Background

The stringent environmental and human health safety regulations and mounting raw material costs have prompted research into reducing the amount of harmful and/or expensive adhesive components and replacing synthetic adhesives with more environmentally-friendly and safer alternatives [1]. Recently, the authors introduced fully green nanotechnology as a gateway to beneficiation of natural cellulose fibers [2–5]. Thermal pretreatment has been conducted in moist environments using hot water or steam at temperatures up to 160 and 240°C, respectively, or in dry environments using inert gases at temperatures up to 240°C [6, 7]. Under these conditions, hemicelluloses are removed, crystallinity index of cellulose is increased, and cellulose degree of polymerization is reduced, while lignin is not considerably affected [7, 8]. It seems clear that a thermochemical pretreatment conducted above lignin melting temperature can cause lignin to coalescence, migrate, and redeposit on biomass cell walls matrix [9]. The softening point of lignin (spherical droplets) after streaming treatment is believed to be lower than that of the original material and this makes it possible for a plastic flow to occur in situ [10]. These lignin droplets were found by hot-water extraction or dilute acid pretreatment of lignocellulosic biomass [9, 11, 12]. Steam pretreatment can cause partial hydrolysis of hemicellulose for both hardwoods and softwoods which markedly increases the compressibility of wood and in turn significantly reduces the build-up of internal stresses in composites during hot pressing [13, 14]. This steam pretreatment process is a very effective method for producing dimensionally stable wood-based composites [13].

It has been proposed that the pressing temperature is one of the most important manufacturing parameters influencing the binderless boards’ properties [15–18]. Okuda et al. [15, 18] proposed that parts of lignin and hemicelluloses were decomposed during hot pressing process. Recently, the mechanism of self-bonding was summarized by Fahmy and Mobarak [2], who demonstrated that the chemical changes take place in two stages during hot pressing. The first stage is a hydrolysis stage, whereby acetic and formic acids and some sugars are liberated through the moisture found in the wood particles and then these acids hydrolyze the hemicelluloses in the lignocellulosic material. Some of the pentoses and hexoses produced during hydrolysis are further dehydrated to furfural and hydroxymethyl furfural, respectively. Simultaneously, the lignin becomes activated, i.e., a part of the lignin–carbohydrate bond becomes cleaved (cracked by the formed acids) resulting in the exposition of new functional groups or sites. The second stage is the recondensation reaction of the activated lignin molecules as well as lignin degradation products (such as phenol, which might be formed at high temperature above 155°C) with furfural. However, the self-bonding mechanism during hydrothermal treatment and hot pressing has not been completely elucidated so far.

Traditionally, milled wood lignin (MWL) is considered to be a representative source of native lignin and has been extensively used for the elucidation of native lignin structure. This is currently the most common procedure for the isolation of lignin from wood. However, the yield of MWL is relatively low as revealed by Björkman [19]. Furthermore, lignin yield is dependent on milling time. Nevertheless, longer milling time leads to chemical modification of the lignin, such as increase in carbonyl content and phenolic hydroxyl content as well as decrease in molecular weight and cleavage of aryl ether linkages [20]. Whiting and Goring concluded that MWL is not representative of the whole lignin in wood but primarily originates in the secondary wall of the cell according to the structural studies of dioxane-extracted lignin from compound middle lamella and secondary wall of spruce [21, 22]. To overcome these limitations, a preparative method combining physical and chemical treatments for lignin isolation and purification, such as milling and enzymatic attack, was recently introduced. Chang et al. [23] isolated lignin by extracting MWL first, and subsequently the residue was treated with cellulolytic enzymes followed by 96% aqueous dioxane extraction to isolate cellulolytic enzyme lignin (CEL). CEL has higher molecular weights and β-O-4′ inter-linkages, but a lower lignin condensation compared to MWL. The higher yield of CEL extracted from wood helps us understand the lignin structure as a whole. CEL is preferred over MWL for studies of lignin structure as it can be isolated in higher yield with less degradation due to ball milling [23, 24].

