NC State
Yao, L., Chen, C., Zheng, X., Peng, Z., Yang, H., and Xie, Y. (2016). "Determination of lignin-carbohydrate complexes structure of wheat straw using carbon-13 isotope as a tracer," BioRes. 11(3), 6692-6707.


To maximize the use of wheat straw as a feedstock for biofuels and other biorefinery products, the structure of lignin-carbohydrate complexes (LCCs) was characterized by injection of 13C isotope-labeled xylose into living wheat straw. Afterwards, lignin-carbohydrate complexes were extracted from the harvested straw by the Björkman method. The extracted LCCs were chemically characterized by Fourier transform-infrared spectroscopy (FT-IR), sugar composition, molecular weight analysis, 13C-NMR, and HSQC. The results showed that LCCs in wheat straw were particularly enriched with xylan and exhibited narrow polydispersity (Mw/Mn < 1.5). NMR analysis showed that the lignin was linked with the carbohydrates through γ-ester, phenyl glycoside, and benzyl ether bonds. In addition to S, G, and H lignin units, p-coumarate and ferulate were also in the LCCs. The substructures in lignin were β-O-4′, β-β’, and β-5′. Quantitative data analysis of 13C-NMR combined with HSQC showed that the lignin in the LCCs of wheat straw contained guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units in the proportion of 5:4:1 (S:G:H). The main lignin substructure, β-O-4′, comprised 71.64% of the isolated lignin. The total LCC linkages (the sum of phenyl glycoside, γ-ester and benzyl ether bonds) were 210.86 /100C9 in 13C-LCC, which was dominated by phenyl glycoside linkages, followed by γ-ester bonds and minor amounts of benzyl ether bonds. Lignin and xylan in the LCCs of wheat straw were mainly linked by benzyl ether bonds and phenyl glycoside linkages.

Download PDF

Full Article

Determination of Lignin-Carbohydrate Complexes Structure of Wheat Straw using Carbon-13 Isotope as a Tracer

Lan Yao,a,b,c Congxin Chen,a Xing Zheng,a Ziqian Peng,Haitao Yang,a,* and Yimin Xie a,*

To maximize the use of wheat straw as a feedstock for biofuels and other biorefinery products, the structure of lignin-carbohydrate complexes (LCCs) was characterized by injection of 13C isotope-labeled xylose into living wheat straw. Afterwards, lignin-carbohydrate complexes were extracted from the harvested straw by the Björkman method. The extracted LCCs were chemically characterized by Fourier transform-infrared spectroscopy (FT-IR), sugar composition, molecular weight analysis, 13C-NMR, and HSQC. The results showed that LCCs in wheat straw were particularly enriched with xylan and exhibited narrow polydispersity (Mw/M< 1.5). NMR analysis showed that the lignin was linked with the carbohydrates through γ-ester, phenyl glycoside, and benzyl ether bonds. In addition to S, G, and H lignin units, p-coumarate and ferulate were also in the LCCs. The substructures in lignin were βO-4′, ββ‘, and β-5′. Quantitative data analysis of 13C-NMR combined with HSQC showed that the lignin in the LCCs of wheat straw contained guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units in the proportion of 5:4:1 (S:G:H). The main lignin substructure, βO-4′, comprised 71.64% of the isolated lignin. The total LCC linkages (the sum of phenyl glycoside, γ-ester and benzyl ether bonds) were 210.86 /100Cin 13C-LCC, which was dominated by phenyl glycoside linkages, followed by γ-ester bonds and minor amounts of benzyl ether bonds. Lignin and xylan in the LCCs of wheat straw were mainly linked by benzyl ether bonds and phenyl glycoside linkages.

Keywords: Isotopelabeled; Wheat straw; Lignincarbohydrate complexes (LCCs); 13CNMR; 2DHSQC

Contact information: a: School of Pulp & Paper Engineering, Hubei University of Technology, 430068, Wuhan, China; b: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology , Guangzhou, 510640 China; c: Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education of China, Jinan, 250353; d: NO.1 Middle School Affiliated to Central China Normal University, 430223, Wuhan, China;

* Corresponding authors:;


Bioethanol production from lignocellulosic materials has drawn worldwide attention due to concern about the exhaustion of fossil fuels. In China, agricultural residues can be used as the feedstock to produce ethanol fuel because of its abundance, high carbohydrate content, and low cost (Yao et al. 2010). Agricultural residue is mainly comprised of cellulose, hemicellulose, and lignin, which is considered for second generation biofuel production (Wörmeyer et al. 2011). Wheat straw is the most attractive low cost feedstock for production of bioethanol because of its abundance (nearly 600 million tons/year), renewability, and low lignin content (ca. 15%) (Buranov and Mazza 2008). The bioconversion of lignocellulosic materials to ethanol involves four steps: pretreatment, enzymatic hydrolysis, fermentation, and concentration (Hendriks and Zeeman 2009). Pretreatment removes some hemicelluloses and lignin or breaks the linkages connecting lignin to the carbohydrates (Chen et al. 2009). Previous studies have shown that lignin is important during enzymatic hydrolysis, as it reduces the effective cellulase activity (Berlin et al. 2006; Pan 2008; Nakagame et al. 2011; Hu et al. 2012). The existence of lignin-carbohydrate complexes (LCCs) in the plant cell wall makes it more difficult to reduce the lignin content of the material during pretreatment. Recent studies revealed that LCCs also impact glucose recovery. There is a correlation between LCCs and enzymatic saccharification, such that LCCs hinder lignocellulosic biodegradation (Min et al. 2014a, b). The structural changes of lignin and LCCs in corn stover after alkaline pretreatment have been examined for their effects on enzymatic saccharification (Min et al. 2014c). As an abundant biomass grown in China, it is particularly important to study the LCC linkages of wheat straw to develop more environmentally compatible technologies for bioethanol production.

