Structural Insights on Recalcitrance during Hydrothermal

Research Article pubs.acs.org/journal/ascecg

Structural Insights on Recalcitrance during Hydrothermal Hemicellulose Extraction from Wood Nicola Giummarella†,‡ and Martin Lawoko*,†,‡ †

Wallenberg Wood Science Center, Royal Institute of Technology, KTH 10044, Stockholm, Sweden Department of Fiber and Polymer Technology School of Chemistry, Royal Institute of Technology, KTH, Teknikringen 56-58, 10044 Stockholm, Sweden

‡

S Supporting Information *

ABSTRACT: Hydrothermal extraction of hemicelluloses from lignocellulosic biomass for conversion to renewable materials or fuels has captured attention. The extraction is however partial and some lignin is codissolved. Herein, we investigated the role of molecular structure in the recalcitrance. Wood meal of Spruce and Birch were subjected to pressurized hydrothermal extraction at 160 °C for 2 h, which extracted 68−75% of the hemicelluloses. 2D heteronuclear single quantum coherence (HSQC) NMR, HSQC-TOCSY, and 13 C NMR were applied for structural studies of both extracts and residues. Subsequent to the known partial hydrolysis of native carbon-2 and carbon-3 acetates in hemicellulose, some acetylation of primary alcohols on hemicelluloses and lignin was observed. Lignin carbohydrate complexes (LCC) were detected in both the extracts and residues. In Spruce extracts, only the phenyl glycoside-type of LCC was detected. Birch extracts contained both the phenyl glycoside and benzyl ether-types. In the hydrothermal wood residues of both species, benzyl ether- and gamma (γ)-ester-LCC were present. Structural changes in lignin included decrease in aryl ether (βO4) content and increases in resinol- (ββ) and phenyl coumaran (β5) contents. On the basis of the overall analysis, the mechanisms and contribution of molecular structure to recalcitrance is discussed. KEYWORDS: Autohydrolysis, Recalcitrance, trans-Acetylation, Lignin carbohydrate complexes (LCC), LCC repolymerization, 2D HSQC, HSQC-TOCSY, 13C NMR

■

INTRODUCTION The utilization of wood polymers as raw material alternatives to fossil-based ones is gaining interests on the basis of sustainability. The extraction of hemicelluloses for this purpose is one example. Several different solvent systems have been investigated although water-based extractions have attracted most attention due to its more benign effect on environment. For such systems, when temperatures below 180 °C are applied without added catalyst, the pre-extraction is incomplete. The amounts extracted depend on the temperature, reaction time,1 pH,2 liquor to wood ratios,3 and particle sizes4,5 among several other factors. For nonbuffered aqueous systems, the consensus is that the extraction is autocatalytic due to the increased dissociation of water at high temperature. The hydrolysissensitive linkages are acetyl groups on hemicelluloses,6 glycosidic linkages in polysaccharides,7,8 and ether linkages in lignin.6,9 The hydrolysis of acetyl groups is of specific interest as acetic acid is produced and catalyzes secondary hydrolysis reactions. At about 170 °C, monosugars and their dehydration products such as furfural and hydroxyl methyl furfural (HMF) have been detected in solution in significant quantities.10 The dissolution of lignin has also been observed and shown to be problematic © 2017 American Chemical Society

by forming sticky substances that are responsible for plugging in processing equipment.11 The proposed dissolution mechanism of lignin includes both hydrolytic- and homolytic-12 cleavages of aryl ether linkages. Pseudolignin, which consists mostly of furan derivatives, is also formed.10 Both lignin and pseudolignin are inhibitors of fermentation of sugars13 and are therefore problematic. In spite of the known molecular events, deeper insights on recalcitrance from a molecular structure viewpoint are lacking. It has been suggested that the covalent bond between lignin and hemicelluloses (lignin carbohydrate complexes) may be responsible for holding back some of the hemicelluloses.14,15 Fundamental knowledge on recalcitrance is therefore of interest for the development of more efficient processes for hemicellulose extractions in higher yield and purity. We selected to investigate recalcitrance at 160 °C for 2 h to minimize the efficient hydrolysis of hemicellulose, which occurs at temperatures above 170 °C.10 Spruce and Birch wood were chosen since these are common softwoods and hardwoods used Received: February 17, 2017 Revised: April 10, 2017 Published: May 2, 2017 5156

DOI: 10.1021/acssuschemeng.7b00511 ACS Sustainable Chem. Eng. 2017, 5, 5156−5165

Research Article

ACS Sustainable Chemistry & Engineering in Sweden for technical production. Furthermore, they have different chemistries with respect to hemicellulose types and lignin structure. The main hemicellulose in softwoods is galactoglucomannan whereas that in hardwoods is glucuronoxylan. These may show different solubility properties. On the analytical front, we recently developed a mild quantitative fractionation protocol aimed at studying the linkages between lignin and carbohydrates.16,17 In the present investigation, the protocol was applied to obtain soluble fractions enabling the quantitative analysis of the extracted wood residues in addition to the hot water extracts. Similar fractions are also obtained from the untreated woods as reference points. Thus, a holistic view of the structural changes occurring during extraction enabled discussions on important structural attributes to recalcitrance.

