Structural characterization of the lignin-carbohydrate complex in biomass pretreated with Fenton oxidation and hydrothermal treatment and consequences on enzymatic hydrolysis

In this study, lignin-carbohydrate complexes (LCCs) were isolated from biomass (raw and pretreated) to investigate the structural changes in biomass pretreated by Fenton oxidation and hydrothermal treatment, and their effect on enzymatic hydrolysis.

Carbohydrate Polymers 270 (2021) 118375 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Structural characterization of the lignin-carbohydrate complex in biomass pretreated with Fenton oxidation and hydrothermal treatment and consequences on enzymatic hydrolysis efficiency So-Yeon Jeong a, Eun-Ju Lee a, b, Se-Eun Ban a, b, Jae-Won Lee a, b, * a b Department of Wood Science and Engineering, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju 61186, Republic of Korea A R T I C L E I N F O A B S T R A C T Keywords: Lignin-carbohydrate complexes (LCCs) Fenton oxidation Hydrothermal treatment 2D HSQC NMR Enzymatic hydrolysis In this study, lignin-carbohydrate complexes (LCCs) were isolated from biomass (raw and pretreated) to inves­ tigate the structural changes in biomass pretreated by Fenton oxidation and hydrothermal treatment, and their effect on enzymatic hydrolysis The composition and structure of the LCCs fractions were investigated via car­ bohydrate analysis, XRD, FT-IR, and 2D HSQC NMR The biomass degradation rate of yellow poplar and larch during Fenton oxidation and hydrothermal treatment was approximately 30% Most of the hemicellulose was degraded during pretreatment, while xylan remained in the yellow poplar, and galactan, mannan, and xylan remained in the larch The fractional yield of glucan-rich LCC (LCC1) in the yellow poplar (raw and pretreated biomass) was high, while that of glucomannan-rich LCC (LCC3) in larch was higher than the yield yellow poplar Phenyl glycoside, γ-ester, and benzyl ether linkages were observed in the LCCs of yellow poplar, while phenyl glycoside and γ-ester were detected in those of larch Following pretreatment, the frequencies of β–β′ , β-5, and γ-ester in the LCCs of larch were found to be higher than in those of yellow poplar The efficiencies of enzymatic hydrolysis for the pretreated yellow poplar and larch were 93.53% and 26.23%, respectively These finding indicated that the β–β′ , β-5, and γ-ester linkages included in the pretreated biomass affected the efficiency of enzymatic hydrolysis Introduction Lignocellulosic biomass, as an alternative resource to fossil fuels, can be converted into bio-based chemicals and biofuels through biorefinery processes, and their optimization has been the focus of various studies (Galbe & Wallberg, 2019) Pretreatment is essential for producing sugar from lignocellulosic biomass, and multiple studies have focused on promoting its utilization Generally, biomass degradation affected by the type of biomass and catalyst, reaction temperature, and reaction time during pretreatment The structural characteristics of pretreated biomass directly affect enzymatic hydrolysis, and the structure of lignin carbohydrate complexes (LCCs) in lignocellulosic biomass plays a crucial role in recalcitrance during biomass processing and fractionation (Jeffries, 1991; Volynets, Ein-Mozaffari, & Dahman, 2017; Zhao et al., 2020) In enzymatic hydrolysis, various structural characteristics, such as lignin-enzyme binding and cellulose crystallinity, hinder access to ´s-Pejo ´, Ballesteros, & enzymes and reduce its efficiency (Alvira, Toma Negro, 2010; Viikari, Vehmaanperă a, & Koivula, 2012; Zhao, Zhang, & Liu, 2012) However, the lignin content and cellulose crystallinity alone are insufficient to directly explain the correlation with enzymatic hy­ drolysis efficiency (Koo et al., 2012; Maeda et al., 2011) The differences in the pretreatment efficiency due to the lignin distribution and location on the cell wall of lignocellulosic biomass have been investigated through reaction kinetics, chemical, and microstructural analysis (Kim & Lee, 2019; Mittal et al., 2015; Pu, Hu, Huang, Davison, & Ragauskas, 2013) However, structural analysis of pretreated biomass must be conducted to fully understand the pretreatment effect and enzyme hy­ drolysis mechanism (Zhao, Zhang, & Liu, 2012) Lignocellulosic biomass contains lignin-carbohydrate bonds that link cellulose, hemicellulose, and lignin by chemical bonds The three types of LCC linkages include phenyl glycoside, benzyl ether, and γ-ester (Zhang et al., 2020) The LCC of hardwood mainly consists of phenyl * Corresponding author at: Department of Wood Science and Engineering, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea E-mail address: ljw43376@chonnam.ac.kr (J.