Traditional chemical degradation methods have been used to study the chemical structure of LCCs, but these methods degrade most native LCC linkages (Watanabe and Koshijima 1988). Solution-state NMR is a powerful tool to characterize the structure of lignin in biomass and in isolated LCC structures (Lu and Ralph 2003; Yelle et al. 2008; Mansfield et al. 2012; Cheng et al. 2013). Information about lignin structures and linkages connecting lignin to carbohydrates can be obtained using one-dimensional 1H- and 13C-NMR and two-dimensional heteronuclear single-quantum coherence (2D-HSQC) NMR spectroscopy. Recently, combined quantitative 13C-NMR and 1H-13C HSQC NMR techniques have been developed (Zhang and Gellerstedt 2007) and effectively applied to measure lignin and LCCs (Balakshin et al. 2011; Wen et al. 2013a, 2013b, 2013c). 13C isotope labeling is a useful tool to study the structure of lignin and LCCs (Yang et al. 2013, 2014; Yao et al. 2015) as it amplifies the signal of 13C-labeled lignin or carbohydrates in the NMR spectra for easier analysis.

In this study, 13C isotope-labeled xylose was injected into living wheat straw to obtain 13C-labeled LCCs, which were isolated using the Björkman technique. FT-IR, HPLC, GPC, and NMR were used to analyze the LCC structures. A more in-depth and complete characterization of the LCCs of wheat straw was acquired to maximize the use of wheat straw as a feedstock for the production biofuels and other biorefinery products.


Administration of 13C Isotope-Labeled Xylose to Wheat Straw

A conventional variety of wheat straw (Triticum aestivum E-mai 352) was chosen. A solution containing the 13C isotope-labeled xylose (2 mg/mL), l-2-aminooxy-3-phenylpropionic acid (AOPP, 0.005 mol/L), and coniferin (2 mg/mL) was injected into the internode sections of the wheat straw over 30 days. For the control samples, a solution containing xylose (2 mg/mL), l-2-aminooxy-3-phenylpropionic acid (AOPP, 0.005 mol/L), and coniferin (2 mg/mL) was injected into the wheat straw. After the injections, the wheat straw was allowed to grow for another 20 days.

Preparation of 13C Isotope-Labeled Xylose Wheat Straw Powder

The air-dried, 13C isotope-labeled wheat straw culms were cut into small pieces, ground, and passed through 80- to 100-mesh screens to obtain a sieved powder. This sieved powder was subjected to Soxhlet extraction with benzene/ethanol (2:1, v/v) for 6 h and then washed with hot water to remove water-insoluble and water-soluble impurities. The extractive-free wheat straw powder was freeze-dried.

Preparation of 13C Isotope-Labeled LCCs

The dried wheat straw powder (extractive-free) was ground in a vibratory ball mill for 72 h. Afterwards, the wheat straw powder was used to obtain LCCs using the Björkman (1957) procedure. LCCs extracted from wheat straw control (injected with xylose) and 13C-labeled were called LCC and LCC-13C.

Component Analyses of LCCs

Lignin and carbohydrate contents were determined according to the method described by NREL (2006). The procedure uses a two-step acid hydrolysis to fractionate the biomass into two forms that are more easily quantified. The lignin fractionates were the acid-insoluble material. Monomeric sugars in the hydrolysis liquid were determined by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) with a refractive index detector (Shimadzu) on an Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) running at a flow rate of 0.6 ml/min at 65 °C, with pure water as the eluent. Chromatography-grade d-glucose, d-xylose, d-galactose, d-mannose, and l-arabinose were used as calibration standards to quantify the polysaccharide content of the LCCs.

FT-IR Spectroscopy

Infrared spectra were determined using a Nicolet Nexus FT-IR (Thermo Scientific, Waltham, USA). Dried preparations (1 to 2 mg) were milled into a powder with a diameter less than 1 mm. The powder was dispersed in spectroscopic grade KBr and subsequently pressed into disks using 10 tons of pressure for 1 min. A total of 32 scans with a 4 cm-1 resolution were collected and averaged. The wavenumber range scanned was 4000 to 500 cm-1.

Molecular Weight Determination

Gel permeation chromatography (GPC) was determined by HPLC (Shimadzu, Kyoto, Japan) using a refractive index detector (Shimadzu) and using a Shim-pack GPC-803D column (Shimadzu) operating at a flow rate of 0.6 mL/min at 40 °C with dimethylformamide (DMF) as the eluent. A sample (2 mg) was dissolved in 1 mL DMF, and 20 μL of this solution was injected into the GPC. Monodisperse polystyrene standards with average molecular weights of 2900; 6100; 47,100; 10,700; and 19,800 g mol-1 were used as calibration standards for determining the molecular weight of lignin-carbohydrate complexes. LC solution software (Shimadzu, Kyoto, Japan) was used for the data analysis.

