Quantitative Structural Characters of Lignins Obtained from Residue after Hydrothermal Pretreatment

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1 Quantitative Structural Characters of Lignins Obtained from Residue after Hydrothermal Pretreatment Jia-Long Wen 1 Bai-Liang Xue 2 Feng Xu 3 Run-Cang Sun 4* 1 PHD student, Institute of Biomass Chemistry and Technology- Beijing Forest University, Beijing, China wenjialonghello@126.com 2 Master student, Institute of Biomass Chemistry and Technology- Beijing Forest University, Beijing, China xuebailiang67@163.com 3 Professor, Institute of Biomass Chemistry and Technology- Beijing Forest University, Beijing, China xfx315@163.com 4 Professor and director, Institute of Biomass Chemistry and Technology- Beijing Forest University, Beijing, China rcsun3@bjfu.edu.cn * Corresponding author Abstract Hydrothermally treated (HTT) process was demonstrated to increase enzymatic hydrolysis efficiency of lignocelluloses in the current bio-ethanol production. In this study, to maximize the utilization of enzymatic hydrolysis lignin (EHL), the quantitative information of its structural features were investigated. The structural features of the lignins obtained from different HTT species (softwood, hardwood and grass species) were characterized by FT-IR spectroscopy, gel permeation chromatography (GPC), quantitative 13 C and 2D HSQC nuclear magnetic resonance (NMR) spectroscopy. The results reveal that the typical structural features of lignin, such as β-o-4, β-β, and β-5 substructures, are preserved during HTT process. However, some condensed lignin structures from different materials were also detected, especially for pine wood. The degrees of lignin condensation (DC) was decreased in the order of pine (0.63) > birch (0.45) > bamboo (0.15). Interestingly, the lignin from HTT bamboo, p-coumaric acid was observed to acylated at γ-position of the lignin, suggested that the HTT process has slight effect on the native ester in the grass lignin. The molecular weights (M w ) of lignin preparations from various HTT biomasses (pine, birch, and bamboo) were 8860, 10840, and 9430 g/mol, respectively. In addition, the lignins isolated from HTT biomass have more uniform molecules, as revealed by low polydispersity values (M w /M n, ). Keywords: Lignin, Quantitative Structure, S/G ratio, β-o-4, HTT Paper EC-4 1 of 11

2 Introduction Biorefineries can provide the production of energy, fuels, chemicals and materials as well as food and feed components to enable a lastingly successfully production on the basis of renewable resources [1]. Motivating by the tendency of biomass refining, lignocellulosic material (LCM) can be fractionated into their main components by sequential treatments to give separate streams that may be used for value-added products exploitation. However, the utilization of LCM as chemical feedstock for fuels and chemicals is hindered by the inefficiency. The toughest obstacle of biomass utilization is tight and complex structure of biomass. Therefore, a number of pretreatments, such as including dilute acid, sulfur dioxide, ammonia expansion, and hydrothermal pretreatments, were develop to alleviate the natural obstacle and increase enzymatic hydrolysis [2]. Among these pretreatments, hydrothermal pretreatments are considered to be economically and environmentally attractive technologies aiming at fractionation and utilization of individual components of LCM. These processes have been used for a variety of biomass types and have been shown to work effectively at a commercial scale. The hemicelluloses-based oligosaccharides were firstly removed and collected in the first step [3]. The hydrothermally-treated wood residues were undergone enzymatic hydrolysis to obtain glucose for ethanol production. Furthermore, the enzymatic hydrolysis residues (EHR) are still rich in lignin and small amount of obstinate cellulose. The EHR should be explored as value-added materials to realize the dream of waste valorization. The objective of this work was firstly to compare qualitative and quantitative structural features of the isolated lignins from hydrothermally pretreated biomass. Materials and Methods Materials. Pinus yunnanensis (Softwood) and Betula alnoides (hardwood) was harvested in October 2010 from Yunnan Province (China). Bambusa rigida sp. (grasses) was obtained from Sichuan, China. The content of Klason lignin in Pinus, Betula, and Bambusa is 23%, 24% and 23%, respectively. All chemical reagents used were analytical grade or best available. Hydrothermal treatment (HTT). The ball-milled wood meals were initially presoaked in deionized water at a solid loading of 5%. Subsequently, the slurry containing water and wood meal was sealed in a 1.0 L high pressure and temperature stainless steel reactor (PARR, America). Then the reactor was purged three times with nitrogen to remove the air/oxygen in the reactor airspace. The ball-milled materials were firstly hydrothermally pretreated at 150 o C for 2 h. After the reaction completed, the reactor was cooled down to room temperature by cool water, which was installed inside of the reactor. Paper EC-4 2 of 11

