Abstract
To elucidate chemical linkages between lignin and polysaccharides, the aqueous mixed solutions of coniferin-[α-13C], syringin-[α-13C], D-glucose-[6-2H], and phenylalanine ammonia-lyase inhibitor were injected into a living wheat stalk. Internode tissues with high abundance of 2H-13C were collected. The milled wood lignin, lignin-carbohydrate complex (LCC), and residual LCC (R-LCC) with enrichment of 2H-13C were isolated. The 13C and 2H abundances showed that the lignin and polysaccharides of internode tissues were labeled by 13C and 2H, respectively. Analysis with carbon-13 nuclear magnetic resonance (13C-NMR) showed that ketal and benzyl ether bonds were formed between α-C of lignin and carbohydrates. The R-LCC and LCC were further treated with enzymes to obtain enzymatic degraded R-LCC (ED-R-LCC) and enzymatic degraded LCC (ED-LCC). 13C-NMR spectra of ED-LCC showed that the α-C of lignin side chain was combined with 6-C of carbohydrates by ether, ester, and ketal linkages. 1H-NMR differential spectra of ED-LCCs revealed an LC linkage of benzyl ether bond. Glucan-lignin (En-R-GL) and xylan-lignin (En-R-XL) complexes were separated from ED-R-LCC by ionic liquid. A part of lignin α-C was linked to cellulose 6-C by benzyl ether and α-ketal linkages. 13C-NMR spectra of En-R-XL showed there were α-benzyl ether and α-ketal bonds between lignin and xylan.
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Elucidation of Lignin and Polysaccharide Linkages in Wheat Straw by 2H/13C Isotopic Tracer
Yimin Xie,a,b,* Xudong Chen,a Kai Zhang,a Sheng Cui,a and Gongxia Zhang a
To elucidate chemical linkages between lignin and polysaccharides, the aqueous mixed solutions of coniferin-[α-13C], syringin-[α-13C], D-glucose-[6-2H], and phenylalanine ammonia-lyase inhibitor were injected into a living wheat stalk. Internode tissues with high abundance of 2H-13C were collected. The milled wood lignin, lignin-carbohydrate complex (LCC), and residual LCC (R-LCC) with enrichment of 2H-13C were isolated. The 13C and 2H abundances showed that the lignin and polysaccharides of internode tissues were labeled by 13C and 2H, respectively. Analysis with carbon-13 nuclear magnetic resonance (13C-NMR) showed that ketal and benzyl ether bonds were formed between α-C of lignin and carbohydrates. The R-LCC and LCC were further treated with enzymes to obtain enzymatic degraded R-LCC (ED-R-LCC) and enzymatic degraded LCC (ED-LCC). 13C-NMR spectra of ED-LCC showed that the α-C of lignin side chain was combined with 6-C of carbohydrates by ether, ester, and ketal linkages. 1H-NMR differential spectra of ED-LCCs revealed an LC linkage of benzyl ether bond. Glucan-lignin (En-R-GL) and xylan-lignin (En-R-XL) complexes were separated from ED-R-LCC by ionic liquid. A part of lignin α-C was linked to cellulose 6-C by benzyl ether and α-ketal linkages. 13C-NMR spectra of En-R-XL showed there were α-benzyl ether and α-ketal bonds between lignin and xylan.
DOI: 10.15376/biores.18.1.550-569
Keywords: Wheat straw; Lignin-carbohydrate complex; Isotope tracer; Deuterium; Carbon-13;
Contact information: a: Research Institute of Pulp & Paper Engineering, Hubei University of Technology, 430068, Wuhan, China; b: Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, 430068, Wuhan, China; *Corresponding author: ppymxie@163.com
INTRODUCTION
The cell wall of lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. Lignin-carbohydrate complexes (LCC) have chemical bonds between carbohydrates and lignin moieties, and these can seriously inhibit the separation of cellulose from lignin and hemicellulose (Björkman 1956). In the process of biorefinery and pulping, cellulose and lignin in plant raw materials are difficult to separate efficiently, which results in huge resource consumption and energy waste (Tribot et al. 2019).