■

MATERIALS AND METHODS

Materials and Chemicals. All chemicals used were of analytical grade and purchased from Sigma-Aldrich. Wood meals were obtained from Norway Spruce (Picea abies) industrial chips (cellulose, 42%; hemicellulose, 28%; lignin, 28%; extractives, 2%) produced at Södra mill in Värö and Birch (Betula) chips (cellulose, 44%; hemicellulose, 29%; lignin, 21%; extractives, 4%) chipped from logs in a laboratory chipper at Innventia, Stockholm, Sweden. In both cases, knots and bark were removed prior to Wiley milling. Accelerated Solvent Extraction (ASE) of Spruce and Birch Wood. Wiley meal (40 mesh) extractive-free wood16 of both species were extracted with water in a Dionex Accelerator Solvent Extractor 350. Stainless steel cells (34 mL) were filled with approximately 5 g of sample using glass fiber (30 mm diameter) filters. One static cycle (160 °C, 120 min, 1500 psi) with a rinse volume of 60% of the cell volume was performed on each cell. Purging time was set to 90 s. The extracts, W1-S160 (Spruce) and W1-B160 (Birch), were collected in diverse 60 mL vials and lyophilized. Quantitative Analytical Fractionation of the Residue. The residues were subjected to a dissolution−precipitation scheme as recently described16,17 and summarized in Figure 1a. Shortly, P1-S160 (Spruce) and P1-B160 (Birch) were obtained by addition of 20% in volume of water after complete dissolution in 1-allyl-3-methylimidazolium chloride ([Amim]Cl)-DMSO (16 h, 70 °C) of ball milled hydrothermal wood residue. Similarly, P2-S160 and P2-B160 were precipitated in ethanol by addition of three times the volume of DMSO-water (VDMSO/Vwater = 1:1). Finally, to the remaining solution, three times its volume of water was added to obtain fractions P3-S160 (Spruce) and P3-B160 (Birch). Carbohydrate and Lignin-Composition Analysis. Monosaccharide composition of the freeze-dried fractions was determined after acid hydrolysis according to Effland19 and analyzed as described by Davis20 by HPAEC/PAD (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-1 column.21 Klason lignin was determined as described by Effland19 and acid soluble lignin by Tappi method 250.22 Uronic Acid Determination. The content of polygalacturonic acid and 4-O-methyl glucuronic acid were determined by methanolysis followed by TFA hydrolysis23−25 and quantified by HPAEC/PAD.21 SEC-DMSO/0.5% LiBr. Size-exclusion chromatography (SEC) of LCCs fractions was obtained with SEC 1260 Infinity (Polymer Standard Services, Germany) coupled with a dual system detector (UV, RI) using DMSO + 0.5% LiBr (w/w) as the mobile phase. The separation system consisted of PSS GRAM Precolumn, PSS GRAM 100 Å, and PSS GRAM 10 000 Å analytical columns thermostated at 60 °C and connected in series. Pullulan standards of 708 kDa, 337 kDa, 194 kDa, 47.1 kDa, 21.1 kDa, 9.6 kDa, 6.1 kDa, 1.08 kDa, and 342 Da were used for standard calibration. Preparation of the samples and experimental setup was as previously described.26 Saponification of Esterified Linkages in Hemicellulose. A 0.2% solution of W1-S160 in 0.4 M NaOH was prepared. Saponification was carried on for 5 min at 80 °C.27 After cooling, the solution was neutralized with Dowex 50WX8 hydrogen form

Figure 1. (a) Quantitative fractionation scheme demonstrated on Spruce. Steps adopted and fractions obtained from Hydrothermal Spruce are reported in red while those regarding Native Spruce in blue. S = Spruce, P = Precipitates, and W = Water extracts with following number indicating the temperature of hydrothermal treatment (80 °C for native Spruce protocol and 160 °C for the hydrothermal one). Ball Milled Native Spruce = BMS; Ball Milled Hydrothermal Spruce = BMHS; EBMS = Extracted Ball Milled Native Spruce. Similarly, the protocol was applied for hydrothermal- and native Birch. (b) Preparation scheme for Milled Wood Lignin (MWL) Spruce as elsewhere described.18 (200−400 mesh, capacity 1.7 mequiv/mL, density ≈ 1.040 g/cm3) to pH 5. The resin, separated by filtration, was washed with water and acetone and the obtained permeate was freeze-dried after removal of acetone by rotoevaporation. 2D HSQC, HSQC TOCSY, and 13C NMR Analyses. An amount of 100 mg of all the studied samples were dissolved in 700 μL of DMSOd6 and CDCl3 or d6-acetone for the acetylated samples. The acetylation was done as reported by Ralph et al.28 For milled wood lignin,18 acetylation was performed as described by Gellerstedt.29 All NMR spectra were recorded and processed as described elsewhere.16 The unsubstituted carbon 2 of aromatic groups was used as an internal standard for the semiquantification.30 When quantitative analysis was possible, the method by Zhang and Gellerstedt, which applies both 13C and 2D HSQC analyses was used.31 The central DMSO (δC/δH = 39.5/2.5 ppm), acetone (δC/δH = 29.8/2.0 ppm) and chloroform (δC/ δH = 77.3/7.2 ppm) signals were used as an internal reference. 2D HSQC-TOCSY experiments were carried out with the Bruker pulse program “hsqcetgpml” setting as relaxation delay D9 = 90 ms and number of scans to 165, which leads to a run time of 20 h.

