Structural changes of lignin in the soda-AQ pulping process studied using the carbon-13 tracer method

Abstract

To reveal the structural changes of lignin during the soda–AQ cooking process and chemical bonds between lignin and xylan of wheat straw, 13C isotope-labeled technology was used in this study. First, 13C isotope-labeled xylose was injected into the living wheat straw. The success of 13C isotope-labeling was confirmed by the results of 13C abundance determination. Then, milled wood lignin-13C was extracted from wheat straw. The wheat straw, which had already been labeled by xylose-13C, was cooked by the soda-AQ process. Soda-AQ Lignin-13C and Residual Lignin-13C were extracted from black liquor and residual pulp. FT-IR, 13C-NMR, and 2D HSQC NMR analyses indicated that all lignin preparations were HGS-type lignin. The main lignin linkages were β-O-4’ units, β–β’ units, β-5’ units, and β-1 units, with the highest content of β-O-4’. Furthermore, 82.8% of β-O-4’ units, 77.2% of β–β’ units, 75.4% of β-5’ units, and 75.4% of β-1 units were degraded during the cooking process. LC bonds between lignin and xylan were at C2 and C5 positions of xylan. It was found that the C-2 position of xylan in wheat straw could be mainly connected to lignin by γ-ester bonds, and C-5 position of xylan in wheat straw was possibly linked with lignin by benzyl ether bonds.

Full Article

Structural Changes of Lignin in the Soda-AQ Pulping Process Studied Using the Carbon-13 Tracer Method

Haitao Yang,^a,b Xing Zheng,^a Lan Yao,^a,b,* and Yimin Xie ^a,*

To reveal the structural changes of lignin during the soda–AQ cooking process and chemical bonds between lignin and xylan of wheat straw, ¹³C isotope-labeled technology was used in this study. First, ¹³C isotope-labeled xylose was injected into the living wheat straw. The success of ¹³C isotope-labeling was confirmed by the results of ¹³C abundance determination. Then, milled wood lignin-¹³C was extracted from wheat straw. The wheat straw, which had already been labeled by xylose-¹³C, was cooked by the soda-AQ process. Soda-AQ Lignin-¹³C and Residual Lignin-¹³C were extracted from black liquor and residual pulp. FT-IR, ¹³C-NMR, and 2D HSQC NMR analyses indicated that all lignin preparations were HGS-type lignin. The main lignin linkages were β-O-4’ units, β–β’ units, β-5’ units, and β-1 units, with the highest content of β-O-4’. Furthermore, 82.8% of β-O-4’ units, 77.2% of β–β’ units, 75.4% of β-5’ units, and 75.4% of β-1 units were degraded during the cooking process. LC bonds between lignin and xylan were at C₂ and C₅ positions of xylan. It was found that the C-2 position of xylan in wheat straw could be mainly connected to lignin by γ-ester bonds, and C-5 position of xylan in wheat straw was possibly linked with lignin by benzyl ether bonds.

Keywords: Isotope-labeled; Lignin; Lignin-carbohydrate complexes (LCCs); ¹³C-NMR; 2D-HSQC

Contact information: a: School 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 authors: yaolislan1982@aliyun.com; ppymxie@scut.edu.cn

INTRODUCTION

Numerous studies have been carried out on the structure of lignin and lignin-carbohydrate complexes (LCCs). Using the traditional analytical methods to study the structure of lignin and LCC will inevitably cause some structural changes during the separation process. Furthermore, the separated products cannot be representative of the precise structure of the plant itself or the residual lignin and LCCs in the pulp. Common chemical degradation methods, such as oxidation (Watanabe and Koshijima 1988), acid-catalyzed hydrolysis (Eriksson et al. 1980), and alkali hydrolysis (Takakashi and Koshijima 1988), are unable to cleave all of the bonds connecting lignin and carbohydrates. In addition, it is likely that some unknown side reactions occur in the process of strong chemical decomposition. Therefore, traditional analytical methods cannot effectively determine the structural characteristics of lignin and LCC.

