Valorization of lignin-carbohydrate complexes from hydrolysates of

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Valorization of lignin-carbohydrate complexes from hydrolysates of Norway spruce: Efficient separation, structural characterization, and antioxidant activity Yongchao Zhang, Singhi Wang, Wenyang Xu, Fang Cheng, Andrey Pranovich, Annika Ingegärd Smeds, Stefan M. Willför, and Chunlin Xu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05142 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Sustainable Chemistry & Engineering

Valorization of lignin-carbohydrate complexes from hydrolysates of Norway spruce: Efficient separation, structural characterization, and antioxidant activity

Yongchao Zhang†, Singhi Wang†, Wenyang Xu†, Fang Cheng‡,§,Andrey Pranovich†, Annika Smeds†, Stefan Willför†, Chunlin Xu†,* †Johan

Gadolin Process Chemistry Centre, c/o Laboratory of Wood and Paper Chemistry,

Åbo Akademi University, Turku FI-20500, Finland ‡School

of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510006,

China §Cell

Biology, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6,

Turku 20520, Finland *Corresponding authors: E-mail: [email protected] (C. Xu)

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ABSTRACT Hot-water extraction has shown a great potential in a green biorefinery, however, efficient separation of lignin and carbohydrates is still a big challenge, especially to lignin-carbohydrate complexes (LCCs). The aim of present study was to develop a facile approach for efficiently fractionating LCCs from hot-water extraction liquor of spruce wood, in order to understand their comprehensive structure and thus to achieve the valorization of fractions. Approximately 93% of the hemicelluloses were first recovered by a selective adsorptive resin from hot-water hydrolysates. The lignin-rich fractions obtained from desorption of the adsorbed compounds were further subjected to a gradient dialysis procedure towards functional fractionation. Linear relationship between lignin and uronic acids as well as xylose contents in graded fractions of all hydrolysates was observed. Further characterization of LCCs by 2D NMR and py-GC-MS showed the large difference of their structures in each graded fractions and suggested ester bonds as the main types of chemical linkages between lignin and carbohydrate. Furthermore, the antioxidant properties of those LCCs as well as hemicellulose fractions will facilitate their applications to a broader spectrum such as cosmetics, pharmaceuticals, and functional packaging. Keywords: Norway spruce, hot-water extraction, lignin-carbohydrate complexes (LCCs), functional fractionation, structure analysis, antioxidant activity

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INTRODUCTION Lignocellulosic biomass, the most abundant natural resource, has become one of the primary feedstocks for the production of biomaterials, energy, and platform chemicals on the basis of renewability and sustainability. It is mainly composed of cellulose, hemicelluloses, and lignin. Recently, biorefinery has been emerged as an important platform to produce new valueadded products from lignocellulosic biomass.1, 2 To achieve an integrated biorefinery process on industrial scale, different pretreatment approaches, such as enzymatic hydrolysis, ammonia pretreatment, organic acid prehydrolysis, and alkaline pretreatment, have been investigated.3-6 Nevertheless, the challenge still lies in the need of an environmentally friendly and economically feasible fractionation process to ensure valorization of all fractions of the lignocellulosic biomass. Hot-water extraction, which is catalyzed by hydronium ions generated by acetic acid produced from the cleavage of acetyl groups, has been considered as an effective process to extract hemicelluloses.7-9 Hot-water extraction affords excellent techno-economic advantages, as no chemicals other than water is added in the whole process.10-12 It was earlier found that 8090% galactoglucomannans (GGMs) could be extracted from spruce wood by pressurized hotwater extraction at temperatures up to 170 °C for 60 min,13 but other compounds, including parts of lignin and lignin-carbohydrate complexes (LCCs) were also liberated into the hot-water extraction liquors.14,

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It was reported that LCCs limit the separation of lignin and

carbohydrates in hot-water extraction liquors and also hinder enzymatic hydrolysis of hemicellulosic sugars for biofuel production.16, 17 However, lignin and LCCs generated during the hydrolysis process can be considered as high added-value compounds, showing potential applications in medicine and cosmetic formulations on the basis of diverse unique pharmacological activities and novel natural antioxidants.17-20