■

RESULTS AND DISCUSSION To gain insight on molecular-structure attributes to recalcitrance during hydrothermal extraction of wood, Spruce and Birch were subjected to accelerated solvent extraction at 160 °C for 2 h. Our investigation focused on a process that extracts mainly polymeric/oligomeric hemicelluloses in fair yields, using unbuffered water as green and sustainable solvent. Extracts 5157


Research Article

ACS Sustainable Chemistry & Engineering

Table 1. Mass Balance and Composition of Extracted- and Precipitated-Fractions Obtained from Hydrothermal Spruce and Bircha

a

For acetyl content, native is compared with hydrothermal. Sugar composition quantified by HPAEC-PAD. Acetyl content by 2D-HSQC. In the table: d = 80% aqueous dioxane extract; - = not detected; n.a. = not analyzed; %Err. = error percentage of the value; * = mole ratio; Ara = Arabinose; Gal = Galactose; Glu = Glucose; Xyl = Xylose; Man = Mannose; Fuc = Fucose; Rha = Rhamnose; GluA = 4-O-methylglucuronic acid; GalA = Galacturonic acid; Cx = position of acetyl group on Carbon x in Mannose and/or Xylose.

wood previously reported.16,17 Crude milled wood lignin was also prepared from the hydrothermal wood residues (Figure 1b) in yields of 25% for Spruce and 85% for Birch, which were significantly higher than those from the original woods (11%, 33%),16,17 respectively. The higher yields of Birch MWL, when compared to Spruce MWL, is due to that Birch wood has a higher content of cleavage prone βO4 than Spruce, which upon hydrothermal- and mechanical treatments yields a higher amount of lignin and LCC fragments that are extractable by dioxane. Structural Studies by 2D HSQC-, HSQC-TOCSY- and 13 C- NMR. Changes in Acetylation Patterns of Hemicelluloses. Native wood hemicelluloses are partially acetylsubstituted at the carbon 2 and carbon 3 hydroxyls of the xylose (in hardwoods) or mannose units (in softwoods).32 Here, we studied how these acetylation patterns changed as a result of pressurized hydrothermal pretreatment at 160 °C for 2 h in order to investigate if this affected the solubility properties. The analysis was performed semiquantitatively, which arises from that the differences in transverse relaxation (T2-relaxation) profiles between the anomeric (C1/H1) and C2/H2, C3/H3 in the HSQC are not corrected for. However, for a more reliable semiquantification, we adopted a strategy to overcome problems associated with signal overlap in the 2D HSQC, by using sugar analysis data obtained by HPAEC analysis (Table 1) in combination with cluster integral of the anomeric region of the carbohydrates from the 2D HSQC, which represents the total amount of each sugar. In the case of xylose, HPAEC data

were freeze-dried prior to characterization. The residues were subjected to analytical fractionation as described in the experimental details (Figure 1a). This scheme, originally developed for fractionation of native ball milled wood at pHneutral conditions and mild temperatures,16,17 gave quantitative yield for autohydrolysis wood as well, demonstrating its universality of application (Table 1). Obtained fractions were studied by NMR spectroscopy for a detailed structure. Mass Balance and Composition. The extraction yields of W1 were 21% for Spruce (S160) and 27.5% for Birch (B160) on a wood basis. In both cases, approximately 30% more yield was obtained when compared to reference ball milled native wood extracted at 80 °C and atmospheric pressure.16,17 The extraction yields of hemicellulose by the hydrothermal treatment are about 75% and 68% for Spruce and Birch, respectively. W1-S160 contained 8.1% of spruce lignin and W1B180 contained 19.8% of the lignin in birch. Major portions of the cellulose and lignin are retained in the residue. Monosugars composition of the hydrothermally treated fractions obtained after 2-stage acid hydrolysis suggests that W1-S160 and W1B160 contain mainly galactoglucomannan- and glucuronoxylan, respectively. Of the fractions obtained by subjecting the residue to analytical fractionation, a cellulose-enriched fraction, P1, accounts for approximately 55% of the mass in both spruce and birch and at least half of the wood lignins, P2 is mainly hemicellulose-based and P3 is enriched in lignin and structurally similar to milled wood lignin. All fractions have compositional similarities with those obtained from native 5158


Research Article


Figure 2. Main interunit linkages and end groups in lignin (1−11), carbohydrates linkages in mannans, xylans, glucans and lignin-carbohydrate identified in the 2D HSQC NMR spectra of Hydrothermal Spruce and Birch (Figures 3 and 4; Supporting Information, Figures S1−S7).

native (80 °C extraction) to the hydrothermally treated wood at 160 °C as observed from the respective fractions (Table 1). In connection with the improved yields therefore, depolymerization caused by the auto- and acid- hydrolyses may have promoted hemicellulose extraction, consistent with the literature.33 The deacetylation is more pronounced in the case of Spruce wood. Interestingly, however, a partial acetylation of mannose at the C6 hydroxyl, clearly observable in the spectra of the W1-S160 fraction, occurred (Figure 3a). C6-hydroxyls in native wood hemicelluloses are not acetylated. Thus, trans-acetylation from C2 or C3 to C6 in mannan was effective. This suggested that acetates of primary alcohols, once formed, were stable at the applied hydrothermal conditions. Such acetyl migration has been observed by Xu et al.34 The ramifications of the favorable acetylation of primary alcohols will be discussed in another section of this paper. From the volume integrals, we estimate the C6 acetylation levels in

from methanolysis-TFA hydrolysis was applied due to higher yield of xylose. The calculation is summarized in eq 1. X C2(Ac) = mol X =

IHSQC[X C2(Ac)] X mole%(HPAEC) × IHSQC[Cluster]Anomeric

(1)

Equation 1 is an example of the calculation of the degree of acetylation on Carbon 2 in xylan expressed as mol % of the xylan unit; Ac = acetylation, X = xylan, and I = integral, [ ] = HSQC signal. The two hemicellulose-enriched fractions, i.e., the W1 watersoluble fractions (Figure 3a,b) and water insoluble fraction P2 from hydrothermally obtained extracts (Figure 3c,d) were compared to analogous native wood fractions in the analysis. In general, deacetylation occurs to significant levels going from 5159


Research Article


Figure 3. 2D HSQC NMR spectra of (a) W1-S160, (b) W1-B160, (c) P2-S160, (d) P2-B160, in DMSO-d6; (e) 2D HSQC and (f) 13C extended spectra of acetylated MWL ball milled hydrothermal spruce (in acetone-d6). Colors, numbering, and abbreviations used are shown in Figure 2. Assignment of main 13C−1H correlation signals of lignin, carbohydrates, and LC bonds are listed in the Supporting Information (Tables S1−S4). In the figure, -Ac = acetylated.

