Nov 2, 2016 - Lignin structure has been considered to be an important factor that significantly influences the biorefinery processes. In this work, the effect of ball milling on the structural components and extractable lignin in enzymatic residues w
Nov 2, 2016 - Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China.
Nov 2, 2016 - These different stretching vibration positions between SCEL and LCEL may be attributed to the difference of steric hindrance, electron cloud density, and field effect among functional groups. For example, Li et al.(55) pointed out that
3. Humphrey, A. E.; Moreira, Î.; Armiger, W.; Zabriskie, D. Biotechnol. Bioeng. Symp. 1977, 7, 45. 4. Su, T.-M.; Paulavicius, I. Appl. Polym. Symp. 1975, 28, 221.
Feb 2, 2017 - Citation data is made available by participants in Crossref's Cited-by Linking service. For a more comprehensive list of citations to this ... Recovery of yerba mate ( Ilex paraguariensis ) residue for the development of PLA-based biona
Feb 2, 2017 - State Key Laboratory of Polymer Materials Engineering, Polymer Research ... Forest Products Biotechnology/Bioenergy Group, Department of ...
Feb 2, 2017 - Chromium(III) acetylacetonate (10 Î¼mol mLâ1) was added to the lignin solution to provide complete relaxation of all nuclei. ..... Richter , A. P.; Bharti , B.; Armstrong , H. B.; Brown , J. S.; Plemmons , D.; Paunov , V. N.; Stoyanov
Feb 2, 2017 - Peaks were assigned by comparison with the literature using Mnova 9 software. ..... Myint , A. A.; Lee , H. W.; Seo , B.; Son , W. S.; Yoon , J.; Yoon , T. J.; Park , H. J.; Yu , J.; Yoon , J.; Lee , Y. W. One pot synthesis of environme
Feb 2, 2017 - Conversion of condensed lignin into value-added products in current lignocellulosic biorefineries has been challenging due to its structure ...
Research Article pubs.acs.org/journal/ascecg
Comparison of the Structural Characteristics of Cellulolytic Enzyme Lignin Preparations Isolated from Wheat Straw Stem and Leaf Bo Jiang,† Tingyue Cao,† Feng Gu,‡ Wenjuan Wu,† and Yongcan Jin*,†,§ †
Jiangsu Co-Innovation Center of Eﬃcient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China ‡ School of Chemistry and Chemical Engineering, Yancheng Institute of Technology, Yancheng 224051, China
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S Supporting Information *
ABSTRACT: Lignin structure has been considered to be an important factor that signiﬁcantly inﬂuences the bioreﬁnery processes. In this work, the eﬀect of ball milling on the structural components and extractable lignin in enzymatic residues was evaluated, and the structural characteristics of the cellulolytic enzyme lignin preparations isolated from wheat straw stem (SCEL) and leaf (LCEL) were comparatively investigated by a combination of nitrobenzene oxidation (NBO), ozonation, infrared spectroscopy, and 1H−13C heteronuclear single quantum coherence nuclear magnetic resonance (2D HSQC NMR). The results showed that 4 h ball-milled samples were good enough for structural analysis with high lignin yield. Both CELs are typical p-hydroxyphenylguaiacyl-syringyl lignins which are associated with pcoumarates and ferulates. However, the structure of lignin in wheat straw stem is rather diﬀerent from that in leaf. Compared to stem lignin, leaf lignin has lower product yields of NBO and ozonation, lower erythro/threo ratio, and higher condensation degree. The analysis of 2D HSQC NMR indicated that the S/G ratio of SCEL was 0.8, which is about twice as much as that of LCEL. The ﬂavone tricin is incorporated into both stem and leaf lignins. The content of tricin in LCEL is higher than that in SCEL. KEYWORDS: Wheat straw, Stem, Leaf, Cellulolytic enzyme lignin (CEL), Structural characteristics