NMR Spectroscopy

All NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer (Fällanden, Switzerland) that was operated at 25 °C utilizing DMSO-d6 as the solvent. For quantitative 13C NMR, 125 mg of the LCC was dissolved in 0.5 mL of DMSO-d6. Quantitative 13C NMR spectra were recorded in the FT mode at 100.6 MHz. An inverse-gated decoupling sequence, which allows quantitative analysis and comparison of the signal intensities, was used with the following parameters: 90° pulse angle; 1.4 s acquisition time; 2 s relaxation delay; 64,000 data point acquisitions; and 30,000 scans. Chromium (III) acetylacetonate (0.01M) was added to the sample solution to provide complete relaxation of all nuclei.

For heteronuclear single quantum coherence (HSQC), LCC (60 mg) was dissolved in 0.5 mL of DMSO-d6. HSQC NMR spectra were recorded. The spectral widths were 5000 and 20,000 Hz for the 1H and 13C dimensions, respectively. The number of collected complex points was 1024 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. Prior to Fourier transformation, the data matrices were zero filled to 1024 points in the 13C dimension. MestReNova software (Mestrelab Research, Santiago de Compostela, Spain) was used for the integration with the acquired 2D HSQC data.


Molecular Weight and Component Determination

The molecular weight distributions of the LCCs were studied with gel permeation chromatography (GPC) connected to a refractive index (RI) detector, using dimethylfomamide (DMF) as the eluent and polystyrene standards for molecular weight calibrations (Table 1). No LCC samples had wide polydispersity, as shown by Mw/Mn< 1.5. The molecular weights of the LCC samples were between 30,000 to 50,000 g∙mol-1. The Mn of the LCC and LCC-13C was almost the same, at about 35,000 g∙mol-1. Compared with LCCs in Arundo donax L., the molecular weights of wheat straw LCCs were higher. This result was attributed to the different species and different mobile phase used with GPC (You et al. 2015). The Mw of the LCC-13C was slightly higher than that of the LCC, indicating that there were more saccharides connected to the lignin in the LCC-13C than in the LCC sample (Yang et al. 2013a); this result corresponded to the results obtained from the carbohydrate analysis (discussed below).

Table 1. Average Molecular Weight of LCCs

The LCCs were composed of lignin and carbohydrate. The sugar and lignin content of wheat straw LCCs are shown in Table 2. Most of the LCCs were carbohydrate, with about 20% lignin. The LCC samples were also analyzed by HPLC for their sugar composition. The results indicated that the isolated wheat straw LCCs were primarily composed of four different monosaccharides, namely, xylose (62.66%), glucose (6.88%), arabinose (7.23%), and mannose (1.17%); there were negligible amounts of galactose. The Ara/Xyl ratio reflects the branching degree of hemicelluloses. The Ara/Xyl ratio of the isolated LCCs was 0.115, which was lower than that of the hemicelluloses isolated from wheat straw (Sun et al. 2005). This observation suggests that the side-chains of the hemicelluloses of LCCs could be degraded during their extraction. The results implied that arabinoxylans are the major hemicellulose fraction in wheat straw LCCs. The glucan content suggested that glucans were associated with the xylans in the LCCs. Generally, there were insignificant differences between the LCC and the LCC-13C data.

Table 2. Chemical Component Analyses of LCCs (%)

FT-IR Determination of LCC

FT-IR spectra of the LCC-13C and LCC are shown in Fig. 1. Strong signals from the aromatic rings were observed at 1515 cm-1 and 1421 cm-1 (Faix 1991), which indicated lignin in the sample. The strong absorption at 1046 cm-1 was from stretching vibrations of C-O and C-C, which indicated the presence of xylans (Sun et al. 2005). Additionally, signals from C=O stretching were observed at 1731 cm-1, which was attributed to esterified phenolic groups and acetyls connected to the hemicelluloses (You et al. 2015). The weak signal at 1630 cm-1 was assigned to the uronic acid group from hemicelluloses. The C-O stretching of syringyl lignin units (S) was observed at 1322 cm-1 (Sun et al. 2005). Signals at 1246 cm-1 were assigned to guaiacyl lignin units (G). The two signals showed that G and S were the main lignin substructures in wheat straw LCCs. The band at 1162 cm-1 is characteristic of carbonyl groups comprising esters, which indicated the presence of ester-linked p-counaric or ferulic acids (Sun et al. 2011). A characteristic peak from a β-glucosidic bond between the xylose in the hemicelluloses was observed at 897 cm-1 (Sun et al. 2005).

Fig. 1. FT-IR spectra of LCCs from wheat straw

There were insignificant differences when comparing the two IR spectra. Thus, the injection of exogenous 13C-xylose did not interfere with the normal metabolism of wheat straw. These observations are in agreement with an earlier study with AOPP and conferin (Yang et al. 2014), where xylose-13C was successfully introduced into the wheat plant.

Fig. 213C-NMR spectra of LCCs from wheat straw

13C-NMR Determination of LCC

The 13C-NMR spectra of the LCC and 13C-LCC are shown in Fig. 2. The assignments of important signals are listed in Table 3. The region between 160 and 103 ppm was used as a reference to compare the other signal shifts.