Mechanical treatment is a prerequisite for dissolution of otherwise impenetrable woody structure and is known to cause homolytic cleavage of labile βO4 linkages in lignin, forming new phenolic hydroxyls and α-carbonyls.16,17,39 Glycosidic linkages in the carbohydrates are also partially cleaved. Both these reactions decrease the degree of polymerization of the wood polymers enhancing solubility in different solvent systems. Effect of Hydrothermal Extraction at 160 °C. Significant decrease in aryl ether linkages (βO4) are observed when comparing the native fractions (80 °C extracted) with those of hydrothermal treatment at 160 °C (Table 2). To appreciate this decrease, we draw attention to the P1 fractions since these contained up to 63.4% of the lignin in Spruce and 48.7% in Birch. The difference in lignin content of the two fractions could be due to that syringyl units, only present in Birch, have different solubility properties from guaiacyl, with the latter more prone to precipitation when water is added to obtain P1 fractions. About 25% of the βO4 are cleaved in both wood species. We attribute part of this to hydrolysis based on the detection of Hibberts ketones (β-carbonyls structures) in the W1-S160 and P2-S160, which are formed in accordance with known mechanisms.40−42 These ketones have Cα/Hα correlations appearing at 44.6/3.64 ppm and Cγ/Hγ at 67.6/4.17 ppm.43

mannose at about 2−3%. As expected, saponification caused the disappearance of the assigned acetate signals at the C2, C3, and C6 positions of the sugar units (Supporting Information, Figure S1). C6 acetylation was not detected in the fractions obtained from the hydrothermal residues and may be explained by the lower reactivity in the solid phase. From the data, no direct correlation between the degree of acetylation of hemicellulose and solubility can be deduced as some of the insoluble fractions had similar acetylation patterns as the soluble ones. However, it can be expected that the clustering or spacing of acetyls along the chains may have effects, something not deductible from our study. Changes in Lignin Structure. 2D HSQC NMR has been used extensively to study lignin interunit linkages.31,35−37 The C/H correlations of the studied interunit linkages are reported in Tables S1 and S3 (Supporting Information). Most of the samples herein were semiquantified except for milled wood lignins from the hydrothermal woods where quantitative 2D HSQC was determined according to the method described by Zhang and Gellerstedt.31 Furthermore, 2D HSQC-TOCSY was applied as proof of connectivity as demonstrated by Ralph and co-workers.38 Effect of Ball Milling. Because most of the fractions we studied have undergone ball milling (except the 160 °C water extracts), distinguishing the effects of ball milling from those of hydrothermal treatment at structural level becomes important. 5160


Research Article


Table 2. Semiquantitative Determination of Interunit Linkages, End Groups in Lignin, and S/G Ratio Calculated by 2D HSQC NMRa

a

For milled wood lignins (MWL) and P1-B160 (80% aqueous dioxane extract), quantitative (Q) and semiquantitative measurements are compared. * = secoisolariciresinol, CA = cinnamyl alcohol, DCA = dehydroconiferyl alcohol, C.ald = cinnamyl aldehyde, ESI = spectra available in the corresponding number figure in the Supporting Information; W = weak signal, S = strong signal; (Q) = data obtained from quantitative analysis; − = nondetected, D = detected but not quantified due to overlapped region; d = 80% aqueous dioxane extract, PG = phenyl glycoside, BE = benzyl ether, GE = γ-esters; S/G ratio = syringyl to guaiacyl ratio.

ppm (Figure 3e,f). These structures include βO4, ββ, dibenzodioxin (DBD), and benzyl ethers (BE). The β5 is quantified separately due to different T2-relaxation. This concept is visualized in Figure 3e,f. The quantification is exemplified in eq 2.

The content of β5 structures in Spruce and ββ structures in Birch increased and may be due to lignin repolymerization through radical coupling reactions.39,44 In summary, both homolytic and hydrolytic cleavage reactions are key reactions leading to changes that reinforce the structural integrity of lignin. In this work, MWL was also studied to be able to assign interunit linkages in lignin. Such assignments can be helpful for identifying lignin structures in heavily overlapped LCC spectra as those seen in this work (Figures 3 and 4). For milled wood lignins (dioxane lignins) both semiquantitative30 and quantitative analyses31 were performed. The quantitative method requires lignin substrates with minimal interference from carbohydrate signals. In this method data from quantitative 13 C NMR is applied to 2D HSQC to obtain quantitative data for individual structures within a cluster. In the quantitative 13C NMR, the integral of the aromatic region (100−160 ppm) is set to a value of 6.12 (includes correction for vinyl groups in this region)45 and represents the total C9/units. This integral is set as a quantitative reference. The other signals are quantified relative to this reference. Because of poor resolution in 13C, most signals are quantified as clusters. The quantitation can then be applied to appropriate 2D HSQC clusters where the individual structure signals are well resolved. An important criterion is that all signals in the cluster have similar transverse relaxation (T2-relaxation) profiles. This criterion applies to the region in the 2D HSQC cluster at C/H of 78.4−86.4/4−5.2