3 Ball-milled Wood HT Residues Hydrothermally treated (HT), 150 o C, 2 h Enzymatic Hydrolysis Residues Incubuted with celluclast 1.5 L and Novozyme 188, ph 4.8, 48 h Extracted with 85%dioxane with 0.05 M HCl, S/L=1:30 Residue Filtrate Lignin preparations Concentrated, precipitated in to water (10 volumes), purified Figure 1. Scheme of lignin isolation from hydrothermally treated residues Preparation of enzymatic mild acidolysis lignins (EMALs). The EMALs were isolated according to a previous report [4]. The hydrothermally treated wood species (pine, birch and bamboo) (5 g) was suspended in acetate buffer (0.05 mol/l, 100 ml, ph 4.8), with the loading of 200 FPU cellulase (Celluclast 1.5 L) and 200 FPU β-glucosidase (Novozyme 188), respectively. The reaction mixture was incubated at 50 ºC in a rotary shaker (150 rpm) for 48 h. After enzymatic hydrolysis, the resulting enzymatic hydrolysis residues (EHRs), which contained carbohydrate impurities, was collected by centrifugation and washed with acidified water (ph=2) followed by freeze-drying. The HERs was then treated with 0.05 M HCl in acidic dioxane-water (85:15, v/v) at 86 o C under nitrogen for 2 hours, respectively. The resulting mixture was filtered and the lignin solution was collected. The solid residue was washed with fresh dioxane until filtrate was clear. The lignin solution and the combined washings were then neutralized with solid sodium bicarbonate. The neutralized solution was finally precipitated in a large quality of acidified water (10 volumes, ph=2) and then the precipitated lignin was collected by centrifugation followed by freeze-drying. To remove the carbohydrate remained in the lignin preparations, the lignins were firstly dissolved in 90% acetic acid, and then the lignin solutions were dropping into 10 volumes of water to induce precipitate. The precipitated lignins were washed with acidified water for several times and then freezedried. Associated Polysaccharides Analysis. The composition of structural carbohydrates was determined using National Renewable Energy Laboratory (NREL) protocol, and analyzed by high-performance anion exchange chromatography (HPAEC) (Dionex, ISC 3000, Paper EC-4 3 of 11