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Nowadays, the interest in lignin and LCCs recovery from hot-water extraction liquor has been increasing. A number of methods for the separation of these aromatic compounds, such as precipitation,14 membrane filtration,21 and adsorption,22-24 have been presented. Among these methods, the adsorption using activated carbon and XAD resins showed high efficiency and selectivity for the purification of complex mixtures. Contrary to activated carbon, XAD resins not only exhibit an efficient adsorption capability, but also allow more effective desorption of the adsorbed compounds with washing solvent, such as acetone, methanol and ethanol.25-28 Schwartz and Lawoko27 achieved 90% removal of acid-soluble lignin from hemicellulose rich hydrolysates of lignocellulose using Amberlite XAD-4 resin, and 85% of the adsorbed lignin could be recovered with 75% acetone. Importantly, previous works found that XAD resins also displayed a strong adsorption capacity for LCCs from biomass hydrolysates.29, 30 Narron et al.31 recovered approximately 90% of the soluble lignin from a hardwood and a non-wood biomassbased autohydrolyzate, and found that the isolated lignin adsorbents featured of about 10 total LCC per 100 aromatic rings using spectroscopic analysis. Structural characterization of the complexes obtained from biorefinery pretreatment hydrolysates, including lignin compounds and LCCs, has been investigated to some extent by modern instrumental analytical approaches, especially two-dimensional nuclear magnetic resonance (2D NMR) which allows direct detection of various LCC linkages.30, 32-34 Furthermore, the antioxidant activity can be evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing activity power (FRAP) methods.32, 35, 36 However, they are still underutilized byproducts of the biorefinery process due to the challenges in separation and applications. In the present study, spruce sawdust was extracted with hot water at different conditions to obtain the extraction liquors. A combined fractionation process, consisting of an XAD resin adsorption followed by gradient dialysis process (Figure 1), was performed for separation of 4


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hemicellulose and lignin-rich fractions and further gradient fractionation of LCCs. The chemical composition of different fractions was analyzed and compared thoroughly to understand the structure correlation. Moreover, structural characterization of LCCs was comprehensively conducted, using different analytical technologies. Evaluation of the antioxidant activities of different fractions was also carried out to assess their potential in highvalue applications. MATERIALS AND METHODS Materials. Wood sawdust (40-60 mesh), which was prepared from Norway spruce (Picea abies Karst) obtained from Southwest Finland, was selected as the raw material in this study. It was extracted with acetone in a Soxhlet apparatus for 4 h. Extractive-free sawdust was airdried and stored in the freezer. The composition of the extractives-free sawdust, which was measured by the TAPPI standard, was cellulose 42.0%, hemicelluloses 26.1%, and lignin 27.8%. XAD-7 Amberlite ® resin was purchased from Sigma Aldrich. Two dialysis membranes: one with a cut-off of 12-14 kDa was purchased from Medicell International Ltd., UK and a cellulose ester membrane with a cut-off of 3.5-5 kDa was purchased from Spectra/Por® Biotech, Spectrum Laboratories, Inc.. All chemicals used were of analytical or reagent grade. Hot-water extraction. The process scheme of the extraction and separation of hemicelluloses and LCCs is depicted in Figure 1. Hot-water extraction was performed using distilled water in an accelerated solvent extractor ASE-300 apparatus (Dionex, Sunnyvale, CA, USA). About 45 g of dry extractives-free sawdust was weighted in a stainless-steel cell equipped with glass microfiber filters (Ahlstrom, Mt. Holy Springs, USA) and extracted with 300 mL distilled water at 120 °C and 160 °C for 20 min and 60 min, respectively. After the reaction, the extract solution was purged out with nitrogen and rinsed with 100 mL distilled water. The obtained water extracts were stored at 4 °C in the dark.