-W Lee) https://doi.org/10.1016/j.carbpol.2021.118375 Received 19 April 2021; Received in revised form June 2021; Accepted 23 June 2021 Available online 26 June 2021 0144-8617/© 2021 The Authors Published by Elsevier Ltd This is (http://creativecommons.org/licenses/by-nc-nd/4.0/) an open access article under the CC BY-NC-ND license S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig Chemical compositions (a) and X-ray diffraction (XRD) patterns (b) of the raw material, Fenton oxidation treated biomass, and Fenton oxidationhydrothermal-treated biomass (RM: raw material, FO: Fenton oxidation, FO/HT: Fenton oxidation and hydrothermal treatment, YP: yellow poplar, and L: larch) Fig Enzymatic hydrolysis yield of the biomass obtained by Fenton oxidation and hydrothermal treatment (a: yellow poplar, b: larch, RM: raw material, FO: Fenton oxidation, FO/HT: Fenton oxidation and hydrothermal treatment, YP: yellow poplar, and L: larch) glycoside linkages, while benzyl ether is the main linkage in softwood LCC Unlike other types of softwood, larch contains benzyl ester and phenyl glycoside linkages The hemicellulose and lignin in lignocellu­ losic biomass cannot be completely removed by pretreatment; thus, the LCC structure remains in the biomass, even after pretreatment (Huang, He, Li, Min, & Yong, 2015; Zhao et al., 2020) The composition and structure of LCC differ depending on the pre­ treatment process and biomass species; thus, the LCC characteristics of the pretreated biomass can affect the efficiency of enzymatic hydrolysis (Tarasov, Leitch, & Fatehi, 2018) The three main linkages in the LCC can decompose during pretreatment, and the decomposition properties differ depending on the pretreatment conditions and species The com­ plex structure of LCCs in the lignocellulosic biomass hinders the access of enzymes to cellulose during enzymatic hydrolysis (Min et al., 2014; Min, Yang, Chiang, Jameel, & Chang, 2014; Zhao et al., 2020) There­ fore, the structural characteristics and degradation behavior of LCC in the lignocellulosic biomass must be understood for effective pretreat­ ment and enzymatic hydrolysis The changes in the LCC structure of sugarcane bagasse due to hydrothermal treatment and acid pretreat­ ment have recently been analyzed, and the cellulase adsorption behavior on pretreated biomass has been investigated (Zhang et al., 2020) It has been confirmed that the adsorption of cellulase to the LCC of pretreated biomass is lower than that of the raw material However, most previous studies on the LCC of biomass mainly focused on the species or isolation protocol, and few studies have been conducted on the structural changes in LCC by biomass pretreatment and its effect on enzymatic hydrolysis (Du, Gellerstedt, & Li, 2013; Zikeli, Ters, Fackler, Srebotnik, & Li, 2016) Fenton oxidation is primarily used to remove organic matter from wastewater and is an eco-friendly oxidation process that can be per­ formed at a low reaction temperature in a short time (Jeong & Lee, 2020) Major components of lignocellulosic biomass are degraded by hydroxide radicals generated during Fenton oxidation process (Gian­ nakis, 2019) In general, many oligomers are generated in the hydro­ lysate when hydrothermal treatment is performed for biomass pretreatment Sequential Fenton oxidation and hydrothermal treatment can reduce oligomer production and improve the biomass hydrolysis process Recently, a two-step pretreatment process including Fenton oxidation and hydrothermal was reported to improve the hydrolysis efficiency of biomass (Jeong & Lee, 2020; Park et al., 2018; Xiao, Song, & Sun, 2017; Zhang, Pei, Wang, Cui, & Liu, 2016) The purpose of this study was to analyze the structural changes in biomass due to Fenton oxidation and hydrothermal treatment and the effect of the LCC struc­ ture in the pretreated biomass on enzyme hydrolysis Finally, we S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Erlenmeyer flask After then, Enzymatic hydrolysis was started by the addition of the cellulose enzyme (Cellic® CTec2; 17.5 FPU/biomass (g)) The reaction was conducted at 50 ◦ C and 150 rpm for 96 h, and samples were extracted every 24 h for glucose analysis The glucose was deter­ mined by HPLC (Waters 2695 system; Alliance, MA, USA) equipped with an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) and a refractive index detector (Waters 2414 system; Alliance, MA, USA) The analysis was conducted with mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min for 55 Table Mass fraction yield of the isolated LCC1, LCC2, and LCC3 from the raw material (RM), Fenton oxidation-treated biomass (FO), and Fenton oxidationhydrothermal-treated biomass (FO/HT) (unit: %) Fraction Yellow poplar Absolute RM FO FO/HT Larch RM FO FO/HT LCC1 57.44 LCC2 9.88 LCC3 14.14 Sum 81.46 LCC1 52.26 LCC2 8.80 LCC3 12.03 Sum 73.10 LCC1 30.22 LCC2 4.84 LCC3 2.43 Sum 37.48 LCC1 LCC2 LCC3 Sum LCC1 LCC2 LCC3 Sum LCC1 LCC2 LCC3 Sum 47.13 27.45 10.19 84.76 43.84 26.69 4.21 74.73 