The 13C-NMR signals from the C1 to C5 of the xylose cyclic ring were observed at 102.2 ppm, 73.1 ppm, 74.5 ppm, 75.9 ppm, and 63.7 ppm; all signals were intensified in the 13C-LCC, which indicated the success of the injections of exogenous 13C-xylose into the wheat straw (Yang et al. 2014). There were deviation of signals from C1-C5 of xylan when compared with purified xylan, which were at 101.8 ppm (C1), 72.5 ppm (C2), 74.0 ppm (C3), 75.4 ppm (C4), and 63.0 ppm (C5) (Cheng 2011), indicating that most of the carbons in xylan were connected with lignin by certain linkages in LCC from wheat straw. Additionally, a cross-peak at 169.7 ppm was assigned to an acetyl group (Sun et al. 2005). The strong signal at 73.1 ppm is characteristic of the C3 atom in 4-O-methyl-d-glucuronic acid groups of xylans.

Table 3. Analysis of 13C-NMR Spectra of LCC from Wheat Straw

2D-HSQC Determination of LCC

2D-HSQC is commonly used to analyze the structures of lignin and LCCs, and it provides evidence for the existence of various linkages between lignin and carbohydrates. This analytical technique can deconvolute the overlapping signals from lignin and carbohydrate in 13C-NMR spectra. The 2D-HSQC spectra of the LCC-13C and LCC are shown in Fig. 3. The main linkages and structural units identified are shown in Fig. 4.

Fig. 3. HSQC spectra of LCCs from wheat straw (f1 and f2 refer to 13C and 1H in the axis)

Fig. 4. Major structures and LCC linkages present in the LCCs of wheat straw: (A) βO-4′ alkyl-aryl ethers; (A’) βO-4′ alkyl-aryl ethers with acetylated γOH; (B) phenylcoumarane; (C) resinols; (D) dibenzodioxocin; (I) cinnamyl alcohol end-groups; (J) cinnamyl aldehyde end-groups; (PCA) p-coumarates; (FA) ferulates; (H) p-hydroxyphenyl units; (G) guaiacyl units; (S) syringyl unit; and (S’) oxidized syringyl units with a Cα ketone

Benzyl ethers (BEs) in LCC linkages

The amount of benzyl ethers in the LCCs was obtained from the signal of CαH in the LCC structure. Benzyl ether structures are composed of linkages between the Cα position of lignin with the primary OH groups (BE1) and secondary OH groups (BE2) of the carbohydrates (Balakshinet al. 2011). Previous studies indicated cross-peaks at 80 to 81/4.4 to 4.6 ppm and 80 to 81/5.0 to 5.2 ppm, respectively (Min et al. 2014c). The linkages between lignin and primary OH groups of carbohydrates primarily occur with the Cposition of glucan, galactan, and mannan, and with the C5 position of arabinan, while linkages between lignin and secondary OH groups of carbohydrates was mostly formed between lignin and xylan (Yuan et al. 2011). It is worth noting that the BE1 signal might overlap γ-acetylated βO-4′ substructures linked to a G lignin unit. The signals of BE2 always overlap the signals of β-1′ lignin substructures. However, these kinds of signals are not observed in the 2D-HSQC spectra.

In the present study, the Cα-Hα correlations in the benzyl ethers of LCC structures were found at δC/δH 82.1/5.0 ppm in the HSQC spectra; this signal suggests a bond between the Cα position of lignin with secondary OH groups of carbohydrates. Signals for benzyl ether bonds at about δC/δH 80 to 81/4.4 to 4.6 ppm from the Cα position of lignin with primary OH groups of carbohydrates were not detected. This observation indicated that the benzyl ethers (BEs) LCC linkages in wheat straw are principally formed between lignin and xylan.

The formulae used to calculate the varying amounts of identified LCC moieties have been proposed by Yuan et al. (2011). To obtain the absolute values from corresponding signals, the resonance for the total aromatic carbons (163.0 to 103.0 ppm in 13C NMR) is assigned a value of 600. The integral values for selected internal standard references and other structural moieties are expressed per 100 aromatic units (Ar). To quantify the amount of LCC linkages, three clusters at δC 103 to 96/δH 5.5 to 3.8 ppm, δC 90 to 78/δH5.7 to 3.0 ppm and δC 65 to 58/δH5.0 to 2.5 ppm were integrated in the 13C NMR spectra; these clusters corresponded to phenyl glycoside, benzyl ether, and γ-esters, respectively (Min et al. 2014c) (Table 4). The number of BEs in the LCC and 13C-LCC were 2.12 and 11.84 per 100 Ar. These values correspond to the values previously reported for the LCC linkages of A. donax L. (You et al. 2015). The difference of BE2 in the LCC and 13C-LCC was due to the 13C-labeling, which indicated that part of the lignin and xylan in wheat straw were connected by benzyl ether bonds.

