Unraveling the Lignin Structural Variation in Different Bamboo Species

The structure of cellulolytic enzyme lignin (CEL) prepared from three bamboo species (Neosinocalamus affinis, Bambusa lapidea, and Dendrocalamus brandisii) has been characterized by different analytical methods. The chemical composition analysis revealed a higher lignin content, up to 32.6% of B. lapidea as compared to that of N. affinis (20.7%) and D. brandisii (23.8%). The results indicated that bamboo lignin was a p-hydroxyphenyl-guaiacyl-syringyl (H-G-S) lignin associated with p-coumarates and ferulates. Advanced NMR analyses displayed that the isolated CELs were extensively acylated at the γ-carbon of the lignin side chain (with either acetate and/or p-coumarate groups). Moreover, a predominance of S over G lignin moieties was found in CELs of N. affinis and B. lapidea, with the lowest S/G ratio observed in D. brandisii lignin. Catalytic hydrogenolysis of lignin demonstrated that 4-propyl-substituted syringol/guaiacol and propanol guaiacol/syringol derived from β-O-4′ moieties, and methyl coumarate/ferulate derived from hydroxycinnamic units were identified as the six major monomeric products. We anticipate that the insights of this work could shed light on the sufficient understanding of lignin, which could open a new avenue to facilitate the efficient utilization of bamboo.


Introduction
As the cell wall component of terrestrial plants, lignin is widely distributed in nature, and its content is second only to (hemi)cellulose [1,2]. Lignin contains a complex structure of the phenolic polymer, which is especially suitable for the production of aromatic chemicals, and its components vary with different plant materials [3][4][5][6][7][8]. However, most biorefinery schemes focus on the use of easy-to-use compositions, while lignin is relatively underutilized [9][10][11]. For example, about one million t/y of lignosulfonate accounted for only 2% of the total production for commercialization [12,13]. Catalytic conversion of lignin for the production of small molecule chemicals or fuels has received much attention because of its high yield and widespread availability [14]. Nevertheless, it is still a significant challenge for lignin valorization in biorefinery owing to its complexity and heterogeneity of the internal macromolecular structure [15,16]. To solve the above-mentioned challenges, it is necessary to gain a broad understanding of the "main" structural characteristics of lignin to provide a theoretical basis for biomass upgrading, pulping, and biorefinery [14,17].
The content and composition of lignin vary with wood type, cell type and single cell wall layer, and environmental conditions [18]. In recent years, herbaceous plants, such as bamboo and hemp, have gradually attracted extensive attention due to their short growth cycle and mild growth conditions. As typical of fast-growing plants, most of the herbaceous plants contain relatively low lignin contents but with high levels of hydroxycinnamic acid, i.e., p-coumaric acid (pCA) and ferulic acid (FA) [19][20][21][22] Generally, cellulolytic enzyme lignin (CEL), which is obtained by extraction of the residue from cellulase treated ballmilled materials with 96% dioxane solvent, is more representative of the total lignin in lignocellulosic biomass [23,24]. The structural characterization of lignin macromolecules isolated from different hardwood and bamboo species [17,18,25] using advanced nuclear magnetic resonance (NMR) technologies, inducing 13 C, 31 P, and 2D HSQC, could facilitate the development of efficient utilization strategies to meet current biorefinery toward a circular economy [26].
In this study, to better unravel the lignin structural variation in three bamboo species (Neosinocalamus affinis, Bambusa lapidea, and Dendrocalamus brandisii), which are widely grown in southwest China, typical CEL preparations were successively isolated from different bamboo species. The composition and structures of the obtained CEL fractions were comprehensively investigated. In order to obtain further insights into their structures, the CEL samples were also analyzed by catalytic hydrogenolysis. The results of this work are important not only for providing new insights into the bamboo lignin characteristics but also for the industrial processing of bamboo for pulp, chemical, or biofuel production.