30.05 15.11 2.06 47.22 2.3 Hydrolysate and biomass chemical analysis The sugars and degradation products in the hydrolysate were analyzed by HPLC under the conditions reported in a previous study (Jeong, Trinh, Lee, & Lee, 2014) The sugars and degradation products were identified by HPLC (Waters 2695 system; Alliance, MA, USA) equipped with an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) and a refractive index detector (Waters 2414 sys­ tem; Alliance, MA, USA) The analysis was conducted with mM H2SO4 as the aqueous mobile phase at a flow rate of 0.6 mL/min for 55 The total phenolic compounds (TPCs) were determined by UV/Vis spec­ trometer following the Folin–Ciocalteu method (Scalbert, Monties, & Janin, 1989) The chemical composition of each type of biomass was determined following the NREL method (Sluiter et al., 2008) An Ami­ nex HPX-87P column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) was used for the analysis of sugars The analysis was performed with deionized water as the mobile phase, at an isocratic flow rate of 0.6 mL/ for 55 2.4 Isolation of lignin carbohydrate complexes (LCCs) from biomass LCC1: glucan-lignin, LCC2: glucomannan-lignin, LCC3: xylan-lignin Yield of LCC (%) = {Recovered LCC (g) / biomass (g)} * 100 LCCs were isolated from each type of biomass (raw material, Fenton oxidation-treated biomass, and Fenton oxidation-hydrothermal-treated biomass) following the method suggested by Li and Du (Du, Geller­ stedt, & Li, 2013; Li, Martin-Sampedro, Pedrazzi, & Gellerstedt, 2011) and the scheme is described in Fig S1 Prior to LCC isolation, extractivefree biomass (20–80 mesh) was prepared by ethanol/benzene (1,2, v/v) extraction for h using Soxhlet extraction apparatus The extractive-free sample was ground to over 100 mesh using a planetary ball mill (PM 100, Retsch GmbH, Haan, Germany) equipped with a 100 mL ZrO2 bowl containing 30 balls will a diameter of cm The ball-milled sample (10 g) was gently mixed with 100 mL of dimethyl sulfoxide (DMSO) and 100 mL of 40% (w/w) tetrabutylammonium hydroxide (TBAH) in H2O at room temperature for h with stirring Following the reaction, 1400 mL of distilled water was added to the reaction mixture and left to precip­ itate glucan-lignin (LCC1) for 24 h at room temperature The precipitate (LCC1) and supernatant (including glucomannan-lignin and xylan-lignin (LCC2 and LCC3, respectively)) were separated by centrifugation (4000 rpm, 15 min) The precipitate was washed with distilled water and neutralized with M HCl, and finally freeze-dried to obtain LCC1 To obtain LCC2 and LCC3, the supernatant was mixed with 1400 mL of saturated 0.2 M Ba(OH)2 and the reaction was conducted for h at room temperature The precipitate (LCC2) and supernatant (LCC3) from the reaction mixture were separated by centrifugation (4000 rpm, 15 min) Neutralization, dialysis (molecular mass cut-off 1000 Da), and freezedrying were conducted sequentially to obtain LCC2 and LCC3 attempted to understand the degradation behavior of LCC during pre­ treatment and the correlation between enzymatic hydrolysis and the LCC structure of pretreated biomass Materials and methods 2.1 Biomass and Fenton oxidation-hydrothermal treatment of biomass Yellow poplar (Liriodendron tulipifera L.) and Larch (Larix kaempferi) chips were used as the raw material in this study The chips were milled to 20–80 mesh and stored at room temperature for further processing The chemical compositions of the raw materials are shown in Table S1 The biomass was pretreated by sequential Fenton oxidation and hydrothermal treatment Based on a previous study, FeSO4⋅7H2O and H2O2 (28%, w/w) were used as the Fenton reagent (Jeong & Lee, 2016) The Fenton reagent (FeSO4⋅7H2O:H2O2) molar ratio was fixed to 1:25 Fenton oxidation was conducted using biomass (30 g, dry weight), distilled water (120 mL), and Fenton reagent (180 mL) in a L Erlen­ meyer flask The pH of the mixture was adjusted to using M NaOH and the reaction was conducted at 50 ◦ C for h with stirring at 150 rpm The Fenton oxidation-treated biomass then underwent hydrothermal treatment The reaction conducted in an EMS reactor (Mode EMV-HT/ HP 600, Gyeonggi-do, Korea) with a biomass/distilled water (w/w) ratio of 1:8 at 170 ◦ C for 10 (Jeong & Lee, 2020) The treated biomass and hydrolysate were then separated by filtration and stored at ◦ C until further processing 2.5 Structure analysis of biomass and LCCs The biomass crystallinity was determined using an X'pert PRO Multipurpose X-ray diffractometer (XRD, PANalitical, the Netherlands) under the following conditions: 2θ = 5–50◦ , 40 kV, and 30 mÅ, and the crys­ tallinity index was calculated following Segal's method (Segal, Creely, Martin Jr, & Conrad, 1959) The structural properties of the LCC were investigated by FT-IR spectrometry (Spectrum 400, PerkinElmer, United Kingdom) The 2.2 Enzymatic hydrolysis