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Fractionation of lignin-carbohydrate complexes. As shown schematically in Figure 1, fractionation of a hemicellulose-rich fraction and LCCs from the extracts was done using a column packed with XAD-7 resin. First, the packed column was rinsed with methanol using 3 volumes of the column and then the column was conditioned by elution with an aqueous solution 2 M HCl prior to each extraction. Extracts were acidified to pH 2 with aqueous HCl and were passed through the column and the respective hydrophilic effluent, i.e. hemicelluloserich fraction (HF), was collected. Then by washing the column with methanol, LCC was obtained from the methanol solution (methanol fraction: MF). HFs were stored in the dark at 4 °C and some droplets of toluene were added as a bacterial inhibitor. Distillated water was added to each MF (1:1 v/v) and aqueous NaOH was used to neutralize the solutions to pH ~5-6. Each MF was condensed to 100-200 mL by evaporating in a rotary evaporator at 45 °C. Further fractionation of LCCs was performed using two dialysis steps. The first step was performed using a membrane with a cut-off of 12 000-14 000 Daltons. Both retentate I (Ret I) and the permeate were collected. The permeates were evaporated in a rotary evaporator at 45 °C and further dialyzed using a membrane with a cut-off of 3 500-5 000 Daltons. Both retentates and permeates were collected and coded as retentate II (Ret II) and permeate II (Perm II), respectively. Characterization. Carbohydrate analysis of different fractions was conducted by gas chromatography (GC) after mehanolysis as reported earlier.37 Briefly, the dried samples were hydrolyzed using water-free acidic methanol at 100 ºC for 5 h. After the acid methanolysis process, 1 mL of 0.1 mg/mL sorbitol and resorcinol were added as the internal calibration and then the samples were evaporated under a nitrogen flow in a 50 ºC water bath. After drying, the samples were silylated using pyridine, hexamethyldisilzane (HMDS) and trimethylchlorosilane (TMCS) reagents and then transferred to GC vials. The silylated samples were analyzed by GCFID. 6


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Lignin and lignin-related substances in the aqueous solutions were measured by ultraviolet-visible (UV-VIS) spectroscopy at 280 nm after dilution with water until the absorbance value was in the range of 0.3–0.7 cm-1. The lignin extinction coefficient used in the measurements was 56 cm mg/L.38 Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) analysis of the fractions was performed with a filament pulse resistance-heated pyrolyser Pyrola 2000 (Pyrol AB, Lund, Sweden) connected to an HP 6890-5973 GC-quadrupole-MSD instrument (Hewlett-Packard, Palo Alto, CA) equipped with a ZB-35 column of 30 m × 0.25 mm i.d. with 0.25 μm film thickness (Phenomenex). The pyrolysis of 100 μg of each sample was performed for 2 s at 580 °C. The GC oven temperature was programmed from 50 °C (0.5 min) to 260 at 6 °C min−1 and then to 300 °C (8 min) at 20 °C min-1. Pure helium was the carrier gas with a constant flow rate of 1 mL/min-1. The 2D Heteronuclear Single-Quantum Correlation (HSQC) spectra of the LCCs were recorded with a 500 MHz Bruker Avance instrument, fitted with a 5-mm broadband probe with a gradient field in the Z-direction at room temperature in deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. 80 mg of sample was placed into a 5 mm NMR tube and dissolved in 0.75 mL of DMSO-d6. A standard Bruker HSQC pulse sequence, “hsqcedegpsisp2.3,” was used. Molar weight distribution was determined by dissolving 3 mg of freeze-dried sample in DMSO and analyzing the solutions by a HPLC instrument (Agilent 1100 Series) equipped with UV and RI detectors (Shimadzu Corp., Tokyo, Japan). A dn/dc value of 0.04 mL/g was used. To estimate the molar mass of the samples, polysaccharides of pullulan were applied as the calibration. All the data were generated by ASTRA software (Wyatt Technology). Antioxidant activity. Antioxidant activities were measured with the Trolox Equivalent Antioxidant Capacity (TEAC) Assay Kit (ABTS) Antioxidant Assay Kit (Sigma., MO, USA) 7



according to manufacturer’s instruction. Trolox Standards were used to make a standard curve, and assays were performed in duplicate. Antioxidant levels of the test samples should fall within the range of the standard curve, and when necessary, the samples were diluted with 1´Assay Buffer prior to the assay to bring the antioxidant level within range. RESULTS AND DISCUSSION Chemical composition of fractions. The XAD resin selectivity for aromatics has previously been demonstrated.27,