carbohydrates and achieve lignin with high yield and purity.5,6 For decades, a lot of work was devoted to understanding the structural features of lignin from a plant cell wall; for example, the monomeric content of lignin polymer and some other structural features were analyzed with diﬀerent chemical degradation methods such as alkaline nitrobenzene oxidation,7 ozonation,8 thioacidolysis,9 and derivatization followed by reductive cleavage that uses acetyl bromide for derivatization and zinc for reductive cleavage.10 These wet chemical methods can be very precise for speciﬁc functional groups and structural moieties. However, each chemical method gives limited information (mainly uncondensed lignin units) and is not able to provide a general picture of the entire lignin structure. In recent years, the analytical methods of nuclear magnetic resonance (NMR) for lignin characterization have been signiﬁcantly improved. NMR has the advantages of the analysis of the whole lignin structure and direct detection of lignin moieties, including the presence of aryl ether, and condensed and uncondensed aromatic and aliphatic carbons.11,12 Addi-
ACS Sustainable Chemistry & Engineering tionally, two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR has been developed to quantify the lignin structures and LCC linkages. The semiquantitative 2D NMR could be an ideal experiment for the estimation of speciﬁc lignin structures, and provides information on the interunit linkages.13,14 For example, del Rı ́o et al.15 investigated milled wood lignin from wheat straw and found it is a G−S−H type lignin associated with p-coumarates and ferulates. Rencoret et al.16 investigated cellulolytic lignin in brewer’s spent grain and found the lignin presents a predominance of G units, and the main substructures present are β−O−4′ followed by β−5′, β−β′, and 5−5′ linkages. The ﬂavone tricin was present in these lignins, as also occurred in other grasses.17,18 Wheat straw has been considered as one of the most important feedstocks for the production of chemicals, materials, and fuels via bioreﬁnery technology. Lignin structure is one of the key factors that inﬂuence the processes of bioreﬁnery such as pretreatment and enzymatic hydrolysis. Research studies indicated that the structures of herbaceous lignin in leaf are rather diﬀerent from that in stem. For instance, Markovic et al.19 studied the structure of acid detergent lignin (ADL) in alfalfa leaf and stem by the attenuated total reﬂectance Fourier transform infrared (FTIR), and the results indicated the spectra of ADL from leaf and stem are similar in frequency of absorption bands, but diﬀerent in their intensities. Min et al.20 investigated the structure of lignin in corn stover and pointed out that stem lignin had higher contents of p-coumaric acid, ferulic acid, and β−O−4′ linkages but with lower contents of β−5′ and β−β′ linkages, and a lower ratio of p-hydroxyphenyl/ guaiacyl (H/G). The alkaline nitrobenzene oxidation (NBO) data showed stem lignin had higher products’ yield and syringaldehyde/vanillin (S/V) ratio than leaf lignin. These ﬁndings suggest that structural diﬀerences between stem and leaf in herbaceous lignin may result in diﬀerent bioreﬁnery processes. As Jin et al.21 reported, the enzymatic sugar recovery of sodium-carbonate-pretreated wheat leaf was higher than that of wheat stem, and the diﬀerent structure of lignin in stem and leaf might be one of the important inﬂuencing factors. In this paper, the CEL protocol was used to isolate lignin preparations from wheat straw stem and leaf. The CEL preparations were characterized by destructive (alkaline nitrobenzene oxidation and ozonation) and nondestructive (2D HSQC NMR and FTIR spectroscopy) methods for understanding the diﬀerences in structural characteristics between lignin in wheat straw stem and leaf.
