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May 16, 2011 - Food Chem. 2011, 59, 6605-6615. ARTICLE pubs.acs.org/JAFC. Structural Characterization of Lignin from Triploid of. Populus tomentosa Carr.
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Dec 15, 2017 - The lignins from different anatomical parts of macaÃºba (Acrocomia aculeata) palm fruit, namely stalks, epicarp, and endocarp, were studied. The lignin from stalks was enriched in S-lignin units (S/G 1.2) and Î²-ether linkages (84% of
Structural Elucidation of Whole Lignin in Cell Walls of Triploid of Populus tomentosa Carr. Sheng Yang,† Tong-Qi Yuan,†,* and Run-Cang Sun†,‡,* †
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
S Supporting Information *
ABSTRACT: To protect adequately the structural features and characterize whole lignin structure of triploid of Populus tomentosa Carr., milled wood lignin (MWL), lignin−carbohydrate complex (LCC), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL) fractions were sequentially isolated from ball-milled wood with mild conditions. The total pure yield of the four lignin fractions could reach up to 92.6% based on Klason lignin content. The structures of the four lignin fractions were quantitatively analyzed. Results showed that the main substructures in the four lignin fractions were β-O-4′ aryl ether and resinol. The LCC fraction contained a high percentage of xylose (96.2%) and numerous β-O-4′ aryl ether linkages (84.4 per 100Ar). The relative high content of phenyl glycoside (PhGlc) linkages between lignin and xylan in the LCC fraction (4.39 per 100Ar) was clearly detected. In addition, some low-molecular-weight lignin fractions were found in the secondary wall. A panorama of whole lignin structure of poplar was proposed. KEYWORDS: Poplar, Cell wall, Whole lignin, HSQC, Structural elucidation
proposed.8,9 In addition, a procedure combining enzymatic and mild acidolysis was developed to produce enzymatic mild acidolysis lignin (EMAL).10 This is more representative of the total lignin in the plant cell wall, and oﬀers higher yields and purities than MWL and CEL.11 Recently, another novel method combined with a mild alkaline treatments and subsequent in situ enzymatic hydrolysis were developed by Wen at al.12 A swollen residual enzyme lignin (SREL) was obtained as the residual lignin, and an extremely high lignin yield of 95% (based on the weight of Klason lignin in the plant cell wall) was achieved. Although the aforementioned lignin preparations played a signiﬁcant role in the structural elucidation of lignin, some structural information is lost during the pretreatment and isolation processes, such as the application of acidic or alkaline pretreatment.11,13 Ideally, the structure of native lignin in the plant cell wall should be identiﬁed in intact samples.13 The in situ characterization of the cell wall can be achieved with advanced nuclear magnetic resonance (NMR) techniques. Generally, prior to in situ NMR characterization, ball-milling and dissolution of plant cell wall materials are required. Until now, there have been two available approaches (whole cell wall dissolution and gel solution systems) to achieve the goal of direct NMR characterization of plant cell wall compo-
ACS Sustainable Chemistry & Engineering nents.14−17 The ability to proﬁle quickly the plant cell wall in a meaningful way is a signiﬁcant advance. However, signiﬁcant signal overlap between cell wall components limits the utilization of these methods in the aspect of quantitative analysis of speciﬁc lignin structure.13 Therefore, considering the requirements of quantitative analysis, characterizing lignin in the plant cell wall with the appropriate isolation method should be more eﬃcient than the in situ one. In addition, the proportions of the three lignin units (G, S, and H) vary by location in the cell wall.16 This inevitably leads to diﬀerences in the spatial distribution of macromolecular structural features of native lignin. This information could also be explored by an appropriate isolation method. The lignin in the plant cell wall is commonly fractionated into MWL, CEL, and residual enzyme lignins (REL). In most cases, only the MWL and CEL fractions are considered to be representative of native lignin in the plant cell wall. Both can be easily dissolved in DMSO-d6 without any modiﬁcation. However, large proportions of lignin in REL are not well analyzed due to their poor solubility in DMSO-d6. It should be noted that removing CEL can improve the accessibility of cellulase to cellulose in the REL. This further increases the eﬃciency of enzymatic hydrolysis.18 Thus, some of the carbohydrates remaining in the REL can be removed by cellulase treatment, improving solubility of ﬁnal residue in DMSO-d6. In addition, part of lignin components were commonly separated along with hemicelluloses due to the presence of lignin−carbohydrate complex (LCC) linkages. Therefore, some important information on lignin structure is missed. To obtain the structural features of whole lignin in the plant cell wall, all the lignins fractions should be considered. In our previous works, the structures of MWL and CEL of triploid of Populus tomentosa Carr. have been fully investigated.19,20 In this study, except for MWL and CEL, an enzymatic hydrolysis residual enzyme lignin (EHREL) was obtained after further enzymatic treatment of REL to eliminate residual carbohydrates. The structural features of lignin in the LCC fraction was also considered. All these four lignin fractions were obtained in mild conditions, without any acidic or alkaline pretreatment, and fully elucidated by composition analysis, twodimensional heteronuclear single-quantum coherence (2D HSQC) NMR spectroscopy, and gel permeation chromatography (GPC).