Composition Analysis of Bamboo
The contents of main constituents (i.e., Klason lignin, acid-soluble lignin, glucan, xylan, arabinan, galactan, rhamnan, mannan, glucuronic acid, galacturonic acid, and ash) in the selected three bamboo species (N. affinis, B. lapidea, and D. brandisii) are summarized in Table 1. Quantitative measurement of lignin is an important aspect of the study of lignin structure [54]. It was observed that the total lignin content (Klason lignin plus acid-soluble lignin) of B. lapidea amounted to 32.6%, which was significantly higher than those of N. affinis (20.8%) and D. brandisii (23.8%). This result was consistent with previously reported Phyllostachys pubescens (26.1~28.2%) [12,[55][56][57] and Dendrocalamus sinicus (28.6%) [58]. After a general analysis of the chemical composition of the three bamboo species, the variations in the isolated CEL preparations were systematically analyzed, especially with the advanced NMR technologies ( 13 C, 31 P, and 2D HSQC).  Figure 1 shows the FT-IR spectra of lignin extracted from three species of bamboo, which can identify the characteristic functional groups [33][34][35]. Obviously, the broad absorption band at 3429 cm −1 is associated with the OH stretching vibration. The bands at 1664 and 1655 cm −1 indicate the unconjugated carbonyl of the keto group and the carbonyl stretch of the conjugated carbonate, respectively. The peaks at 1599, 1511, and 1426 cm −1 correspond to aromatic backbone vibrations and C-H deformations, and the methoxy group at 1460 cm −1 is the asymmetric C-H vibration. The analysis of the spectra of the three lignins demonstrated that the aromatic skeleton of the lignin structure was kept well during the isolation process [12]. Syringyl and condensed guaiacyl absorb the band at 1239 cm −1 , and the fused G-unit with the C-O stretching at 1253 cm −1 [58][59][60]. The 1125 and 834 cm −1 peaks dominate as unambiguous signals for HGS lignin, which proves that the three bamboo lignins exhibit the pattern of typical grass lignin.   Figure 1 shows the FT-IR spectra of lignin extracted from three species of bamboo, which can identify the characteristic functional groups [33][34][35]. Obviously, the broad absorption band at 3429 cm −1 is associated with the OH stretching vibration. The bands at 1664 and 1655 cm −1 indicate the unconjugated carbonyl of the keto group and the carbonyl stretch of the conjugated carbonate, respectively. The peaks at 1599, 1511, and 1426 cm −1 correspond to aromatic backbone vibrations and C-H deformations, and the methoxy group at 1460 cm −1 is the asymmetric C-H vibration. The analysis of the spectra of the three lignins demonstrated that the aromatic skeleton of the lignin structure was kept well during the isolation process [12]. Syringyl and condensed guaiacyl absorb the band at 1239 cm −1 , and the fused G-unit with the C-O stretching at 1253 cm −1 [58][59][60]. The 1125 and 834 cm −1 peaks dominate as unambiguous signals for HGS lignin, which proves that the three bamboo lignins exhibit the pattern of typical grass lignin.

Molecular Weight Distribution and Thermal Stability
The molecular weight distribution of the CEL preparations after acetylation was determined by gel permeation chromatography (GPC). It was observed that CEL N displayed the lowest weight average molecular weight (M w ) of 8.13 kDa in comparison with those of CEL B (9.08 kDa) and CEL D (9.55 kDa) in Figure 2. However, all these data were comparable with previously reported bamboo CELs [61][62][63]. Moreover, the polydispersity index (M w /M n , Ð) of the three CELs was found to be relatively narrow (Ð < 3.0) and with no significant differences (