% β O4 = =

IHSQC[1] IHSQC[1 + 3 + 5 + 11]

β O4 × 100 C9 units × I13C[1 + 3 + 5 + 11] × 100 (2)

Equation 2 is an example of quantitative measurement of signal 1 (βO4) as described elsewhere.31 I = integral, [ ] = HSQC signal of the interunit linkages, 3 = resinol structure, 5 = dibenzodioxin, 11 = spirodienone as in Figure 2 and Figure 3e,f. From the comparisons of semi- and quantitative measurements, there are clear differences (Table 2), which however do not affect the comparative analysis between different fractions based on the semiquantitative method. Lignin Carbohydrate Complexes (LCC). Lignin carbohydrate complexes (LCCs) have been proposed to play a role in recalcitrance during hot water extraction of wood hemicelluloses.14,15 However, no linkage analysis was provided in the support. The predominant types of native LCC are phenyl glycosides (PG), benzyl ethers (BE), and gamma esters (GE).46 5161


Research Article


Figure 4. 2D HSQC NMR spectra in DMSO-d6 of (a) P3-B160, (b) HSQC-TOCSY of P3-B160, (c) P1-S160d, and (d) P1-B160d, where d = 80% aqueous dioxane extract. (e,f) Measure of role of LCC in recalcitrance of hemicellulose extraction in Spruce and Birch residues, respectively. Values are calculated as shown in eq 3.

weak lignin- and hemicellulose-related signals, and absence of lignin carbohydrate bond signals were observed (Supporting Information, Figure S2a,b). Thus, for detailed analysis, enrichment of lignin and LCC was achieved by extracting the original (nonacetylated) fractions with 80% aqueous dioxane. Subsequent 2D HSQC analysis of the recovered dioxanesoluble lignin yielded spectra with unique features, i.e., a spectra of large and small molecules visualized in the broad (lighter) respective narrower and dense (darker) contours. This observation was also consistent with the molar mass distribution of the P1 fractions which were broad (Supporting Information, Figures S8 and S9) with bimodal tendencies. The main sugar detected in the P1-B160 dioxane-fraction following methanolysis-TFA hydrolysis was xylose (Table 1), which was in agreement with the 2D NMR analysis (Figure 4d; Supporting Information, Figure S3). For the P1-S160 dioxane fraction, the detected sugars in the 2D NMR were mannose (main), galactose, xylose, and glucose (Figure 4c). Galacturonic- and glucuronic acids were also present (Table 1) in both fractions. Benzyl ethers (BE) and γ-esters (GE) were also detected (Figure 4c,d), suggesting that both the sugars and uronic acids were linked to lignin through benzyl ether- and γester-bonds, respectively. Not all the γ-esters were however formed between lignin and uronic acids. Two classes were identified. (i) γ-Esters with Cγ/Hγ resonances at 62.5/3.82 and 4.22 ppm: The presence of methyl cross signals from acetyl groups at 20/1.93, 2.01 ppm, in the fractions suggested these were

2D HSQC NMR spectroscopy is so far the most effective tool in studying LCC.47−51 In the water extract of Spruce, only PG was detected (Figure 3a), but in the case of Birch, PG, and BE were both present (Figure 3b). The C1/H1 correlations at 4.85/101.3 ppm and 4.88/102.6 ppm are assigned to PG in guaiacyl and syringyl units, respectively.49 It is worth noting that internal anomeric signals from rhamnan originating from the 1-2 linkage between rhamnose and galacturonic acid in pectins at 100.8/5.02 ppm, and the anomeric carbon in esterified glucuronic acid at 100.1− 101/4.6−4.747,50 ppm, may overlap with some PG signals. The residues of the hydrothermal treatments at 160 °C were also analyzed after subjection to analytical fractionation (Figure 1a). Benzyl ethers were detected in the P3-S160 (Supporting Information, Figure S2c) and P3-B160 (Figure 4a,b), which were completely soluble in DMSO-d6 solvent without derivatization. In Table S1 (Supporting Information), the C/ H correlation of BEs are shown. The cross signal of the βO4 in BE structures (1BE) is at 85.6/4.34 ppm. The Cγ/Hγ correlation of BEs in a γ-hydroxylated βO4 unit should be between 61.0 and 62.0/3.40−3.90 ppm. This region is heavily overlapped. HSQC TOCSY (Figure 4b) of P3-B160 however resolved a correlation at 61.2/3.79 ppm (Signal 1γ BE) which aligned with the weak BE signal at 82.3/5.20 ppm substantiating that BE was in βO4 structure. Because of poor solubility, the P1 fractions were acetylated prior to the HSQC analysis. As expected from their compositions (Table 1), enhanced signals from cellulose, 5162


Research Article


Concluding Discussion. In this work, we investigated the role of molecular structure on recalcitrance during hemicellulose extraction from wood. We suggest native lignin carbohydrate (LC) bonds of the benzyl ether- and γ-ester type play important roles in recalcitrance. LC bonds of the phenyl glycoside type, on the other hand, were only present in the water extracts. Further, we identify some important reactions within LCC matrix leading to structural changes contributing to hemicellulose retention. (i) Acetyl-transfer from acetates of secondary alcohols in native hemicellulose to the primary alcohol in the lignin moiety of LCC. This may cause precipitate formation and redeposition on the basis of increased hydrophobicity of the LCC matrix. (ii) LCC repolymerization through radical coupling- or condensation reactions within the lignin moiety result in high molar mass and increased hydrophobicity. From a viewpoint of molecular structure therefore, the stability of the linkage between lignin and hemicelluloses may play a central role in hemicellulose recalcitrance during hydrothermal extraction.