With the development of science and technology, more experimental technologies have been successfully used to study the structure of lignin and LCCs. Karlsson et al. (2001) applied molecular exclusion chromatography to isolate components from pine sulfate pulp after treatment with enzyme, and showed that it had characteristic absorption of lignin and carbohydrates in ultraviolet spectra, thus proving that chemical bonds existed between lignin and sugar units. Then, Lawoko et al. (2003) adopted enzymolysis with chemical treatment methods to systematically fractionate unbleached softwood kraft pulp (fiber); the lignin and sugar content of the degraded products were also detected. The coexistence of lignin and sugar in some fractions indirectly showed the presence of bonds between lignin and carbohydrates in softwood kraft pulp. Although researchers have determined there are covalent bonds connecting lignin and carbohydrates, it is still unknown what types of bonds bind these components together.

In the 1980s, ¹³C-NMR technology began to be widely used in the study of the bonds connecting lignin and lignin-carbohydrates complexes. However, lignin macro-molecules and carbohydrate compounds are extremely complicated. It is impossible to distinguish the bonds connecting lignin and carbohydrates from the ordinary ¹³C-NMR spectra without the additional use of special analytical methods, such as ¹³C isotope labeling techniques. The use of isotope labeling technology can overcome the chemical destruction of the native structure of LCCs in plants during isolation, making the results more accurate and representative. Some researchers (Su et al. 2002; Xie et al. 2003; Wang et al. 2006; Gu et al. 2002a,b) have used isotope-labeled coniferin as a lignin precursor to bio-synthesize dehydrogenated polymers (DHPs) with xylan, holo-cellulose, cellulose, or pectin. It was found that lignin and glycan can form LCCs, which were connected by ether, ester, and acetal bonds, as detected by ¹³C-NMR. On this basis, various investigators (Yang et al. 2004; Gu et al. 2002a,b; Xie and Terashima 1991; Xie et al. 2000) have successfully used the ¹³C isotope tracer technique combined with ¹³C-NMR to analyze lignin and LCC bonds in plants such as straw and ginkgo. Recently, 2D-HSQC has been commonly used to analyze the structure of lignin and LCC, which can overcome the overlapping problem from lignin and carbohydrate. But, this approach evaluates only relative proportions of various signals and is unable to provide with the absolute values. A combination of HSQC and ¹³C-NMR, as suggested by Zhang and Gellerstedt (2007), is a method to obtain the absolute values of various signals. This method is very effective to analyze the content of various lignin structures and has been widely applied (Balakshin et al, 2011; Wen et al, 2013a,2013b,2013c; Yuan et al. 2011).

In this study, ¹³C isotope-labeled xylose was efficiently injected into living wheat straw, according to the results of ¹³C abundance detection. The structural characteristics of ML extracted from wheat straw were studied using the ¹³C-NMR spectra technique. Then, ¹³C isotope-labeled wheat straw was pulped using the soda-AQ method. Finally, the changes in lignin before and after cooking were analyzed by FT-IR, ¹³C-NMR and 2D-HSQC.

EXPERIMENTAL

Administration of ¹³C Isotope-Labeled Xylose to Wheat Straw

A conventional variety of wheat straw, Triticum aestivum E-mai 352, was planted in the field in March 2012. A mixed 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 2^nd, 3^rd, and 4^th sections of the internodes (starting from the root) of the wheat straw within 30 days. After the injection, the wheat straw was allowed to grow for 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, then ground and sieved to obtain an 80- to 100-mesh fraction. This fraction was subjected to extraction with benzene/ethanol (2:1, v/v) in a Soxhlet apparatus for 6 h and hot water successively. Finally, the extractive-free wheat straw powder was freeze-dried.

Preparation of ML (¹³C Isotope-Labeled Xylose Wheat Straw and Natural Wheat Straw)

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 prepare the ML using the procedure of Björkman (1956).

Preparation of Soda-AQ Lignin and Residual Lignin

Soda-AQ pulping was performed under the following conditions: 15% NaOH, 0.05% AQ, 5:1 liquor-to-wheat straw ratio. Wheat straw black liquor lignin was obtained by acid precipitation (Yuan et al. 2009). Residual lignin was prepared and purified according to the procedures of Kapareju and Wu (Kaparaju and Felby 2010; Wu and Argyropoulos 2003).

