond to form oligomer and a intermediate; (5) [AlCl₄]⁻ of stable complex was eliminated to form carbocation of C_β position; (6) afterwards, a series of typical processes for the intermediate occurred, such as hydrolysis, electron transfer, dehydrogenation, and oxidization, which would produce acetic acid, vanillin and syringaldehyde; (7) eventually, diverse phenols were obtained in the following processes. Therefore, a better understanding of the AlCl₃-catalyzed mechanism could efficiently control the product distribution during lignin depolymerization process.

Fig. 7 Proposed catalytic mechanism of AlCl₃ catalysis.

3.9. Implications

In this study, AlCl₃ was used as catalyst to transform and degrade the SREL in aqueous systems, however, increasing condensed structures occurred during the process, which indeed decrease the liquid yield. The ideal object of the catalysts in hydrothermal degradation of lignin is to cleave the bond among lignin units and to inhibit the condensation.⁴⁴ In fact, in a real lignin degradation system, besides the catalysts, solvent system and degradation temperature is also important to the final yield and product distribution of degraded products. It was reported that decomposition of lignin in hot organic solvent–water solution would promote its final degradation because that the addition of organic solvent in hydrothermal system improves the solubility of lignin and its decomposition products, additionally, the reaction temperature of decomposition of lignin can be reduced by the presence of alcohols or organic acids due to their low critical point of the supercritical condition.⁴⁴ In short, AlCl₃-based hydrothermal degradation has some advantages due to its required relative low temperature, and syringaldehyde and vanillin can be selectly prepared from lignin. Further studies on the development of selective catalysts and synergic degradation systems should be conducted to optimize the production of degraded products.

4. Conclusions

In the present study, structural changes and depolymerization of swollen residual enzyme lignin (SREL) in aqueous AlCl₃ at mild condition (140–180 °C, 60 min) were applied and comprehensively investigated. The results showed that the depolymerization reaction occurred under the mild conditions (140–150 °C), while repolymerization reaction took place at higher temperatures (160–180 °C), as revealed by the changes of molecular weight, chemical composition, substructures and phenolic hydroxyl groups. In addition, aldehydes, ketones, carboxylic acids, esters, phenolics etc. could be detected in the bio-oil, in which 31.70% syringaldehyde and 8.26% vanillin could be obtained. In short, understanding of the effects of aqueous AlCl₃ pretreatment on lignin chemistry will improve the pretreatment methodology. Furthermore, the efficient depolymerization system could be an alternative approach for further utilization of this abundant, unique, and renewable natural aromatic resource.

Acknowledgements

The authors wish to express their gratitude for the financial support from Natural Science Foundation of China (31430092, 31500486, and 31110103902), International Science & Technology Cooperation Program of China (2015DFG31860).