Figure 1. Isolation procedure for milled wood lignin (MWL), lignin− carbohydrate complex (LCC), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL). solvents until the ﬁltrate was clear. Such operations were repeated twice. The combined ﬁltrates were ﬁrst concentrated at reduced pressure and then precipitated in 3 volumes of 95% ethanol. A pellet rich in hemicelluloses was recovered by ﬁltering, washing with 70% ethanol, and freeze-drying to obtain the LCC fraction. After the ethanol was evaporated, the 96% dioxane soluble lignin was obtained by precipitation at pH 1.5−2.0 to obtain the MWL fraction. The residue after extraction of MWL and LCC was washed with water and treated with a cellulolytic enzyme in an acetate buﬀer solution (pH 4.5) at 45 °C for 48 h. Cellulase was added at 35 FPU/g substrate with 2% solid loading. After enzymatic hydrolysis, the solution was centrifuged and the enzyme-treated residue was washed with buﬀer solution and water in turn. This was followed by repeated extraction (2 × 24 h) with dioxane/water (96:4, v/v) with a solid-toliquid ratio of 1:10 (g/mL) in the dark, under a nitrogen atmosphere. The supernatant was collected by centrifugation and concentrated, followed by freeze-drying, to obtain the CEL fraction. To investigate eﬀectively the structural features of the lignin in the residue after CEL isolation, the ﬁnal residue was further treated with a cellulolytic enzyme in an acetate buﬀer solution (pH 4.5) at 45 °C for 48 h. In this step, cellulase was added at 75 FPU/g substrate with 1% solid loading. After the enzymatic hydrolysis, the solution was centrifuged and the enzyme-treated residue was thoroughly rinsed with hot water (∼80 °C), followed by freeze-drying, to obtain the EHREL fraction. Structure Elucidation of Whole Lignin. The associated carbohydrates of the four lignin fractions were determined by hydrolysis with dilute sulfuric acid according to a procedure in a previous work.22 The weight-average (Mw) and number-average (Mn) molecular weights of the acetylated lignin preparations (MWL-Ac, LCC-Ac, CEL-Ac, and EHREL-Ac) were determined by GPC on a PLgel 10 mm Mixed-B 7.5 mm i.d. column based on previous literature.22 Acetylation of MWL, LCC, and CEL was conducted according to Pan et al.23 Acetylation of the EHREL fraction was conducted as previously described.16 The 2D HSQC spectra were recorded at 25 °C on a Bruker AVIII 400 MHz spectrometer. A quantitative method based on 2D HSQC spectra24 was used to calculate the relative amount of lignin−lignin and LCC linkages of the four lignin fractions. The representative integrating range of lignin−lignin and LCC linkages was chosen according to previous literature.20,25
MATERIALS AND METHODS
Materials. Wood sample was obtained from the triploid of P. tomentosa Carr., a fast-growing 6 year-old poplar tree harvested from Shandong province, China. It was extracted with toluene/ethanol (2:1, v/v) in a Soxhlet instrument for 6 h. The Klason lignin content was analyzed according to the standard of National Renewable Energy Laboratory (NREL),21 which amounts to 19.9% of the dry wood. The air-dried extractive-free wood sample was then milled with a planetary ball mill (Fritsch, Germany) in a 500 mL ZrO2 bowl with mixed balls (10 balls of 2 cm diameter and 25 balls of 1 cm diameter). Milling was conducted according a previous work.8 The cellulolytic enzyme used in this study was Celluclast 1.5 L, containing hemicellulase activities, kindly supplied by Novozymes, with a ﬁlter paper activity of 70 FPU g−1. All chemicals used were of analytical grade or better, and were directly used as purchased without further puriﬁcation. Fractionation Procedure. MWL, LCC, CEL, and EHREL fractions were prepared according to the procedure shown in Figure 1. The ball-milled wood sample was suspended in 96% dioxane with a solid-to-liquid ratio of 1:10 (g/mL) at room temperature for 24 h. The extraction procedure was conducted in the dark, under a nitrogen atmosphere. The mixture was ﬁltered and washed with the same