The objective of this study was to unravel the structural characteristics of lignins of Eucalyptus wood and their behaviors during hydrothermal pretreatment and hot pressing in the production of self-bonding binderless. Because polysaccharides and lignin strongly contribute to self-bonding. This would be also important to understand the differences in mechanical properties of boards from hydrothermal pretreated fibers prepared under various conditions. Moreover, an exhaustive chromatographic and spectroscopic characterization of the isolated CELs was provided as a preliminary investigation.

Results

Chemical composition

The chemical compositions of the untreated and pretreated E. grandis as well as the corresponding binderless boards are presented in Table 1. The untreated Eucalyptus feedstock had a typical composition of hardwood with 43.4% cellulose (determined as glucan), 22.3% hemicelluloses (determined as galactan, arabian, rhamnan, and glucuronic acid), and 31.7% lignin (determined as Klason lignin and acid-soluble lignin). These results were comparable with those found in woods from different eucalypt species [25]. The solid yield was slightly affected by the treatment severity and varied from 86.9% to 96.6%. This was due to the solubilization of amorphous cellulose and the degradation of hemicelluloses or fragile pentoses and hexoses during hydrothermal pretreatment. On average, the Klason lignin contents in the spent solids varied in the range 29.3 – 33.2%, and increased up to 16% of the Klason lignin of the control under harsh conditions. It is worth noting that the Klason lignin content in the binderless boards showed a significant decrease from 33.2% to 28.0% as depicted in Table 1. The reduction of lignin content of the Eucalyptus wood after hot pressing resulted in a decrease in the glass transition temperature. It is believed that the softening point of lignin after hydrothermal pretreatment decreased and a possible plastic flow in situ may occur, making the lignin more accessible to the fiber surface [10, 13]. Thus the thermal softening of lignin plays an important role in the self-bonding mechanism. Other studies have suggested the possibility of the contribution of the cleavage of inter-monomer linkages in lignin, such as β aryl ether bonds, thus decreasing its molecular weight during the hot pressing process [15, 18]. Simultaneously, an increase in glucan content was observed in binderless panels as compared to the pretreated Eucalyptus wood.

Table 1 Solid yield and chemical composition of untreated Eucalyptus globule , hydrothermal pretreated fibers, and manufactured binderless boards from Eucalyptus grandis
Full size table

Mechanical properties of the binderless boards

Although the primary effect of the study was not to improve the mechanical properties of binderless boards, the effect of hot pressing on the mechanical properties was also investigated. It should be noted that the control panel had so poor mechanical properties that the internal bond (IB) could not be tested. It can be seen from Figure 1 that most of the samples obtained in the present study meet the minimum of requirements of Japanese Industrial Standard A 5908, Type-8, (0.15 MPa) [26] but still lower than the minimum requirements of Chinese Industrial Standard GB/T 11718–2009, (0.40 MPa) [27]. Table 2 shows a summary of the obtained IB values of binderless boards obtained by hydrothermal pretreatment of different lignocellulosic materials at various process conditions. Compared to the summarized results in the previous data in Table 2, in general, the binderless boards in this work demonstrated a comparative promising result. Future work will be conducted to improve the mechanical properties of binderless boards to higher values and thus provide better insights into the understanding of the mechanism during hot pressing.

Figure 1
figure 1

Average internal bond values of the binderless boards manufactured from Eucalyptus globulu .

Full size image
Table 2 Binderless boards prepared from hydrothermal pretreated fibers by various process conditions and the obtained internal bond (IB)
Full size table

Generally, it was found that the higher IB of binderless board was obtained under harsh pretreatment severities. This high IB value made from the hydrothermal pretreated E. grandis could be explained based on intermolecular hydrogen bonding between the cellulose and lignin molecule [28]. In accordance with this, a board absorption band appearing in the 3312 – 3344 cm−1 region in the hydrothermal pretreated fibers and binderless panels indicated OH stretching in the Fourier-transform infrared (FT-IR) spectra in the Supporting information of Additional file 1: Figure S1 and the data shown in Table 3.