Figure 1. Isolation procedure of cellulolytic enzyme lignin from wheat straw stem and leaf. Nanjing Nanda Instrument Plant, China) at a ﬁxed frequency of 600 rpm. Two 100 mL zirconium dioxide bowls with 16 zirconium dioxide balls (1 cm diameter) in each bowl were used in the milling. The milling time was 2−6 h to obtain milled straw samples with diﬀerent milling degrees. An interval of 5 min was set between every 15 min of milling to prevent overheating. After ball milling, the straw powder (MS and ML for stem and leaf, respectively) was carefully collected and dried under vacuum. The ball-milled sample (5 g) was suspended in 100 mL of acetate buﬀer at pH 4.8, and an enzyme cocktail mixed by NS 50013, NS 50014, and NS 50010 with a ratio of 1 FPU:1.2 FXU:1 CBU was added in a 250 mL Erlenmeyer ﬂask and then incubated in a shaker (DZH-2102, Jinghong, Shanghai, China) at 180 rpm and 50 °C. The charge of mixed enzyme based on cellulase activity was 60 FPU/g cellulose. After 72 h of enzymatic hydrolysis, the mixture was centrifuged to remove the supernatant. The residue was washed by centrifugation for 3 times using sodium acetate buﬀer and deionized water, respectively. The washed enzymatic residues of ball-milled stem and leaf (EMS and EML) were freeze-dried and then extracted twice (2 × 24 h) with 50 mL of 96% aqueous dioxane (v/v) under nitrogen atmosphere. The supernatants were combined, and the solvent was removed by vacuum evaporation. The dried crude lignin samples were puriﬁed by 90% (w/w) acetic acid.22 The obtained CEL preparations from stem and leaf were named SCEL and LCEL, respectively. No further puriﬁcation was performed for the preservation of the structural features of the lignin preparations. Extractable Lignin Measurement. Extractable lignin23 was used to evaluate the solubility of lignin in enzymatically hydrolyzed residues. A 20 mg sample was suspended in 10 mL of 96% (v/v) aqueous dioxane. The mixture was magnetically stirred for 48 h at room temperature. The extract was separated by centrifugation, and 5 mL of the supernatant was reduced with 1 mg of NaBH4 in 1 mL of 0.05 M NaOH for 24 h, and then was neutralized with 4 mL of glacial acetic acid. The same process was duplicated on 96% (v/v) aqueous dioxane without sample suspension for the preparation of reference. The UV absorbance at 280 nm was measured to calculate the amount of lignin using 13 L/(g cm) from sweetgum23 as the gram absorptivity. Analytical Methods. Lignin and sugar content of the samples were analyzed using the NREL protocol.24 The Klason lignin (KL) content was taken as the ash-free residue after acid hydrolysis. The hydrolysate was collected for the determination of the acid-soluble lignin (ASL) and the structural sugars. The ASL was measured by absorbance at 205 nm in a UV−vis spectrometer (TU-1810, Beijing Puxi, China) and 110 L/(g cm) as absorptivity value was used which is an average of several reported values. The monomeric sugars were quantitatively measured with high performance liquid chromatography (HPLC, Agilent 1200 Series, Santa Clara, CA) equipped with the
MATERIALS AND METHODS
Materials. Wheat straw (Triticum aestiuium) was collected from Yancheng, Jiangsu, China, in May 2011. The materials were classiﬁed into stem and leaf (sheath included) by hand, and then were ground using a Wiley mill. The particles passed through 20 mesh (0.85 mm) sieve were collected. The straw meals were extracted with ethanol/ benzene (1:2, v/v) for 48 h to obtain extractive-free samples. No speciﬁc step was carried out to remove protein. The extracted samples were air-dried and subsequently vacuum-dried. Cellulase from Trichoderma reesei (NS 50013, 84 FPU/mL), βglucosidase from Aspergillus niger (NS 50010, 350 CBU/mL), and xylanase (NS 50014, 850 FXU/mL) were generously provided by Novozymes (Novo Nordisk A/S). The chemicals used in this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and/or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Isolation of CEL. The procedure for the isolation of CEL from wheat straw stem and leaf is illustrated in Figure 1. The vacuum-dried straw (2 g in each bowl) was milled in a planetary ball mill (QM-3SP2, 343
Data are the mean of two measurements. bThe contents of benzene−ethanol extractives from wheat straw stem and leaf were 4.6% and 6.5%, respectively. EMS: enzymatic residue of the ball-milled stem (2−6 h). EML: enzymatic residue of the ball-milled leaf (2−6 h). SCEL: CEL isolated from stem. LCEL: CEL isolated from leaf. cSR: solid recovery on the basis of starting material.