LCC linkage plays a crucial role in wood structure, since all lignin moieties in softwoods and 47 to 66% of lignin fragments in hardwoods (Henriksson et al. 2007) are bound to carbohydrates, mainly to hemicellulose (Lawoko et al. 2005). Lignin and xylose in softwood are mainly linked by ether bonds, while lignin and mannose are mainly linked by phenyl glycosidic bond. Xyloses in hardwood LCC are mainly linked with lignin by benzyl ether bonds (Koshijima and Watanabe 2003), while lignin structures are linked to 30% glucuronic acid through the benzyl ester bond (Takahahsi et al. 1988). LCC from grass is structurally different from that in woods due to the incorporation of hydroxycinnamates into the cell wall (You et al. 2015). It is well established that ferulic acid is ester-linked to carbohydrates and ether-linked to lignin in the cell wall of grass, forming a “lignin–ferulate–polysaccharide” (LFP) complex (Buranov and Mazza 2008). The LCC in wheat straw were particularly enriched with xylan and exhibited narrow polydispersity (Yao et al. 2015). Although previous work has studied many wood LCC and wheat LCC, the chemical linkage between cellulose and lignin in wheat LCC is still worthy of in-depth study, which may help develop the appropriate process to break the lignin-carbohydrate bond for efficient and selective separation of cellulose from biomass.
Many studies have shown that the chemical linkages of LCC belong to the following types: benzyl ether bond, benzyl ester bond, ketal bond, and phenyl glycosidic bond, etc. (Xie et al. 2000). The quinone methide intermediates in lignin biosynthesis couples with carbohydrate to form benzyl ether and benzyl ester bonds. The hydroxyl groups of alcohol and phenol on lignin moieties can be easily combined with the hydroxyl groups of glycosides on carbohydrates to form phenyl glycosidic bonds. Acidification (Eriksson et al. 1980), alkaline hydrolysis (Takahashi and Koshijima 1988), and other degradative methods can be used to study the structure of LCC. However, during its treatment process, it is inevitable to cause damage to the structure of lignin and carbohydrates. Therefore, non-destructive methods are required to better understand LCC structure without cleavage of lignin-carbohydrate (LC) bonds, and thus to extract lignocelluloses effectively and selectively (Tarasov et al. 2018).
Isotope labeling technique has a wide application in exploring the structure of LCC. Xie et al. (1991, 1993, 1994a,b) synthesized the lignin precursors of coniferin-[α-13C], coniferin-[β-13C], and coniferin-[γ-13C] and successively carried out selective 13C enrichments of ginkgo and rice straw lignin side chains (Cα, Cβ, and Cγ). The 13C abundance of Cα of newly-formed xylem of ginkgo wood was 3.5 times more than that of natural abundance. Milled wood lignin (MWL) was prepared from the xylem of ginkgo, and the 13C-NMR spectrum of lignin was analyzed. The results showed that the structure contained α-carbonyl, α-aldehyde, Cγ-carbonyl, Cγ-carboxyl, methylene, and phenylcoumaran. The connection between lignin and carbohydrates was also found. Xiang et al. (2013) studied the cellulose precursor, i.e., uridine diphosphate glucose-[6-13C], which was synthesized and injected into a living ginkgo tree together with a lignin inhibitor L-α-aminooxy-β-phenylpropionic acid (AOPP), and an exogenous lignin precursor. The 13C-enriched LCC was isolated from the newly-formed xylem of ginkgo shoots. Then, it was degraded by cellulase and hemicellulase. Thus, the lignin-rich fractions, which were called enzyme-degraded LCC (ED-LCC), were obtained. Through determining their carbon-13 nuclear magnetic resonance (13C NMR) spectra, the bond formation between C6 position of glucose units in cellulose and carbons of lignin side chain were confirmed to be benzyl ether linkage. However, Xiang’s study was able to prove the existence of LC bonds from the cellulose side only.
Enzymatic hydrolysis cuts the long chains of carbohydrates without changing the structure of the LC bonds due to its high selectivity and mild conditions (Karlsson et al. 2001). At present, researchers have proposed a general classification process for extracting LCC from lignocellulosic biomass, i.e., the non-extractable biomass is first ground by ball milling and dissolved in DMSO/Tetra-Butyl-Ammonium Hydroxide (TBAH) solution, and three LCC fractions are further extracted as follows: a glucan-enriched fraction (glucan-lignin, GL), a mannan-enriched fraction (GML), and a xylan-enriched fraction (xylan-lignin, XL) (Du et al. 2013).
The inhibitor of phenylalanine ammonia lyase (PAL), i.e., (carboxymethyl methoxy amine hydrochloride, AOA) was applied. The AOA can inhibit the transformation of D-glucose-[6-2H2] to lignin as shown in Fig. 1. The coniferin-[α-13C] was degraded into the corresponding coniferyl alcohol by β-glucosidase in cells and deposited in the biosynthesis of lignin macromolecules.