RESULTS AND DISCUSSION Yield and Composition. Table 1 displays the yields and compositions of the four lignin fractions isolated from triploid of Populus tomentosa Carr. As can be seen, the yield of the MWL fraction was16.6%, based on the weight of Klason lignin in the plant cell wall. This was lower than the value in our previous study (22.1%), although the same ball milling and
ACS Sustainable Chemistry & Engineering Table 1. Yields and Carbohydrate Contents of Lignin Fractions carbohydrate contentb (%)
yield (%) samples
sugar content (%)
MWL LCC CEL EHREL
16.6 7.7 49.9 27.6
16.1 5.2 45.5 25.8
3.18 32.55 8.76 6.49
0.20 (7.1) 0.60 (1.9) 0.42 (5.3) NDc (0.0)
Ara 0.09 0.26 0.31 0.82
(3.1) (0.8) (3.9) (13.6)
0.05 0.11 0.67 1.72
(1.8) (0.3) (8.5) (28.5)
Glc 0.14 0.25 3.27 0.66
(5.0) (0.8) (41.3) (10.9)
0.18 (6.4) NDc (0.0) 0.15 (1.9) 0.61 (10.1)
2.15 (76.5) 30.58 (96.2) 3.09 (39.1) 2.23 (36.9)
0.23/0.14 0.75/ND 0.85/ND 0.39/0.06
Based on Klason lignin of wood. bRha = rhamnose, Ara = arabinose, Gal = galactose, Glc = glucose, Man = mannose, Xyl = xylose, GlcA = glucuronic acid, GlaA = galacturonic acid. cNot detected.
EHREL fraction. As shown in Table 1, the yield of the ﬁnal EHREL fraction was 27.6%, based on the weight of Klason lignin in the plant cell wall. The sugar content was 6.49% in the EHREL fraction. This was signiﬁcantly lower than that (24.68%) of the REL fraction reported in our previous work.19 Thus, an extra enzymatic treatment could eﬀectively remove residual carbohydrates in the REL fraction. The relatively high purity of the EHREL fraction in this study was suitable for subsequent NMR analysis. After the amounts of associated carbohydrates were calibrated, the total yield of the four lignin fractions was 92.6%, which was close to the yield (95%) of SREL reported by Wen et al.12 However, some structural features, which were sensitive to alkaline treatment, such as ester linkages, could be reserved in the present study. In light of these results, it was concluded that the combination of the four lignin fractions in this study can better represent whole lignin in the poplar cell wall. 2D HSQC NMR Analysis. Two-dimensional 1H 13C NMR (2D NMR) can provide important structural information and facilitate resolution of otherwise overlapping resonances in either the 1H or 13C NMR spectra.28 In the present study, to understand whole lignin structure of the poplar cell wall, the four lignin fractions were characterized by 2D HSQC NMR techniques. The side-chain (δC/δH 50−90/2.5−6.0) and aromatic (δC/δH 100−135/5.5−8.5) regions of the HSQC spectra of the four lignin fractions are shown in Figures 2 and 3, respectively. Main substructures are depicted in Figure 5. HSQC cross-signals of lignin and associated carbohydrates were assigned by comparison with the published literature.15,20,28−31 Table S1 lists the main lignin and associated carbohydrate cross-signals assigned in the HSQC spectra. Lignin Side-Chain Regions. As shown in Figure 2, all of the four spectra showed prominent signals corresponding to methoxyls (δC/δH 55.6/3.73) and β-O-4′ aryl ether linkages. The Cα−Hα correlations in β-O-4′ substructures were observed at δC/δH 71.8/4.86, whereas the Cβ−Hβ correlations corresponding to the erythro and threo forms of the S-type β-O-4′ substructures (A) can be distinguished at δC/δH 85.9/4.12 and 86.8/3.99, respectively. These correlations shifted to δC/δH 83.9/4.29 in structure A, linked to G/H lignin units. The Cγ− Hγ correlations in structure A were observed at δC/δH 59.5− 59.7/3.40−3.63. The Cγ−Hγ correlations in γ-acylated lignin units (A′ and A″) were observed at δC/δH 63.2/4.33−4.49 in the spectra of the CEL fraction. These signals indicated that lignins in triploid of Populus tomentosa Carr. cell wall were partially acylated at the γ-carbon in β-O-4′ aryl ether linkages of the side chains. These cross-signals could also be found at lower contour levels in the side-chain region of the spectra of MWL and LCC fractions (not shown). However, they were absent in the spectra of the EHREL fraction. In addition, a signal at δC/