¹³C Abundance Determination

Sample (0.1 mg) stable isotopic values (δ¹³C) were analyzed in duplicate, using a continuous flow system isotope ratio mass spectrometry (Isoprime 100) coupled with an elemental analyzer (Vario Pyro Cube), after being wrapped in a tin boat. The average standard deviations of the measurements were ±0.15‰ for δ¹³C. Values of δ¹³C are expressed in standard delta notation relative to the Pee Dee Belemnite (PDB) standard. The sample δ¹³C was corrected using the two-point correction method according to the international standards IAEA-CH3 and IAEA-601. The ¹³C abundances (%) of samples were calculated using the δ¹³C values and the ¹³C/¹²C ratio of the PDB standard (¹³C/¹²C = 11237.2±90*10^-6).

¹³C-NMR Spectroscopy

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

2D-HSQC Spectroscopy

Lignin (60 mg) was dissolved in 0.5 mL of DMSO-d₆. 2D HSQC NMR spectra were recorded in the HSQC experiments. 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 ¹J_CH used was 145 Hz. Prior to Fourier transformation, the data matrices were zero filled to 1024 points in the ¹³C dimension.

RESULTS AND DISCUSSION

¹³C/¹²C Isotopic Ratio

The ¹³C content (Vienna Pee Dee Belemnite (VPDB)  ¹³C scale) of the xylose-¹³C₅ (all five carbons in xylose were isotope-labeled) enriched wheat straw was significantly higher than the control (Table 1). These results showed that with the AOPP and coniferin, the xylose-¹³C₅ was successfully introduced into the wheat straw and took part in the plant’s normal metabolism.

Table 1. ¹³C Abundance of ¹³C Isotope-Labeled Wheat Straw

• • • 1. 
      2. FT-IR Determination of Lignin Samples

The FT-IR spectra of the lignin samples are shown in Fig. 1. The spectrum of the ML-¹³C was quite similar to that of ML, soda-AQ lignin, and residual lignin. The signals at 1600 cm^-1, 1510 cm^-1, and 1420 cm^-1, corresponding to the aromatic ring (Yang et al. 2013), were obvious in the spectra, which indicated that the basic lignin structure was not changed during the cooking process.

Fig. 1. FT-IR spectra of lignin samples (A-ML; B-ML-¹³C; C-Soda-AQ Lignin; D-Soda-AQ Lignin-¹³C; E-Residual Lignin ; F-Residual Lignin-¹³C; ML was extracted from wheat straw; Soda-AQ lignin was extracted from black liquor; Residual lignin was extracted from pulp.)

The signal at 1740 cm^-1 was from non-conjugated carbonyl groups (Faix 1991). In lignin samples, that was mainly from the carboxyl or ester linkage of Cγ of lignin side chain. The intensity of this signal in ML-¹³C was stronger than that of ML. It may be due to the length of time of ball milling during ML extraction, resulting in oxidation of partial structures. Furthermore, by comparing the six spectra, it was found that residual lignin had the lowest content of non-conjugated carbonyl groups, indicating the dissolution of most of this structure in black liquor.

The FT-IR spectra of ML and ML-¹³C were almost identical. Thus, it was concluded that the injection of xylose-¹³C₅ did not interfere with the normal metabolism of wheat straw. Compared with soda-AQ lignin, the signals at 2920 and 2850 cm^-1 from the C-H stretching vibration of methyl and methylene units were intensified in the residual lignin, indicating the breakdown and rearrangement of chemical bonds between lignin monomer units. The signal from H-type lignin was at 834 cm^-1. The intensity of this signal was also reduced in residual lignin, compared with that of ML and soda-AQ lignin, indicating the dissolving of H-type lignin during the cooking process.

¹³C NMR Determination of Lignin Samples

¹³C-NMR spectra of lignin samples are shown in Fig. 2. The region between 160 and 103 ppm was used as a reference to compare other signal shifts.