Molecular Weight Distribution and Thermal Stability
The molecular weight distribution of the CEL preparations after acetylation was determined by gel permeation chromatography (GPC). It was observed that CELN displayed the lowest weight average molecular weight (Mw) of 8.13 kDa in comparison with those of CELB (9.08 kDa) and CELD (9.55 kDa) in Figure 2. However, all these data were comparable with previously reported bamboo CELs [61][62][63]. Moreover, the polydispersity index (Mw/Mn, Ð) of the three CELs was found to be relatively narrow (Ð < 3.0) and with no significant differences ( Table 2).  As depicted in Figure 3, the percentages of residues at different stages and the corresponding temperatures (Tm) at the maximum rate of quality loss were obtained from the TG and DTG curves of the three bamboo CELs. Among them, CELN showed the lowest residual amount, whereas CELB and CELD were in an incremental residual amount, indicating that CELN was lower in thermal stability. Moreover, a three-stage weight loss process was observed in the TG curve. The first stage was a slight weight loss due to the volatilization of bound water and loss of the residue extracts at 50~200 °C. The pyrolysis rate was high in the second stage at 200~500 °C, which was due to the pyrolysis of carbohydrates and lignin. The curve in the third stage gradually flattened, which was mainly attributed to the degradation of tar and coke. As discussed above, lignin was easier to convert into char due to its highly agglomerated properties [64,65]. The cleavage of the β-O-4′ bonds mainly occurred at 250~350 °C [66]. By analyzing the DTG curves, CELN exhibited the representative lowest decomposition peak, which might be related to the content of β-O-4′ linkages in it. Therefore, it could be concluded that the thermal stabilities of CELB and CELD were relatively higher than that of CELN.  As depicted in Figure 3, the percentages of residues at different stages and the corresponding temperatures (T m ) at the maximum rate of quality loss were obtained from the TG and DTG curves of the three bamboo CELs. Among them, CEL N showed the lowest residual amount, whereas CEL B and CEL D were in an incremental residual amount, indicating that CEL N was lower in thermal stability. Moreover, a three-stage weight loss process was observed in the TG curve. The first stage was a slight weight loss due to the volatilization of bound water and loss of the residue extracts at 50~200 • C. The pyrolysis rate was high in the second stage at 200~500 • C, which was due to the pyrolysis of carbohydrates and lignin. The curve in the third stage gradually flattened, which was mainly attributed to the degradation of tar and coke. As discussed above, lignin was easier to convert into char due to its highly agglomerated properties [64,65]. The cleavage of the β-O-4 bonds mainly occurred at 250~350 • C [66]. By analyzing the DTG curves, CEL N exhibited the representative lowest decomposition peak, which might be related to the content of β-O-4 linkages in it. Therefore, it could be concluded that the thermal stabilities of CEL B and CEL D were relatively higher than that of CEL N .

NMR Characterization
The 13 C NMR spectroscopy provides valid evidence for the analysis of the chemical structure of bamboo lignin [41][42][43]. As shown in Figure  1-118 ppm, which originated from C-9, C-4, C-2/C-6, C-3/C-5, and C-β in the structures, respectively. Additionally, we assigned the above spectral regions to functional groups and quantified them by integration (Table S1). The analysis results of quantitative 13 C NMR spectroscopy further revealed that the bamboo CEL was a typical HGS-type lignin, which was in good accordance with the aforementioned results of FT-IR.
The aliphatic-oxygenated region of the spectra (Figure 5b) provided information about the different interunit linkages present in the lignin. In this region, correlation peaks from methoxyls and side chains in β-O-4′ substructures (A, 61.2%) were the most prominent in the HSQC spectra of the isolated CELN. Other substructures were also visible in the HSQC spectrum of the CELN, including signals for phenylcoumarans (B, 2.3%) and resinols (C, 4.6%). Notably, a large predominance of β-O-4′ linkages (up to 48.6% of all linkages) was observed at δC/δH 63.5/4.21, which was assigned to the γ-acylated lignin units A′. This indicated that the structure of native lignin was highly remarkable, being extensively acylated (acetylated and/or p-coumaroylated) in bamboo [69]. The main crosssignals from S, G, and H units are visible in the aromatic region of the HSQC spectra (Figure 5c), which correspond to the benzenic rings of lignin units. The prominent signal at δC/δH 103.9/6.58 ppm was associated with C2,6-H2,6 in S-type units. The peaks at δC/δH