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Herein, Figure 2a depicts separation efficiency of

hemicelluloses by XAD-7 resin sorption from hot-water extraction liquor obtained under different conditions. As seen, the recovery rate of carbohydrates from extract solutions (ETH) was from 77% to 93%. 93% of the hemicelluloses were recovered from ETH at higher temperature as well as longer residence time. At 120 °C after 20 min, extraction of spruce sawdust with water extracted only 3.51 mg/g wood while extraction at 160 °C for 60 min favored the dissolution of non-cellulosic carbohydrates, reaching a yield of 122.52 mg/g wood. Previous studies have shown that using hot water at a higher temperature can promote the depolymerization of hemicelluloses.7,

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The main constituent of the HF was mannose,

approximately 39.5–50.7 wt% of the total carbohydrates, but other sugars, such as xylose, glucose, arabinose and galactose, were also present. However, the methanol fractions (MFs) still contained a part of the carbohydrates, which are most likely covalently attached to lignin moieties since pure carbohydrates cannot be adsorbed by the resin. Figure 2b shows the components and contents of carbohydrates, which were obtained from MFs after the sorption on XAD-7 resin. The MFs were separated by a two-step dialysis process. The carbohydrates in different fractions after dialysis were mainly composed of mannose and uronic acids, which increased significantly with the extraction time and temperature. After a gradient process using dialysis, approximately 47–83% of the carbohydrates obtained from the MFs was contained in the Ret I fractions, while only 1.7–7.5% were present in the Ret II fractions under various 8


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extraction time and temperature. Simultaneously, the major amount of lignin fractions were collected in the Perm II fractions, and the percentage of lignin contents increased with the decrease of dialysis area as compared with carbohydrates (Figure 2c). As can be seen in Figure 2d, the content of lignin in different fractions increased with extraction temperature and time. Though the sorption on XAD-7 resin showed a good preference for lignin structures, an appreciable amount of lignin containing compounds was found in the hemicellulose fraction. The percentage of lignin in the hemicelluloses fraction was nevertheless negligible. We assumed that a certain amount of LCCs is present in the methanol fractions and could be further fractionated by dialysis process according to their size. To confirm this hypotheses, linear correlation analysis was used to investigate the relationship between the lignin and carbohydrates contents in the Ret I, Ret II, and Perm II fractions. There is a linear relationship between lignin and the carbohydrate content in the dialysis process if the existing LCCs can be fractionated into different fractionations. As can be seen in Figure 3, the lignin content was significantly positively correlated with the uronic acid content and strong positive correlations with the xylose content clearly indicate that during the dialysis process the LCCs present in MF was fractionated according to their size. Galacturonic acid showed a higher correlation coefficient with lignin at most extraction conditions. Linear relationships between lignin and xylose content show a higher correlation coefficient (R2) of 99.63% (Figure 3b). Figure 3d shows that the correlation coefficients (R2) between lignin and glucuronic acid or galacturonic acid were 97.72% and 91.10%, respectively. Overall, the linear correlation analysis confirmed our hypotheses and suggested that xylan and uronic acid, including glucuronic acid and galacturonic acid, are the important carbohydrates linked in LCCs. A previous study by Giummarella and Lawoko also demonstrated that LCCs structures contained a large amount of uronic acids and xylan.30 It is also noticed that correlation coefficient of lignin and xylose

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increased over extraction time at each temperature, indicating that xylans which were not directly linked with lignin were hydrolyzed with time and did not sorb on XAD-7 resin. Structural characterization Pyrolysis GC-MS Analysis. To facilitate discussion, fractionated samples were labelled using number1, 2, 3, 4,which is placed behind the sample abbreviation name, e.g. Ret I-4, to represent their extraction conditions at 120 °C for 20 min, 120 °C for 60 min, 160 °C for 20 min and 160 °C for 60 min, respectively. After two-step dialysis fractionation process, structures of the lignin-rich fractions were identified by analytical pyrolysis coupled to gas chromatography and mass spectrometry detection. In general, the first part of the pyrograms in the range 3–7 min is dominated by the pyrolysis products of carbohydrates, while a complex mixture of phenolic products from lignin thermal cleavage are present in the range of 7–27 min. The total pyrograms of different fraction samples are shown in Figure 4. The identification of the peaks and relative abundances of all pyrolyzed products are listed in Table 1. The attribution of the origin of each chromatographic peak was based on previously published data.33, 41 Lignin structures could be characterized by the various phenol (P) and guaiacol (G) unit fragments detected from different fractions. Upon comparison of the relative abundance of fragmentation patterns between different dialysis processes, a large increase in the G/P ratio could be noticed, being 4.61, 19.56 and 27.51 in Ret I-4, Ret II-4 and Perm II-4 fractions, respectively. Moreover, there is no noticeable change in the G/P ratio of the Perm II fractions obtained from different extraction conditions. As can be seen from Figure 4, the pyrolyzed LCC fractions present a higher content of phenolic -OH group due to the cleavage of the ether bonds in the lignin structure during the pyrolysis, resulted in new phenolic hydroxyl groups were released. Among the pyrolytic products, the major products presented in different fraction samples were generated from the G lignin-units, but the abundances of the various pyrolytic lignin fragments were quite different 10