Esters in LCC linkages

There was no signal detected at δC/δH 75/6.1 ppm for a benzyl ester (α-ester) (You et al. 2015). Signals from Cγ in the γ-ester were found in the range of δC/δH 65 to 62/4.0 to 4.5 ppm. Signals from lignin, which was γ-acetylated, overlap the γ-ester; thus, it was not feasible to assign this type of signal to the γ-ester linkages (Del et al. 2012a). However, this issue could be resolved by a cryoprobe, as the signals in the range of δC/δH 62 to 63/4.0 to 4.5 ppm and 63 to 64/4.0 to 4.5 ppm were ascribed to γ-acetylated lignin and Cγ in the LCC γ-ester, respectively (Balakshin et al. 2011). After the analysis, the γ-ester (including γ-acetylated βO-4′ aryl ether substructures) in the LCC and 13C-LCC were 13.95 and 15.28 per 100 Ar, respectively. These results are consistent with the report by Min et al. (2014c) examining the LCCs of corn stover. The similarity of γ-ester linkages in the LCC and 13C-LCC suggested that the number of this kind linkage between lignin and xylan in wheat straw was slight.

Phenyl glycoside (PhGly) in LCC linkages

Phenyl glycoside produced signals at δC/δH 100 to 103/4.0 to 4.8 ppm (Min et al. 2014c). The classic Björkman LCC extraction method is an appropriate analysis for these linkages. A cross-peak from C1-H1 of the carbohydrates connected to lignin by PhGly linkages occurred at δC/δH 101.9/4.25 ppm. The phenyl glycoside in the LCC and 13C-LCC were 50.17 and 183.74 per 100 Ar, respectively. Compared with the analysis of You et al. (2015), which examined the LCCs of A. donax, the amount of phenyl glycoside the linkages seen here, 251 per 100 monomeric lignin units, was lower. This result was attributed to the higher amount of glucan (28.9%) in the LCCs of A. donax. The amount of PhGly linkages in the 13C-LCC was three times higher than LCC, which indicated that part of lignin and xylan in the wheat straw were connected by PhGly linkages. Based on the HSQC data of LCC model compound (Miyagawa et al. 2014), signals at δC/δH 100.1/5.01, 100.3/4.96 and 102.1/5.04 ppm were assigned to p-hydroxybenzaldehyde (G, H and S, respectively) derivative β-xylosides. Signals attributed to monolignol β-xylosides and dihydromonolignol β-xylosides were not detected, which indicated that those compounds had been destroyed during the ball milling process to extract LCC.

Associated carbohydrates

Carbohydrate signals in the LCC were also noteworthy. The strong signals from the C1, C2, C3, C4, and C5 positions in the β-d-xylose were at δC/δH 101.9/4.24 ppm, 72.8/3.05 ppm, 74.1/3.24 ppm, 75.6/3.49 ppm, and 63.1/3.14 ppm, respectively (Wen et al. 2010). Signals from the C2-H2position in 2-O-acetyl-β-d-xylopyranoside, as well as the C3-H3 and C1-H1 positions in 3-O-acetyl-β-d-xylopyranoside, were at δC/δH 73.5/4.49 ppm, 75.1/4.79 ppm, and 101.9/4.25 ppm, respectively (Peng et al. 2014). A signal at δC/δH 80.4/3.32 ppm was assigned to C4-H4 in 4-O-methyl glucuronic acid (You et al. 2015). The signal indicated that the side chains of most carbohydrates in the wheat straw LCCs are 4-O-methyl glucuronic acid, which was partially acetylated at the C2 and C3 positions. Additionally, cross-peaks at δC/δH 92.3/4.89 ppm and 98.1/4.26 ppm were ascribed to (1→4)-α-d-xylose (αX1) and (1→4)-β-d-xylose (βX1) (Peng et al. 2014).

Major lignin structures

In the region of δC/δH 50 to 90/2.5 to 6.0 ppm, signals from -OCH3 (δC/δH 55.7/3.72 ppm) and βO-4′ were detected. Signals from Cγ-Hγ and acylated Cγ-Hγ positions in the βO-4′ were at δC/δH 60.9/3.56 and 63.2/3.86 ppm, respectively (Rencoret et al. 2009). Signals at δC/δH 71.9/4.74, 86.2/3.96 and 82.4/4.20 ppm were assigned to Cα-Hα and Cβ-Hβ positions in the βO-4′ (Del et al. 2012a). In addition, signals from ββ‘, β-5′ and 5-5′ were detected. A signal at δC/δH 83.3/4.80 ppm was ascribed to the Cα-Hα position in the ββ‘. A weak signal at δC/δH 71.4/3.84 ppm was assigned to the Cγ-Hγ position in the ββ‘ (Martínez et al. 2008). Signals from Cα-Hα in the β-5′ were detected at δC/δH 87.4/5.52. A signal from the Cγ-Hγ position of β-5′ overlapped that of the C5-H5 position in xylose. Signals assigned to Cα-Hα in the 5-5′ were observed at δC/δH 83.8/4.90 ppm (Del et al. 2012a).

