Impact of Steam Explosion on the Wheat Straw Lignin Structure

Article pubs.acs.org/JAFC

Impact of Steam Explosion on the Wheat Straw Lignin Structure Studied by Solution-State Nuclear Magnetic Resonance and Density Functional Methods Harri Heikkinen,*,† Thomas Elder,‡ Hannu Maaheimo,† Stella Rovio,† Jenni Rahikainen,† Kristiina Kruus,† and Tarja Tamminen† †

VTT Technical Research Centre of Finland, Biologinkuja 7, Espoo, FI-02044 VTT, Finland United States Department of Agriculture (USDA) Forest Service Southern Research Station, 2500 Shreveport Highway, Pineville, Louisiana 71360, United States

‡

ABSTRACT: Chemical changes of lignin induced by the steam explosion (SE) process were elucidated. Wheat straw was studied as the raw material, and lignins were isolated by the enzymatic mild acidolysis lignin (EMAL) procedure before and after the SE treatment for analyses mainly by two-dimensional (2D) [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] and 31P nuclear magnetic resonance (NMR). The β-O-4 structures were found to be homolytically cleaved, followed by recoupling to β-5 linkages. The homolytic cleavage/recoupling reactions were also studied by computational methods, which verified their thermodynamic feasibility. The presence of the tricin bound to wheat straw lignin was confirmed, and it was shown to participate in lignin reactions during the SE treatment. The preferred homolytic β-O-4 cleavage reaction was calculated to follow bond dissociation energies: G−O−G (guaiacyl) (69.7 kcal/mol) > G−O−S (syringyl) (68.4 kcal/mol) > G−O−T (tricin) (67.0 kcal/mol). KEYWORDS: wheat straw, lignin, 2D NMR, 31P NMR, steam explosion, tricin

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INTRODUCTION In contrast to other wood components, lignin is by far the most complex heterogeneous polymer. The chemical structure of lignin is composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units originating from p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, respectively. Their proportions vary depending upon the plant origin, with softwood lignin mainly composed of G units, together with a small amount of H units, whereas both G and S units are abundant in hardwood. In grass lignins, e.g., in wheat straw, H units are also present.1 The most abundant interunit linkages are β-O-4′ (aryl ether), β-5′ (phenylcoumaran), β-β′ (pinoresinol), β-1′ (spirodienone), 5-5′-O-4 (dibenzodioxocin), and 4-O-5′.2 Recently, α,β-diarylether structures have been detected in wheat straw lignin.3 Covalently bound heteropolysaccharides may be present in isolated lignin samples, as residues of the original lignin− carbohydrate complexes (LCC) in the secondary plant cell wall. These carbohydrates may be bound to lignin through phenyl glycoside, ether, or ester linkages.4 Their possible presence needs to be taken into account when characterizing the lignin heterogeneous structure. Nuclear magnetic resonance (NMR) spectroscopy is an effective analytical tool for directly characterizing the heterogeneous structure of lignin. In particular, two-dimensional (2D) 1 H−13C NMR experiments [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] allow for the detection of different lignin units and interunit linkages, with the exception of the 4-O-5′ subunit.5 NMR is also the only analytical method available for unambiguously detecting dibenzodioxocin and spirodienone structures.6−8 Even though several other methods exist [e.g., pyrolysis−gas © 2014 American Chemical Society

chromatography/mass spectrometry (GC/MS), permanganate and nitrobenzene oxidation, derivatization followed by reductive cleavage (DFRC), thioacidolysis] to reveal H/G/S composition of lignin polymers, they all rely on the backbone cleavage of lignin, and hence, calculations are based on fragments produced therein.9,10 Therefore, quantitative 1H−13C HSQC 2D NMR has become a rather powerful and widely used tool for lignin structure elucidation.11−13 Various pretreatment methods can be used in modern biorefineries, in which the biomass components are used as raw material for value-added biomaterials and chemicals. Steam explosion (SE) is the most traditional and established pretreatment method, enhancing the reactivity of lignocellulosic biomass toward enzymatic hydrolysis for the production of fermentable sugars.14 In SE, biomass is heated with high-pressure steam, followed by rapid decompression, which disrupts the hemicellulose−lignin and cellulose networks, so that mainly the hemicelluloses are solubilized and the lignin and cellulose are largely left as solid material. Steam explosion is also an autohydrolysis process, catalyzed by the organic acids released from the biomass components, especially via hydrolysis of acetyl groups in hemicelluloses.15 Cellulases, used in the enzymatic hydrolysis of biomass, tend to adsorb on the lignin surfaces, thus making the lignin act as an inhibitory agent, retarding the hydrolysis process. A recent study showed that SE pretreated lignins from spruce and wheat Received: Revised: Accepted: Published: 10437