isolation producers were used.20 The diﬀerence in yield of the two MWL fractions may be due to the fact that a 6 year-old Populus tomentosa Carr. was used in this study whereas a 3 yearold one was used in our previous study.21 Isolation of the LCC fraction in this study was actually a classical puriﬁcation process for the cured MWL fraction.26 Most carbohydrates in the crude MWL fraction were moved to the LCC fraction during this step. However, due to the covalent linkages between lignin units and carbohydrates,3 part of the lignin was also removed with the carbohydrates and recovered in LCC fraction. As shown in Table 1, the yield of the LCC fraction was 7.7%, based on the weight of Klason lignin in the plant cell wall. It was carbohydrate-rich (32.55%), but still contained substantial amounts of lignin. The LCC fraction contained a larger percentage of xylose (96.2%) among the total sugars and uronic acids. Other sugars, such as glucose, galactose, arabinose, and rhamnose, were observed in noticeable amounts. These results indicated that the linear hemicelluloses (xylans) in the plant cell wall were easily released by ball milling. Although a signiﬁcant amount of xylose was moved into LCC fraction, it was still the main sugar in the MWL fraction. Therefore, it could be deduced that it was diﬃcult to isolate the lignin associated with more branched hemicelluloses from the cell wall during ball-milling. These results accorded with that of a previous study.19 It should be noted that both the MWL and LCC fractions had a low glucose content. This indicated that the MWL and LCC fractions still contain some components composed of glucose units. Especially for the LCC fraction, the low levels of glucose may originate from other wood-based glucans. The CEL fraction was isolated from the residual wood meal after extraction of crude MWL. The yield of the CEL fraction was 49.9%, signiﬁcantly higher than the other lignin fractions in this study. Some carbohydrates (8.76%) still remained in this fraction, due to the existence of lignin−carbohydrate complex linkages. Glucose and xylose were the major component sugars in the CEL fraction, comprising 41.3% and 39.1% of total sugars, respectively. The high glucose content of the CEL fraction was likely due to residual fragments of high crystallinity cellulose as well as the glucose linked to the lignin through covalent bonds. The high yield of the CEL fraction and the high glucose content may indicate that the CEL fraction mainly originated in the secondary wall. It has been reported that xylans are the predominant hemicellulosic components in the cell walls of fast-growing poplar wood, and that most of hemicelluloses are located in secondary wall.27 This could explain the high proportion of xylose in the CEL fraction. The elimination of lignin in the plant cell wall could improve the accessibility of cellulase to cellulose.18 Therefore, the residue after extrication of CEL was further treated with cellulase again in this study to isolate C
the spectra of the four lignin fractions were β−β′ (resinol, B), β-5′ (phenylcoumaran, C), and β-1′ (spirodienone, D). Strong signals for resinol substructures B were observed with their Cα−Hα, Cβ−Hβ, and double Cγ−Hγ correlations at δC/δH 84.8/ 4.65, 53.5/3.06, and 71.0/3.82 and 4.18, respectively. Phenylcoumaran substructures C were found in lower amounts. Signals for their Cα−Hα and Cβ−Hβ correlations were discovered at δC/δH 86.8/5.46 and 53.3/3.46, respectively. However, the Cγ−Hγ correlations were overlapped with other signals at around δC/δH 62.5/3.73. Moreover, small signals corresponding to spirodienone substructures (D) could also be found in the spectra of MWL, LCC, and CEL fractions at lower counter levels (not shown). Their C α−Hα and C β−Hβ correlations were detected