refractive index detector (RID). The HPLC analysis was carried out using a Bio Rad Aminex HPX-87H 20n exclusion column (300 mm × 7.8 mm, Bio-Rad Laboratories, Hercules, CA) with a Cation-H Reﬁll Cartridge guard column (30 mm × 4.6 mm, Bio-Rad Laboratories, Hercules, CA). The ash content was determined by combustion at 575 °C. Alkaline nitrobenzene oxidation and ozonation were carried out according to the procedure reported by Chen25 and Akiyama et al.,8 respectively. The 2D NMR spectra of the CELs were recorded at 25 °C on an AVANCE III 600 MHz instrument (Bruker, Switzerland) equipped with a cryogenically cooled 5 mm TCI z-gradient triple-resonance probe. The lignin preparations (50 mg) were dissolved in 0.5 mL of deuterated dimethyl sulfoxide (DMSO-d6) according to the method previously described.26,27 The central solvent peak was used as the internal reference (δC/δH 39.5/2.50). The HSQC experiments used Bruker’s “hsqcetgpsp.2” adiabatic pulse program with spectral widths from 0 to 16 ppm (9615 Hz) and from 0 to 165 ppm (24 900 Hz) for the 1H- and 13C-dimensions. The number of collected complex points was 2048 for the 1H-dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time increments were recorded in the 13C-dimension. The 1JCH used was 145 Hz. Processing used typical matched Gaussian apodization in the 1H-dimension and squared cosine-bell apodization in the 13C-dimension. Prior to Fourier transformation, the data matrices were zero-ﬁlled to 1024 points in the 13 C-dimension. FTIR spectra of SCEL and LCEL were recorded using a VERTEX 80 V FTIR spectrometer (Bruker, Germany). Around 2 mg of lignin samples was mixed with 400 mg of KBr, and then determined after grinding and tabletting. The scan resolution was 4 cm−1, and the scan area was 4000−400 cm−1.
the cleavage of β−O−4′ bonds and the increase of a α-carbonyl group in a certain degree. Ikeda et al.30 investigated the eﬀect of ball milling on lignin structure and found the drops of etheriﬁed β−O−4′ linkages and the increases of phenolic β−O−4′ linkages occurred during the ball-milling process. However, the eﬀect of ball milling on total lignin was diﬀerent from isolated lignin preparations;31 in particular, the amount of β−O−4′ in the total lignin decreases progressively with ball milling, but it is rather constant in MWL and CEL.6,32 Furthermore, Capanema et al.32 compared MWL and CEL preparations from three kinds of hardwood and pointed out that the yield of the lignin preparations increases linearly with the milling time in the interval of 2.5−12.5 h, and the yields of CEL preparations are about twice as high as those of the corresponding MWLs. In contrast, the S/G ratio does not change in the total lignin, but ﬂuctuates in MWL and CEL depending on the yield. Ball milling in this work helps improve substrate enzymatic digestibility because more saccharides, especially hemicelluloses, were released. Fujimoto et al.23 and Hu et al.6 described that if the extractable lignin yields from milled woods are the same, the structural changes of lignin caused by the ball milling are similar regardless of the diﬀerence in milling conditions and apparatus. In this study, extractable lignin was introduced as a general criterion to evaluate the milling degree and the potential yield of isolated lignin. Figure 2 shows the extractable lignin yield of enzymatically hydrolyzed stem and leaf. Extractable lignin yield of stem was signiﬁcantly improved when the ball-milling time
RESULTS AND DISCUSSION Eﬀect of Ball-Milling Time on Structural Components and Extractable Lignin in Enzymatic Residues. The chemical composition of wheat straw stem was rather diﬀerent from that of leaf as shown in Table 1. After enzymatic hydrolysis, the glucan and xylan contents were very low in the hydrolyzed residues of both wheat straw stem and leaf. However, the target products, SCEL and LCEL, still contained a certain amount of xylan after 1,4-dioxane/water extraction. The removal of xylan was less than that of glucan; it indicated that the lignin and hemicelluloses are present in cell walls not only as a simple mixture but through chemical linkages.28 Ball milling leads to the structural modiﬁcation of total lignin, such as the increase of carbonyl content, the decrease of molar mass, and cleavage of aryl ether bonds. Lu and Ralph29 pointed out that ball milling destroys the side-chain structure of lignin by
Figure 2. Eﬀects of ball-milling hours on the yield of extractable lignin on the basis of lignin in raw materials.