of biomass Each type of biomass (2 g, raw material, Fenton oxidation-treated biomass, and Fenton oxidation-hydrothermal-treated biomass) were mixed with 50 mM sodium citrate buffer (pH 4.8, 20 mL) in a 125 mL S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig Total carbohydrate and lignin contents (a, b), and carbohydrate composition (c, d) of the yellow poplar and larch LCCs (LCC1: glucan-lignin, LCC2: glucomannan-lignin, LCC3: xylan-lignin, RM: raw material, FO: Fenton oxidation, FO/HT: Fenton oxidation and hydrothermal treatment, YP: yellow poplar, and L: larch) spectra were recorded in a range from 4000 to 400 cm− at a resolution of cm− with 32 scans Prior to NMR analysis, LCC samples were acetylated to increase their solubility by dissolving 200 mg of the LCC sample in 12 mL of DMSO/Nmethylimidazole (2:1, v/v) for h at room temperature with stirring (Lu & Ralph, 2003) Acetic anhydride (4 mL) was then added to the mixture, and the reaction was performed for h at room temperature with stir­ ring The pH of reaction mixture was adjusted to with M HCl to precipitate the LCCs The precipitated residue was separated via centrifugation using a 0.45 μm nylon membrane filter The residue was then washed with distilled water and freeze-dried to obtain acetylated LCCs The 2D HSQC NMR spectra were recorded using an AVANCE 600 spectrometer (Bruker, Germany) at 25 ◦ C To obtain the NMR spectra, 50 mg of the acetylated LCC sample was dissolved in 500 μL of dimethyl sulfoxide (DMSO‑d6, 99.8%) The spectral widths for HSQC were 7200 and 36,000 Hz for the 1H and 13C-dimensions, respectively The scan­ ning time, acquisition time between transients, and relaxation time were 32, 0.07 s, and a s, respectively Additionally, the 1JC-H was 150 Hz Prior to Fourier transformation, the data matrices were filled from to 1024 points in the 13C-dimension, in which 512 times increments were recorded The data were processed using standard Bruker Topspin-NMR software from biomass (yellow poplar and larch) Fenton oxidation are shown in Table S2 Small amounts of sugars and acetic acid were detected in the liquid fraction The pH and degradation rate were similar for both spe­ cies, suggesting that biomass degradation due to Fenton oxidation occurred regardless of the species This is because Fenton oxidation in­ duces random biomass degradation by hydroxyl radicals (Giannakis, 2019) Following Fenton oxidation, sequential biomass hydrothermal treatment was conducted, and the hydrolysate analysis results are pre­ sented in Table S3 Unlike Fenton oxidation, there were significant differences in the sugars and degradation products of the two species The hydrolysate mainly contained sugars and degradation products generated from hemicellulose and lignin In the yellow poplar, xylose (10.41 g/L), acetic acid (4.27 g/L), and TPC (3.65 g/L) were detected as major compounds, while galactose (4.58 g/L) was the major compound obtained from the larch This was due to the differences in the hemi­ cellulose and lignin structures between softwood and hardwood The hemicellulose in yellow poplar and larch is mainly composed of glu­ curonoxylan and arabinogalactan, respectively; thus, the composition of the sugars produced after hydrothermal treatment differed depending on the type of biomass (Peng, Peng, Xu, & Sun, 2012; Sjostrom, 1993) Most of the xylose residue in the yellow poplar contained an acetyl group at C-2 or C-3, which could be easily cleaved by acids or alkalis (Peng, Peng, Xu, & Sun, 2012; Sjostrom, 1993) Therefore, the acetic acid concentration in the yellow poplar was higher than that in larch The lignin in larch is mostly composed of guaiacyl units, and that in yellow poplar contains guaiacyl and syringyl units (Nitsos, CholiPapadopoulou, Matis, & Triantafyllidis, 2016) Generally, the guaiacyl unit contains a methoxyl group at C-3 position on the aromatic ring, forming a C–C bond with other phenylpropane units; thus, Results and discussion 3.1 Properties of biomass pretreated by Fenton oxidant and hydrothermal treatment The sugars and degradation products in the liquid fraction obtained S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig FT-IR spectra of the LCCs from yellow poplar (YP, a) and larch (L, b) (LCC1: glucan-lignin, LCC2: glucomannan-lignin, LCC3: xylan-lignin, RM: raw material, and FO/HT: Fenton oxidation and hydrothermal treatment) hydrothermal treatment could not easily achieve decomposition How­ ever, the syringyl unit contained two methoxy groups at the C-3 and C-5 positions on the aromatic ring; thus, the syringyl units were linked to other phenylpropane units by ether linkages Therefore, the syringyl unit in the lignin could decompose more easily than the guaiacyl unit (Sequeiros & Labidi, 2017) As given in Table S3, the TPC concentration was high in the yellow poplar and covalent linkages existed between lignin