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(Table 1). The most abundant lignin pyrolytic fragments were guaiacol (G, 17.26%) and 4vinylguaiacol (G-vinyl, 16.84) for Ret I-4 fractions, and guaiacol (G, 30.83%) and 4vinylguaiacol (G-vinyl, 19.02%) for Ret II-4 fractions, and 4-vinylguaiacol (G-vinyl, 7.46) vanillin, (G-CHO, 20.44%), dihydroconiferyl alcohol (G-C-C-C-OH, 16.47) and coniferyl aldehyde (G-C═C-CHO, 25.98%) for Perm II-4 fractions. It has been reported that the relative content of 4-methylguaiacol, which was formed as a result of C-C cleavage in side chains, could be used for the evaluation of the content of CH2 groups in the α-position of the side-chains of phenylpropane units.42 From the lignin fragments, a significant increase in the amounts of all Cα carbonyl structures was observed, reaching 24.36% in Perm II-4 fractions compared with 7.36% and 10.06% in the Ret I-4 and Ret II-4 fractions, respectively (Table 1). However, Perm II-1 fractions, which were originated from relative mild extraction conditions at the hot-water extraction stage, contains only small amounts of Cα carbonyl structures (11.32%) compared with Perm II-4 fractions. Here these carbonyl groups could be formed from the corresponding phenylpropane units as a result of the cleavage of Cα-Cβ or Cβ-Cγ bonds.43 Previous studies had shown that the above-mentioned structural descriptors could be used for evaluation of the antioxidant activity of different lignin-rich fractions.42 On the basis of the observations from this study and as reported in the literature,42 we could conclude that the differences in content of pyrolysis products structures imply that there are differences in the antioxidant activity of the original structures present in the sample. Moreover, the various pyrolytic fragments derived from carbohydrates were observed in the Ret I-4 fractions, and several carbohydrate fragments were also noticed in the Ret II-4 fractions during the pyrolysis, but no such signals were detected in Perm II fractions. There was also difference in the abundance of the pyrolytic products derived from carbohydrates, such as methyl acetate (7.51%) and 2-furanmethanol (7.85%) in the Ret I-4 fractions, and methyl

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ethanoate (6.01%) in the Ret II-4 fractions. These results also reveal the efficient and selective separation of LCCs, which may contribute to obtaining well-defined and fractionated products. 2D-HSQC NMR spectra of the LCCs. To further decipher structural differences and possible linkages between lignin and carbohydrates in the lignin-rich fractions, the 2D-HSQC NMR technique was applied. The HSQC spectra are presented in Figure 5(b-g) and the main substructures are depicted in Figure 5a. The main cross-signal assignments in both side-chain regions and aromatic regions are identified by comparison with literature data.34, 44, 45 As shown in the Figure 5, the spectrum of the Ret I-4 fractions presents strong and relatively complete signals as compared to the Perm II fractions (Perm II-1 and Perm II-4), and the spectra of four Perm II fractions were considerably similar to each other (Figure S1). In the side-chain region (Figure 5b, d, f), the cross-signals of methoxyl groups (OMe, δC/δH=71.8/4.86 ppm) and β-O-4 substructures (A) were the most prominent, as reported by a previous study.31, 32 The Cα-Hα (Aα, δC/δH=71.8/4.86 ppm), Cγ-Hγ (Aγ, δC/δH=60.10-60.52/3.20-3.40 ppm) and Cβ-Hβ (Aβ, δC/δH=84.2/4.30 ppm) correlations in β-O-4′ substructures (A) were observed in the side-chain region of the HSQC spectrum. Phenylcoumaran substructures (C) were indicated by the signals of Cα-Hα and Cβ-Hβ correlations at δC/δH=87.48/5.44 and 53.62/3.47. There was also a small amount of signals corresponding to resinol (β-β, B) correlations at δC/δH=54.11/3.06 and 53.68/3.47 ppm. According to previous studies, predominant signals corresponding to various types of chemical linkages between lignin and carbohydrates in LCCs can be identified from 2D HSQC cross-signals, and the main types are phenyl glycoside, benzyl ether, and ester linkages.30, 31, 34, 44