May 23, October October October

2014 7, 2014 7, 2014 7, 2014

dx.doi.org/10.1021/jf504622j | J. Agric. Food Chem. 2014, 62, 10437−10444

Journal of Agricultural and Food Chemistry

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Figure 1. (A) Side-chain region (δC/δH, 50−90/2.5−5.7 ppm) on contour-plotted 1H−13C HSQC NMR spectrum of wheat straw EMAL, (B) aromatic/unsaturated region (δC/δH, 88−160/6.05−7.65 ppm) on contour-plotted 1H−13C HSQC NMR spectrum of wheat straw EMAL, (C) sidechain region (δC/δH, 50−90/2.5−5.7 ppm) on contour-plotted 1H−13C HSQC NMR spectrum of SE-treated wheat straw EMAL, and (D) aromatic/unsaturated region (δC/δH, 88−160/6.05−7.65 ppm) on contour-plotted 1H−13C HSQC NMR spectrum of SE-treated wheat straw EMAL.

coupling reactions.17−19 Dependent upon the SE conditions, the formation of higher molecular weight lignins has been observed, explained by competing depolymerization and repolymerization reactions.19 The aim of the present study was to elucidate the chemical changes within the enzymatic mild acidolysis lignin of wheat straw structure, caused by the SE process. NMR techniques using state-of-the-art pulse variations [quantitative 2D 1H−13C HSQC, 2D 1H−13C HMBC, and one-dimensional (1D) 31P NMR]

straw bound more of the hydrolytic enzymes (Trichoderma reesei Cel7A and Cel7A-core) compared to the non-pretreated analogues.16 This indicates that chemical changes that are detrimental to the saccharification process take place within the lignin structure during the SE process. Several studies regarding the impact of SE on the lignin structure have been reported. The major effects are the decrease in the level of β-O-4′ units and an increase in the level of C−C condensed structures and other interunit linkages (β-1′ and β-5′) formed via radical 10438



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Figure 2. Main lignin structures detected: (A) β-O-4′, (Aacyl) β-O-4′ alkyl-aryl ethers with acylated γ-OH, (Aox) Cα-oxidized β-O-4′ structure, (B) phenylcoumaran (β-5′), (C) resinol β-β′, (D) dibenzodioxocin (5-5′-O-4′), (E) α,β-diarylether, (F) spirodienone (β-1), (I) cinnamyl alcohol, (J) cinnamyl aldehyde, (PCA) p-coumarate, (FA) ferulate, (H) p-hydroxyphenyl unit, (G) guaiacyl unit, (S) syringyl unit, and (S′) Cα-oxidized S unit. the treatment was terminated by fast release of pressure. Isolation of EMAL lignin from non-treated and SE treated wheat straw involved the following four main steps: extraction of acetone-soluble material, ball milling, enzymatic hydrolysis of cell wall carbohydrates, and lignin extraction with a mildly acidic dioxane/water mixture. The lignin isolation procedure is explained in more detail in the literature.16 4′,5,7Trihydroxy-3′,5′-dimethoxyflavone (tricin) was purchased from SelectLab Chemicals GmbH (Bönen, Germany) and used as received. 2D 1H−13C HSQC and HMBC NMR Measurements. For the 2D 1 H−13C HSQC and HMBC experiments, 50 mg of the lignin sample was dissolved in 1 mL of dimethyl sulfoxide (DMSO)-d6, and the experiments were carried out at 30 °C on a 600 MHz Bruker Avance III NMR spectrometer equipped with a CPQCI cryoprobe. The quantitative HSQC spectra were acquired using echo/anti-echo time proportional phase incrementation (TPPI) selection, and matched sweep adiabatic pulses optimized for 13C sweep width of 200−50 ppm were used for all 180° 13C pulses to compensate for the differences in the 1JCH coupling constants.22 The HMBC experiments were optimized for 8 Hz (62.5 ms) long-range couplings. Matrices of 2048 × 256 (HSQC) or 2048 × 512 (HMBC) data points were collected and