and carbohydrate (mainly hemicellulose), which included benzyl ether, benzyl ester, and phenyl glucoside linkages The composition of the linkages differed between the biomass species The benzyl ether and phenyl glycoside linkages degraded easily under acidic conditions, while ester linkages could be degraded under alkaline conditions (Brunow & Lundquist, 2010; Giummarella, Pu, Ragauskas, & Lawoko, 2019; Kosh­ ijima & Watanabe, 2013; Lawoko, Deshpande, & van Heiningen, 2009; Tarasov, Leitch, & Fatehi, 2018) It has been reported that phenyl glycoside linkages decompose more easily than other linkages during hydrothermal treatment Therefore, the lignin degradation rate of larch, which contains benzyl ester and phenyl glycoside linkages, by hydro­ thermal treatment should be lower than that of yellow poplar (Tarasov, Leitch, & Fatehi, 2018) The chemical composition and crystallinity of the different biomass (raw material, Fenton oxidation-treated biomass and Fenton oxidationhydrothermal-treated biomass) are shown in Fig During Fenton oxidation, hemicellulose was slightly degraded (Fig 1a) This is consistent with the result presented in Table S2 After Fenton oxidation and hydrothermal treatment, the biomass degradation rates between species were similar, with values of 29.05% and 28.59% for yellow poplar and larch, respectively The glucan and lignin contents also increased with hemicellulose degradation in both species The lignin content in larch was significantly higher (45.59%) compared to that of S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 hydrothermal treatment did not affect the crystalline cellulose in the biomass, and the crystallinity of the biomass increased due to the degradation of the amorphous hemicellulose (Nitsos, CholiPapadopoulou, Matis, & Triantafyllidis, 2016) The degradation rate (28.59%) in larch, which had a relatively low crystallinity (35.12–53.35%), was similar to that in the yellow poplar (29.05%) after Fenton oxidation-hydrothermal treatment; however, the enzymatic hy­ drolysis yield was as low as 26.23% after 96 h (Fig 2b) The enzymatic hydrolysis yield is influenced by various factors, such as the pore size, specific surface area, lignin content, and lignin structure, as well as the cellulose crystallinity (Zhao, Zhang, & Liu, 2012) Generally, softwood has a lower specific surface area than hardwood, and a high content of lignin involved in irreversible enzyme adsorption (Nitsos, CholiPapadopoulou, Matis, & Triantafyllidis, 2016; Palonen, Thomsen, Ten­ kanen, Schmidt, & Viikari, 2004) Softwood lignin mainly consists of guaiacyl units; thus, it is more difficult to degrade than that in hardwood (Nitsos, Choli-Papadopoulou, Matis, & Triantafyllidis, 2016; Palonen, Thomsen, Tenkanen, Schmidt, & Viikari, 2004) Therefore, the lignin of larch was a major factor affecting enzymatic hydrolysis Hemicellulose also affects factor on enzymatic hydrolysis as it exists in the surrounding cellulose and acts as a physical barrier that is highly resistant to enzyme attack (Palonen, Thomsen, Tenkanen, Schmidt, & Viikari, 2004) Compared with yellow poplar, relatively high concentration of hemi­ cellulose remained after the Fenton oxidation-hydrothermal treatment of larch, which reduced the enzyme hydrolysis yield The lignin in biomass forms an LCC structure by forming chemical bonds with cellu­ lose and hemicellulose, which the can cause irreversible adsorption with enzymes during enzymatic hydrolysis (Huang, He, Li, Min, & Yong, 2015; Tarasov, Leitch, & Fatehi, 2018; Zhao et al., 2020) The compo­ sition and structure of the LCC changed according to the biomass pre­ treatment conditions; thus, the enzyme hydrolysis yield may have been affected by the composition and structure of the LCC (raw material, Fenton oxidation-treated biomass, and Fenton oxidation-hydrothermaltreated biomass) Table Assignments of the 13C/1H correlation signals observed in the 2D HSQC spectra of the LCC fractions isolated from yellow poplar and larch Label δC/δH (ppm) Lignin cross-signals Bβ 54.3/3.06 MeO 56.31/3.74 Aγ 60.2/(3.40; 3.71) Aα Aβ(G) 71.9/4.87 84.5/4.29 Bα Aβ(S) 85.5/4.63 86.5/4.11 Cα 87.4/5.51 S2,6 104.7/6.70 G2 G5 G6 111.8/7.00 115.9/6.77 119.5/6.76 Polysaccharide cross-signals Xyl5 63.7/(3.17; 3.88) Xyl2 73.2/3.06 74.4/3.28 Xyl3 Xyl4 75.9/3.51 Xyl1 102.4/4.27 100.1/4.69 Glu1 Glu2 71.7/4.56 72.4/5.06 Glu3 Glu4 76.5/3.72 Glu5 72.2/3.78 62.5/(4.03; 4.26) Glu6 Man1 97.1/4.94 Man2 68.8/(4.90;5.22) 70.6/(4.81;5.12) Man3 Man4 72.9/3.86 LCC linkage Phenyl glycoside Benzyl ether γ-Ester Assignment Cβ/Hβ in β–β′ resinol substructures (B) C/H in methoxyls (MeO) Cγ/Hγ in γ-hydroxylated β-O-4′ substructures (A) Cα/Hα in β-O-4′ substructures (A) Cβ/Hβ in β-O-4′ substructures linked to a G unit (A) Cα/Hα in β-β′ resinol substructures (B) Cβ/Hβ in β-O-4′ substructures linked to a S unit (A) Cα/Hα in phenylcoumaran