Signals in the region of δC/δH 100 to 135/5.5 to 8.5 ppm were principally from S, G, and H lignin units. Signals of C2-H2/C6-H6 aromatic positions in S and in oxidized S were at δC/δH 104.1/6.66 and 106.7/7.26ppm, respectively (Wen et al. 2013). Signals at δC/δH 111.0/6.92, 114.2/6.80, and 119.1/6.75 ppm were from C2-H2, C5-H5, and C6-H6 aromatic positions in G lignin units. Signals assigned to C2-H2/C6-H6 aromatic positions in H units were at δC/δH 128.5/7.05 ppm. Additionally, a signal from C2-H2 in ferulic acid (FA2) was at δC/δH 111.3/7.33 ppm (Wen et al. 2013). Signals from p-coumaric acid (PCA2 and PCA6) and end groups in p-coumaryl alcohol and cinnamaldehyde were also detected. Combining HSQC with 13C-NMR is an analytical method to obtain the absolute quantities of various substructures (Min et al. 2014a). The absolute contents of important signals were calculated from the spectra data and are summarized in Table 4. The analysis showed that wheat straw LCCs were mostly bonded by ferulate linkages, which was followed by γ-ester, phenyl glycoside, and benzyl ether linkages. Lignin was mostly connected by benzyl ether linkages to the xylans. The main substructure of the lignin was the βO-4′ (71.64%), which was followed by the ββ‘ (14.93%) and 5-5’ (11.94%), with minor amounts of β-5′ (1.49%). The S/G ratio was 1.28, which was the same as that observed from milled lignin from wheat straw (Yang et al. 2014). This observation implied that the structural units were the same in milled lignins and LCCs. However, these weak signals were attributed to the degradation of the lignin during the isolation process.

Table 4. Characteristics of LCCs from Quantitative NMR Method

a: amount of specific interunit linkage was expressed as number per 100 Ar


  1. HPLC and GPC analysis showed that the major polysaccharide in the LCCs was xylan. LCCs had narrow polydispersity. There were insignificant differences between the LCC and the LCC-13C in sugar composition and molecular weight.
  2. FT-IR and 13C-NMR results showed that lignin and carbohydrate coexist in the LCCs. The injection of exogenous xylose was successfully introduced into the wheat plant.
  3. Quantitative analysis showed that the total LCC linkages were 210.86 /100Cin 13C-LCC, which was dominated by phenyl glycoside linkages, followed by γ-ester bonds and minor amounts of benzyl ether bonds. Compared with the HSQC data of LCC model compound, it was found that xylan and lignin were mainly connected as p-hydroxybenzaldehyde derivative β-xylosides. The main lignin substructures in the LCCs of wheat straw were βO-4′, with small amounts of ββ‘ and 5-5’, and trace amounts of β-5′. In addition to S, G, and H lignin units, there were also certain amounts of p-coumarate and ferulate in the LCCs. The lignin structural features in the LCCs were quite similar to that of milled straw lignin.


The authors are grateful for the support from the Natural Science Foundation of China (No. 31500496), the China Scholarship Council (No. 2011842330), the State Key Laboratory of Pulp and Paper Engineering (No. 201510), and the Foundation (KF201409) of Key Laboratory of Paper Science & Technology of the Ministry of Education.


Buranov, A. U., and Mazza, G. (2008). “Lignin in straw of herbaceous crops,” Ind. Crop. Prod.28(3), 237-259. DOI: 10.1016/j.indcrop.2008.03.008

Balakshin, M., Capanema, E., Gracz, H., Chang, H.-m., and Jameel, H. (2011). “Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy,” Planta 233(6), 1097-1110. DOI: 10.1007/s00425-011-1359-2

Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., and Saddler, J. (2006). “Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations,” J. Biotechnol. 125(2), 198-209. DOI: 10.1016/j.jbiotec.2006.02.021

Björkman, A. (1957). “Studied on finely divided wood. Part 3. Extraction of lignin- carbohydrate complexes with neutral solvent,” Sven. Papperstidn. 60(3), 243-251. DOI: 1010.1007/BF00392053

Chen, M., Zhao, J., and Xia, L. M. (2009). “Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility,” Biomass Bioenerg. 33(10), 1381-1385. DOI: 10.1016/j.biombioe.2009.05.025

Cheng, H. L. (2011). “Isolation, characterization and sulfation of corn stover hemicellulose,” Doctoral Thesis, South China University of Technology, 90-91. (Chinese)

Cheng, K., Sorek, H., Zimmermann, H., Wemmer, D. E., and Pauly, M. (2013). “Solution-state 2D NMR spectroscopy of plant cell walls enabled by a dimethylsulfoxide-d6/1-ethyl-3-methylimidazolium acetate solvent,” Anal. Chem. 85(6), 3213-3221. DOI: 10.1021/ac303529v

del Río, J. C., Prinsen, P., Rencoret, J., Nieto, L., Barbero, J. J., Ralph, J., Martínez, A. T., and Gutierrez, A. (2012a). “Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems,” J. Agric. Food. Chem. 60(14), 3169-3634. DOI: 10.1021/jf300099g

del Río, J. C., Rencoret, J., Prinsen, P., Martínez, A. T., Ralph, J., and Gutierrez, A. (2012b). “Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods,” J. Agric. Food. Chem. 60(23), 5922-5935. DOI: 10.1021/jf301002n

Faix, O. (1991). “Classification of lignins from different botanical origins by FT-IR spectroscopy,” Holzforschung 45(s1), 21-27. DOI: 10.1515/hfsg.1991.45.s1.21,

Hendriks, A. T. W. M., and Zeeman G. (2009). “Pretreatments to enhance the digestibility of lignocellulosic biomass,” Bioresour. Technol. 100(1), 10-18. DOI: 10.1016/j.biortech.2008.05.027