Table 3 Hydrogen bond intensity and cellulose crystallinity ratios
Full size table

Structural changes during hydrothermal pretreatment and hot pressing

The hydrogen bond intensity (HBI) of cellulose is closely related to the crystal system and the degree of intermolecular regularity, that is, crystallinity, as well as the amount of bound water [29]. The ratio of the absorbance bands at 3400 and 1323 cm−1 was used to study the cellulose samples HBI [29, 30]. As quantitative indices for evaluation of the overall crystallinity of cellulose, the lateral order index (LOI) and total crystallinity index (TCI) were determined based on FT-IR spectra. Higher values of TCI and LOI indicate that the materials have a higher crystallinity and more ordered structure of cellulose [31, 32]. Low LOI values are indicative of less cellulose type I present [32]. The results obtained are shown in Table 3.

It can be seen from Table 3 that the hydrothermal pretreated fibers and manufactured binderless boards from E. grandis showed higher numbers of HBI, TCI, and LOI than those of the control. This reveals that the cellulose of Eucalyptus wood has a highly ordered crystalline structure and the crystallinity of cellulose increases due to degradation of the amorphous cellulose, which provides a great stability to the cellulose chains and protects them against acid attack during hydrolysis. This finding was in consistent with the results reported in the literature [33]. However, this pretreatment process was unable to completely break apart the inter- and intra- chain hydrogen bondings [34]. The cellulose crystallinity index (CrI) values were also calculated from the corresponding X-ray diffraction (XRD) patterns (Additional file 1: Figure S2) and solid-state cross polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) patterns (Additional file 1: Figure S3), and the data are listed in Table 3. As the data shown, they were consistent with the conclusion from the FT-IR analysis, and further confirmed that hydrothermal pretreatment and hot pressing can change cellulose crystalline structures by disrupting inter/intra hydrogen bonding of cellulose chains.

FT-IR spectra analysis of the isolated lignins

The FT-IR spectra of the three CELs are presented in Figure 2, and the IR absorption assignments are based on the literature values [35–38]. It can be seen that the characteristic bands of lignin skeleton and functions of these lignins are very similar. All spectra present typical lignin patterns, including bands of aromatic ring vibrations at 1593 cm−1, 1504 cm−1, and 1423 cm−1. Other bands are assigned to ring breathing of syringyl (S) (1326 cm−1) and guaiacyl (G) units (1265 cm−1 shoulder), aromatic in-plane and bending in S (1122 cm−1) and G units (1029 cm−1), and out-of-plane C-H bending of G (918 cm−1) and S units (829 cm−1). Moreover, the bands at 1721 cm−1 and 1655 cm−1 correspond to the stretching of non-conjugated and conjugated carbonyls, respectively. No significant difference was detected between the lignin fractions, suggesting little effect of the hydrothermal treatment and hot pressing on the distribution of functional groups in the lignins.

Figure 2
figure 2

FT-IR spectra of the CEL samples.

Full size image

2D HSQC NMR of the isolated lignins

The two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR spectra of the isolated CELs were well resolved as shown in Figure 3. The signals assignments are based on the literature and summarized in Table 4 [39–41], and the main lignin structures identified are shown in Figure 4. The spectra showed mainly lignin moieties, whereas minor polysaccharides, such as hemicelluloses, were present. This is anticipated as most of these carbohydrates were digested due to the enzymatic hydrolysis. In the side region of the HSQC NMR spectra, the cross-signals of methoxyl groups and side-chains in β-O-4′ substructures in lignin were the most prominent ones. The C-H correlations in substructures (A) were well resolved for Cγ-Hγ, Cα-Hα, and Cβ-Hβ (A γ , A α , and A β , respectively). The Cβ-Hβ correlations gave two different signals corresponding to β-O-4′ subunit which linked to G and S units (A β(G) and A β(S) , respectively). The presence of resinol (β-β’) substructures (B) was evidenced by C/H correlations for α-, β-, and γ-C positions centered at δC/δH 85.1/4.66, 53.4/3.06, and 71.7/3.81 and 4.18 ppm, respectively. Lignin phenylcoumaran (β-5′) substructure (C) was also confirmed by its C-H correlations at around 86.8/5.45 (Cα-Hα), 53.1/3.47 (Cβ-Hβ), and 63.2/3.70 (Cγ-Hγ) ppm. Finally, small signals corresponding to spirodienone Cγ-Hγ in p-hydroxycinnamyl alcohol end groups (F) could be also found in the spectra, its Cβ-Hβ correlations being at 61.3/4.09 ppm.