substructures (C) C2/H2 and C6/H6 in etherified syringyl units (S) C2/H2 in guaiacyl units (G) C3/H3 in guaiacyl units (G) C6/H6 in guaiacyl units (G) C5/H5 in β-D-xylopyranoside C2/H2 in β-D-xylopyranoside C3/H3 in β-D-xylopyranoside C4/H4 in β-D-xylopyranoside C1/H1 in β-D-xylopyranoside C1/H1 in β-D-glucopyranoside C2/H2 in β-D-glucopyranoside C3/H3 in β-D-glucopyranoside C4/H4 in β-D-glucopyranoside C5/H5 in β-D-glucopyranoside C6/H6 β-D-glucopyranoside C1/H1 in β-D-mannopyranoside C2/H2 in β-D-mannopyranoside C3/H3 in β-D-mannopyranoside C4/H4 in β-D-mannopyranoside 99.8;100.1/4.92;5.11 γ-Ester LCC linkages 80.7/4.62 Cα-Hα in benzyl ether (secondary OH) linkages γ-Ester LCC linkages 62.2; 63.5; 64.2/ 4.09;4.17;4.27 3.3 LCC yield and carbohydrate composition LCCs were obtained from the different types of biomass (raw mate­ rial, Fenton oxidation-treated biomass, and Fenton oxidationhydrothermal-treated biomass), and the yields are shown in Table The LCC yields of the raw materials (84.76% and 81.46% for larch and yellow poplar, respectively) were similar to those in previous studies, and there was no significant difference between the species (Monot, Chirat, Evangelista, & Brochier-Salon, 2017) However, the LCC composition differed between larch and yellow poplar The yield of LCC2 from larch was 15.11–27.45%, which was higher than that of yellow poplar (4.84–9.88%) In LCCs, lignin is associated with cellulose and hemicellulose by hydrogen and covalent bonds, and the structure inhibits enzymatic hydrolysis Therefore, the high LCC yield in larch following Fenton oxidation-hydrothermal treatment would have nega­ tively affected enzymatic hydrolysis (Zhao et al., 2020) With Fenton oxidation-hydrothermal treatment, the LCC yields decreased in both species Yellow poplar contained large amounts of LCC1 and low amounts of LCC2, while LCC1 (30.22%) was predominant in the biomass after Fenton oxidation-hydrothermal treatment, and the yield of LCC2 and LCC3 were only 4.84% and 2.43%, respectively These results sug­ gest that hemicellulose cross linked with lignin was degraded during Fenton oxidation-hydrothermal treatment Larch had more LCC2 and less LCC3 than yellow poplar due to differences in the chemical com­ positions of softwood and hardwood The yield of LCCs decreased after Fenton oxidation-hydrothermal treatment, while that of LCC2 was higher than that of LCC3 Therefore, the hemicellulose was not suffi­ ciently degraded by Fenton oxidation-hydrothermal treatment There was no significant difference in LCC yields in both species after Fenton oxidation Therefore, Fenton oxidation alone cannot degrade biomass components, which is consistent with previous research results (Jeong & the raw material (34.98%) Additionally, a large amount of hemicellu­ lose remained in the larch because the benzyl ester linkages (between lignin and hemicellulose) in larch were not easily decomposed by hy­ drothermal treatment The biomass XRD patterns were analyzed to investigate the changes of crystallinity between the biomass types and treatment processes (Fig 1b) Although the crystallinity of the raw materials (45.63% and 35.12%) differed, the crystallinity increased in both species according to the treatment processes (Fenton oxidation or hydrothermal treatment) Treatment induced the degradation of hemi­ cellulose and the amorphous region of cellulose, thereby increasing the crystallinity of the treated biomass (Fenton oxidation or hydrothermal treatment) (Jeong & Lee, 2016) 3.2 Enzymatic hydrolysis of Fenton oxidation-hydrothermal-treated biomass The enzymatic hydrolysis yields of the biomass are shown in Fig The raw material and Fenton oxidation-treated biomass yields of yellow poplar were 7.11% and 23.02% after 96 h of enzymatic hydrolysis, respectively (Fig 2a), while the yield of Fenton oxida­ tion–hydrothermal-treated biomass reached 93.53% Generally, the enzymatic hydrolysis yield decreased with increasing biomass crystal­ linity; (Zhu, O'Dwyer, Chang, Granda, & Holtzapple, 2008) however, there was no correlation between crystallinity and the enzymatic hy­ drolysis yield in this study Therefore, Fenton oxidation and S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig 2D HSQC NMR spectra of the LCCs from yellow poplar (LCC1: glucan-lignin, LCC2: glucomannan-lignin, LCC3: xylan-lignin, RM: raw material, and FO/HT: Fenton oxidation and hydrothermal treatment) Lee, 2016) The carbohydrate and lignin contents of the LCCs were determined, and the analytical results are shown in Fig The carbohydrate and lignin contents of the LCCs of yellow poplar were similar to those after Fenton oxidation However, the lignin content of LCC1 of Fenton oxidation-hydrothermal-treated yellow poplar decreased, while that of the LCC2 and LCC3 increased (Fig 3a) In larch, the lignin content of the LCCs increased significantly after Fenton