a Data are the mean of two measurements. bMS: 4 h ball-milled stem. ML: 4 h ball-milled leaf. EMS: enzymatic residue of MS. EML: enzymatic residue of ML. SCEL: CEL isolated from stem. LCEL: CEL isolated from leaf. cS = syringaldehyde + syringic acid. V = vanillin + vanillic acid. H = phydroxybenzaldehyde + p-hydroxybenzoic acid.
biomass lignin in softwood6 and hardwood,32 this is apparently not the case for nonwood lignins, speciﬁcally for the wheat straw ones in this work, as indicated by the S/V/H ratio data of CELs versus total lignin (Table 2). This is likely due to more heterogeneous structure of nonwood lignins. The ozonation product yield and E/T ratio of lignin decreased with the ball-milling hours. For example, for 4 h ball-milled stem, the decrease of ozonation yield and E/T ratio were 7.1% and 3.2%, respectively. However, the ozonation products’ yield of LCEL (0.46 mmol/g lignin) was higher than that of the raw material (0.33 mmol/g lignin). In a comparison with that of stem, leaf had lower ozonation yield. It might be caused by all of these yields being based on the sum of Klason lignin and acid-soluble lignin, while the Klason procedure cannot discriminate between true lignin and lignin-like materials in leaf.35 Salamanca et al.33 also pointed out that the Klason lignin may include both lignin and other nonhydrolyzable products. In untreated leaf, the Klason lignin residue originated from components that are highly resistant to degradation by H2SO4, and the Klason lignin residue greatly overestimated the real lignin content of leaf. Some lignin-like materials in leaf contribute to the amount of Klason lignin residue,36,2 and these materials were easily removed during the lignin isolation process;36 as a result, the extracted leaf lignin showed both higher NBO and ozonation product yield than those of lignin in original leaf and its enzymatic hydrolysis residue. The E/T ratio of leaf lignin was lower than that of stem lignin, in good agreement with the result of NBO analysis which showed that leaf lignin had low S unit content. The ratio of syringyl to guaiacyl stereochemically governed the proportion of erythro and threo forms of β−O−4′ structures during lignin formation.8 1 H−13C HSQC NMR Analysis. 1H−13C NMR is a powerful tool to probe structures of lignin and its derivatives. The signals relate to the structural units, and various linkages between units of lignin in 2D NMR spectra can be assigned according to the published literatures.12,15−17,37 The NMR spectra of CEL preparations from wheat straw stem and leaf are illustrated in Figure 3, and the detailed assignments of the main peaks of SCEL and LCEL in NMR spectra (δC/δH 150−50/8.0−2.5) are listed in Table S1. Figure 4 depicts the major lignin substructures shown in Figure 3. A semiquantitative analysis based on HSQC signals was performed using Bruker’s Topspin 2.1 processing software, and the integral method was according to the method described by del Rı ́o et al.15
improved from 2 to 4 h. The increase of extractable lignin yield leveled oﬀ when the ball-milling time was over 4 h. This result indicated that 4 h of ball milling is good enough for isolating lignin by 96% 1,4-dioxane/water extraction, and the yield of extractable lignin in stem, on the basis of lignin in raw material, could reach 68.3%. On the basis of lignin in the enzymatic residue, more than 85% of the lignin in 4 h ball-milled stem could be extracted after enzymatic hydrolysis. The extractable lignin of leaf was much lower than that of stem. This was potentially caused by the structural diﬀerences between leaf lignin and stem lignin, or by the more nonlignin components in leaf.33 Fujimoto et al.23 studied the quantitative evaluation of milling eﬀects on lignin structure during the isolation process of milled wood lignin (MWL) by ozonation. The results indicated that the proportion of β−O−4′ linkages showed a declining trend with the extension of ball-milling time. The ozonation products’ yield and erythro to threo ratio (E/T) decreased with the increase of extractable lignin yield. The structure of erythro form β−O−4′ was broken preferentially in the process of ball milling, but the degree would not exceed 25%.6 The degree for 4 h ball-milled wheat straw stem and leaf in this work was only 3.2% and 4.7%, respectively. Therefore, CEL preparations obtained through 4 h of ball-milling time in this work were representative for investigation of lignin interunit linkages. Structural Characteristics of Lignin During CEL Isolation. Alkaline nitrobenzene oxidation and ozonation were performed to investigate the eﬀects of ball milling and enzymatic hydrolysis on the structural characteristics of lignin in wheat straw stem and leaf. The results are given in Table 2. The diﬀerence in NBO products’ yield between stem (2.23 mmol/g lignin) and leaf (1.60 mmol/g lignin) suggested that the condensation degree of lignin in leaf was higher than that in stem. In a comparison to lignin in raw materials and enzymatic residues, the isolated CELs had higher NBO products’ yield. This implied that wheat straw CEL featured a lower condensation degree than that of the original lignin. Due to the high proportion of condensed guaiacyl units, only about 30% of them are converted to vanillin. On the contrary, the conversion of syringyl units to syringaldehyde may be as high as 90% due to the low proportion of condensation.25,34 After 4 h of ball milling, the decrease of NBO product yield was 1.3% for stem, while it was 6.3% for leaf. The eﬀects of ball-milling time in this work (2−6 h) on the aromatic structure of lignin were not signiﬁcant since no obvious changes in the S/V/H ratio were observed. However, while CEL represents well the total 345
glycoside and is one of the main factors causing recalcitrance of bioreﬁning.41 In the process of lignin puriﬁcation by 90% (w/w) acetic acid, LCC substructures, especially benzyl ether and phenyl glycoside structures, were most removed which reduced the signal overlap of lignin and carbohydrate in the 2D NMR spectra of lignins. The relative amounts of the main lignin interunit linkages and the molar abundances of the diﬀerent lignin units (H, G, and S), p-coumarates, and ferulates, and the molar S/G ratios of the lignins in wheat straw, estimated from volume integration of contours in the HSQC spectra, are given in Table 3. The percentage of these structural units was calculated by referring these structural unit signals to the total number of aromatic rings (H + G + S). The data indicated that the main substructures present are β−O−4′ alkyl−aryl ethers both in SCEL (64%) and LCEL (56%), while other linkages referred to as the condensed structures (β−β′ resinols, β−5′ phenylcoumarans, and β−1′ spirodienones) were present in minor amounts. In particular, the contents of β−β′, β−1′ interunit linkages were similar, but the content of β−5′ substructures in LCEL was higher than that in SCEL, that was caused by the higher condensation degree of leaf lignin, which may correlate with the S/G ratio of stem and leaf lignins. Furthermore, tricin and its derivatives were believed to protect plants from pathogens;42 the high amounts of tricin in wheat straw are remarkable (12−17%) which may induce the isolation and puriﬁcation of tricin for potential application such as food and medicine ﬁelds even though the physiological function of tricin in plants remains poorly understood. Comparatively, in the aromatic/unsaturated region of the HSQC spectra, the signal of G units is obviously more intensive than H and S units both in SCEL and LCEL. The result of LCEL from semiquantitative 2D NMR analysis is consistent with the results of NBO. However, the data of SCEL from NMR are not consistent with the NBO results because the condensation degree of G units is higher than that of S units. Additionally, the content of G units in LCEL (71%) was higher than that in SCEL (54%) which had more S unit content; it induced a signiﬁcant diﬀerence on S/G ratio between SCEL (0.8) and LCEL (0.4). The NBO results showed that the leaf lignin exhibits a higher degree of condensation (Table 2). The more branched and condensed G units such as 5−5′, 4−O−5′ units not only acted as a surface barrier, but restricted the swelling of lignocellulose and reduced the accessible surface area available to the enzyme.43 However, the linear lignin contained more S units that could adsorb on the cellulose surface more tightly which blocked the accessibility of cellulose dramatically.44 Comprehensively, the eﬀects of lignin on lignocellulosic sacchariﬁcation may depend on the S/G ratio as reports pointed out that lignin with high S/G ratio is negative on biomass enzymatic digestibility in Miscanthus.45,46 Therefore, the lower S/G ratio in leaf lignin may explain why the enzymatic sugar recovery of sodium-carbonate-pretreated21 and green-liquor-pretreated47 wheat leaf was higher than that of wheat stem. In a comparison of the ratio of S/G/H in 2D HSQC NMR spectra with the ratio of S/V/H in nitrobenzene oxidation, apparent diﬀerences were observed, and that was because the p-coumarates and ferulates in wheat straw contributed to the H and V units under alkaline nitrobenzene oxidation condition, respectively.48 Alkaline nitrobenzene oxidation can only detect the noncondensed guaiacyl, syringyl, and p-hydroxyphenyl units, and it measures the S/V of releasable syringaldehyde (minor syringic acid) and vanillin
Figure 3. Signals of 2D HSQC NMR spectra in side chain (δC/δH 50− 90/2.5−6.0) and aromatic (δC/δH 90−150/6.0−8.0) regions of SCEL and LCEL.