at δC/δH 81.2/5.07 and 59.7/2.77, respectively. Except for these linkages, Cγ−Hγ correlations (at δC/δH 61.4/4.10) in p-hydroxycinnamyl alcohol end groups (I) could also be clearly observed in the spectra of the four lignin fractions. This was consistent with previous studies.19,20 Aromatic Regions. In the aromatic regions of the 2D HSQC spectra of the four lignin fractions, the S units showed a prominent signal for the C2,6−H2,6 correlation at δC/δH 103.8/ 6.71. The G units showed diﬀerent correlations for C2−H2, C5−H5, and C6−H6 at δC/δH 110.9/6.98, 114.9/6.77, and 119.0/6.80, respectively. Signals corresponding to C2,6−H2,6 correlations in Cα-oxidized S units (S′) (δC/δH 106.2/7.23 and 7.07) were present in all 2D HSQC spectra of the four lignin fractions. Generally, the C2,6−H2,6 aromatic correlation for the H units was located at δC/δH 127.9/7.19. This signal was weak in all of the four spectra, and could only be observed at lower counter levels in the 2D HSQC spectra of the MWL, LCC, and CEL fractions (not shown). The C3,5−H3,5 correlations of H units were overlapped with those of the C5 position of G units and the p-hydroxybenzoate substructure (PB) C3,5−H3,5 correlations. The C2,6−H2,6 correlations of PB were observed as a strong signal at δC/δH 131.2/7.67 in the spectra of the MWL and CEL fractions. This signal was relatively weak in the spectra of the LCC and EHREL fractions. It has been reported that PB exclusively acylates the γ-position of lignin side chains, which is analogous to p-coumarates (pCA) in grasses.29,35 Other signiﬁcant signals in the aromatic regions of the 2D HSQC spectra of the four fractions were assigned to phydroxycinnamyl alcohol end groups (I) and cinnamaldehyde end groups (J), as well as spirodienone substructures (D). Except for the signal of J structure in the spectrum of the MWL fraction, these cross-signals could only be observed at lower counter levels (not shown). Associated Carbohydrates. The side-chain regions of the 2D HSQC spectrum of the LCC fraction showed strong crosssignals belonging to the associated carbohydrates, such as a 2O-Ac-β-D-Xylp C2−H2 correlation at δC/δH 73.2/4.49 and a 3O-Ac-β- D -Xylp C 3−H3 correlation at δC/δH 74.7/4.80. Furthermore, the signals from β-D-Xylp were clearly observed, with its C2−H2, C3−H3, and C4−H4 correlations at δC/δH 72.5/ 3.02, 73.7/3.22, and 75.4/3.60, respectively. The C5−H5 correlations of β-D-Xylp were also observed at δC/δH 62.6/ 3.40 and 3.72. However, part of this signal was overlapped with Cγ−Hγ correlations of phenylcoumaran substructures (C). Its corresponding anomeric correlation (C1−H1) was found at δC/ δH 103.2/4.20 (shown in Figure 4). In addition, an obvious signal of C1−H1 correlation in 4-O-methyl-α-D-GlcUA (U) was observed in this spectrum. In fact, acetylated 4-O-methylgluconoxylan has been found to be a major hemicellulosic component in hardwoods, and acetyl groups frequently acylate
Figure 2. Aliphatic (side-chain) regions of 2D 13C 1H correlation (HSQC) spectra of milled wood lignin (MWL), lignin−carbohydrate complex (LCC), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL). Symbols are taken from Figure 5.
Figure 3. Aromatic regions of 2D 13C 1H correlation (HSQC) spectra of milled wood lignin (MWL), lignin−carbohydrate complex (LCC), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL). Symbols are taken from Figure 5.
δH 83.1/5.21, which was assigned to the Cβ−Hβ correlations in oxidized (CαO) β-O-4′ substructures (F), could be found at lower contour levels in the side-chain region of the spectra of MWL, LCC, and CEL (not shown). The α-oxidized β-O-4′ substructures may be caused by the oxidation reaction occurred in α-position of lignin unit during ball milling process.32−34 Besides β-O-4′ ether substructures, other linkages observed in D
Figure 4. Anomeric regions of 2D 13C 1H correlation (HSQC) spectra of milled wood lignin (MWL), lignin−carbohydrate complex (LCC), and cellulolytic enzyme lignin (CEL).