4 Sunnyvale, CA, USA) on a CarboPac PA 20 analytical column (4 250 mm) with pulsedamperometric detection. FT-IR Analyses. FT-IR spectra of lignin fractions were conducted using a Thermo Scientific Nicolet in10 FT-IR Microscope (Thermo Nicolet Corporation, Madison, WI) equipped with a liquid nitrogen cooled MCT detector. NMR Spectra of EMALs. NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer at 25 o C in DMSO-d 6. For the quantitative 13 C NMR experiments, 140 mg of lignin was dissolved in 0.5 ml DMSO-d 6. The quantitative 13 C NMR spectra were recorded in the FT mode at MHz. The inverse gated decoupling sequence (C13IG sequence from Bruker Standard Library), which allows quantitative analysis and comparison of signal intensities, was used with the following parameters: 30 o pulse angle; 1.4 s acquisition time; 2 s relaxation delay; 64 K data points, and 30,000 scans. To achieve sufficient relaxation in a feasible time, 20 μl of chromium (III) acetylacetonate (0.01 M) was added as a relaxation agent for the quantitative 13 C spectrum to reduce the relaxation delay according to a previous report [5]. Results and Discussion To understand the quantitative structure and thermal properties of lignins obtained from residue after hydrothermal pretreatment, the purified lignin should be obtained. Herein, the EMAL was isolated according to the existing literature [4]. The carbohydrate contents of EMALs isolated from hydrothermally treated lignocellulosic materials (pine, birch, and bamboo) are given in Table 1. It was found that the carbohydrate contents of the lignin preparations varied with different raw-materials. However, the carbohydrate contents in the lignins are low, which will not hinder the structural analysis of lignin in the subsequent sections. Table 1. Yield and carbohydrate contents of lignin preparations Samples Sugar (%) Ara Gal Glu Xyl Man GlcA Pine 1.63 Tr Birch 1.29 Tr Bamboo 3.46 Tr Tr 0.29 FT-IR Characterization. The fingerprint region of FT-IR spectra of the EMALs (Figure 2) after HTT exhibits typical lignin patterns. The bands at 1593, 1507, and 1421 cm -1, corresponding to aromatic skeletal vibrations and the C H deformation combined with aromatic ring vibration at 1462 cm -1, are present in these three spectra [6]. For HTT softwood (pine), the composed band system between 1175 and 1065 cm -1 showed a maximum at 1138 cm -1. This feature is typical only for G lignins. Another, for hardwood (birch), the same band system showed a maximum at about 1125 cm -1. This is sensitive criterion for GS lignins, indicating the HTT birch lignin belongs to typical GS lignin. However, a band around at 1167 cm -1 (C=O vibration of esters) is additionally present in the spectrum of bamboo. Moreover, the whole C=O range between cm -1 is Paper EC-4 4 of 11

5 intense in the spectrum of bamboo. Another intense band at 834 cm -1 further indicated that typical HGS lignins of the bamboo. Figure 2. FT-IR spectra of HTT lignins from pine, birch, and bamboo Estimation of Different Lignin Moieties from Quantitative 13 C NMR Spectra. The integral of the ppm region was set as the reference, assuming that it includes six aromatic carbons and 0.12 vinylic carbons [7]. It follows that the integral value divided by 6.12 is equivalent to one aromatic ring. Figure 3 displays the quantitative 13 C NMR spectra of the HTT lignins, and Table 2 gives the quantification of the most important lignin moieties. The methoxy groups of the lignins determined by quantitative 13 C NMR spectra showed 0.91 (pine EMAL), 1.37 (birch EMAL), and 1.18 (bamboo EMAL) methoxy groups per aromatic ring. Compared with the existing results for methoxy groups of hardwood, softwood and grass, the data presented here is slightly lower, the decrease of aromatic methoxy content is attributed to the demethoxylation during HTT process. In addition, lignin structural moieties, such as aromatic C-O, aromatic C-C, and aromatic C-H, were determined based on quantitative 13 C NMR spectra. Furthermore, the content (expressed by per Ar) of lignin inter-linkage β-o-4, β-β, and β-5 were calculated. It was observed that the content of β-β and β-5 is higher in pine than that of in birch and bamboo, suggested that more condensed lignin units presented in the HTT pine EMAL. The degrees of lignin condensation (DC) were also calculated according to Capanema et al. [8, 9]. The DC values were 0.63, 0.45, and 0.15 for the lignins of pine, birch and bamboo, respectively. This also suggested that pine lignin is more condensed. Paper EC-4 5 of 11

6 Figure 3. Quantitative 13 C-NMR spectra of HTT lignins from pine, birch, and bamboo Table 2. Main chemical shift assignments in the quantitative 13 C-NMR spectra of nonacetylated HTT lignins Quantification δ (ppm) Assignments Pine Birch Bamboo Aromatic C-O Aromatic C-C Aromatic C-H OCH β-o C β in β-β, β a DC a DC, Degree of condensation. For pine wood, DC was calculated by ( h-units) - [(I ) + M + 2 I]; For birch and bamboo, s + g + h=1 (S, G, and H units were calculate from 2D), I theor CAr-H = (2s + 3g + 2h), DC=I theor CAr-H -I ppm. Paper EC-4 6 of 11