As shown in Figure 3, signals from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were observed clearly in the isolated SCEL and LCEL. The prominent signals corresponding to p-coumarate (PCA) and ferulate (FA) structures were observed which were typically identiﬁed in gramineous plant lignin.12,16,17,27,37 The NMR spectra indicated that β−O−4′ are the main interunit linkages of lignin, followed by phenylcoumarans and other minor linkages, such as resinols, spirodienones, dibenzodioxocins, and α,β-diaryl ethers. The signals of the β−5′ structure in wheat straw is much more intensive than that of the β−β′ structure. It indicates that the condensation degree of G units is higher than that of S units, which could be used to explain the reason for the S/V increment in isolated lignin samples.38 The intensive signals derived from tricin (T) were detected which acted as antioxidants and antimicrobial and antiviral agents in vascular plants.18 Tricin is considered to be fully compatible with ligniﬁcation reactions and is an authentic lignin monomer.17 Tricin linked to lignin units via 4′−O−β-ether bonds had been reported.17,39 Polysaccharide signals (for X), mainly originated from hemicellulose, were found in the spectra, including xylan correlations in the range δC/δH 65−80/2.5−4.5, which partially overlapped with some lignin signals. As shown in Figure 3, the polysaccharide cross-peak signals of X2 (δC/δH 72.9/3.14), X3 (δC/δH 74.1/3.32), X4 (δC/δH 75.6/3.63), and X5 (δC/δH 63.2/ 3.26 and 3.95) were assigned to β-D-xylopyranoside.40 These polysaccharide signals evidenced that lignin was mainly linked with xylan via covalent bonds which caused diﬃculties in eﬀective separation of components on a technical scale. LCC is primarily composed of γ-ester, benzyl ether, and phenyl 346
Figure 4. Main structures present in the lignins of wheat straw: (A) β−O−4′ alkyl−aryl ethers; (A′) β−O−4′ alkyl−aryl ethers with acylated γ-OH; (B) phenylcoumarans; (C) resinols; (F) spirodienones; (I) cinnamyl alcohol end-groups; (J) cinnamyl aldehyde end-groups; (FA) ferulates; (PCA) p-coumarates; (G) guaiacyl units; (S) syringyl units; (H) p-hydroxyphenyl units; (T) tricin units connected with lignin polymer through β−O−4′ linkage.