the C-2 and C-3 positions.36 The intensities of cross-signals of the associated carbohydrates were signiﬁcantly lower in the spectra of MWL and CEL fractions. Only weak signals of β-DXylp were observed in the side-chain regions of the 2D HSQC spectra of MWL and CEL fractions. The aforementioned results observed in 2D HSQC spectra accorded well with composition analysis results. However, it was interestingly found that the signals of associated carbohydrates disappeared in the 2D HSQC spectrum of the EHREL fraction, although 6.49% of associated carbohydrates remained. In this study, the MWL, LCC, and CEL fractions were easily dissolved in the DMSO-d6 before NMR analysis. However, the solubility of the EHREL fraction in DMSO-d6 was poor. Only a gel-state mixture could be obtained after adding the EHREL fraction in DMSO-d6. Fortunately, the presence of these carbohydrates take no problem to obtain a high quality 2D HSQC spectrum of the EHREL fraction. Figure 4 displays the well-resolved anomeric correlations (δC/δH 90−115/3.5−6.0) of the associated carbohydrates of MWL, LCC, and CEL fractions. No signals were found in the anomeric region of the EHREL fractions, even at lower counter levels. Thus, the anomeric region of EHREL fraction was not shown in this study. The assignments of the anomeric correlations were based on the published literature.29,31 The corresponding anomeric correlations of β-D-xylopyranoside units acetylated at C-2 (X2), C-3 (X3), and both positions (X23) were clearly observed at δC/δH 99.4/4.52, 101.6/4.32, and 98.9/4.71, respectively, in the spectrum of LCC fraction. However, the intensities of these signals were relatively low in the spectra of the MWL and CEL fractions. The corresponding anomeric correlations of β-D-xylopyranoside units (X1) were found at δC/δH 103.2/4.20 in the spectra of MWL, LCC, and CEL fractions, whereas the minor anomeric correlations of β-Dglucopyranoside units (Glc1) may overlap with it in this region. The anomeric correlations from the reducing end of (1→4)-αD-xylopyranoside (αX1) and (1→4)-β-D-xylopyranoside (βX1) units were found at δ C/δ H 92.2/4.88 and 97.4/4.26, respectively. In addition, anomeric correlations from 4-Omethyl-α-D-GlcUA (U1) and (1→2)-α-L-rhamnopyranoside units (R1) were clearly observed at δC/δH 97.2/5.18 and 98.8/5.12, respectively, in the spectra of the LCC and CEL fractions. According to previous literature,31 other correlations at δC/ δH 100.6/4.65 and 101.5/4.79 in the anomeric regions belong to phenyl glycoside linkage (PhGlc) units. These cross-signals were labeled PhGlc2 and PhGlc3, respectively. They were obvious in the spectra of the LCC fraction, but relatively weak in the spectra of the MWL and CEL fractions. Except for the
phenol glycoside linkages, the other types of native LCC linkages in plants are believed to be mainly γ-esters (Est) and benzyl ethers (BE).30 In this study, BE and Est were detected in the aliphatic regions at lower counter levels in the 2D HSQC spectra of MWL, LCC, and CEL (not shown). The main LCC linkages are given in Figure 5. The BE structures can be divided into two types: (a) BE1, linkages between the α-position of lignin and primary −OH groups of carbohydrates (at C-6 of Glc, Gal, and Man, and C-5 of Ara); and (b) BE2, linkages between the α-position of lignin and secondary −OH groups of carbohydrates, mainly of the lignin−xylan type.38−40 The signal of BE2α, which commonly overlaps with that of Dα at δC/δH 81.3/5.07 and the proportion of this structure, was signiﬁcantly lower than that of BE1α in the plant cell wall.36 Signals for Est bonds were located at δC/δH 63.2/4.33−4.49. However, the signals of these bonds overlapped with that of Cγ-Hγ in γacylated β-O-4′ substructures.17,40 According to the spectra shown in this study, the basic structural features of the four lignin fractions resembled each other, despite some diﬀerences in intensities of the cross-signals. Therefore, quantiﬁcation of lignin structures and LCC linkages is essential. Quantiﬁcation of Whole Lignin. Table 2 displays the relative abundance of G and S lignin units and those of the main linkages (referred to as per 100 aromatic units). This was calculated from the 2D HSQC spectra of the lignin samples, based on a previous study.24 The three main LCC linkages were also quantitatively investigated. Interestingly, the LCC fraction had the highest β-O-4′ linkage content (84.5 per 100Ar) among the four lignin fractions. The CEL fraction revealed secondary β-O-4′ linkage content (55.9 per 100Ar). The MWL and EHREL fractions had similar β-O-4′ linkage content, amounting to 45.7 and 45.8 per 100Ar, respectively. It is well-known that the β-O-4′ linkages in hardwood lignin are mainly formed by S units. However, Table 2 indicated that the S/G ratio of the LCC fraction was not the highest of the four lignin fractions. This implied that most of the G units in the LCC fraction just connected with other lignin units through βO-4′ linkages. The low content of the β-5′ linkage of the LCC fraction (0.6 per 100Ar) further revealed the particularity of the G units in this fraction. The S/G ratio of MWL fraction, amounting to 1.74, was the lowest among the four lignin fractions. This was likely the reason that the MWL fraction had the lowest β-O-4′ linkage content. In fact, it has been reported that the MWL fraction was mainly from the middle lamella of the plant cell wall, and more G units were located in this region.41,42 The high content of β-5′ linkage of the MWL fraction (2.8 per 100Ar) could conﬁrm this, because the G unit was the premise for obtaining the β-5′ linkage.24 E
Figure 5. Key structural details of lignin and carbohydrates of milled wood lignin, lignin−carbohydrate complex, cellulolytic enzyme lignin and residual enzyme lignin: (A) β-O-4′ aryl ether linkages with a free −OH at the γ-carbon; (A′) β-O-4′ aryl ether linkages with acetylated −OH at γcarbon; (A″) β-O-4′ aryl ether linkages with p-hydroxybenzoated −OH at γ-carbon; (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; (D) spirodienone substructures formed by β-1′ and α-O-α′ linkages; (F) Cα-oxidized β-O-4′ substructures; (I) p-hydroxycinnamyl alcohol end groups; (J) cinnamaldehyde end groups; (PB) phydroxybenzoate substructures; (H) p-hydroxyphenyl units; (G) guaiacyl units; (S) syringyl units; (S′) oxidized syringyl units with a Cα ketone; (G′) oxidized guaiacyl units with a α-ketone; (PhGlc) phenyl glycoside; (Est) γ-ester; and (BE) benzyl ether.