Figure 3
figure 3

Expanded aliphatic oxygenated (δ C –δ H 2.5–6.0 and 50 – 90 ppm; top) and aromatic (δ C –δ H 6.0 – 7.8 and 95 – 135 ppm; bottom) regions of the HSQC NMR spectra of the CEL samples. The spectra are aligned to represent each sample treatments as follows: (a and d) Sample control; (b and e) hot-compressed water pretreated of sample F4-a; (c and f) hot-compressed water pretreated followed by hot pressing of sample F4-b.

Full size image
Table 4 Assignments of main lignin and polysaccharide 13 C- 1 H correlation signals in the HSQC spectra the lignin fractions
Full size table
Figure 4
figure 4

Main substructures present. A, β-aryl ether structure formed by β-O-4′ linkages; B, resinol including β-β’ linkages; C, phenylcoumaran including β-5′ linkages; F, p-hydroxycinnamyl alcohol end groups; G, Guaiacyl unit; S, Syringyl unit; S’, Oxidized syringyl units bearing a carboxyl group at Cα.

Full size image

The main cross-signals observed in the aromatic region of the HSQC NMR spectra belong to the aromatic rings of lignin monomers. Correlations from S and G lignin units could be observed in the spectra of the CELs. Strong C2,6-H2,6 correlations for syringyl (S 2,6 ) and Cα-oxidized S-lignin units (S’) were observed at 103.9/6.69 ppm and 106.2/7.19 ppm, respectively. For guaiacyl (G) units, G 2 correlation was observed at 111.1/6.96 ppm and correlations for G 5 /G 6 were observed at 115.0/6.64 and 6.91 ppm, and 119.0/6.82 ppm.

Summary of changes in lignin structure as revealed by 2D HSQC NMR

The relative abundances of the main lignin inter-unit linkages and the S/G ratio, calculated from the HSQC spectra of the CELs, are shown in Table 5. All the lignins showed a predominance of β-O-4′ units (A; 79 – 81% of the total side chains) followed by β-β’ resinol type units (B; 16 – 17%). The β-5′ phenylcoumaran type lignin unit (C α ) ranged from 3.2% to 3.6%. The slight decrease amount of β-O-4′ was anticipated. Under high temperature and pressure water causes an auto-catalyzed cleavage of glycosidic bond in hemicelluloses and lignin-hemicellulose linkages, thus producing acetic acid from the acetyl groups. Therefore, the acidic environment during hydrothermal processes probably resulted in the partial hydrolysis of ether linkages [42, 43]. On the other hand, the relative content of C-C linkages (such as β-β’ linkages) increased, demonstrating that the lignin became more condensed.