have been applied for the structural characterization of the lignins. Density functional theory calculations have been used to support the proposed routes in which coupling reactions are followed by homolytic cleavage of the β-O-4′ linkages. The structure of lignin in wheat straw has been elucidated previously. Also, the incorporation of tricin in the lignin via β-O-4 in G units has been reported.3 In this study, quantitative data about the removal of tricin during SE were obtained by a novel 31 P NMR method. Homolytic cleavage of β-O-4 substructures in the lignin during SE was also elucidated using theoretical calculations, and a comparison was carried out using model systems (G−O−G, G−O−S, and G−O−T).

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MATERIALS AND METHODS

Lignins and Lignin Model Compounds. Lignins were isolated from raw wheat straw (MTT Agrifood Research, Jokioinen, Finland) before and after SE treatment, employing the enzymatic mild acidolysis lignin (EMAL) isolation method.20,21 The milled wheat straw sample was pretreated with steam at 200 °C using a 10 min residence time, and 10439



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Table 1. Assignment of Selected 13C/1H Chemical Shifts Observed in HSQC NMR Spectra of Wheat Straw Lignina δC/δH (ppm) 52.9/3.43 53.4/3.03 55.4/3.71 59.4/3.7 and 59.7/3.6 61.3/4.07 62.7/3.77 63.9/4.15 and 4.21 70.9/4.71 70.9/3.8 and 4.1 71.6/4.83 79.1/5.54 80.7/4.50 82.5/4.87 82.6/5.11 82.9/4.45 83.8/4.26 84.6/4.62 84.8/7.46 85.4/3.84 85.9/4.09 86.7/5.42 94.1/6.56 98.7/6.21 103.7/6.67 103.9/7.29 104.5/7.02 106.3/7.30 110.8/6.96 113.6/6.25 114.4/6.67 and 115.4/6.76 118.8/6.75 123.5/7.18 126.2/6.74 127.7/7.16 128.4/6.20 128.4/6.42 130.0/7.44 144.9/7.56 153.3/7.58 191.1/9.78 194.1/9.57 a

Table 2. Amount of Main Interunit Linkages and Structural Characteristics Detected in Wheat Straw before (EMAL) and after the SE Treatment from the Integration of 1H−13C Correlation Signals in HSQC NMR Spectra

assignment Cβ−Hβ in phenylcoumaran substructures (Bβ) Cβ−Hβ in β-β′ resinol substructures (Cβ) C−H in metoxyl group (−OCH3) Cγ−Hγ in β-O-4′ substructures (Aγ) Cγ−Hγ in cinnamyl alcohol end groups (Iγ) Cγ−Hγ in phenylcoumaran substructures (Bγ) Cγ−Hγ in γ-acylated β-O-4′ substructures (Aacyl) Cα−Hα in β-O-4′ linked to a G unit (Aα(G)) Cγ−Hγ in β-β′ resinol substructures (Cγ) Cα−Hα in β-O-4′ linked to a S unit (Aα(S)) Cα−Hα in α-O-4′-linked substructures (Eα) Cβ−Hβ in γ-acylated β-O-4′ structures linked to a G unit (Aacylβ) Cα−Hα in dibenzodioxocin substructures (Dα) Cβ−Hβ in α-oxidized β-O-4′ substructures (Aoxβ) Cβ−Hβ in β-O-4′ linked to a H unit (Aβ(H)) Cβ−Hβ in β-O-4′ linked to a G unit (Aβ(G)) Cα−Hα in β-β′ resinol substructures (Cα) Cα′−Hα′ in spirodienone substructures (Fα′) Cβ−Hβ in dibenzodioxocin substructures (Dβ) Cβ−Hβ in β-O-4′ linked to a S unit (Aβ(S)) Cα−Hα in phenylcoumaran substructures (Bα) C8−H8 in tricin (T8) C6−H6 in tricin (T6) C2−H2 and C6−H6 in etherified S units (S2,6) C2′−H2′ and C6′−H6′ in tricin (T2′,6′) C3−H3 in tricin (T3) C2−H2 and C6−H6 in α-oxidized S units (S′2,6) C2−H2 in G units (G2) Cβ−Hβ in p-coumarate (PCAβ) and ferulate (FAβ) C5−H5 in G units (G5) C6−H6 in G units (G6) C6−H6 in ferulate (FA6) Cβ−Hβ in cinnamyl aldehyde end groups (Jβ) C2−H2 and C6−H6 in H units (H2,6) Cβ−Hβ in cinnamyl alcohol end groups (Iβ) Cα−Hα in cinnamyl alcohol end groups (Iα) C2−H2 and C6−H6 in p-coumarate (PCA2,6) Cα−Hα in p-coumarate (PCAα) and ferulate (FAα) Cα−Hα cinnamyl aldehyde end groups (Jα) Cα−Hα in Ar−CαHαO (ArCHO) Cγ−Hγ in cinnamyl aldehyde end groups (Jγ)