compared with the MWL, LCC, and CEL fractions, the β−β′ linkage content of EHREL fraction was relatively low. This was contradicted with its relatively high S unit proportion. This implied that there were less β−β′ linkages between S units in the EHREL fraction than the other three lignin fractions. In the other words, the condensation degree of S units in EHREL fraction was relatively low. The contents of the β-1′ linkages and oxidized (CαO) β-O-4′ substructures (F) were low in the four lignin fractions, and both did not occur in the EHREL fraction. The contents of Est in the MWL, LCC, and CEL fractions were 4.5, 0.7, and 4.4 per 100Ar, respectively. However, it should be emphasized that the amount of γ-ester LCC linkages
In this study, the relatively high S unit proportion and carbohydrate content of the LCC fraction implied that part of this fraction may have originated from the secondary wall. The S/G ratio of the CEL fraction was the highest among the four lignin fractions, amounting to 2.18. This could explain the relatively high amount of β-O-4′ linkages in the CEL fraction. The EHREL fraction also has a relatively high S/G ratio (1.99). This indicated that this fraction could also be obtained from secondary wall of the plant cell wall, as with the CEL fraction. All of the four lignin fractions had relatively high β−β′ linkage content, amounting to 11.7, 10.3, 10.4, and 5.6 per 100Ar, respectively. The high proportion of β−β′ linkages in the four fractions was likely caused by the high S unit content.24 As F
Sum of LCC γ-ester (Est) and γ-acylated β-O-4′ aryl ether substructures (A′ and A″). bNot detected. a
calculated was the sum of LCC γ-ester (Est) and γ-acylated βO-4′ aryl ether substructures (A′ and A″). The exact amount of this linkage was diﬃcult to determine in this study. The contents of BE of the MWL, LCC, and CEL fractions were 2.0, 0.6, and 1.1 per 100Ar, respectively. No PhGlc linkages were found in the MWL fraction. This indicated that the main LCC linkages in the MWL fraction were BE. This result was diﬀerent with our previous study.20 This may be due to the properties of the diﬀerent materials.43 The content of PhGlc in the LCC fraction was the highest among the four lignin fractions, amounting to 4.4 per 100Ar. The other two LCC linkages in this fraction were quite low. Therefore, the PhGlc should be the main LCC linkage of the LCC fraction. It was worthy to emphasize that, according to the results of the composition analysis, the PhGlc linkages in the LCC fraction may be mainly formed between lignin and xylan. The content of the PhGlc linkage in the CEL fraction was only 0.6 per 100Ar. It has been reported that most phenyl glycoside moieties in LCC linkages are apt to cleave during enzyme treatment.32 In addition, the Est was also easily broken during enzyme treatment.44 The content of Est in the CEL fraction should also be very low. Thus, the relatively high calculation value (4.4 per 100Ar) of the CEL fraction should be mainly assigned to the γ-aclated βO-4′ structure. It should be noted that no signals of LCC linkages were found in the spectrum of the EHREL fraction, even at lower counter levels. This indicated that lignins and carbohydrates may be connected through physical combination in this fraction. Molecular Weight Distribution. Table 3 displays the values of the weight-average (Mw) and number-average (Mn)
Figure 6. Molecular-weight distribution curves of acetylated lignin fractions.