acetyl esters. The presence of a signal at 81/4.62 ppm (Figure 4c,d; Signal 1γ ester) suggested they were esters in the γposition of a βO4 subunit. The absence of signals related to acetylated polysaccharides in these fractions substantiated lignin acetylation. As discussed earlier with reference to mannose, the acetyl-transfer to primary alcohol was also effective in lignin, i.e., a partial acetylation of primary alcohol (at the γ position in lignin) had occurred. (ii) γ-Esters with Cγ/Hγ resonances at 5.3/4.12 and 4.33 ppm: We tentatively assign these to uronic acid esterified lignins (Figure 4c,d; Signal GE) due to that these fractions were also enriched in uronic acids and that the esters fall within the assigned region for γ-esters (62−65/4.0−4.5 ppm).50 These esters existed both as small and large molecules (broad and narrow contours, the latter overlay the former). Some of these esters were probably formed during hydrothermal treatment through esterification of uronic acids to γ-hydroxyls on the same basis as the aforementioned acetylation. The stability of native- and formed lignin-carbohydrate esters may in part be responsible for retention of xylans and pectins. The acetylation of primary alcohol in lignin moiety of an LCC matrix should enhance the hydrophobicity making it insoluble and may also in part explain retention of hemicelluloses. HSQC-TOCSY of P1-B160 (Supporting Information, Figure S3) showed a clear bond correlation of the terminal anomeric in α-xylose at 90.5/4.65 ppm with the corresponding C2 and C3 correlations at 69.8/3.18 and 69.8/3.60 ppm. On Recalcitrance. Evidently, the hydrothermal treatment at 160 °C had limited effects, if any, on LC bond cleavage. By calculating the amount of LC linkages and normalizing it for hemicellulose content of wood (native and hydrothermal), the linkage enrichment in the residue (with respect to hemicellulose fraction) can be studied. The equation applied (eq 3) is used to plot a figure of the LC content in the native and hydrothermal woods (normalized for the hemicellulose content) as shown in Figure 4e,f. It is observed that for both wood species, benzyl ethers and γ-esters are indeed enriched but the phenyl glycosides disappear during the hydrothermal treatment. This indicates that hydrophobic lignin may indeed hold back bound hemicellulose during the hydrothermal treatment. Thus, if lignin carbohydrate bonds play a role in retaining hemicellulose in the hydrothermal wood then the stability of the benzyl ether- and γ-esters linkages are responsible. I

■

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00511. 2D HSQC and 13C spectra, 2D HSQC assignments, and SEC chromatograms (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail:[email protected]. Phone: +46 8 7908047, +46 73 4607647. Fax: +46 8 7908066. ORCID

Martin Lawoko: 0000-0002-8614-6291 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Knut and Alice Wallenberg Foundation and are gratefully acknowledged for financial support to the Wallenberg Wood Science Center.

■

[LC]

HSQC ∑ I [Anomeric region] mol(LC/Carbs) HSQC = Hemicellulose in wood Hemicellulose content %

ASSOCIATED CONTENT

ABBREVIATIONS [Amim]Cl, 1-allyl-3-methylimidazolium chloride; BMW, ball milled wood; MWL, milled wood lignin; 4OMGA, 4-O-methyl glucuronic acid; BMS, ball milled native spruce; BMHS, ball milled hydrothermal spruce; EBMS, extracted ball milled native spruce; LCC, lignin carbohydrate complex

(3)

Equation 3 is the calculation for recalcitrance measurements used in Figure 4e (Spruce) and Figure 4f (Birch). IHSQC = HSQC integral, LC = lignin carbohydrate linkage of phenyl glycoside (PG) or benzyl ether (BE) or γ- ester (GE) types. Carbs = Carbohydrates. Molar Mass Distribution. Size exclusion chromatography (Supporting Information, Figure S8 and S9) showed that the hot water extracts were of lower hydrodynamic volume than the polymers in the residues. Considering that the polymers in the hydrothermal residues incurred some depolymerization during the prerequisite ball milling, the true molar masses could be much higher than reported here. Thus, the recalcitrance due to restrictive solubility imposed by high molar mass cannot be excluded.

■

REFERENCES

(1) Tunc, M. S.; van Heiningen, A. R. P. Hemicellulose Extraction of Mixed Southern Hardwood with Water at 150 °C: Effect of Time. Ind. Eng. Chem. Res. 2008, 47 (18), 7031−7037. (2) Reyes, P.; Mendonca, R. T.; Rodriguez, J.; Fardim, P.; Vega, B. Characterization of the hemicellulosic fraction obtained after prehydrolysis of Pinus Radiata wood chips with hot water at different initial pH. J. Chil. Chem. Soc. 2013, 58, 1614−1618. (3) Tunc, M. S. Effect of Liquid to Solid Ratio on Autohydrolysis of Eucalyptus globulus Wood Meal. BioResources 2014, 9 (2), 4214− 4225.