Table 5 Lignin structural characteristics from integration of 13 C- 1 H correlation signals in the HSQC spectra the lignin fractions
Full size table

It was found that the S/G ratio and the signal of Cα-oxidized S-lignin units (S’) increased. The CEL of the untreated sample E. grandis which had a proportion of S/G ratio at around 1.7 (Table 5), became a S-rich lignin after hydrothermal pretreatment and hot pressing. The result is consistent with our recent work in which we observed that the S/G ratio of shrub wood lignin, analyzed by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and 2D HSQC NMR, increased with the increment of hydrothermal pretreatment severity [44]. Similar results have been reported by Okuda et al. [15] who found that the S/V ratio increased with increasing pressing temperature. This result could have been due to the preferential degradation of low molecular weight of no-condensed G-type lignin during the treatment. Higher thermal stability of the β-aryl ether linkages of syringyl present as compared to guaiacyl-type lignin in hardwood make the later one lost during hydrothermal pretreatment and hot pressing [1]. Thus a more pronounced decrease in G units compared to S units likely accounted for the increased S/G ratio of lignin from 1.7 (Control) to 2.4 (F4-b). Moreover, a recent work has demonstrated that the presence of these oxidized S’ units in eucalypt wood might be an artifact of the milling process [45, 46]. However, all the samples in the present study were planetary ball milled under the same conditions, demonstrating that the variation was due to the differences in the composition of the biomass. After electron transfer and aryl cation radical formation, aromatic ring oxidation has been reported in model degradation by laccase-HBT, whereas the Cα attack followed by alkyl-aryl ether breakdown predominates, thus resulted in an increase in the amount of Cα-oxidized S-lignin [47, 48]. The proposed mechanism was through the Baeyer-Villiger reaction which may be similar to the mechanism of hot pressing in the present study. Furthermore, this can be rationalized due to the basis that non-phenolic lignin compounds was oxidized and cleaved during hot pressing process and that the action of hot pressing couple proceeds via Cα-hydrogen atom abstraction [49].

31P NMR spectra of the lignin polymers

To further investigate the structural changes of lignin during hydrothermal pretreatment and hot pressing process, quantitative 31P NMR analysis was performed using 2–chloro–4,4,5,5–tetramethyl–1,3,2–dioxaphospholane (TMDP) as phosphorylation reagent [50]. Data from the quantitative 31P NMR spectra of the CELs samples are presented in Table 6 and Figure 5. The concentration of each hydroxyl functional (in mmol/g) was calculated on the basis of the hydrolysis content of the internal standard and its integrated peak area.

Table 6 Concentrations of functional groups and molecular weight distributions of the lignin fractions
Full size table
Figure 5
figure 5

Quantitative 31 P NMR of the CEL samples.

Full size image

It can be seen from Table 6, a slight increase in aliphatic OH concentration was observed after hydrothermal pretreatment. This was because higher pretreatment severity led to greater reductions in β-O-4 structures, resulting increase in a higher amount of free phenolic groups [51]. Moreover, a post-treatment with hot pressing process was accompanied by a slight decrease in the aliphatic OH but an increase of S-OH. This was most likely as a result of acid-catalyzed elimination reactions thus led to a cleavage of β-O-4 linakges [52]. The fact that the more syringyl units are produced during hydrothermal treatment, such as steam explosion treatment, most likely as a result of scission of β-O-4 bonds, has already been supported by Granata and Argyropoulos [50]. In addition, loss of carboxylic hydroxyl group can be observed in the resulting CELs obtained from the hydrothermal treatment and hot pressing.

Molecular weight distribution of the isolated lignins

To investigate the effect of hydrothermal and hot pressing treatment process on the molecular weights of lignin fractions, the gel permeation chromatography (GPC) elution curves of the three CEL samples were recorded as depicted in Figure 6 and the dada are summarized in Table 6. It was observed that CEL-F4-a had a higher Mw than that of the control. This observation confirmed that lignin repolymerization occurred during hydrothermal pretreatment, which was due to acid-catalyzed condensation between the aromatic C6 or C5 and a carbonium ion at a higher severity [43, 53]. In this regard, during the hydrothermal pretreatment of Eucalyptus wood, acid-catalyzed cleavage of β-O-4′ linkages and ester bonds were the major mechanisms of lignin cleavage. After hot pressing process, the Mw was decreased to 16600 g/mol, which was slightly lower than that of the control. This suggested that hot pressing process degraded the lignin macromolecular polymer in the hydrothermal pretreated fibers to a noticeable extent, which confirmed the aforementioned reduction contents of β-O-4′ linkages as determined by 2D HSQC and 31P NMR.