wheat straw interunit linkage

a

β-O-4′ (G + S) β-O-4′ (S) β-O-4′ (G) β-5 β-β 5-5′-O-4′ β-1 (including SD) α-oxidized β-O-4′b α,β-diaryl ethers cinnamyl alcohol cinnamyl aldehyde aromatic unitsa S/G ratio PCA FA PCA/FA ratio tricin

EMAL

SE

66 41 25 10 5 8 trace 6 3 0.7 1.4

51 26 25 16 3 2 trace 2 1 0.6 1.5

0.6 11 2.7 4.0 13

0.4 5 1.9 2.6 2

Expressed as the number per 100 C9 units (0.5S2,6 + G2). bHβ−Cβ correlation signal integral. a

lignins were dissolved in 0.1 M NaOH (1 mg/mL) solutions overnight, followed by filtration through a 0.45 μm polytetrafluoroethylene (PTFE) syringe filter. Results were calculated relative to polystyrenesulfonate sodium salt standard using Waters Empower 3 software. Density Functional Calculations. All calculations were performed with Gaussian 09, C.01 using the facilities of the Alabama Supercomputer Center in Huntsville, AL. All structures were optimized at the M06-2X level of theory, using the 6-311++G(d,p) basis set and the fine grid consisting of 75 radial shells and 302 angular points per shell. Frequency calculations were performed on the stationary points identified. Bond dissociation enthalpies were determined as the difference between the enthalpy of the reactant and products.

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RESULTS AND DISCUSSION The amounts of the lignin units and the detected interunit linkages in wheat straw EMALs before and after steam explosion treatment were determined by 2D 1H−13C HSQC NMR experiments (Figure 1). The lignin subunits detected are presented in Figure 2, and the spectroscopic assignments based on the literature are presented in Table 1. The quantitation was based on the assumption that the C2 position in G units and the C2 and C6 positions in the S units are not substituted. Thus, (G2 + S2,6/2) was used as an internal standard. The interunit linkages were determined by careful integration of the Hα−Cα correlation signals in the HSQC NMR spectra (with the exception of Hβ−Cβ in the α-oxidized β-O-4′ structure) and comparing to the total C9 unit integral. Hγ−Cγ correlation signals originating from resinol structures were shown only as a trace in the HSQC NMR spectra. Calculated amounts of the main interunit linkages and structural characteristics are shown in Table 2. It was observed that the S-type β-O-4′ interunit linkages were cleaved preferentially (36% decrease), whereas no cleavage on β-O-4′ in the G type was observed. Recently, incorporation of tricin into the wheat straw lignin via β-O-4′ in the G unit has been reported.3 Accordingly, also, these linkages were cleaved during the SE treatment, leading to significant loss of tricin