5163


Research Article


(25) Morais de Carvalho, D.; Martínez-Abad, A.; Evtuguin, D. V.; Colodette, J. L.; Lindström, M.; Vilaplana, F.; Sevastyanova, O. Isolation and characterization of acetylated glucuronoarabinoxylan from sugarcane bagasse and straw. Carbohydr. Polym. 2017, 156, 223− 234. (26) Duval, A.; Vilaplana, F.; Crestini, C.; Lawoko, M. Solvent screening for the fractionation of industrial kraft lignin. Holzforschung 2015, 70 (1), 11−20. (27) Chokboribal, J.; Tachaboonyakiat, W.; Sangvanich, P.; Ruangpornvisuti, V.; Jettanacheawchankit, S.; Thunyakitpisal, P. Deacetylation affects the physical properties and bioactivity of acemannan, an extracted polysaccharide from Aloe vera. Carbohydr. Polym. 2015, 133, 556−566. (28) Lu, F.; Ralph, J. Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR. Plant J. 2003, 35, 535−544. (29) Gellerstedt, G. Gel permeation chromatography. In Methods in Lignin Chemistry; Lin, S.Y., Dence, C.W., Eds.; Springer-Verlag: Heidelberg, Germany, 1992. (30) Sette, M.; Wechselberger, R.; Crestini, C. Elucidation of Lignin Structure by Quantitative 2D NMR. Chem. - Eur. J. 2011, 17, 9529− 9535. (31) 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. (32) Dumitriu, S. Polysaccharides: Structural Diversity and Functional Versatility, 2nd ed.; CRC Press: Boca Raton, FL, 2004. (33) Garrote, G.; Domínguez, H.; Parajó, J. C. Study on the deacetylation of hemicelluloses during the hydrothermal processing of Eucalyptus wood. Holz als Roh- und Werkstoff 2001, 59 (1), 53−59. (34) Xu, C.; Leppänen, A.-S.; Eklund, P.; Holmlund, P.; Sjöholm, R.; Sundberg, K.; Willför, S. Acetylation and characterization of spruce (Picea abies) galactoglucomannans. Carbohydr. Res. 2010, 345, 810− 816. (35) Kim, H.; Ralph, J.; Akiyama, T. Solution-state 2D NMR of ballmilled plant cell wall gels in DMSO-d6. BioEnergy Res. 2008, 1, 56−66. (36) Rencoret, J.; Marques, G.; Gutierrez, A.; Nieto, L.; Santos, J. I.; Jimenez-Barbero, J.; Martinez, A. T.; del Rio, J. C. 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 2009, 63, 691−698. (37) Ralph, S. A.; Ralph, J.; Landucci, L. L. NMR Database of Lignin and Cell Wall Model Compounds, 2009; www.glbrc.org/databases_ and_software/nmrdatabase/. (38) Marita, J. A.; Ralph, J.; Hatfield, R. D.; Chapple, C. NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc. Natl. Acad. Sci. U. S. A. 1999, 96 (2), 12328−12332. (39) Li, S.; Lundquist, K. Cleavage of arylglycerol β-aryl ethers under neutral and acid conditions. Nord. Pulp Pap. Res. J. 2000, 15 (4), 292− 299. (40) Lundquist, K.; Parkås, J. Important Issues and Results from Knut Lundquist’s Lignin Research at Chalmers. J. Wood Chem. Technol. 2015, 35 (1), 3−7. (41) Yokoyama, T.; Matsumoto, Y. Revisiting the mechanism of b-O4 bond cleavage during acidolysis of lignin. Part 1: Kinetics of the formation of enol ether from non-phenolic C6-C2 type model compounds. Holzforschung 2008, 62, 164−168. (42) Samuel, R.; Cao, S.; Das, B. K.; Hu, F.; Pu, Y.; Ragauskas, A. J. Investigation of the fate of poplar lignin during autohydrolysis pretreatment to understand the biomass recalcitrance. RSC Adv. 2013, 3, 5305−5309. (43) Miles-Barrett, D.; Neal, A.; Hand, C.; Montgomery, J.; Panovic, I.; Ojo, O. S.; Lancefield, C. S.; Cordes, D. B.; Slawin, A. M. Z.; Lebl, T.; Westwood, N. J. The synthesis and analysis of lignin-bound Hibbert ketone structures in technical lignins. Org. Biomol. Chem. 2016, 14 (42), 10023−10030. (44) Leschinsky, M.; Zuckerstätter, G.; Weber, H. K.; Patt, R.; Sixta, H. Effect of autohydrolysis of Eucalyptus globulus wood on lignin