Fig. 1. Inhibition of the transformation of D-glucose-[6,6-2H2] to lignin and metabolism of coniferin-[α-13C]
This research used 13C-enriched lignin and 2H-enriched carbohydrate precursors, respectively. The internode tissues with high abundances of 2H-13C from wheat straw were collected. As compared with previous studies, this research applied the Björkman’s method (1957) and enzymatic hydrolysis to remove the fragments rich in hemicellulose, thereby enriching the fragments with LC bonds between cellulose and lignin. Then, the linkages between lignin and cellulose were elucidated clearly by 13C-NMR and 1H-NMR differential spectral analysis.
EXPERIMENTAL
Materials
Wheat Emai 596 was provided by Hubei Academy of Agricultural Sciences (Wuhan, China). Sodium acetate-1-13C and D-glucose- [6-2H2] were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The AOA was purchased from McLin (Shanghai, China). All other chemicals used were of analytical grade.
Fig. 2. Chemical structures of coniferin-[α-13C] (I) and syringin-[α-13C] (II)
Synthesis of Isotope Labeled Lignin Precursors
According to the method of Xie et al. (1991, 1993, 1994a, and 1994b), Sodium acetate-1-13C was used as the starting material in the synthesis of coniferin-[α-13C] and syringin-[α-13C].
Administration of Lignin and Carbohydrate Precursors
Two batches of growing wheat Emai 596 were selected as the experimental plant because of its wide internode cavities. Before wheat heading in early March, the aqueous solutions of D-glucose-[6-2H2] (5 mg/mL) mixed with coniferin-[α-13C] (5 mg/mL) or syringin-[α-13C] (5 mg/mL) were injected into the internodal cavities of the first, second, and third sections from the root to the top of two batches of wheat, as shown in Table 1,, Fig. 3 (a) and Fig. 3 (b). To inhibit the conversion of deuterium-labeled glucose to lignin, a solution of AOA (0.35 mg/mL), which was the inhibitor of phenylalanine ammonia-lyase, was also injected. After the injection, the plant was allowed to grow for 30 days (Imai and Terashima 1992).
Fig. 3. Administration of Lignin and Carbohydrate Precursors: (a) Flow chart showing the injections of lignin precursors, carbohydrate precursor, and AOA into wheat internode cavities and the subsequent processing; (b) Stalk internodes from root to top
Table 1. Concentration of the Solution Injected to Wheat Internode
Preparation of Extractive-free Wheat Straw Mill
Wheat stalks fully absorbed the two types of lignin precursors and D-glucose-[6-2H2] samples and were harvested after their complete maturity. The internode tissues of group C, group G, and group S were collected. The wheat straw samples were then fully air-dried and ground using a Wiley mill to pass through 60-mesh screen. The milled straw was extracted by ethanol-benzene mixture (1/2, v/v) and hot water, and then air-dried.
Determination of 13C and Deuterium Abundances
The 13C and 2H isotope abundances in 1.0 mg wheat tissue samples were determined by an elemental analyzer – isotope ratio mass spectrometer (EA-IRMS) equipped with FLASH2000 elemental analyzer (Thermo Fisher Scientific GmbH, Dreieich, Germany) and Delta V isotope mass spectrometer (Thermo Fisher Scientific GmbH, Dreieich, Germany). Then, the value of 13Cα/12Cα was calculated using Eqs.1 to 3, while D6/H6 was calculated by Eqs. 4 to 5:
13C/12C = 1.11802% × (1 + δ13C ÷ 1000) (1)
(13Cα/12Cα)G = 1.07252% + (13C/12C－1.07252%) ÷ 0.2073 × 10 (2)
(13Cα/12Cα)S = 1.07252% + (13C/12C－1.07252%) ÷ 0.2073 × 11 (3)
In Eqs. 1 to 3, 13C/12C is the ratio of 13C and 12C abundances in the sample; δ13C is the relative 13C isotope abundance value of the sample (Vienna Pee Dee Belemnite, VPDB‰); 1.11802% is the 13C isotope abundance of the standard sample of VPDB; 13Cα/12Cα is the 13C and 12C isotopic ratios of Cα position in the lignin structural units of the sample; 1.07252% is the natural abundance of 13C isotope of wheat straw; 0.2073 is the lignin content in wheat straw; 10 is the ratio of total 13C content to α-13C content in guaiacyl propane structural unit, 11 is the ratio of total 13C content to α-13C content in syringyl propane structural unit. The relative abundances of D are shown in Eqs. 4 and 5:
D/H = 0.015575% × (1 + δD ÷ 1000) (4)
D6/H6 = (D/H – 0.01317%) ÷ 0.445 × 5 + 0.01317% (5)
In Eqs. 4 and 5: D/H is the ratio of D and H abundances in the sample; δD is the relative D isotope abundance value of the sample (Vienna Standard Mean Ocean Water, VSMOW‰); 0.015575% is the D isotope abundance of the standard sample VSMOW, D6/H6 is the abundance ratio of D to H on glucose 6-C position in the sample; 0.01317% is the natural abundance of D isotope of wheat straw; 0.445 is the cellulose content in wheat straw; 5 is the ratio of total D content in glucose unit to D content on 6-C.