NMR Characterization
The 13 C NMR spectroscopy provides valid evidence for the analysis of the chemical structure of bamboo lignin [41][42][43]. As shown in Figure 4, the peaks of S-type were identified by signals at 151.1-155. 1-118 ppm, which originated from C-9, C-4, C-2/C-6, C-3/C-5, and C-β in the structures, respectively. Additionally, we assigned the above spectral regions to functional groups and quantified them by integration (Table S1). The analysis results of quantitative 13 C NMR spectroscopy further revealed that the bamboo CEL was a typical HGS-type lignin, which was in good accordance with the aforementioned results of FT-IR.
The aliphatic-oxygenated region of the spectra (Figure 5b) provided information about the different interunit linkages present in the lignin. In this region, correlation peaks from methoxyls and side chains in β-O-4 substructures (A, 61.2%) were the most prominent in the HSQC spectra of the isolated CEL N . Other substructures were also visible in the HSQC spectrum of the CEL N , including signals for phenylcoumarans (B, 2.3%) and resinols (C, 4.6%). Notably, a large predominance of β-O-4 linkages (up to 48.6% of all linkages) was observed at δ C /δ H 63.5/4.21, which was assigned to the γ-acylated lignin units A . This indicated that the structure of native lignin was highly remarkable, being extensively acylated (acetylated and/or p-coumaroylated) in bamboo [69]. The main cross-signals from S, G, and H units are visible in the aromatic region of the HSQC spectra (Figure 5c), which correspond to the benzenic rings of lignin units. The prominent signal at δ C /δ H 103.9/6.58 ppm was associated with C 2,6 -H 2,6 in S-type units. The peaks at δ C /δ H 110.9/6.93, 114.4/6.69, and 118.9/6.80 ppm were assigned to C 2 -H 2 , C 5 -H 5 , and C 6 -H 6 of the G-type unit, respectively. The C 2,6 -H 2,6 of the H-type hydroxyphenyl building block appeared at δ C /δ H 127.8/7.28 ppm. In addition, the pCA and FA units were detected in the HSQC spectra. The series of signals at δ C /δ H 130.0/7.35, 115.8/6.81, and 113.8/6.20 ppm were associated with C 2,6 -H 2,6 , C 3,5 -H 3,5 , and C β -H β in pCA, respectively. The crossover signals of C 2 -H 2 and C β -H β in FA appeared at δ C /δ H 110.9/7.25 and 115.3/6.29 ppm. In addition, the HSQC spectra showed signals of C 8 -H 8 and C 6 -H 6 at δ C /δ H 94.3/6.62 and 98.9/6.15 ppm, respectively, which were attributed to the grass-specific tricin (T) lignin units [72,73].
To further investigate the differences in the chemical structures of the three bamboo lignin species, quantitative 31 P NMR technique was employed ( Figure S4), and the quantitative data on the distribution of different OH groups in the CELs are listed in Table S3 [57]. The results of phenolic OH content revealed no significant differences among the three CELs. However, the highest content of aliphatic hydroxyl groups was detected in CELB, and the lowest content of total hydroxyl groups in CELN.  To further investigate the differences in the chemical structures of the three bamboo lignin species, quantitative 31 P NMR technique was employed ( Figure S4), and the quanti-tative data on the distribution of different OH groups in the CELs are listed in Table S3 [57]. The results of phenolic OH content revealed no significant differences among the three CELs. However, the highest content of aliphatic hydroxyl groups was detected in CEL B , and the lowest content of total hydroxyl groups in CEL N .