In the ¹³C-NMR spectra of lignin samples, it was easy to find the signals from p-coumaric acid (Scalbert et al. 1985) at 167.1 ppm (C₉), 160.6 ppm (C₄), 130.9 ppm (C₂/C₆), 125.6 ppm (C₁), and 116.5ppm (C₃/C₅). The weak signal between 90 and 102 ppm indicated the low carbohydrate content in lignin samples.

The signals assigned to guaiacyl (Sun et al. 2005) were at 149.7 ppm (C₃, etherified), 148.1 ppm (C₃), 145.9 ppm (C₄, etherified), 119.9 ppm (C₆), 115.3 ppm (C₅), and 111.6 ppm (C₂). The strong signals from syringyl (Kim and Ralph 2010) at 152.8 ppm (C₃/C₅, etherified), 138.7 ppm (C₄, etherified), 134.8 ppm (C₁, etherified), 133.6 ppm (C₁, non-etherified), 106.9 ppm (C₂/C₆, S with α-CO), and 104.8 ppm (C₂/C₆) could also be seen. The signal from C₂/C₆ of p-hydroxyphenyl (H) was at 128.6 ppm (Wen et al. 2013a). Based on these signals, it could be concluded that the lignin of wheat straw was HGS type.

In the 90 to 50 ppm region, the main signals were from methoxyl (-OCH₃) at 56.5 ppm and β-O-4′, β–β‘, and β-5′ units. The signals assigned to β-O-4′ (Nimz et al. 1981) were at 85.7 ppm (C_β in S type β-O-4′ units) and 84.2 ppm (C_β in G type β-O-4′ units), 72.8 ppm (C_α, β-O-4′), 63.8 ppm (C_γ in G type β-O-4′ units with α-C=O), and 60.7 ppm (C_γ, β-O-4′). The signals at 84.3 ppm and 71.1ppm were from C_α and C_γ of β–β‘ (Sun et al. 1996). And the signal of C_α from β-5′ was at 86.7 ppm.

The intensities of some signals in ML-¹³C were stronger than those of ML. Examples include the signals at 101.8 ppm (C₁ of xylan), 75.4 ppm (C₄ of xylan), 74.0 ppm (C₃ of xylan), 73.4 ppm (C₂ of xylan), and 62.5 ppm (C₅ of xylan). Compared with soda-AQ lignin, there were differences of signals from C₁-C₅ of xylan at 101.4 ppm (C₁), 75.1 ppm (C₄), 73.7 ppm (C₃), 72.5 ppm (C₂), and 62.9 ppm (C₅) for soda-AQ lignin-¹³C. The increased signals indicated that xylose-¹³C₅ had been successfully introduced into wheat straw and synthesized to xylan. It was also found that the locations of signals of C₂, C₅ from xylan in ML-¹³C, and C₅ of xylan in soda-AQ lignin-¹³C were different from the purified xylan (Cheng 2011). This indicated that C₂ and C₅ of xylan were possibly linked with lignin by benzyl ether bonds or γ-ester bonds in wheat straw. As is well known, γ-ester linkages are unstable in the process of alkali treatment. Therefore, it was suggested that C₂ position of xylan in wheat straw could be mainly connected to lignin by γ-ester bonds, and the C₅ position of xylan in wheat straw was possibly linked with lignin by benzyl ether bonds.

In comparison to ML, some signals of the soda-AQ lignin and residual lignin were weakened. The signals at 72.8, 85.7, and 60.7 ppm from α, β, γ of β-O-4′ units were all weakened (Fig. 2). The chemical shifts between 103.6 and 163 ppm were set as 600, the corresponding values of α, β, γ of β-O-4′ units for ML, soda-AQ lignin, and residual lignin were then obtained as 17.08, 2.99, 29.30 (ML), 11.37, 0.34, 13.07 (soda-AQ lignin), and 6.04, 0.48, 3.71 (residual lignin), respectively. These data indicated that the degradation of β-O-4′ units were the main reaction during soda-AQ cooking process, with most of β-O-4′ units dissolving in black liquor, minor left in residual pulp. Meanwhile, the intentions of signals from C_α of β–β‘ units and β-5′ units were also decreased from 1.85, 16.61 (ML) to 0.44, 2.05 (soda-AQ lignin) and 0.50, 2.82 (residual lignin), indicating of the degradation of the two structures in the process of pulping.