Determination of CP/MAS 13C-NMR Spectrum of Milled Wheat Straw
An Avance III HD 600 MHz wide-cavity solid-state NMR spectrometer (Bruker, Billerica, MA, USA) was used. The samples were continuously scanned at 150.6 MHz to obtain 13C-NMR by conventional cross polarization (CP) and magic angle spinning (MAS) methods. Experimental conditions were as follows: temperature 25 °C, pulse delay 3 s, acquisition time of 0.05 s, pulse width 75 kHz, and 5000 scans.
Preparation of LCC and R-LCC
As shown in Fig. 4，the extractives in milled wheat straw (20 g ground sample with a Wiley mill and sieved using 100-mesh) were removed. The extracted sample was dried in vacuo with P2O5 for 2 weeks. The extractive-free milled straw was further ground by a water-cooling vibration ball mill for 72 h, and it was extracted three times with aqueous dioxane (96/4, v/v).
The dioxane solution was subjected to rotary evaporation and freeze-drying to obtain crude MWL. A filtrate was obtained by dissolving in acetic acid aqueous (9/1, v/v) and followed by filtration. The filtrate was added dropwise to deionized water and followed by centrifugation. The obtained precipitate was dissolved in dichloroethane-ethanol (2/1, v/v) and centrifugated. The filtrate was added dropwise to absolute ether and then centrifugated. The precipitate was washed twice with absolute ether, and then it was washed once with petroleum ether. After vacuum drying, MWL 0.8 g was obtained with a yield of 4 %.
The Residue Ι was extracted with acetic acid-water (1/1, v/v) for three times, and the resulting precipitate was R-LCC 15.6 g with a yield of 78%.
The obtained acetic acid-water solution was freeze-dried by rotary evaporation, extracted with DMF, and added dropwise to dichloroethane-ethanol (2/1, v/v). After centrifugation, the precipitate was washed once with dichloroethane-ethanol (2/1, v/v) and washed three times with absolute ether. After vacuum drying, the product was dissolved in acetic acid-water (1/1, v/v), and added dropwise to acetone. The precipitate was obtained by centrifugation and washed once with acetone-acetic acid (96/1, v/v), three times with absolute ether, and once with petroleum ether. After vacuum drying, 2.66 g LCC was obtained with a yield of 13.3%.
Fig. 4. Preparation processes of MWL, LCC and R-LCC
Enzymatic Hydrolysis of LCC and R-LCC
Cellulase (Onozuka RS, Yakult Pharmaceutical Industry Co., Nishinomiya, Japan, 16,000 units/g), hemicellulase (from Aspergillus niger, sigma, ≥ 1500 units/g), and xylanase (from Thermomyces canuginosus, sigma, ≥ 2500 units/g) were completely dissolved in 120 mL 0.5 M acetic acid/sodium acetate buffer (pH=4.5). The enzyme solution was filtered by a G4 glass filter and stored at 5 °C.
Approximately 2 g of LCC was added to 20 mL of the above enzyme solution and 80 mL of the acetic acid/sodium acetate buffer, and a little toluene was then dropped as a protective agent. The mixture was shaken in a water bath at 50 ℃ for 48 h, and centrifuged. After washing with deionized water 4 times, and then freeze -dried, ED-LCC 0.2 g was obtained with a yield of 10% (Lin et al. 1992).