References

1. P. Alvira, E. Tomas-Pejo, M. Ballesteros and M. J. Negro, Bioresour. Technol., 2010, 101, 4851–4861.
2. N. Mosier, C. Wyman, B. Dale, R. Elander, Y. Y. Lee, M. Holtzapple and M. Ladisch, Bioresour. Technol., 2005, 96, 673–686.
3. X. Ouyang, X. Qiu and P. Chen, Colloids Surf., 2006, 282–283, 489–497.
4. T. Saito, R. H. Brown, M. A. Hunt, D. L. Pickel, J. M. Pickel, J. M. Messman, F. S. Baker, M. Keller and A. K. Naskar, Green Chem., 2012, 14, 3295.
5. E. Ämmälahti, G. Brunow, M. Bardet, D. Robert and I. Kilpeläinen, J. Agric. Food Chem., 1998, 46, 5113–5117.
6. J. Ralph, Phytochem. Rev., 2010, 9, 65–83.
7. R. Shu, J. Long, Z. Yuan, Q. Zhang, T. Wang, C. Wang and L. Ma, Bioresour. Technol., 2015, 179, 84–90.
8. A. V. Maldhure and J. D. Ekhe, J. Environ. Chem. Eng., 2013, 1, 844–849.
9. F. P. Bouxin, A. McVeigh, F. Tran, N. J. Westwood, M. C. Jarvis and S. D. Jackson, Green Chem., 2015, 17, 1235–1242.
10. Q. Song, F. Wang, J. Cai, Y. Wang, J. Zhang, W. Yu and J. Xu, Energy Environ. Sci., 2013, 6, 994.
11. A. Rahimi, A. Ulbrich, J. J. Coon and S. S. Stahl, Nature, 2014, 515, 249–252.
12. M. P. Pandey and C. S. Kim, Chem. Eng. Technol., 2011, 34, 29–41.
13. A. K. Deepa and P. L. Dhepe, RSC Adv., 2014, 4, 12625.
14. Q. Song, F. Wang and J. Xu, Chem. Commun., 2012, 48, 7019–7021.
15. J. A. Onwudili and P. T. Williams, Green Chem., 2014, 16, 4740–4748.
16. M. Nagy, K. David, G. J. P. Britovsek and A. J. Ragauskas, Holzforschung, 2009, 63, 513–520.
17. M. R. Sturgeon, M. H. O'Brien, P. N. Ciesielski, R. Katahira, J. S. Kruger, S. C. Chmely, J. Hamlin, K. Lawrence, G. B. Hunsinger, T. D. Foust, R. M. Baldwin, M. J. Biddy and G. T. Beckham, Green Chem., 2014, 16, 824–835.
18. T. Omotoso, S. Boonyasuwat and S. P. Crossley, Green Chem., 2014, 16, 645–652.
19. H. Ohta, B. Feng, H. Kobayashi, K. Hara and A. Fukuoka, Catal. Today, 2014, 234, 139–144.
20. H. Konnerth, J. Zhang, D. Ma, M. H. G. Prechtl and N. Yan, Chem. Eng. Sci., 2015, 123, 155–163.
21. S. Y. Jia, B. J. Cox, X. W. Guo, Z. C. Zhang and J. G. Ekerdt, Ind. Eng. Chem. Res., 2011, 50, 849–855.
22. X. H. Zhang, Q. Zhang, J. X. Long, Y. Xu, T. J. Wang, L. L. Ma and Y. P. Li, BioResources, 2014, 9, 3347–3360.
23. J. L. Wen, S. L. Sun, T. Q. Yuan, F. Xu and R. C. Sun, J. Agric. Food Chem., 2013, 61, 11067–11075.
24. X. J. Shen, B. Wang, P.-L. Huang, J.-L. Wen and R.-C. Sun, Bioresour. Technol., 2016, 206, 57–64.
25. J. L. Wen, S.-L. Sun, T.-Q. Yuan and R.-C. Sun, Green Chem., 2015, 17, 1589–1596.
26. J. L. Wen, S. L. Sun, B. L. Xue and R. C. Sun, J. Agric. Food Chem., 2013, 61, 635–645.
27. A. Granata and D. S. Argyropoulos, J. Agric. Food Chem., 1995, 43, 1538–1544.
28. C. Crestini and D. S. Argyropoulos, J. Agric. Food Chem., 1997, 45, 1212–1219.
29. N. Mahmood, Z. Yuan, J. Schmidt and C. C. Xu, Bioresour. Technol., 2015, 190, 416–419.
30. W. Jiang, G. Lyu, Y. Liu, C. Wang, J. Chen and L. A. Lucia, Ind. Eng. Chem. Res., 2014, 53, 10328–10334.
31. C. Wang, F. Zhou, Z. Yang, W. Wang, F. Yu, Y. Wu and R. A. Chi, Biomass Bioenergy, 2012, 42, 143–150.
32. J. Li, G. Henriksson and G. Gellerstedt, Bioresour. Technol., 2007, 98, 3061–3068.
33. J. L. Wen, B.-L. Xue, F. Xu, R.-C. Sun and A. Pinkert, Ind. Crops Prod., 2013, 42, 332–343.
34. O. Faix, in Holzforschung – International Journal of the Biology, Chemistry, Physics and Technology of Wood, 1991, p. 21.
35. J. L. Wen, S.-L. Sun, B.-L. Xue and R.-C. Sun, Materials, 2013, 6, 359–391.
36. R. Parthasarathi, R. A. Romero, A. Redondo and S. Gnanakaran, J. Phys. Chem. Lett., 2011, 2, 2660–2666.
37. R. Singh, A. Prakash, S. K. Dhiman, B. Balagurumurthy, A. K. Arora, S. K. Puri and T. Bhaskar, Bioresour. Technol., 2014, 165, 319–322.
38. Z. Yuan, S. Cheng, M. Leitch and C. Xu, Bioresour. Technol., 2010, 101, 9308–9313.
39. E. Adler, Wood Sci. Technol., 1977, 11, 169–218.
40. J. Rencoret, G. Marques, A. Gutiérrez, L. Nieto, J. Jiménez-Barbero, Á. T. Martínez and J. C. del Río, Ind. Crops Prod., 2009, 30, 137–143.
41. M. Sette, R. Wechselberger and C. Crestini, Chemistry, 2011, 17, 9529–9535.
42. Y. Pu, S. Cao and A. J. Ragauskas, Energy Environ. Sci., 2011, 4, 3154–3166.
43. G. Chatel and R. D. Rogers, ACS Sustainable Chem. Eng., 2014, 2, 322–339.
44. S. Kang, X. Li, J. Fan and J. Chang, Renewable Sustainable Energy Rev., 2013, 27, 546–558.
45. M. F. Li, S.-N. Sun, F. Xu and R.-C. Sun, Chem. Eng. J., 2012, 179, 80–89.
46. E. Jakab, O. Faix and F. Till, J. Anal. Appl. Pyrolysis, 1997, 40–41, 171–186.
47. T. Faravelli, A. Frassoldati, G. Migliavacca and E. Ranzi, Biomass Bioenergy, 2010, 34, 290–301.
48. J. L. Wen, B.-L. Xue, S.-L. Sun and R.-C. Sun, J. Chem. Technol. Biotechnol., 2013, 88, 1663–1671.
49. H. P. Yang, R. Yan, H. P. Chen, D. H. Lee and C. G. Zheng, Fuel, 2007, 86, 1781–1788.
50. W. Diono, M. Sasaki and M. Goto, Chem. Eng. Process., 2008, 47, 1609–1619.
51. Z. Tang, Y. Zhang and Q. Guo, Ind. Eng. Chem. Res., 2010, 49, 2040–2046.
52. T. D. Matson, K. Barta, A. V. Iretskii and P. C. Ford, J. Am. Chem. Soc., 2011, 133, 14090–14097.
53. L. Zhang, H. Yu, P. Wang, H. Dong and X. Peng, Bioresour. Technol., 2013, 130, 110–116.
54. M. Schwiderski, A. Kruse, R. Grandl and D. Dockendorf, Green Chem., 2014, 16, 1569.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra08945c

---