oxidation-hydrothermal treatment (Fig 3b), and the carbohydrate compositions differed depending on the LCCs of the biomass Glucan and xylan were the major LCC components of the LCCs in yellow poplar (Fig 3c), and the xylan content decreased after Fenton oxidation-hydrothermal treatment LCC1 and LCC2 in larch contained higher contents of hemicellulose than those in yellow poplar (Fig 3d) The contents of xylan and mannan decreased after Fenton oxidation-hydrothermal treatment (Jeong & Lee, 2020) Nevertheless, the concentration of hemicellulose in the LCCs was higher than that in yellow poplar Therefore, the enzyme hydrolysis yield of larch may have been lower than that of yellow poplar (Nassar & MacKay, 2007) 3.4 LCC structural analysis The FT-IR spectra of the LCCs are shown in Figs and S2 The LCC1 in the Fenton oxidation-hydrothermal-treated yellow poplar exhibited similar bands as those of the raw material at 3340 and 1030 cm− 1, which were assigned to O–H stretching and C–O stretching in cellulose, respectively, while bands related to hemicellulose and lignin did not ´zquez et al., 2020) clearly appear (Chen, Tang, & Ju, 2015; Flores-Vela This is due to the low lignin content in LCC1 The intensity of celluloserelated bands increased after Fenton oxidation-hydrothermal treatment In the LCC2 of the raw material and Fenton oxidation-treated biomass, intense signals were observed at 1235, 1368, 1423, 1510, and 1590 cm− 1, which corresponded to lignin and hemicellulose The statement implied that hemicellulose was degraded by Fenton oxidation and hy­ drothermal treatment which led to a relative increase in lignin and cellulose content (Chang et al., 2017; Ishola et al., 2012; Li et al., 2010) This peak pattern was clearly observed in the LCC3, indicating that hemicellulose was degraded by Fenton oxidation-hydrothermal treat­ ment, which corresponds to the results of the carbohydrate composition S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig 2D HSQC NMR spectra of the LCCs from larch (LCC1: glucan-lignin, LCC2: glucomannan-lignin, LCC3: xylan-lignin, RM: raw material, and FO/HT: Fenton oxidation and hydrothermal treatment) analysis (Fig 3c) The bands for larch were similar to those of yellow poplar, and the intensity of bands related to lignin in all LCCs was higher than that in yellow poplar These results are consistent with those in Table and Fig The important compositional and structural information of the LCCs was obtained by 2D HSQC NMR spectroscopy (Table 2, Figs 5–8, and S3–4) Glucan was the major carbohydrate component in LCC1 for all samples Peaks related to mannan and glucan were observed in the spectra for LCC2, while xylan was mainly observed in LCC3 (Figs 5, 6, and S3) The main substructures are presented in Fig S4 Methoxy group signals were observed at δC/δH 56.31/3.74 ppm in all LCCs (Table 2) β-D-Glucopyranoside exhibited prominent signals at C1–H1 (δC/δH 100.1/4.69 ppm), C2–H2 (δC/δH 71.7/4.56 ppm), C3–H3 (δC/δH 72.4/ 5.06 ppm), C4–H4 (δC/δH 76.5/3.72 ppm), C5–H5 (δC/δH 72.2/3.78 ppm), and C6–H6 (δC/δH 62.5/(4.03;4.26) ppm) in the LCC1 of yellow poplar and larch (Table 2) (Du, Gellerstedt, & Li, 2013) Additionally, Fenton oxidation and hydrothermal treatment did not significantly affect the LCC1 structure of yellow poplar and larch Therefore, glucan was not significantly affected by Fenton oxidation and hydrothermal treatment The LCC2 of yellow poplar and larch contained β-D-glucopyranoside and β-D-mannopyranoside The β-D-mannopyranoside signals corre­ sponded to C1–H1 (δC/δH 97.1/4.94 ppm), C2–H2 (δC/δH 68.8/ (4.90;5.22) ppm), C3–H3 (δC/δH 70.6/(4.81;5.12) ppm), and C4–H4 (δC/δH 72.9/3.86 ppm) Additionally, guaiacyl lignin units (G2, G5, and G6) were observed at C2–H2 (δC/δH 111.8/7.00 ppm), C5–H5 (δC/δH 115.9/6.77 ppm), and C6–H6 (δC/δH 119.5/6.76 ppm) in the LCC2 of the raw materials Bβ (β–β′ resinol) and syringyl units (S2,6) were detected in the LCC2 of yellow poplar, but they were not observed in the LCC2 of larch (Du, Gellerstedt, & Li, 2013; Zikeli, Ters, Fackler, Sre­ botnik, & Li, 2016) β-D-Xylopyranoside was detected in the LCC3 of yellow poplar and larch, corresponding to C1–H1 (δC/δH 102.4/4.27 ppm), C2–H2 (δC/δH 73.2/3.06 ppm), C3–H3 (δC/δH 74.4/3.28 ppm), C4–H4 (δC/δH 75.9/ 3.51 ppm), and C5–H5 (δC/δH 63.7/(3.17; 3.88) ppm) Additionally, S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig Amplified anomeric regions of γ-esters (Est) in the LCC1 and LCC3 of yellow poplar (LCC1: glucan-lignin, LCC3: xylan-lignin, RM: raw material, FO: Fenton oxidation, and FO/HT: Fenton oxidation and hydrothermal treatment) guaiacyl lignin units (G2, G5, and G6), syringyl lignin units (S2,6), β-aryl ether structures [β-O-4′ , Aα, Aβ (G), Aβ (S), Aγ], and β–β resinol structure (Bα and Bβ) were detected in the LCC3 of raw materials β-5 phenyl­ coumaran structures (Cα) were not