Hu, F., Jung, S., and Ragauskas, A. (2012). “Pseudo-lignin formation and its impact on enzymatic hydrolysis,” Bioresour. Technol. 117(Aug. 2012), 7-12 DOI: 10.1016/j.biortech.2012.04.037

Lam, T. B. T., Iiyama, K., and Stone, B. A. (1992). “Cinnamic acid bridges between cell wall polymers in wheat and phalaris internodes,” Phytochem. 31(4), 1179–1183. DOI: 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E10.1016/0031-9422(92)80256-E101016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E10.1016/0031-9422(92)80256-E

Lu, F., and Ralph, J. (2003). “Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR,” Plant J. 35(4), 535-544. DOI: 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E10.1016/0031-9422(92)80256-E10.1046/j.1365-313X.2003.01817.x

Mansfield, S. D., Kim, H., Lu, F., and Ralph, J. (2012). “Whole plant cell wall characterization using solution-state 2D NMR,” Nat. Protoc. 7(9), 1579-1589. DOI: 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E 10.1016/0031-9422(92)80256-E10.1016/0031-9422(92)80256-E10.1038/nprot.2012.064

Martínez, A. T., Rencoret, J., Marques, G., Gutierrez, A., Ibarra, D., Barbero, J. J., and del Río, J. C. (2008). “Monolignol acylation and lignin structure in some nonwoody plants: A 2D NMR study,” Phytochemistry 69(16), 2831-2843. DOI: 10.1016/j.phytochem.2008.09.005

Min, D.-y., Yang, C., Chiang, V., Hasan, J., and Chang, H.-m. (2014a). “The influence of lignin-carbohydrate complexes on the cellulose-mediated saccharification II: Transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.),” Fuel 116(15 Jan.2014), 56-62. DOI: 10.1016/j.fuel.2013.11.047

Min, D.-y., Li, Q., Chiang, V., Jameel, H., Chang, H.-m., and Lucia, L. (2014b). “The influence of lignin-carbohydrate complexes on the cellulase-mediated saccharification I: Transgenic black cottonwood (western balsam poplar, California poplar) P. trichocarpa including the xylan down-regulated and the lignin down-regulated lines,” Fuel 119(1 March 2014), 207-213. DOI: 10.1016/j.fuel.2013.11.047

Min, D.-y., Jameel, H., Chang, H.-m., Lucia, L., Wang, Z. G., and Jin, Y. C. (2014c). “The structural changes of lignin and lignin-carbohydrate complexes in corn stover induced by mild sodium hydroxide treatment,” RSC Adv. 4(21), 10845-10850. DOI: 10.1039/C3RA47032F

Miyagawa, Y., Mizukami, T., Kamitakahara, H., and Takano, T. (2014). “Synthesis and fundamental HSQC NMR data of monolignol β-glycosides, dihydromonolignol β-glycosides and p-hydroxybenzaldehyde derivative β-glycosides for the analysis of phenyl glycoside type lignin-carbohydrate complexes (LCCs),” Holzforschung 68(7), 747-760.

Nakagame, S., Chandra, R. P., Kadla, J. F., and Saddler, J. N. (2011). “The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose,” Bioresour. Technol. 102(6), 4507-4517. DOI: 10.1016/j.biortech.2010.12.082

Pan, X. (2008). “Role of functional groups in lignin inhibition of enzymatic hydrolysis of cellulose to glucose,” J. Biobased Mater. Bioenerg. 2(1), 25-32. DOI: 10.1166/jbmb.2008.005

Peng, H., Luo, Q., Ruan, R., Zhang, J. S., and Liu, Y. H. (2014). “Structural features of lignin and lignin-carbohydrate complex from bamboo (Phyllostachys pubescens Mazel),” BioResources9(1), 1276-1289. DOI: 10.15376/biores.9.1.1276-1289

Ralph, J. (2010). “Hydroxycinnamates in lignification,” Phytochem. Rev. 9(1), 65-83. DOI: 10.1007/s11101-009-9141-9

Rencoret, J., Marques, G., Gutierrez, A., Nieto, L., Santos, J. I., Jimenez, B. J., and del Rio, J. C. (2009). “HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state,” Holzforschung 63(6), 691-698. DOI: 10.1515/HF.2009.070

NREL. (2006). “Biomass Program, Standard Method for Determination of Total Solids in Biomass,” (

Sun, R.-C., Xiao, B., and Lawther, J. M. (1998). “Fractional and structural characterization of ball-milled and enzyme lignins from wheat straw,” J. Appl. Polym. Sci. 68(10), 1633-1641. DOI: 10.1002/(SICI)1097-4628(19980606)68:10<1633::AID-APP12>3.0.CO;2-Y

Sun, R.-C., Sun, X.-F., and Zhang, S.-H. (2001). “Quantitative determination of hydroxycinnamic acids in wheat, rice, rye, and barley straws, maize stems, oil palm frond fiber, and fast-growing poplar wood,” J. Agric. Food Chem. 49(11), 5122-5129. DOI: 10.1021/jf010500r

Sun, X.-F., Sun, R.-C., Fowler, P., and Baird, M S. (2005). “Extraction and characterization of original lignin and hemicelluloses from wheat straw,” J. Agric. Food Chem. 53(4), 860-870. DOI: 10.1021/jf040456q