Signal identification was carried out according to the literature.3,25

zero-filled once in F1, and a π/2-shifted squared sine bell weighting function was applied in both dimensions prior to the Fourier transformation. 31 P NMR Measurements and Size-Exclusion Chromatography (SEC) Analyses. For 31P NMR analyses, the lignin samples and tricin were phosphorylated and the freshly prepared samples were measured with 31P NMR at room temperature. The experimental procedure has been explained in detail previously.23 A 500 MHz Bruker Avance III NMR spectrometer equipped with 5.0 mm broadband observe (BBO) probe was used for the measurements. Chemical shifts are relative to the reaction product of water with tetramethyldioxaphospholane, which gives a sharp signal in pyridine/CDCl3 at 132.2 ppm. The following NMR parameters were used: scans, 512; pulse delay, 5 s; Lorentzian line broadening, 4 Hz. SEC analyses were carried out using a HPLC system (Waters Corp., Milford, MA) equipped with 8 × 3000 mm MCX 1000 and 100 000 Å columns (Polymer Standard Services, Mainz, Germany) and a Waters 2998 UV detector (Waters Corp., Milford, MA) set at 280 nm using 0.1 M NaOH eluent (0.5 mL/min flow rate). For the SEC analysis, 10440



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formation of the new linkage. To evaluate if the conclusions made based on the NMR analyses are sound from the theoretical point of view, calculations were performed to elucidate the thermodynamic feasibility of the rearrangement reaction. The results also indicated that the SE treatment of wheat straw caused a substantial decrease of cinnamic acids, particularly p-coumaric acid, which acylates a portion of γ-OH of the lignin side chain in grasses, and may thus be hydrolytically cleaved during the autocatalytic SE treatment. The higher levels of the p-coumarates (11 per C9) in comparison to the value presented in the literature [4% in milled wood lignin (MWL) wheat straw] may be explained by the milder hydrolytic conditions applied in the EMAL process compared to MWL isolation. The wheat straw lignin samples were analyzed by 31P NMR to obtain more information on the hydroxyl groups on lignin. Because the lignin was known to contain tricin, it was necessary to apply a model compound for signal verification in the 31 P NMR spectrum. As a consequence, a novel quantitative

(85% decrease). The result is in accordance with the literature,19 where the cleavage of β-O-4′ structures during SE is depicted as one of the most predominant depolymerization reactions in lignin. Our results show that various β-O-4′ substructures differ in the tendency to cleave. The SE treatment also caused an increase in the average lignin molar mass (Mw) from 3600 to 6100 as measured with SEC (Figure 3).16 This was contradictory with the observed rather high degree of cleavage of the aryl ethers but can be explained by competing repolymerization reactions. From Table 2, it can be seen that SE alters the lignin structure in several ways. Besides the decrease in the content of β-O-4′, an increase in the amount of phenylcoumaran (β-5′) interunit linkages after the SE treatment was also detected. Upon heating lignin with water, rearrangement from alkyl-aryl ether (β-O-4′) to phenylcoumaran (β-5′) has been previously reported.24 It has been proposed that this rearrangement is initiated by the homolytic cleavage of β-O-4′, followed by the

Figure 3. Calculated SEC results of wheat straw lignins before (purple) and after (green) the SE treatment. Both Mn and Mw of the wheat straw lignin increased after the SE treatment, alongside with the polymerization degree (PD).

Figure 4. (A) 31P NMR spectrum of wheat straw EMAL, (B) 31P NMR of SE-treated wheat straw EMAL, and (C) structure of tricin with 31P NMR showing three different −OH groups in the molecule. 10441



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Figure 5. (A) Stacked HSQC (gray) and HMBC (violet) NMR spectra of wheat straw EMAL and (B) stacked HSQC (gray) and HMBC (violet) NMR spectra of the tricin model compound, showing the main long-range correlations recorded with Δ = 62.5 ms. Long-range correlation signals not originating from tricin are shown in orange.