Catalytic Hydrogenolysis of Lignin
Catalytic hydrogenolysis was developed to produce aromatic products and phenolic moieties from lignin. Typically, alkyl aryl ether linkages in the lignin biomacromolecules are cleaved during this process. Moreover, secondary (benzylic) alcohols are removed, and aliphatic double bonds are reduced, which provides more additional information regarding the characteristics of the lignin side chain. On treatment of bamboo CEL N with 10 wt% of Ru@NC at 240 • C and 3 MPa of H 2 in MeOH for 4 h in a Parr autoclave, a brown soluble oily product was obtained after extraction with CH 2 Cl 2 ( Figure S1). This catalytic hydrogenolysis process afforded a monophenol yield of 26.6 wt% (Table 1), and the detailed monomer distribution is depicted in Figure S5. Both syringyl-and guaiacyl-derived phenols were detected with an S/G ratio of 2.2, slightly higher than that of S/G monomer composition in the original lignin (1.8). Among the monomers, 4-propyl-substituted syringol (S1, 12.2 wt%) and guaiacol (G1, 4.1 wt%) were identified as the two major products, corresponding to 61.9 mol% selectivity of total monomers. Small quantities of 4-n-propanol guaiacol/syringol (G2/S2, 5.1 wt%) were also detected ( Table 3). As a typical herbaceous species, bamboo lignin features hydroxycinnamic acid, which is bonded with α-OH or γ-OH of β-O-4 moieties through ester or ether linkages [74,75]. Accordingly, two specific phenolic monomers (3, 4.1 wt%; 4, 1.1 wt%) were also generated from the pCA and FA units, which amounted to 20.9 mol% selectivity of total monomers. Moreover, the analysis of the oily product (CELO N ) and by GPC revealed a significant decrease in molecular weight (M w 0.66 kDa) relative to the initial CEL N (M w 8.13 kDa) ( Table 2). These results demonstrated the successful scission of most C-O bonds under such a catalytic condition.