Figure 6
figure 6

Molecular weight distributions of the CEL samples.

Full size image

Conclusions

The result showed that during the hydrothermal pretreatment of Eucalyptus wood, acid-catalyzed cleavage of β-O-4′ linkages and ester bonds were the major mechanisms of lignin cleavage. This degradation pathway led to a more condensed lignin that has a high average molecular weight and more phenolic hydroxyl groups than those of the control. The hot pressing process resulted in the binderless boards with reduced lignin contents and decreased the glass transition temperature, thus making the lignin more accessible to the fiber surface. CEL isolated from the binderless boards showed an increased syringyl to guaiacyl propane (S/G) ratio but a lower molecular weight than that of the untreated Eucalyptus wood and the hydrothermal pretreated fibers. Moreover, Eucalyptus wood has the potential to be used to manufacture binderless boards. Based on the finding of this study, it is suggested that the combination of hydrothermal pretreatment and hot pressing process is a good way for conditioning hardwood sawdust for the production of binderless boards. The thermal softening of lignin, rich in phenolic hydroxyl groups, and increased condensed lignin structure contributed to the self-bonding formation of lignocellulosic materials. Future work will be conducted to optimize the pretreatment conditions and improve the performance of binderless board to higher values and commercial uses.

Methods

Materials

Eucalyptus grandis x E. urophylla, five years old, was manually collected from Hezhou City, Guanxi province, China. The dried Eucalyptus stems were ground to pass a 40-mesh screen. All chemical reagents were analytical grade or best available. The acid xylanase and cellulase with declared activities of 100,000 international units (IU) per gram of protein and 145 filter paper units (FPU)/g were kindly provided by Shanghai Youtell Biochemical Co., Ltd. (Shanghai China).

Hydrothermal pretreatment of Eucalyptuswood

The wood meals of Eucalyptus (100 g dry basis per batch) were added into a batch reactor (1 litter volume). For hot water extraction, the sample was treated under desired temperature 150°C and digestion periods (1, 4, and 8 h). For steam pretreatment, the sample was treated under desired temperature 160°C and digestion periods (1.5, 3, 4.5, and 6 h). Defibrillated fibers were collected to prepare binderless boards and denoted as F1-a, F4-a, F8-a, Q1-a, Q2-a, Q3-a, and Q4-a, respectively.

Binderless board preparation and mechanical characterization of the boards

Each air-dried sample of powders was hand-formed into homogeneous single-layered mats using a forming box (50 × 50 cm2) and pressed at a pressure of 15 MPa at 160°C for 20 min, by adjusting the target board thickness to 5.0 mm and the density to 1.0 g/cm3. Two boards were prepared for each powder. The hydrothermal pretreated Eucalyptus fibers were dried to 10% moisture content under air-dried condition. The binderless boards were prepared on a laboratory sale by the standard techniques under controlled conditions. The manufactured binderless boards from the defibrillated fibers were denoted as F1-b, F4-b, F8-b, Q1-b, Q2-b, Q3-b, and Q4-b, respectively.

The properties of the binderless boards were evaluated in accordance with China National Standard for fiberboards [27, 54]. The five specimens of 25.4 × 25.4 × 1.25 cm3 were prepared. The internal bonding strength (IB) test, regarded as the index of self-bonding, was carried out by using a universal testing machine.