sample before the SE treatment, confirming the presence of tricin in wheat straw lignin and its partial cleavage in the SE pretreatment. The quantitative results of tricin contents prior to and after the SE treatment are 0.15 and 0.02 mmol/g of lignin, respectively, determined using the tricin peak of C7−OH for quantitation. Furthermore, the signal of the 4′-OH group was significantly less intense than the two others, indicative of its role in linking to lignin. Tricin incorporation into the lignin network through β-O-4′ ether linkages was thus confirmed both by 31P NMR and based on the tricin C4′ carbon chemical shift (δ13C, 139.6 ppm). Figure 5 presents the long-range 13C−1H correlations of the tricin moiety between the wheat straw EMAL sample and the tricin model compound. Interestingly, C7−OH correlation (δ1H, 10.7 ppm in model compound) is missing in the wheat straw

method for tricin was developed. In addition, a comparison of the model compound and the wheat straw lignin showed evidence of 4′-OH of tricin being the linking site to lignin. The 31 P NMR spectra of the phosphitylated lignins before and after SE pretreatment along with a pure tricin model compound sample are shown in Figure 4. As seen from the 31P NMR spectrum (Figure 4C), the pure tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone) molecule shows three distinct chemical shifts at 136.40, 137.67, and 141.96 ppm. We have tentatively assigned these to 7-OH, 5-OH, and 4′-OH hydroxyl groups, respectively. In the literature, the chemical shift at 136.40 ppm has been previously detected and hydroxyl in unknown structures.25 reported as phenol ́́ The tricin signals found in the wheat straw EMAL lignin 31 P NMR spectrum (Figure 4A) are much more intense in the 10442



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Figure 6. Homolytic β-O-4 cleavage reaction in G−O−G with formed phenoxy radical tautomers. The illustrations represent spin densities.

Figure 7. Schematic presentation of two routes (A and B) for the β-O-4 to β-5 transformation.

EMAL spectrum, even though C5−OH (δ1H, 12.9 ppm in wheat straw and model compound) is easily detectable. This may be due to higher acidity of C7−OH. Some of the long-range correlations of the tricin are also missing from the wheat straw EMAL spectrum, indicating the impact of the presence of lignin and carbohydrates toward the visibility of long-range correlations. We also noticed that the tricin T8 1H−13C correlation signal (δC/δH, 94.1/6.56) shows a small shift upfield (δC/δH, 93.8/6.38) after SE treatment, whereas the T6 signal was not shifted. The feasibility of the postulated lignin reactions during the SE treatment were evaluated by comparing the bond dissociation energies. The β-O-4 bond dissociation energies for the homolytic cleavage reactions were calculated for G−O−G-, G−O−S-, and G−O−T-type β-O-4 structures. G−O−G showed the highest dissociation energy (69.7 kcal/mol);

G−O−S showed intermediate dissociation energy (68.4 kcal/mol); and G−O−T showed the lowest dissociation energy (67.9 kcal/mol). These values are in accordance with the 2D NMR data (Table 2), which indicated the order of stability of β-O-4′ structures to be G−O−G > G−O−S > G−O−T. Figure 6 presents an example of the homolytic β-O-4 cleavage, including G−O−G moieties with relevant tautomerization of the phenoxy radical and including the spin densities of the alternative radical forms. The feasibility of possible reaction routes leading to the homolytic β-O-4 cleavage was evaluated on the basis of their energetic requirements. Two possible reaction routes (A and B) for the cleavage, followed by transformation to the β-5 substructure are presented in Figure 7. The first route (A) requires and includes the formation of a tautomer from the phenoxy radical, while the second route (B) 10443



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couples the phenoxy radical and β-O-4 radical without the need for tautomerization. Altogether, the transformation from β-O-4 to β-5 (+water) substructure requires energy of around 15 kcal/mol, which could be adequately provided by the SE process. It is worth pointing out that the β-5 dilignol is actually a more stable form than the original β-O-4 dimer by 11.6 kcal/mol. In conclusion, homolytic cleavage of the β-O-4 linkages and recoupling to β-5 linkages were identified as the main reaction types of lignin in SE treatment. The β-O-4 bond dissociation energies for the homolytic cleavage reactions were calculated for G−O−G (69.7 kcal/mol), G−O−S (68.4 kcal/mol), and G−O−T (67.9 kcal/mol) type β-O-4 structures. This was confirmed by 2D 1H−13C HSQC and 1H−13C HMBC NMR data, indicating nearly complete removal of tricin and significant reduction of G−O−S units. We also postulated means to identify and measure the amount of tricin in the lignin sample with the aid of a 1D 31P NMR experiment. The development of biomass pretreatment methods in the field of biorefinery is of vital importance for generating valueadded biomaterials and chemicals. The presence of tricin as part of the lignin structure may generate more diversity in the use of lignin as a feedstock for value-added products. The cleavage of tricin from lignin may also enhance saccharification, even if the mechanism for this cannot be explained on the basis of the present results.