Catalytic Hydrogenolysis of Lignin
Catalytic hydrogenolysis was developed to produce aromatic products and phenolic moieties from lignin. Typically, alkyl aryl ether linkages in the lignin biomacromolecules are cleaved during this process. Moreover, secondary (benzylic) alcohols are removed, and aliphatic double bonds are reduced, which provides more additional information regarding the characteristics of the lignin side chain. On treatment of bamboo CELN with 10 wt% of Ru@NC at 240 °C and 3 MPa of H2 in MeOH for 4 h in a Parr autoclave, a brown soluble oily product was obtained after extraction with CH2Cl2 ( Figure S1). This catalytic hydrogenolysis process afforded a monophenol yield of 26.6 wt% (Table 1), and the detailed monomer distribution is depicted in Figure S5. Both syringyl-and guaiacylderived phenols were detected with an S/G ratio of 2.2, slightly higher than that of S/G monomer composition in the original lignin (1.8). Among the monomers, 4-propylsubstituted syringol (S1, 12.2 wt%) and guaiacol (G1, 4.1 wt%) were identified as the two major products, corresponding to 61.9 mol% selectivity of total monomers. Small quantities of 4-n-propanol guaiacol/syringol (G2/S2, 5.1 wt%) were also detected ( Table 3). As a typical herbaceous species, bamboo lignin features hydroxycinnamic acid, which is bonded with α-OH or γ-OH of β-O-4′ moieties through ester or ether linkages [74,75]. Accordingly, two specific phenolic monomers (3, 4.1 wt%; 4, 1.1 wt%) were also generated from the pCA and FA units, which amounted to 20.9 mol% selectivity of total monomers. Moreover, the analysis of the oily product (CELON) and by GPC revealed a significant decrease in molecular weight (Mw 0.66 kDa) relative to the initial CELN (Mw 8.13 kDa) ( Table 2). These results demonstrated the successful scission of most C-O bonds under such a catalytic condition. The as-obtained oily product was further characterized by 2D HSQC NMR spectroscopy ( Figure 6). All signals of monomeric phenols were assigned based on the comparison with authentic samples. Notably, no detectable signals for lignin linkages remained in Figure 6b, such as β-aryl ether (β-O-4′, A), phenylcoumaran (β-5, B), and resinol (β-β, C), indicating efficient depolymerization of lignin over the Ru@NC catalyst. The cross peaks at δC/δH 13.5/0.85, 24.5/1.53, and 36.6/2.48 ppm ascribed to the propyl end chains in G1/S1 could be easily observed (Figure 6c). A family of cross peaks located at δC/δH 31.8/2.51, 34.6/1.71, and 61.3/3.25 emerged, relating to the propyl end chain in G2/S2. In addition, signal at δC/δH 51.0/3.58 was attributed to the ester group (CO2Me) in the product (Figure 6b). In the aliphatic region, the signal peaks of the C7, C8, and C9 belonging to G-and S-products were found, while the signal peaks of 3 and 4 were mainly found in  The as-obtained oily product was further characterized by 2D HSQC NMR spectroscopy ( Figure 6). All signals of monomeric phenols were assigned based on the comparison with authentic samples. Notably, no detectable signals for lignin linkages remained in Figure 6b, such as β-aryl ether (β-O-4 , A), phenylcoumaran (β-5, B), and resinol (β-β, C), indicating efficient depolymerization of lignin over the Ru@NC catalyst. The cross peaks at δ C /δ H 13.5/0.85, 24.5/1.53, and 36.6/2.48 ppm ascribed to the propyl end chains in G1/S1 could be easily observed (Figure 6c). A family of cross peaks located at δ C /δ H 31.8/2.51, 34.6/1.71, and 61.3/3.25 emerged, relating to the propyl end chain in G2/S2. In addition, signal at δ C /δ H 51.0/3.58 was attributed to the ester group (CO 2 Me) in the product (Figure 6b). In the aliphatic region, the signal peaks of the C 7 , C 8 , and C 9 belonging to G-and S-products were found, while the signal peaks of 3 and 4 were mainly found in the aromatic region (Figure 6a,c). The characteristic of the monomers was further verified by the addition of the 3D version in Figure 6d. the aromatic region (Figure 6a,c). The characteristic of the monomers was further verified by the addition of the 3D version in Figure 6d.
We further evaluated the catalytic hydrogenolysis performance of the other two bamboo lignin species over the Ru@NC catalyst ( Figures S2 and S3). Notably, CELB afforded a relatively higher yield of phenolic monomer (26.5 wt%) than that of CELD (16.5 wt%), which was due to the high proportion of β-O-4′ linkages in CELB [15,71]. As expected, both CELB and CELD gave G1 and S1 as the dominant products (60.5~64.6% selectivity). Moreover, the 2D HSQC NMR spectroscopies of lignin oils from those two lignin preparations indicated that there were no β-O-4′ structures existing, illustrating the fullest cleavage of C-O linkages, which was in good accordance with the GPC analysis in Table 2    We further evaluated the catalytic hydrogenolysis performance of the other two bamboo lignin species over the Ru@NC catalyst ( Figures S2 and S3). Notably, CEL B afforded a relatively higher yield of phenolic monomer (26.5 wt%) than that of CEL D (16.5 wt%), which was due to the high proportion of β-O-4 linkages in CEL B [15,71]. As expected, both CEL B and CEL D gave G1 and S1 as the dominant products (60.5~64.6% selectivity). Moreover, the 2D HSQC NMR spectroscopies of lignin oils from those two lignin preparations indicated that there were no β-O-4 structures existing, illustrating the fullest cleavage of C-O linkages, which was in good accordance with the GPC analysis in Table 2 and Figure 2.