As compared to Perm II fractions, the spectrum of Ret I-4 fractions shows predominant

signals corresponding to various types of chemical linkages between lignin and carbohydrate in LCC structures. It can be noted that the cross-signals of xylans in ester structures were clearly observed through the C1-H1, C2-H2, C3-H3, C4-H4, and C5-H5 correlations from β-D12


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xylopyranoside (X1, X2, X3, X4, and X5) in the Ret I-4 fractions. It has been found that the same linkages in LCCs structures from poplar are also more abundant than other linkages.33, 41 The cross-signals of C1-H1, C3-H3 and C4-H4 correlations from α-L- arabinofuranoside (Ara1, Ara3 and Ara4), and C1-H1 correlations from β-D-mannopyranoside (Man1) could be detected. There were also some small amount of signals corresponding to β-D-glucopyranoside (Glc1) and 4-O-methyl-α-D-glucuronic acid (GlcA1). Previous publications also reported that the LCCs structures contained a higher amount of ester linkages between lignin and hemicellulose than the ether linkages.31, 32 However, the intensive cross-signals, such as X2, X3, Ara3 and Man1, could not be detected anymore due to their low-frequency, and only a part of signals of X4 and X5 were found in the spectra of the Perm II-4 fractions. It suggested that Perm II-4 fractions contained less LCCs. The signals of the C2-H2 correlations from 2-O-acetyl-β-Dxylopranoside (X22) and C3-H3 correlations from 3-O-acetyl-β-D-xylopranoside (X33) were observed in the HSQC spectra of Perm II-4 fractions, while these signals could not be found in Ret I-4 fractions. These results reveal that the LCC structures with the multi-linkages were remained in the Ret I-4 fractions by dialysis process. In addition, the signals of the C2-H2, C5H5 and C6-H6 correlation in G lignin units (G2, G5 and G6) were observed in the aromatic regions of the spectra in the Ret I-4 and Perm II-4 fractions. The molecular weights of different fractions were determined by gel-permeation chromatography. The weight-average (Mw) and retention time are listed in Table 2. As compared to the weight-averaged molecular weight (Mw) of the Perm II fractions (Mw = 455738 g/mol) at different hydrolysis conditions, Ret I fractions (Mw = 1645-6659 g/mol) and Ret II (Mw = 743-1224 g/mol) remained the macromolecular compounds in lignin-rich fractions ascribed to the dialysis processes. This result further confirms the effective gradient fractionation using the multi-dialysis process according to their physical size.

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Antioxidant capacity of different fractions. In order to stimulate the use of the hot-water extraction, including both hemicellulose fractions and LCCs, their antioxidant capacity was investigated. It has been reported that LCCs isolated from bamboo exhibited pronounced antioxidant activities for scavenging the DPPH radical and hydroxyl radicals.32 Among their arising applications, the valorization in cosmetic industries is thought to be promising due to their antioxidant activity.46 During this work, the antioxidant activities of different fractions have been extensively investigated using the ABTS method. As shown in Figure 6, the hemicellulose fraction presented a higher antioxidant capacity as compared to lignin-rich fractions (Ret I, Ret II and Perm II). Previous reports indicated that the strong antioxidant activity of hemicellulose may be related to monosaccharide composition, molecular weight distribution and structure of polysaccharides.47,