parts of composing units (G and H units) were overlapped with the contours of aromatic parts of p-coumarate and ferulate esters, the value of the S/G ratio was still calculated by using the reported assignments of the aromatic contour of each composing unit.26 Even with the inclusion of the ferulates in G units and p-coumarates in H units when correlating 2D NMR data with the NBO ones, the discrepancies were still considerably obvious. Santos et al.49 pointed out that wood lignin has a good linear relation between S/G and S/V ratios, and the S/G ratio of lignin can be predicted by multiplying the S/V value by a constant (0.806). However, an earlier reported value of 0.59 may be more reasonable.50 In this work, the value of the constant was 0.62 and 0.50 for SCEL and LCEL, respectively, which was reasonable in consideration of structural diﬀerences between nonwood and wood lignin. The diﬀerent forms of ferulic acid and lignin-like materials36 in stem and leaf lignins may cause diﬀerent contents of V and S units, or the diﬀerence was simply derived from the enrichment of certain types of subunits in the free phenolic groups. FTIR Spectroscopy. The FTIR spectra of LCEL and SCEL in wheat straw are shown in Figure S1. The assignments of the main absorption bands51−54 are listed in Table S2. The spectra showed some common features but also vibrations that were speciﬁc to each lignin. The stretching vibration of the S unit at 1330 cm−1 in SCEL was apparently stronger than that in LCEL which means stem lignin has more S unit content than that does leaf lignin, which is consistent with the data of NBO products and the analysis of 2D NMR. In contrast, the bending vibration of C−H and C−O at 1085 cm−1 in LCEL is shown
Table 3. Structural Characteristics (Lignin Interunit Linkages, Aromatic Units, and S/G Ratio, p-Coumarate/ Ferulate, Tricin) from Integration of C−H Correlation Peaks in the HSQC Spectra of the SCEL and LCEL SCEL Lignin Interunit Linkagesb (%) β−O−4′ substructures (A/A′) β−5′ phenylcoumaran substructures (B) β−β′ resinol substructures (C) β−1′ spirodienones (F) Lignin Aromatic Unitsa H (%) G (%) S (%) S/G ratio p-Hydroxycinnamatesb p-coumarates (%) ferulates (%) p-coumarates/ferulates ratio Flavonoidb (%) tricin (T) a
64 2 <1 2
56 5 <1 1
4 54 42 0.8
3 71 26 0.4
11 3 3.7
9 2 4.5
Molar percentages (H + G + S = 100). Interunit linkages, with pcoumarate, ferulate, and tricin molar contents as percentages of lignin content (H + G + S).
(minor vanillic acid) monomers from oxidative cleavage of the side chain.20 NMR does proﬁle the “entire” lignin (the isolated lignin), in principle including the condensed and the noncondensed parts of lignin. Although some contours of aromatic 347
clearly but is almost undetectable in SCEL. These diﬀerent stretching vibration positions between SCEL and LCEL may be attributed to the diﬀerence of steric hindrance, electron cloud density, and ﬁeld eﬀect among functional groups. For example, Li et al.55 pointed out that the S unit is characterized with more methoxy groups on the benzene ring which generates a steric hindrance eﬀect. The oxygen atoms in phenolic hydroxyl and methoxy groups have shown strong electronegativity. The unshared p electron pairs can form a p−π conjugated system with the π electron cloud of the benzene ring.
CONCLUSIONS The structure of lignin in wheat straw stem is rather diﬀerent from that in leaf. Wet chemistry results showed that leaf lignin has a lower product yield of nitrobenzene oxidation and ozonation, a lower erythro/threo ratio, and a higher condensation degree than stem lignin. The 2D HSQC NMR spectra and ozonation results showed that the stem lignin contains more β−O−4′ linkages which are mainly composed by the erythro form. Besides, leaf lignin has less syringyl (S) content and more guaiacyl (G) content along with similar hydroxyphenyl (H) content, which means there is a lower ratio of S/G in leaf lignin. The ﬂavone tricin is incorporated into both stem and leaf lignins. The content of tricin in leaf lignin is higher than that in stem lignin. The analysis of FTIR spectra also showed that the functional groups are structurally diﬀerent between wheat straw stem and leaf lignins. The diﬀerence in structural characteristics between stem and leaf lignins in herbaceous plants may be the main inﬂuencing factors for sacchariﬁcation of lignocellulosic biomass which results in diﬀerent bioreﬁnery processes. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01710. FTIR spectra of LCEL and SCEL, assignment of the 1 H−13C correlation peaks in the 2D HSQC spectra of SCEL and LCEL, and positions and assignments of absorption peaks in CELs (PDF)
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Laboratory of Wood Chemistry, Department of Paper Science and Technology, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China. Funding
This work was supported by the National Key Technology Research and Development Program of China (Grant 2015BAD15B09), the National Natural Science Foundation of China (Grants 31370571, 31400514), the China Postdoctoral Science Foundation (Grant 2016M591853), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China. Notes
The authors declare no competing ﬁnancial interest. 348
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