Elemental Composition of Bamboo, Lignin, and Lignin Oil
The elemental compositions of the three bamboo species, CELs, and the corresponding lignin oily products were investigated, which is summarized in Table S4 and depicted in Figure 8 as the van Krevelen diagram [32,78]. The solid, dashed, and dotted lines in the diagram represent the processes of demethanation, decarboxylation, and dehydration reactions, respectively. Notably, the H/C ratio of BambooB was slightly lower than that of the other two bamboo samples (BambooN and BambooD) because of the lower content of carbohydrates, which was consistent with the compositional analysis results in Table 1. It was found that the average H/C molar ratios of lignin oil were significantly higher than those of CELs. This was reasonable as (hemi)celluloses were removed in the preparation of CEL samples, which was further verified by the O/C ratios between bamboo and CEL samples. Moreover, it was found that the average O/C molar ratio of lignin oil products (0.26~0.30) was close to those of CEL samples (0.33~0.35) but much lower than those of bamboo materials (0.57~0.61). The results of reduced oxygen content but increased hydrogen content further indicated the efficient C-O bond scission in the catalytic hydrogenolysis of lignin, which led to deoxygenation and decarboxylation process.

Elemental Composition of Bamboo, Lignin, and Lignin Oil
The elemental compositions of the three bamboo species, CELs, and the corresponding lignin oily products were investigated, which is summarized in Table S4 and depicted in Figure 8 as the van Krevelen diagram [32,78]. The solid, dashed, and dotted lines in the diagram represent the processes of demethanation, decarboxylation, and dehydration reactions, respectively. Notably, the H/C ratio of Bamboo B was slightly lower than that of the other two bamboo samples (Bamboo N and Bamboo D ) because of the lower content of carbohydrates, which was consistent with the compositional analysis results in Table 1. It was found that the average H/C molar ratios of lignin oil were significantly higher than those of CELs. This was reasonable as (hemi)celluloses were removed in the preparation of CEL samples, which was further verified by the O/C ratios between bamboo and CEL samples. Moreover, it was found that the average O/C molar ratio of lignin oil products (0.26~0.30) was close to those of CEL samples (0.33~0.35) but much lower than those of bamboo materials (0.57~0.61). The results of reduced oxygen content but increased hydrogen content further indicated the efficient C-O bond scission in the catalytic hydrogenolysis of lignin, which led to deoxygenation and decarboxylation process.

Materials
Three bamboo species (N. affinis, B. lapidea, and D. brandisii) were harvested from Yunnan Province, China. Methanol (MeOH), dichloromethane (DCM), and tetrahydrofuran (THF) were purchased from Energy Chemical (Shanghai, China). The commercial cellulase was kindly provided by Novozymes (Beijing, China) Biotechnology Co., Ltd. Lignin model samples for catalytic degradation were synthesized independently. Dimeric lignin model compounds were prepared following previously reported procedures with modifications [49,52,79]. The Ru@NC catalyst used in this work was prepared and described in detail as previously described [53].

Preparation of Cellulolytic Enzyme Lignin
The bamboo raw materials were smashed into sawdust (40~60 mesh), dried in an oven at 60 °C, and then extracted with ethanol/toluene (1:2, v/v) using a Soxhlet extractor for 10 h. The preground and extracted bamboo samples were then planetary ball milled (Fritsch GmbH, Idar-Oberstein, Germany) at 400 rpm for 4 h with zirconium dioxide (ZrO2) vessels containing ZrO2 ball bearings (10 mm × 30). One cycle of the ball-milling condition consists of a 10 min milling and a 10 min cooling cycle. Subsequently, the ballmilled samples were subjected to digestion (72 h × 2) to obtain enzyme lignin samples by cellulose at 50 °C in NaOAc buffer (pH 4.8). After that, the solid residue was obtained after centrifugation (5000 rpm for 5 min), washing three times with deionized water, lyophilization, and finally ladled as CEL.

Chemical Components Analysis
The structural carbohydrates and lignin, as well as ash in the dewaxed bamboo sawdust, were determined according to the standard analytical procedures (NREL/TP-510-42618 and NREL/TP-510-42622) [80,81].