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Among the LCCs fractions, the Perm II

fractions obtained from different hydrolysis conditions exhibited a higher ABTS scavenging activity in the range from 3.7 to 4.3 mol of Trolox/g, while the Ret I and Ret II fractions displayed a lower antioxidant activity than the other lignin-rich fractions. Combining the results of the composition of carbohydrates and lignin in different fractions (Figure 2), it can be concluded that the LCCs fraction containing low carbohydrates exhibited higher antioxidant activity, which was in agreement with the previous literature.32 Therefore, all lignin-rich fractions showed antioxidant activity to different extents, ranging from 2.7 to 4.3 mol of Trolox/g. CONCLUSION An integrated process combining XAD-7 resin adsorption and gradient dialysis for the fractionation of hot-water extraction liquors was developed. After XAD-7 resin sorption, 93% of the hemicelluloses can be recovered. The obtained lignin-rich fractions, which were separated by gradient molecular weight, showed large difference in their structures, especially the main linkages in LCCs. The process was demonstrated to enable efficient separation of 14


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hemicelluloses and lignin-rich fractions from prehydrolysis liquor, and a functional fractionation of lignin-rich fractions was achieved. The antioxidant activity of different fractions exhibits a great potential to develop their high-value applications in e.g. cosmetics. ACKNOWLEDGEMENT The authors would like to acknowledge financial support from the China Scholarship Council and Fortum Foundation. This work is also part of the activities within Johan Gadolin Process Chemistry Centre, a Center of Excellence appointed by Åbo Akademi University during 2015-2018. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 2D HSQC NMR spectra of Perm II-2 fraction and Perm II-3 fraction. REFERENCES 1. Galkin, M. V.; Samec, J. S., Lignin valorization through catalytic lignocellulose fractionation: a fundamental platform for the future biorefinery. ChemSusChem 2016, 9 (13), 1544-1558, DOI 10.1002/cssc.201600237. 2. Parsell, T.; Yohe, S.; Degenstein, J.; Jarrell, T.; Klein, I.; Gencer, E.; Hewetson, B.; Hurt, M.; Im Kim, J.; Choudhari, H., A synergistic biorefinery based on catalytic conversion of lignin prior to cellulose starting from lignocellulosic biomass. Green Chem. 2015, 17 (3), 1492-1499, DOI 10.1039/C4GC01911C. 3. Aita, G.; Salvi, D.; Walker, M., Enzyme hydrolysis and ethanol fermentation of dilute ammonia pretreated energy cane. Bioresour. Technol. 2011, 102 (6), 4444-4448, DOI 10.1016/j.biortech.2010.12.095.

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Figure captions Figure 1. Flowchart for the extraction and separation of hemicellulose-rich fraction and lignincarbohydrate complexes from spruce sawdust. Figure 2. (a) Extracted total hemicellulose (ETH) and recovered hemicellulose fractions (HF) after sorption with XAD-7 resins under different conditions. (b) Composition of carbohydrates in different fractions. (c) Total amount of carbohydrates and lignin (Inset: Percent of carbohydrates and lignin contents). (d) Lignin contents obtained from different fractions. Figure 3. Linear relationship between lignin and selected carbohydrates content in Ret I, Ret II and Perm II fractions obtained from different extraction conditions: (a) 120 °C, 20 min; (b) 120 °C, 60 min; (c) 160 °C, 20 min; (d) 160 °C, 60 min. Figure 4. Pyrogram of Py-GC-MS of different fractions: (a) Ret I-4 fraction; (b) Ret II-4; (c) Perm II-4 fraction; (d) Perm II-1 fraction. See Table 1 for peak assignments. Figure 5. (a) Main substructures of LCCs identified by 2D HSQC NMR. 2D HSQC NMR spectra of the structures in the side-chain regions and aromatic regions of (b), (c) Ret I-4 fraction, (d), (e) Perm II-4 fraction, and (f), (g) Perm II-1 fraction. Figure 6. ABTS antioxidant activity of different fractions obtained from the whole process.