The intensities of the signals at 10 to 34 ppm from the aliphatic side chain of the phenylpropane units were the strongest in the residual lignin. Aliphatic carbon signals may come from the degradation of β-aryl ether linkages or from the condensation reaction of methyl and methylene units. This result was in accordance with the analysis of FT-IR spectra.

Fig. 2. ¹³C-NMR spectra of lignin samples

Analysis of 2D-HSQC Spectra of Lignin Samples

The 2D-HSQC spectra of lignin samples were shown in Fig. 3. Main substruc-tures of lignins of wheat straw are shown in Fig. 4. The assignments of various signals are shown in Table 2. The main signals of the aliphatic side chains (δC/δH 50-90 /2.5- 6.0) were from -OCH₃ (δC/δH 55.6 /3.69) and β-O-4’ unit. The signal from C_γ-H_γ of the β-O-4’ structure of the lignin samples was approximately at δC/δH 60.6/3.41 (Rencoret et al. 2009). Acylated Cγ from β-O-4’ structures of ML and of residual lignin was at δC/δH 63.8/4.14. However, this signal was not observed in soda-AQ lignins. The C_α-H_α signal from β-O-4’ structures was at δC/δH 72.3/4.81 (Del et al. 2012a,) for all lignin, and the signals from C_β-H_β were at δC/δH 86.1/4.08 (β-O-4’ connected with a S unit) and δC/δH 84.3/4.29 (β-O-4’connected with a G unit). The C_α-H_α signal from β–β’ structures (Martinez et al. 2008) was at approximately δC/δH 84.3/4.64, while the signals of C_γ-H_γ were at δC/δH 71.4/4.17 and δC/δH 71.8/3.80. The signals from β-5’ structures were obvious to see in all lignin samples’ spectra. C_α-H_αof β-5’ was at δC/δH 87.1/5.45 (Yuan et al. 2013). The C_γ-H_γ signal (δC/δH 62.5/3.73) of β-5’ structures was overlapped with the signals of C₅-H₅ from xylan. The C_α-H_α signals from β-1’ structures were at δC/δH 81.8 /5.05 (Del et al. 2012b; Wen et al. 2013c) in the residual lignin-¹³C. Furthermore, the signals at δC/δH 10-30/0.5-1.5 of the residual lignin were from methyl and methylene groups. It can also be seen clearly that there were signals from C₁-C₅ of xylan in all lignins, which were located at δC/δH 101.8/4.28, δC/δH 72.8/3.06, δC/δH 74.0/3.27, δC/δH 75.4/3.52, δC/δH 62.7/3.41, respectively.

In the 2D-HSQC spectra of the lignin samples, the C_2/6-H_2/6 signal from S units (Yuan et al. 2011; Wen et al., 2013c) was at δC/δH 104.3/6.66, and the C_2/6-H_2/6 signal of C_α-oxidized S units was at δC/δH 106.4/7.22. C₂-H₂, C₅-H₅, and C₆-H₆ signals from G units were at δC/δH 111.4/6.94, δC/δH 116.0/6.73, and δC/δH 118.6/6.75, respectively (Del et al. 2012b). In the 2D-HSQC spectra of ML and soda-AQ lignin, the signal from C₂-H₂ of (FA₂) (Wen et al. 2013c) was found at δC/δH 111.7/7.31. This result was consistent with the results of FI-IR that the ferulic acid structure from wheat straw was dissolved in the black liquor in the process of pulping. The C_2,6-H_2,6 signal from H units was at δC/δH 127.5/7.02 and C_3,5-H_3,5 signal from H units was overlapped with the C₅-H₅ signal from G units. The relative content of H-type lignin in ML, soda-AQ lignin, and residual lignin were 15.79%, 10.42%, 5.81% respectively, indicating the degradation of H-type lignin during cooking process. Besides, the signals from p-coumaric acid (PCA_2,6), end groups of cinnamyl alcohol and end groups of cinnamaldehyde (J) were also found.