Materials
Three bamboo species (N. affinis, B. lapidea, and D. brandisii) were harvested from Yunnan Province, China. Methanol (MeOH), dichloromethane (DCM), and tetrahydrofuran (THF) were purchased from Energy Chemical (Shanghai, China). The commercial cellulase was kindly provided by Novozymes (Beijing, China) Biotechnology Co., Ltd. Lignin model samples for catalytic degradation were synthesized independently. Dimeric lignin model compounds were prepared following previously reported procedures with modifications [49,52,79]. The Ru@NC catalyst used in this work was prepared and described in detail as previously described [53].

Preparation of Cellulolytic Enzyme Lignin
The bamboo raw materials were smashed into sawdust (40~60 mesh), dried in an oven at 60 • C, and then extracted with ethanol/toluene (1:2, v/v) using a Soxhlet extractor for 10 h. The preground and extracted bamboo samples were then planetary ball milled (Fritsch GmbH, Idar-Oberstein, Germany) at 400 rpm for 4 h with zirconium dioxide (ZrO 2 ) vessels containing ZrO 2 ball bearings (10 mm × 30). One cycle of the ball-milling condition consists of a 10 min milling and a 10 min cooling cycle. Subsequently, the ball-milled samples were subjected to digestion (72 h × 2) to obtain enzyme lignin samples by cellulose at 50 • C in NaOAc buffer (pH 4.8). After that, the solid residue was obtained after centrifugation (5000 rpm for 5 min), washing three times with deionized water, lyophilization, and finally ladled as CEL.

Chemical Components Analysis
The structural carbohydrates and lignin, as well as ash in the dewaxed bamboo sawdust, were determined according to the standard analytical procedures (NREL/TP-510-42618 and NREL/TP-510-42622) [80,81].

Catalytic Hydrogenolysis of Lignin or Lignin Model Compounds
Typically, CEL (50 mg) or lignin model compounds (15 mg), Ru@NC (5 mg), and MeOH (10 mL) were charged into an autoclave (50 mL, Parr Instrument Company, Moline, IL, USA), which was then flushed with N 2 for three times and pressurized with 3 MPa H 2 at room temperature. Afterwards, the mixture was stirred at 800 rpm and heated to the desired temperature. After the reaction, the autoclave was cooled and depressurized carefully. The reaction mixture was filtered through a nylon 66 membrane filter (0.22 µm), and the insoluble fraction was washed with DCM. Lignin oily product was obtained after removing DCM under a vacuum condition. An external standard (1,3,5-trimethoxybenzene) was added to the lignin oily solution in DCM.

Conclusions
In summary, the structure characterization of CEL isolated from three bamboo species (N. affinis, B. lapidea, and D. brandisii) was investigated. The chemical composition analysis revealed a higher lignin content, up to 32.6% of B. lapidea in comparison with that of N. affinis (20.7%) and D. brandisii (23.8%). The results showed that bamboo lignin was an H-G-S lignin associated with p-coumarates and ferulates, indicating typical characteristics of herbaceous lignin. Moreover, advanced NMR analyses displayed that CEL was extensively acylated at the γ-carbon of the lignin side chain (with either acetate and/or p-coumarate groups). A predominance of S over G lignin moieties was found in CELs of N. affinis and B. lapidea, with the lowest S/G ratio observed in D. brandisii lignin. The catalytic hydrogenolysis of lignin provided deep information on the well-defined low-molecularweight phenols, giving evidence on the relative abundances of the various C-O bonds and the type of units involved in each of the linkage types. Six major monophenols, e.g., 4-propyl-substituted syringol/guaiacol and propanol guaiacol/syringol derived from β-O-4 moieties, and methyl coumarate/ferulate derived from hydroxycinnamic units, were generated in the range of 16.5~26.6 wt% yields. A sufficient understanding of the structural characteristics of lignin macromolecules in bamboo will facilitate the subsequent utilization of lignocellulosic biomass in an integrated biorefinery.