Isolation of CEL

To investigate the original lignin polymers of the untreated and pretreated Eucalyptus wood as well as the binderless board, cellulolytic enzyme lignin (CEL) were isolated according to the procedure developed by Chang et al. [23] and modified by Hu et al. [24]. Specifically, ball milled wood (5 g) was suspended in citrate buffer (250 mL, pH 4.8) and incubated for 24 h at 50°C. The cellulase and acid xylanase were used at a loading of 50 FPU/g substrate and 200 IU/g substrate, respectively, to hydrolyze the cellulose and hemicelluloses. The enzyme treatment was repeated twice. The residue was collected by centrifugation, washed with hot distilled water (200 mL), centrifuged, and freeze-dried. Then the freeze-dried residue was extracted twice (2 × 24 h) with 100 mL of dioxane/water (96:4, v/v) under a nitrogen atmosphere. After each extraction, the supernatant was collected, combined and poured into a 250 mL volumetric flask. Subsequently, the solution was concentrated, precipitated in water, and freeze-dried to produce the CEL.

Physicochemical characterization of the Eucalyptuswood, hydrothermal pretreated fibers, and binderless boards

The chemical compositions of the extractive-free Eucalyptus wood, hydrothermal pretreated fibers and binderless boards were determined according to National Renewable Energy Laboratory Analytical Procedures [55]. FT-IR spectra were acquired using a Thermo Scientific Nicolet iN10 FT-IR Microscope as previously described [44]. The crystallinities of the samples were measured using a XRD-6000 instrument (Shimadzu, Japan) [11]. CP/MAS 13C NMR spectra of the different samples were obtained using a Bruker AVIII 400 M spectrometer (Germany) [11].

Characterization of the lignin polymers

FT-IR spectra of the CELs were performed the same as raw material. The weight average molecular weight (Mw) and number average molecular weight (Mn) of the CELs were performed with GPC after acetylation according to previous publications [56, 57]. Briefly, about 20 mg of dry lignin was dissolved in a 1:1 mixture of acetic anhydride/pyridine (1.00 mL) and stirred at room temperature for 24 h. Ethanol (25.0 mL) was added to the reaction mixture, left for 30 min, and removed with a rotary evaporator. The addition and removal of ethanol was repeated until all traces of acetic acid were removed from the sample. The residue was dissolved in chloroform (2.0 mL) and drop-wise to diethyl ether (100.0 mL) followed by centrifugation. The precipitate was washed three times with diethyl ether and dried under vacuum prior to GPC analysis. The acetylated lignin was dissolved in tetrahydrofuran (THF) (2 mg/mL), and the solution was filtered through a 0.45 μm filter. The filtered solution (20.0 μL) was injected into the HPLC system and detected using an UV detector set at 240 nm. THF was used as the mobile phase and the flow rate was 0.5 mL/min. Standard narrow polystyrene samples were used for calibration, as described previously [44].

For 2D HSQC spectra, the Bruker standard pulse program hsqcetgpsi was used for HSQC experiments as previously described [44]. 31P NMR spectra were acquired according to a previous paper with minor modifications [50]. A total of 20 mg lignin was dissolved in 0.5 mL of anhydrous pyridine and deuterated chloroform (1.6:1, v/v) under stirring. This was followed by the addition of 0.1 μL of cyclohexanol (10.85 mg/mL) as an internal standard, and 0.1 μL of chromium(III) acetylacetonate solution (5 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v), as relaxation reagents. Finally, the mixture was reacted with 0.1 μL of phosphitylating reagent (2-chloro-1,3,2-dioxaphospholane) for 15 min and then was transferred into a 5 mm NMR tube for subsequent NMR analysis. Acquisition conditions were as described previously [50]. The content of hydroxyl groups in lignin was obtained by integration of the following spectral regions as described by Cateto et al. [58] and Monteil-Rivera et al. [59] with miner modification: aliphatic hydroxyls (149.0 – 146.0 ppm), syringyl (S) phenolic hydroxyls (144.5 – 143.2 ppm), condensed phenolic units (difference between the integrals of the peaks at 143.2 – 142.2 ppm and those at 142.2 – 141.4 ppm, guaiacyl (G) phenolic hydroxyls (140.2 – 138.7 ppm), p-hydroxyphenyl (H) phenolic hydroxyls (138.2 – 137.1 ppm), and carboxylic acids (135.5 – 134.2 ppm).