(4) Song, T.; Pranovich, A.; Holmbom, B. Hot water extraction of Ground Spruce Wood of Different Particle Size. BioResources 2012, 7 (3), 4214−4225. (5) Li, Z.; Qin, M.; Xu, C.; Chen, X. Hot Water Extraction of Hemicelluloses from Aspen Wood Chips of Different Sizes. BioResources 2013, 8 (4), 5690−5700. (6) Yelle, D. J.; Kaparaju, P.; Hunt, C. G.; Hirth, K.; Kim, H.; Ralph, J.; Felby, C. Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis. BioEnergy Res. 2013, 6, 211. (7) Visuri, J. A.; Song, T.; Kuitunen, S.; Alopaeus, V. Model for Degradation of Galactoglucomannan in Hot Water Extraction Conditions. Ind. Eng. Chem. Res. 2012, 51 (31), 10338−10344. (8) Ibbett, R.; Gaddipati, S.; Davies, S.; Hill, S.; Tucker, G. The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal−analytical and complementary studies. Bioresour. Technol. 2011, 102 (19), 9272−9278. (9) Zhu, M.-Q.; Wen, J.-L.; Su, Y.-Q.; Wei, Q.; Sun, R.-C. Effect of structural changes of lignin during the autohydrolysis and organosolv pretreatment on Eucommia ulmoides Oliver for an effective enzymatic hydrolysis. Bioresour. Technol. 2015, 185, 378−385. (10) Nitsos, C. K.; Choli-Papadopoulou, T.; Matis, K. A.; Triantafyllidis, K. S. Optimization of Hydrothermal Pretreatment of Hardwood and Softwood Lignocellulosic Residues for Selective Hemicellulose Recovery and Improved Cellulose Enzymatic Hydrolysis. ACS Sustainable Chem. Eng. 2016, 4, 4529−4544. (11) Borrega, M.; Nieminen, K.; Sixta, H. Effects of hot water extraction in a batch reactor on the delignification of birch wood. BioRes. 2011, 6 (2), 1890−1903. (12) Li, S.; Lundquist, K. Reactions of the b-Aryl Ether Lignin Model 1-(4-Hydroxy-3-Methoxyphenyl)-2-(2 Methoxyphenoxy)-1-Propanol on Heating in Aqueous Solution. Holzforschung 2001, 55, 296−301. (13) Schwartz, T. J.; Lawoko, M. Removal of acid-soluble lignin from biomass extracts using Amberlite XAD-4 Resin. BioRes. 2010, 5 (4), 2337−2347. (14) Chen, X.; Lawoko, M.; van Heiningen, A. R. P. Kinetics and mechanism of autohydrolysis of hardwoods. Bioresour. Technol. 2010, 101, 7812−7819. (15) Tunc, M. S.; Lawoko, M.; van Heiningen, A. R. P. Understanding the limitations of removal of hemicelluloses during autohydrolysis of a mixture of southern hardwoods. BioRes. 2011, 5 (1), 356−371. (16) Giummarella, N.; Zhang, L.; Henriksson, G.; Lawoko, M. Structural features of mildly fractionated Lignin Carbohydrate Complexes (LCC) from Spruce. RSC Adv. 2016, 6, 42120−42131. (17) Giummarella, N.; Lawoko, M. Structural Basis for the Formation and Regulation of Lignin−Xylan Bonds in Birch. ACS Sustainable Chem. Eng. 2016, 4 (10), 5319−5326. (18) Björkman, A. Studies on finely divided wood. Part 1. Extraction of lignin with neutral solvents. Svensk. Papperstidn. 1956, 59, 477−485. (19) Effland, M. J. Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi 1977, 60 (10), 143−144. (20) Davis, M. W. A Rapid Modified Method for Compositional Carbohydrate Analysis of Lignocellulosics by High pH AnionExchange Chromatography with Pulsed Amperometric Detection (HPAEC/PAD). J. Wood Chem. Technol. 1998, 18 (2), 235−252. (21) Azhar, S.; Henriksson, G.; Theliander, H.; Lindstrom, M. Extraction of hemicelluloses from fiberized spruce wood. Carbohydr. Polym. 2015, 117, 19−24. (22) TAPPI UM 250, Acid-soluble lignin in wood and pulp. In 1991 TAPPI Useful Methods; TAPPI: Atlanta, GA, 1991. (23) Appeldoorn, M. M.; Kabel, M. A.; Van Eylen, D.; Gruppen, H.; Schols, H. A. Characterization of oligomeric xylan structures from corn fiber resistant to pretreatment and simultaneous saccharification and fermentation. J. Agric. Food Chem. 2010, 58, 11294−11301. (24) Bertaud, F.; Sundberg, A.; Holmbom, B. Evaluation of acid methanolysis for analysis of wood hemicelluloses and pectins. Carbohydr. Polym. 2002, 48, 319−324. 5164


Research Article

ACS Sustainable Chemistry & Engineering structure. Part 1: Comparison of different lignin fractions formed during water prehydrolysis. Holzforschung 2008, 62 (6), 645−652. (45) Robert, D.; Chen, C.-L. Biodegradation in spruce wood by Phanerochaete chrysosporium: quantitative analysis of biodegraded spruce lignins by 13C NMR spectroscopy. Holzforschung 1989, 43, 323−332. (46) Freudenberg, K.; Neish, A. C. Constitution and Biosynthesis of Lignin; Springer-Verlag, Berlin, Heidelberg, Germany, 1968. (47) Du, X.; Perez-Boda, M.; Fernandez, C.; Rencoret, J.; del Río, J. C.; Jimenez-Barbero, J.; Li, J.; Gutierrez, A.; Martinez, A. T. Analysis of lignin−carbohydrate and lignin−lignin linkages after hydrolase treatment of xylan−lignin, glucomannan−lignin and glucan−lignin complexes from spruce wood. Planta 2014, 239, 1079−1090. (48) Balakshin, M. Y.; Capanema, E. A.; Chang, H-m MWL fraction with a high concentration of lignin carbohydrate linkages: Isolation and 2D NMR spectroscopic analysis. Holzforschung 2007, 61, 1−7. (49) Miyagawa, Y.; Mizukami, T.; Kamitakahara, H.; Takano, T. Synthesis and fundamental HSQC NMR data of monolignol βglycosides, dihydromonolignol β-glycosides and p-hydroxybenzaldehyde derivative β-glycosides for the analysis of phenyl glycoside type lignin-carbohydrate complexes (LCCs). Holzforschung 2014, 68 (7), 747−760. (50) Balakshin, M. Y.; Capanema, E. A.; Gracz, H.; Chang, H-m.; Jameel, H. Quantification of lignin−carbohydrate linkages with highresolution NMR spectroscopy. Planta 2011, 233, 1097−1110. (51) Toikka, M.; Sipilä, J.; Teleman, A.; Brunow, G. Lignin− carbohydrate model compounds. Formation of lignin−methyl arabinoside and lignin−methyl galactoside benzyl ethers via quinone methide intermediates. J. Chem. Soc., Perkin Trans. 1 1998, 1, 3813−3818.

5165


Structural Insights on Recalcitrance during Hydrothermal

Recommend Documents