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(10) Evtuguin, D.; Neto, C. P.; Silva, A. M. S.; Domingues, P. M.; Amado, F. M. L.; Robert, D.; Faix, O. Comprehensive study on the chemical structure of dioxane lignin from plantation Eucalyptus globulus wood. J. Agric. Food Chem. 2001, 49, 4252−4261. (11) Heikkinen, S.; Toikka, M.; Karhunen, P.; Kilpeläinen, I. Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: Application to wood lignin. J. Am. Chem. Soc. 2003, 125, 4362−4367. (12) Zhang, L.; Gellerstedt, G. Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references. Magn. Reson. Chem. 2007, 45, 37−45. (13) Sette, M.; Wechselberger, R.; Crestini, C. Elucidation of lignin structure by quantitative 2D NMR. Chem.Eur. J. 2011, 17, 9529− 9535. (14) Ramos, L. P.; Breuil, C.; Kuschner, D. J.; Saddler, J. N. Steam pre-treatment conditions for effective enzymatic hydrolysis and recovery yields of Eucalyptus viminalls wood chips. Holzforschung 1992, 46, 149−154. (15) Jakobsons, J.; Hortling, B.; Erins, P.; Sundquist, J. Characterization of alkali soluble fraction of steam exploded birch wood. Holzforschung 1995, 49, 51−59. (16) Rahikainen, J. L.; Martin-Sampedro, R.; Heikkinen, H.; Rovio, S.; Marjamaa, K.; Tamminen, T.; Rojas, O. J.; Kruus, K. Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption. Bioresour. Technol. 2013, 133, 270−278. (17) Li, S.; Lundquist, K. Cleavage of arylglycerol β-aryl ethers under neutral and acid conditions. Nord. Pulp Pap. Res. J. 2000, 15, 292−299. (18) Robert, D.; Bardet, M.; Lapierre, C. Structural changes in aspen lignin during steam explosion treatment. Cell. Chem. Technol. 1988, 22, 221−230. (19) Li, J.; Henriksson, G.; Gellerstedt, G. Lignin depolymerization/ repolymerization and its critical role for delignification of aspen wood by steam explosion. Bioresour. Technol. 2007, 98, 3061−3068. (20) Guerra, A.; Filpponen, I.; Lucia, L. A.; Saquing, C.; Baumberger, S.; Argyropoulos, D. S. Toward a better understanding of the lignin isolation process from wood. J. Agric. Food Chem. 2006, 54, 5939− 5947. (21) Wu, S.; Argyropoulos, D. S. An improved method for isolating lignin in high yield and purity. J. Pulp Pap. Sci. 2003, 29, 235−240. (22) Zwahlen, C.; Legault, P.; Vincent, S. J. F.; Greenblatt, J.; Konrat, R.; Kay, L. E. Methods for measurements of intermolecular NOEs by multinuclear NMR spectroscopy: Application to a bacteriophage λ Npeptide/boxB RNA complex. J. Am. Chem. Soc. 1997, 119, 6711−6721. (23) Granata, A.; Argyropoulos, D. S. 2-Chloro-4,4,5,5-tetramethyl1,3,2-dioxaphospholane, a reagent for the accurate determination of the uncondensed and condensed phenolic moieties in lignins. J. Agric. Food Chem. 1995, 43, 1538−1544. (24) Nimz, H. A new type of rearrangement in the lignin field. Angew. Chem. 1966, 5, 843. (25) Yang, Q.; Wu, S.; Lou, R.; Gaojin, L. V. Structural characterization of lignin from wheat straw. Wood Sci. Technol. 2011, 45, 419−431.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +358400156497. Fax: +358207227026. E-mail: harri.heikkinen@vtt.fi. Notes

The authors declare no competing financial interest.

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REFERENCES

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Impact of Steam Explosion on the Wheat Straw Lignin Structure

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