The R-LCC (10 g) was added to 100 mL of the above enzyme solution and 400 mL of the acetic acid/sodium acetate buffer. Then the above enzymatic hydrolysis steps were repeated. After centrifugation and freeze-drying, 1.2 g En-R-LCC was obtained with a yield of 12%.
Classification of En-R-LCC Components by Ionic Liquid
As shown in Fig. 5, En-R-LCC (1.0 g) was added to the ionic liquid composed of DMSO/TBAH (5 mL/5 mL). The mixture was stirred continuously for 12 h until the sample was completely dissolved. The solution was then dropped into 100 mL deionized water with stirring and centrifuged to obtain a precipitate and supernatant. The precipitate was fully washed with deionized water to neutral. After freeze drying, the glucan-lignin fraction (En-R-GL) 150 mg was obtained with a yield of 15%. The supernatant was neutralized with dilute HCl, dialyzed (1000 Da) and freeze-dried to obtain 250 mg of xylan-lignin fraction (En-R-XL) with a yield of 25 %.
Fig. 5. Classification of wheat straw En-R-LCC with ionic liquid
Acetylation of Samples
A total of 150 mg of En-R-LCC or En-R-GL was added to the mixture of DMSO/N-methylimidazole (3 mL/1 mL). The mixture was stirred for 5 h until the sample was completely dissolved. Then, 0.6 mL of acetic anhydride was added and allowed to react for 1 h. The solution was dropped into 50 mL deionized water with stirring. After complete precipitation, the mixture was centrifuged, washed 4 times with water, and freeze dried to give acetylated products (Ac-En-R-LCC and Ac-En-R-GL).
Determination of Solution NMR Spectrum
A total of 90 mg of sample was completely dissolved in 0.6 mL DMSO-d6 using a φ5-mm NMR tube. The NMR spectrum was recorded by a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 294 K and scanned at 150 MHz for 13C-NMR. Analysis conditions were as follows: pulse delay 2 s, acquisition time 0.9 s, and 20,000 scans. Spectrum with 2 Hz line broadening was used for processing.
1H-NMR spectrum recording parameters included scanning at 600 MHz, pulse delay of 1.0 s, acquisition time of 2.7 s, and 500 scans with 12k acquisition data points.
RESULTS AND DISCUSSION
Analysis of 13C/2H Abundances in Wheat Internode Tissues
As shown in Tables 2 and 3, the abundances of 13C and D (2H) in the labeling groups were noticeably increased. The 13Cα/12Cα ratio of milled wheat straw sample labeled with 13C and D was about three times that of natural isotope ratio. The D6/H6 ratio was about eight times that of natural isotope ratio, indicating that exogenous coniferin-[α-13C], syringin-[α-13C], and D-glucose-[6-2H2] were effectively absorbed and metabolized during the growth of wheat internode tissues. This resulted in lignin in the cell wall of wheat straw that was labeled with 13C, while the polysaccharides were labeled with deuterium.
Table 2. 13C Abundance of Milled Wheat Straw Administered with Coniferin-[α-13C], Syringin-[α-13C], and D-Glucose-[6,6-2H2]
Table 3. 2H Abundance of Milled Wheat Straw Administered with Coniferin-[α-13C], Syringin-[α-13C], and D-Glucose-[6,6-D2]
Solid-State CP/MAS 13C-NMR Analysis of Wheat Straw Mill
As shown in Fig. 6, the CP/MAS 13C-NMR spectra of milled wheat straw administered with syringin-[α-13C], wheat straw mill administered with coniferin-[α-13C], and natural wheat straw were analyzed. The results showed that there was no remarkable difference in the aromatic regions (δ 110 ppm to 160 ppm).
Fig. 6. CP/MAS 13C-NMR spectra of milled wheat straw: a: administered with syringin-[α-13C], b: administered with coniferin-[α-13C], c: control group, d: difference spectrum obtained by subtracting spectrum (c) from spectrum (a), and e: difference spectrum obtained by subtracting spectrum (c) from spectrum (b)
To distinguish the signals of lignin side chain carbons labeled with 13C isotope from that in unlabeled samples, the CP/MAS 13C-NMR spectra of milled wheat straw were differentiated. In the differential spectra as shown in Fig. 6, all the other carbon signals were eliminated except for the 13C enhanced signal. Therefore, the signals could be assigned more accurately to different types of α-C according to the chemical shifts. The peak of -OCH3 at δ 56.0 ppm (No. 11) with stable content was used as the internal reference, and the tentative assignment of each signal is shown in Table 4. An enhanced signal at δ 100.5 to 110.2 ppm (No.1’) is the signal of α-C with ketal linkages to carbohydrates (Xie et al. 2000). The peak at δ 93.1 to 80.7 ppm (No. 2’) primarily arises from α-C in β-5, β-β, and benzyl ether linkage to carbohydrates (Xiang et al. 2014). The strong signal at δ 80.1 to 67.9 ppm (No. 3’) are assigned to the α-C in the β-O-4 structure and α-C with ester linkage to carbohydrates (Xie et al. 1991), while the signal at δ 67.9 to 58.0 ppm (No. 4’) arises from α-C in the β-1 structure between lignin moieties (Hafrén et al. 2002). Therefore, the connection between lignin structural units and LC bonds can be elucidated.