Watanabe, T., and Koshijima, T. (1988). “Evidence for an ester linkage between lignin and glucuronic acid in lignin-carbohydrate complexes by DDQ-Oxidation,” Agric. Biol. Chem. 52(11), 2953-2955. DOI: 10.1080/00021369.1988.10869116

Wen, J.-L., Sun, Z., Sun, Y.-C., Sun, S.-N., Xu, F., and Sun, R.-C. (2010). “Structural characterization of alkali-extractable lignin fractions from bamboo,” J. Biobased Mater. Bioenerg.4(4), 408–425. DOI: 10.1166/jbmb.2010.1111

Wen, J.-L., Sun, S.-L., Xue, B.-L., and Sun, R.-C (2013a). “Quantitative structures and thermal properties of birch lignins after ionic liquid pretreatment,” J. Agric. Food Chem. 61(3), 635-645. DOI: 10.1021/jf3051939

Wen, J.-L., Sun, S.-L., Xue, B.-L., and Sun, R.-C. (2013b). “Recent advances in characterization of lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology,” Materials6(1), 359-391. DOI: 10.3390/ma6010359

Wen, J.-L., Xue, B.-L, Xu, F., Sun, R.-C., and Pinkert, A. (2013c). “Unmasking the structure features and property of lignin from bamboo,” Ind. Crop Prod. 42(March 2013), 332-343. DOI: 10.1016/j.indcrop.2012.05.041

Wörmeyer, K., Ingram, T., Saake, B., Brunner, G., and Smirnova, I. (2011). “Comparison of different pretreatment methods for lignocellulosic materials. Part II: Influence of pretreatment on the properties of rye straw lignin,” Bioresour. Technol. 102(5), 4157-4164. DOI: 10.1016/j.biortech.2010.11.063

Yao, L., Yue, J., Zhao, J., Dong, J., Li, X., and Qu, Y. (2010). “Application of acidic wastewater from monosodium glutamate process in pretreatment and cellulose production for bioconversion of corn stover-feasibility evaluation,” Bioresour. Technol. 101(22), 8755-8761. DOI:  10.1016/j.biortech.2010.04.104

Yao, L., Yang, H., Xu, J., Zhang, N., Chen, Y., and Xie, Y. (2015). “Elucidation of the structure of lignin and lignin carbohydrate complex of Gramineae,” Cell. Chem. Technol. 49 (3-4), 259-266.

Yang, D., Zhong, L.-X., Yuan, T.-Q., Peng, X.-W., Sun, R.-C. (2013a). “Studies on the structural characterization of lignin, hemicelluloses and cellulose fractionated by ionic liquid followed by alkaline extraction from bamboo,” Ind. Crop Prod. 43(May 2013), 141-149. DOI: 10.1016/j.indcrop.2012.07.024

Yang, H., Zheng, X., Yao, L., Gu, R., and Xie, Y. (2013b). “Elucidation of the bonds between cellulose and dehydrogenation polymer with carbon-13 isotopic tracer method,” Cell. Chem. Technol. 47(3-4), 143-151.

Yang, H., Zheng, X., Yao, L., and Xie, Y. (2014). “Structural changes of lignin in the Soda-AQ pulping process studied using the carbon-13 tracer method,” BioResources 9(1), 176-190. DOI: 10.15376/biores.9.1.176-190

Yelle, D. J., Ralph, J., and Frihart, C. R. (2008). “Characterization of nonderivatized plant cell walls using high-resolution solution-state NMR spectroscopy,” Magn. Res. Chem. 46(6), 508-517. DOI: 10.1002/mrc.2201

You, T.-T., Zhang, L.-M., Zhou, S.-K., and Feng, X. (2015). “Structural elucidation of lignin-carbohydrate complex (LCC) preparations and lignin from Arundo donax Linn,” Ind. Crop Prod.71(Sept. 2015), 65-74. DOI: 10.1016/j.indcrop.2015.03.070

Yuan, T.-Q., Sun, S.-N., Xu, F., and Sun, R.-C. (2011). “Characterization of lignin structures and lignin-carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR spectroscopy,” J. Agric. Food Chem. 59(19), 10604-10614. DOI: 10.1021/jf2031549

Zeng, J., Helms, G. L., Gao, X., and Chen, S. (2013). “Quantification of wheat straw lignin structure by comprehensive NMR analysis,” J. Agric. Food Chem. 61(46), 10848−10857. DOI: 10.1021/jf4030486

Zhang, A., Lu, F., Sun, R., and Ralph, J. (2009). “Ferulate-coniferyl alcohol cross-coupled products formed by radical coupling reactions,” Planta 229(5), 1099-1108. DOI: 10.1007/s00425-009-0894-6

Zhang, L., and Gellerstedt, G. (2007). “Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references,” Magn. Res. Chem. 45(1), 37-45. DOI: 10.1002/mrc.1914

Zhang, X. M., Meng, L. Y., Xu, F., and Sun, R.-C. (2010). “Pretreatment of partially delignified hybrid poplar for biofuels production: Characterization of organosolv hemicelluloses,” Ind. Crop Prod. 33(2), 310-316. DOI: 10.1016/j.indcrop.2010.11.016

Article submitted: February 23, 2016; Peer review completed: May 22, 2016; Revised version received and accepted: May 31, 2016; Published: June 28, 2016.

DOI: 10.15376/biores.11.3.6692-6707