7 2D-HSQC NMR analysis. The inter-linkage and aromatic regions of the HSQC NMR spectra of the three EMALs are shown in Figures 4 and 5. The side-chain region (δ C /δ H 50-90/ ) of the 2D HSQC NMR spectra provided useful information about the inter-coupling bonds present in lignin (β-o-4, β-β, β-5, etc.). As shown in Figure 4, the side-chain regions of the three EMALs in the HSQC spectra were similar except for the associated carbohydrates. The important correlations, such as those from substructures of β-ether (β-o-4) A, resinol (β-β) B, and phenylcoumaran (β-5) C can be readily assigned according to the recent literatures [10]. Table 3. Assignments of 13 C- 1 H cross signals in the HSQC spectra of HTT lignins Lable δ C /δ H (ppm) Assignments C β 53.1/3.46 C β H β in phenylcoumaran substructures (C) B β 53.5/3.07 C β H β in β-β (resinol) substructures (B) OCH /3.70 C H in methoxyls A γ 59.9/3.35 C γ H γ in β-o-4 substructures (A) A γ 59.9/3.80 C γ H γ in β-o-4 substructures (A) A γ' 63.1/4.28 C γ H γ in γ-acylated β-o-4 substructures (A ) B γ 71.8/3.81 C γ H γ in β-β resinol substructures (B) B γ 71.8/4.17 C γ H γ in β-β resinol substructures (B) A α 72.0/4.85 C α H α in β-o-4 substructures linked to a S unit (A) A β (G/H) 83.5/4.32 C β H β in β-o-4 substructures linked to a G unit (A) B α 84.7/4.64 C α H α in β-β (resinol) substructures (B) A β (S) 86.0/4.11 C β H β in β-o-4 substructures linked to S (A, erythro) A β (S) 86.8/3.96 C β H β in β-o-4 substructures linked to S (A, threo) C α 87.6/5.50 C α H α in phenylcoumaran substructures (C) S 2, /6.72 C 2,6 H 2,6 in syringyl units (S) S 2, /7.27 C 2,6 H 2,6 in oxidized (C α OOH) syringyl units (S ) G 2 G / /6.71 C 2 H 2 in guaiacyl units (G) C 5 H 5 in guaiacyl units (G) G /6.72 C 6 H 6 in guaiacyl units (G) H 2, /7.17 C 2,6 H 2,6 in H units (H) PCE 3, /6.83 C 3,5 H 3,5 in p-coumarate PCE 2, /7.48 C 2,6 H 2,6 in p-coumarate PCE /7.51 C 7 H 7 in p-coumarate PCE /6.29 C 8 H 8 in p-coumarate The composition of the lignins is clearly evidenced in the aromatic region of these 2D- HSQC NMR spectra (Figure 5). For birch and bamboo, a significant correlation for the syringyl units (S) was observed at δ C /δ H 103.8/6.69 ppm, while their oxidized (α-ketone) structures S' appeared at δ C /δ H 106.2/7.28 ppm. With the exception of S units, all of the guaiacyl components which are in the lignins can be expressly distinguished. The correlation for the G 2 -position is at δ C /δ H 110.7/6.96 ppm. In addition, some condensed G 2 was also found at lower field (labeled as condensed structural in Figure 5) [11]. For pine EMAL, G 2 and G 6 was observed to be more condensed. The observations were in agreement with the results obtained from 13 C-NMR spectra, in which the degrees of Paper EC-4 7 of 11

8 lignin condensation was decreased in the order of pine (0.63) > birch (0.45) > bamboo (0.15). Specially, the assignments were listed in Table 4 and the major structural features are depicted in Figure 6. Figure 4. The side-chain regions in 2D HSQC NMR spectra of the lignins Figure 5. The aromatic regions of 2D HSQC NMR spectra of the lignins Paper EC-4 8 of 11