Fig. 7. Linkages between lignin structural units and LCC
Fig. 8. 13C-NMR spectra of ED-LCCs prepared from wheat straw
Legend: a: Sample administered with syringin-[α-13C] and D-glucose-[6,6-2H2].; b: sample administered with coniferin-[α-13C] and D-glucose-[6,6-2H2].; and c: control sample
Table 4. Tentative Assignment of Signals from CP/MAS 13C-NMR Difference Spectrum of Wheat Straw Mill Administered with Syringin-[α-13C] and Coniferin-[α-13C], and D-Glucose-[6,6-2H2]
Analyses of 13C-NMR Spectra of ED-LCCs
The 13C-NMR spectra of wheat straw ED-LCCs are shown in Fig. 8, and the tentative assignments of the signals are shown in Table 5.
Table 5. Assignments of 13C-NMR Signals of ED-LCCs from Wheat Straw
The -OCH3 peak with stable content at δ 55.8 ppm (No. 25) was used as the internal reference. An enhanced signal at δ 191.5 ppm (No. 1) is from α-CHO. After α-13C labeling, an enhancement signal can be observed at δ 101.8 ppm (No. 17) and assigned to α-C of lignin linked to the carbohydrates by ketal bond (Zhang et al. 2021). The enhanced signal at δ 87.2 ppm (No. 18) is from α-C in phenylcoumaran. The signal at δ 85.2 ppm (No. 20) is from α-C in pinoresinol. After labeling with α-13C, a weak signal at δ 82.5 ppm (No. 21) is enhanced and assigned to the α-C of the lignin side chain linked to the carbohydrates via benzyl ether bond (Gu et al. 2001). An enhanced signal at δ 73.9 ppm (No. 22) is from α-C connected with carbohydrates through ester bond (Xie et al. 2020). The resonance signal at δ 72.8 ppm (No. 23) is the α-C in the structure of β-O-4 linkage. A peak at δ 60.1 ppm (No. 25) comes from the γ-C in the β-O-4 substructure (Xiang et al. 2014).
Analyses of 1H-NMR Differential Spectra of ED-LCCs
To further understand the connection between lignin side chain α-C and glucan C6 from polysaccharide side, 1H-NMR differential spectra of the ED-LCCs administered with D-glucose-[6-2H2] together with coniferin-[α-13C] or syringin-[α-13C] were analyzed. A proton on the lignin aromatic ring (δ 7.2 ppm) was used as the internal standard. The 1H-NMR differential spectrum of ED-LCC was obtained by subtracting the 1H-NMR spectrum of the D-labeled group from that of control group, as shown in Fig. 9. The tentative signal assignment is shown in Table 6.
Fig. 9 1H-NMR differential spectra of EDLCCs
Legend: a: Control group subtracted wheat straw labeled with syringin-[α-13C] and D-glucose-[6-2H2]; b: Control group minus wheat straw labeled with coniferin-[α-13C] and D-glucose-[6-2H2]
A pair of peaks appeared at δ 4.65 ppm (No. 2) and 4.53 ppm (No. 3), which arise from a pair of H signals on the 6-C of the polysaccharide connected with the lignin side chain α-C by benzyl ether bond. The characteristic signals at δ 4.22 ppm (No. 4) and 3.97 ppm (No. 5) were assigned to H on 6-C of carbohydrates connected with ester bond to lignin side chain (Nishida et al. 1984). A signal at δ 3.63 ppm arises from carbohydrates 6-H,H’ without linkage with lignin, which indicates that some carbohydrates were linked with lignin not through C-6 position.
Table 6. Assignments of signals of 1H-NMR differential spectra of ED-LCCs from Wheat Straw