Conclusions from the 2D-HSQC spectra analysis results are as follows: the main substructures of lignin were H, G, and S units; and that the main lignin linkages were β-O-4’ units, β–β’ units, β-5’ units, and β-1’ units.

From the quantitative analysis of the ¹³C-NMR combination with 2D-HSQC, the absolute contents of β-O-4′ units, β-β’ units, β-5′ units and β-1′ units were obtained (Wen et al. 2013b). The four substructures contents for ML, soda-AQ lignin, and residual lignin were shown in Table 3. The results showed that the highest content substructure was β-O-4′ units, then was β-β’ units, with minor content of β-5′ units and β-1′ units. Furthermore, it was found that 82.8% of β-O-4’ units, 77.2% of β–β’ units, 75.4% of β-5’ units, and 75.4% of β-1’ units were degraded during the cooking process, and 15.8% of β-O-4’ units, 17.3% of β–β’ units, 22.8% of β-5′ units, and 18.0% of β-1’ units were left in the residual pulp.

Fig. 3 (top two). 2D HSQC NMR spectra of lignin samples (ML, Soda-AQ lignin, Residual lignin)

Fig. 3. 2D HSQC NMR spectra of lignin samples (ML, Soda-AQ lignin, Residual lignin)

From Table 3, it was found that the S/G ratio of the lignin samples were 1.28 (ML), 1.42 (Soda-AQ lignin), and 1.41 (Residual lignin).

The other method of calculating the S/G ratio was from analysis of ¹³C-NMR (Wen et al. 2013c). The chemical shifts between 103 and 160 ppm were set as 600, while the corresponding values of methoxyl (55-57 ppm) for the lignin samples were 457.29/600 (ML), 519.63/600 (soda-AQ lignin), and 407.23/600 (residual lignin), respectively.

The results from the two methods were coincident. The S/G ratio of soda-AQ lignin and residual lignin were higher than that of ML, indicating that it was easier to dissolve G. That was due to more methoxyl groups of the S-type lignin than G-type lignin, resulting in more stereospecific blockade.

Table 2. Assignments of the Lignin ¹³C-¹H Correlation Peaks in the 2D HSQC NMR Spectra of lignin samples (ML, Soda-AQ lignin, Residual lignin)

Table 3. Quantitative Characteristics of the Lignin Preparations from Quantitative NMR Method

Fig. 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) resinols; (C) phenylcoumarane; (D) spirodienones; (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；(S’) oxidized syringyl units with a Cα ketone

CONCLUSIONS

1. ¹³C abundance detection showed that ¹³C isotope-labeled xylose was incorporated into the wheat straw in vivo and took part in the plant’s normal metabolism.
2. The analysis results from FT-IR showed the lowest content of p-hydroxyphenyl units in residual lignin, indicating that it was the easiest for p-hydroxyphenyl units to dissolve in black liquor during cooking process.
3. By analyzing the ¹³C-NMR spectra of ML-¹³C and Soda-AQ lignin-¹³C, it was found that the C₂ position of xylan in wheat straw could be mainly connected to lignin by γ-ester bonds, and C₅ position of xylan in wheat straw was possibly linked with lignin by benzyl ether bonds.
4. From the analysis results from ¹³C-NMR combination with 2D-HSQC, it was concluded that the main sub-structures of lignin were H, G, and S units and that the main lignin linkages were β-O-4’, β–β’, β-5’, and β-1, with the highest content of β-O-4′. Furthermore, it was found that most of β-O-4’, β–β’, β-5’, and β-1 were dissolved in black liquor during cooking process, with minor left in residual pulp. From the results of S/G ration, it indicated of easier dissolving of G than S during cooking process.

ACKNOWLEDGMENTS

The authors are grateful for the support of the NSFC (No. 30901140 and 31070521), Key lab of pulp and paper science and technology of ministry of education of China (0806), and State Key Laboratory of Microbial Technology of Shandong University (No. M 2010-01 and M 2012-07).

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Article submitted: August 12, 2013; Peer review completed: October 1, 2013; Revised version received and accepted: November 1, 2013; Published: November 12, 2013.