9 Figure 6. Main lignin substructures in the treated bamboo samples (A) β-o-4 linkages; (A') acylated β-o-4 substructures; (B) resinol structures formed by β-β/α-o-γ/γ-o-α linkages; (C) phenylcoumarane structures formed by β-5/α-o-4 linkages; (S) syringyl unit; (S') oxidized syringyl unit linked a carbonyl or carboxyl group at Cα (phenolic); (H) p-hydroxyphenyl units; (F) p-hydroxycinnamyl alcohol end groups; (G) guaiacyl unit; (G') oxidized guaiacyl units with a Cα ketone; (FA) ferulate; (p-ca) p-coumarate. Molecular Weights Analysis Table 4. Weight-average (M w ) and number-average (M n ) molecular weights (g/mol) and polydispersity (M w /M n ) of the lignin preparations M w M n M w /M n Pine Birch Bamboo Paper EC-4 9 of 11

10 Table 4 shows the weight-average (M w ) and number-average (M n ) molecular weights and polydispersity index (M w /M n ) of lignin preparations isolated from HTT biomass. The molecular weights (M w ) of lignin preparations from various HTT biomasses (pine, birch, and bamboo) were 8860, 10840, and 9430 g/mol, respectively. In addition, the lignin preparations isolated from HTT biomass have more uniform molecules, as indicated by low polydispersity values (M w /M n, ). Conclusion Results obtained reveal that the typical structural features of lignin, such as β-o-4, β-β, and β-5 substructures, are preserved during HTT process. However, some condensed lignins from different materials were detected, especially for pine wood. The degrees of lignin condensation (DC) was decreased in the order of pine (0.63) > birch (0.45) > bamboo (0.15). Interestingly, the lignin from HTT bamboo, p-coumaric acid was observed to acylated at γ-position of the lignin, suggested that the HTT process has slight effect on the native ester in the grass lignin. The molecular weights (M w ) of lignin preparations from various HTT biomasses (pine, birch, and bamboo) were 8860, 10840, and 9430 g/mol, respectively. In addition, the lignins isolated from HTT biomass have more uniform molecules, as revealed by low polydispersity values (M w /M n, ). The well-characterized lignins will prompt us to utilize the residue after enzymatic hydrolysis. References 1. Lyko, H., Deerberg, G., Weidner, E Coupled production in biorefineriescombined use of biomass as a source of energy, fuels and materials. Journal of Biotechnology.142: Yang, B., Wyman, C.E Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioproducts & Biorefining-Biofpr. 2: Tunc M, Sefik., van Heiningen Adriaan, R. P Hemicellulose Extraction of Mixed Southern Hardwood with Water at 150 C: Effect of Time. Industrial & Engineering Chemistry Research. 47 (18): Jaaskelainen, A.S., Sun, Y., Argyropoulos, D.S., Tamminen, T., Hortling, B The effect of isolation method on the chemical structure of residual lignin. Wood Science and Technology. 37 (2): Yuan, T.Q., Sun, S.N., Xu, F., Sun, R.C Characterization of lignin structures and lignin-carbohydrate complex (LCC) linkages by quantitative 13 C and 2D HSQC NMR. Journal of Agricultural and Food Chemistry. 59: Faix, O. (1991) Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 45: Chen, C.L. Characterization of milled wood lignins and dehydrogenative polymerisates from monolignols by carbon-13 NMR spectroscopy. In Lignin and Lignan Biosynthesis; ACS Symposium Series 697; Lewis, N., Sarkanen, S., Eds.; American Chemical Society: Washington, DC, 1998; pp Capanema, E.A., Balakshin, M.Y., Kadla, J.F A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. Journal of Agricultural and Food Chemistry. 52, Paper EC-4 10 of 11

11 9. Capanema, E.A., Balakshin, M.Y., Kadla, J.F Quantitative characterization of a hardwood milled wood lignin by nuclear magnetic resonance spectroscopy. Journal of Agricultural and Food Chemistry. 53, Kim, H., Ralph, J Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d 6 /pyridine-d 5. Organic & Biomolecular Chemistry. 8: Shuai, L., Yang, Q., Zhu, J.Y., Lu, F.C., Weimer, P.J., Ralph, J., Pan, X.J Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production. Bioresource Technology. 101: Paper EC-4 11 of 11