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Table 1. Peak assignment and relative molar abundances of the phenolic compounds released from different fractions after py-GC-MS. peak No.

phenolic compounds

1, 3 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17

methyl acetate 3-hydroxypropanal n-propanal methyl 2-oxopropanoate 3-furanone 2-furanmethanol 1,2-ethanediol, diacetate 2-cyclopentene-1,4-dione 2-methylcyclopent-2-en-1-one 2-acetylfuran 2(3h)-furanone, dihydro2(5h)-furanone 2-hydroxy-2-cyclopenten-1-one 5-methyl-2-furfuraldehyde phenol 2-hydroxy-3-methyl-2cyclopentene-1-one 2-methylphenol (o-cresol) 4-methylphenol (p-cresol) guaiacol 2,5-dimethylphenol 2,4-dimethylphenol p-ethylphenol 3-methylguaiacol 4-methylguaiacol catechol 4-vinylphenol 4-ethylguaiacol 4-vinylguaiacol 4-propenylphenol eugenol 4-propylguaiacol vanillin cis-isoeugenol trans-isoeugenol homovanillin acetovanillone guaiacyl acetone cis-coniferyl alcohol propiovanillone guaiacyl vinyl ketone alpha-oxo-propio-guaiacone dihydroconiferyl alcohol 4-((1e)-3-hydroxy-1-propenyl)2-methoxyphenol ethyl homovanillate coniferyl aldehyde trans-coniferyl alcohol phenol units (P) guaiacol units (G)

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

type

relative content (%) Ret I-4

Ret II-4 6.01 0.52 0.62 1.03 2.2 1.15

Perm II-4 0.71 -

Perm II-1 0.49 -

CH3COOCH2CH3 OHC-C-C-OH C3H6O C-C-CO-CO-O-C C4H4O2 C5H6O2 C-COO-C-COO-C C5H4O2 C6H8O C6H6O2 C5H8O2 C4H4O2 C5H6O2 C6H6O2 Ar-OH (P) C₆H₈O₂

7.51 3.81 3.46 3.83 2.63 7.85 1.74 0.65 1.02 1.14 0.66 1.78 3.21 0.70 5.19 5.61

P-Me P-Me G C-P-C C-P-C P-C-C G-Me G-Me HO-Ar-OH P-vinyl G-Et G-vinyl P-C-C-C G-C-C═C G-C-C-C G-CHO G-C═C-C(cis) G-C═C-C (trans) G-C-CHO G-CO-C G-C-CO-C G-C═C-C-OH (cis) G-CO-C-C C-O-Ar-CO-C═C G-CO-C-C G-C-C-C-OH G-C═C-C-OH

2.92 4.25 17.26 0.56 2.10 2.80 0.68 4.91 6.09 16.84 2.97 0.98 5.43 8.92 5.19 1.93 2.13 3.63 -

0.96 1.15 30.83 0.59 4.37 0.63 2.99 19.02 0.48 3.67 8.30 2.75 7.99 5.23 1.76 2.27 2.28 -

0.39 0.49 9.05 0.31 2.06 1.40 0.71 7.46 0.82 20.44 2.31 4.53 1.31 1.36 0.37 2.61 16.47 -

0.22 0.38 5.37 0.33 0.17 1.71 0.84 0.19 0.64 6.07 0.15 1.12 0.17 8.70 0.71 3.19 5.06 1.85 0.54 0.55 0.77 2.10 5.19 1.94

G-C-COOC-C G-C═C-CHO G-C═C-C-OH (trans)

5.23 23.81 17.81 82.19

1.72 4.94 96.65

25.98 3.50 96.30

0.63 6.29 17.34 3.96 71.19

Table 2. Weight-average molecular weights (Mw) of different fraction samples 24


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No. samples

HF

Ret I

Ret II

Perm II

Mw, g/mol 1

120 °C, 20 min

6163

1653

743

632

2

120 °C, 60 min

2051

1645

1005

738

3

160 °C, 20 min

6197

9691

1224

715

4

160 °C, 60 min

3002

6659

975

455

25



Figure 1.

26


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Figure 2.

27



Figure 3.

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Figure 4.

29



Figure 5.

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Figure 6.

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Abstract Graphic:

A combined biorefinery process based on resin sorption and gradient dialysis procedure achieves functional fractionation of hydrolysates.

33



Figure 1 254x190mm (96 x 96 DPI)


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Figure 2 181x147mm (300 x 300 DPI)



Figure 3


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Figure 4 115x71mm (300 x 300 DPI)



Figure 5


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Figure 6 272x208mm (300 x 300 DPI)


Valorization of lignin-carbohydrate complexes from hydrolysates of

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