molecular weight of the component with the highest content in the LCC fraction was higher than that in the MWL fraction. This may be due to the associated carbohydrates (hemicelluloses) linked with lignin through LCC bonds. The high carbohydrate content of the LCC fraction was conﬁrmed by the composition analysis. It has been reported that the carbohydrate chains linked to lignin can increase the hydrodynamic volume of lignin, thereby increasing the apparent molar mass of the lignin as measured by GPC.29 Thus, the apparent molecular weight of the LCC fraction obtained in this study could not provide the appropriate structural information on lignin in the LCC fraction. The low carbohydrate content of the MWL fraction would not signiﬁcantly obstruct the determination the molecular weight of the lignin. Thus, the molecular weight of the MWL fraction could appropriately reﬂect the structural feature of the lignin in the MWL fraction. The CEL fraction had the highest molecular weight among the four lignin fractions, amounting to 20 610 g/mol. The polydispersity of this lignin fraction was 3.77, higher than that of MWL fraction. This was consistent with the previous study.19 It could be observed that the peak in the curve of CEL fraction located at the high-molecular-weight region. This may be related to the high proportion of S units in this fraction. In fact, the β-O-4′ linkages in the secondary wall of the plant cell wall may not be fully broken under the ball milling conditions used in this study. Most β-O-4′ linkages still remained in the CEL fraction. These has been conﬁrmed by the 2D HSQC analysis. Thus, the molecular weight of the CEL fraction was higher than those of the other three lignin fractions. In addition, numerous lignin components with very high molecular weight (more than 20 000 g/mol) were found in the CEL fraction. Results of the composition analysis indicated that this was caused by cellulose fragments connecting to lignin through covalent bonds. Thus, the apparent molecular weight of the CEL fraction here could also not provide the appropriate structural information on lignin in this fraction.
molecular weights calculated from the GPC curves and the polydispersity (Mw/Mn) of the four lignin fractions. It is evident that the MWL and LCC fractions had the same weight-average molecular weight, 9720 g mol−1, whereas the LCC fraction exhibited wider molecular-weight distributions than the MWL G
Figure 7. Panorama of whole lignin in the plant cell wall of poplar wood (Carb: carbohydrate).
It was surprisingly found that the EHREL fraction had the lowest molecular weight of the four lignin fractions, amounting to 4430 g/mol. This contradicted somewhat with the relative high carbohydrate content (6.49%) of the EHREL fraction. However, 2D HSQC analysis revealed no LCC linkages between lignin and carbohydrates in this fraction. Lignins and carbohydrates may be coexisted or connected through physical combination in this fraction. Thus, the remaining carbohydrate content would not increase the hydrodynamic volume of lignin. The molecular weight of lignin in this fraction could be clearly identiﬁed. The polydispersity of the EHREL fraction was 5.27, the highest of the four lignin fractions. As shown in Figure 6, two obviously peaks belong to low-molecular-weight components were found in the curve of EHREL fraction. This
indicated that there was a large proportion of low-molecularweight lignins in the EHREL fraction. Thus, it could be deduced that there may be some low-molecular-weight lignin fragments in the secondary wall of poplar wood. According to the 2D HSQC results, these low-molecular-weight lignin fragments may be mainly formed by S units. On the other hand, there were also some high-molecular-weight components in the EHREL fraction. The high-molecular-weight components in the EHREL fraction may be mainly formed by the highly condensed G unit linkages (5−5′ and β-5′). In summary, the four lignin fractions (MWL, LCC, CEL, and EHREL), have been used to study whole lignin in the poplar wood cell wall. The heterogeneity of the lignin macromolecular structure in diﬀerent fractions of the plant cell wall have been H
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quantitatively elucidated. A seeming panorama of whole lignin structure in the triploid of Populus tomentosa Carr. cell wall, was proposed, and the results are shown in Figure 7. The methodology introduced in this study could improve the understanding of whole lignin in the plant cell wall. This would be beneﬁcial for the reasonable utilization of lignocelluloses and improve the development of bioreﬁnery industry.
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b01075. Assignment of main lignin and carbohydrate 13C−1H cross-signals in the HSQC spectra of lignin fractions (PDF).
The authors declare no competing ﬁnancial interest.
ACKNOWLEDGMENTS We are grateful for the ﬁnancial support of this research from the National Natural Science Foundation of China (31400296 and 31430092), State Forestry Administration (201404617), Beijing Municipal Commission of Education (20131002201), and the open fund project of State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, China (201364).
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