observed in the LCC3 of yellow poplar, while they were detected in that of larch, because of the dif­ ferences in the hemicellulose and lignin structures of softwood and hardwood (Du, Gellerstedt, & Li, 2013; Zikeli, Ters, Fackler, Srebotnik, & Li, 2016) Following Fenton oxidation, syringyl and guaiacyl lignin unit signals were observed in the LCC2 and LCC3 of yellow poplar at lower con­ centrations than those of the raw materials (Figs S3 and 5) Guaiacyl lignin unit signals were completely removed from the LCC by hydro­ thermal treatment The guaiacyl lignin unit content of the larch was similar to that of the raw material as softwood lignin is more difficult to degrade than hardwood The β–β′ and β-5 linkages of lignin, which interfere with enzymatic hydrolysis, were removed by Fenton oxidation and hydrothermal treatment (Huang et al., 2017) This was confirmed by the LCC2 and LCC3 structures of the yellow poplar Meanwhile, β–β′ and β-5 linkages of lignin were detected in the LCC3 of larch (Fig 6) The LCCs of yellow poplar contained glucan and lignin as most of the hemicellulose was removed by Fenton oxidation and hydrothermal treatment However, the LCCs of larch contained large amounts of mannan, guaiacyl units, β-β′ , and β-5 linkages, which were difficult to degrade Therefore, larch had a lower enzyme hydrolysis yield than yellow poplar due to the differences in the LCC structures between softwood and hardwood Figs and show the linkage types in the LCC1 and LCC3 of yellow poplar and larch, as linkages were not detected in LCC2 Phenyl S.-Y Jeong et al Carbohydrate Polymers 270 (2021) 118375 Fig Amplified anomeric regions of γ-esters (Est) in the LCC1 and LCC3 of larch (LCC1: glucan-lignin, LCC3: xylan-lignin, RM: raw material, FO: Fenton oxidation, and FO/HT: Fenton oxidation and hydrothermal treatment) glycoside and γ-ester linkages were detected in the δC/δH 104–99/ 4.8–5.2 and δC/δH 65–62/4.0–4.5 regions, respectively, while benzyl ether linkages were observed at δC/δH 81–80/4.5–4.7 (BE1) (Xu et al., 2019) The linkage types in the LCC differed depending on the biomass species Phenyl glycoside, benzyl ether, and γ-ester were observed in yellow poplar, while phenyl glycoside and benzyl ether were not detected in larch (Figs and 8) Most of the LCC bonds in the biomass were removed after Fenton oxidation and hydrothermal treatment, while some phenyl glycoside and benzyl ether bonds remained in the biomass Clear phenyl glycoside signals appeared after the larch was pretreated by Fenton oxidation and hydrothermal treatment, indicating that pretreatment produced acid-resistant phenyl glycoside linkages (Zhang et al., 2020) Additionally, the amount of phenyl glycoside linkages may have been increased by the condensation of degradation products derived from lignin and carbohydrates during hydrolysis (Tarasov, Leitch, & Fatehi, 2018) γ-Ester linkages remained in larch after Fenton oxidation and hydrothermal treatment, suggesting that the LCC distribution in larch was higher than that in yellow poplar There­ fore, the LCC linkages in the LCC1 and LCC3 of larch after Fenton oxidation hydrothermal treatment acted as inhibitors for enzymatic hydrolysis Conclusions The LCC structures of raw materials and biomass pretreated by Fenton oxidation and hydrothermal treatment were investigated and their effect on enzymatic hydrolysis were analyzed The LCC fractions were isolated from each type of biomass, and the differences in their 10 Carbohydrate Polymers 270 (2021) 118375 S.-Y Jeong et al composition and structure were analyzed Following pretreatment, the hemicellulose chemical composition and concentration differed be­ tween the LCCs of yellow poplar and larch High amounts of guaiacyl units, β-β′ , and β-5 were detected in the pretreated larch The degrada­ tion of hemicellulose was relatively low during pretreatment due to the γ-ester linkages in larch; therefore, bonds between hemicellulose and lignin were firmly formed Enzymatic hydrolysis was conducted on the pretreated biomass, and the enzymatic hydrolysis yield of larch was lower than that of yellow poplar In summary, high hemicellulose, guaiacyl unit, β-β′ , β-5, and γ-ester linkage concentrations in the LCC of larch may have had a negative effect on enzymatic hydrolysis These results can improve our understanding of the LCC structures and link­ ages in the pretreated biomass, and this study can aid in developing more effective biomass deconstruction or depolymerization strategies for biorefineries Giummarella, N., Pu, Y., Ragauskas, A J., & Lawoko, M (2019) A critical review on the analysis of lignin carbohydrate bonds Green Chemistry, 21(7), 1573–1595 Huang, C., He, J., Li, X., Min, D., & 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the X-ray diffractometer Textile Research Journal, 29(10), 786–794 CRediT authorship contribution statement So-Yeon Jeong: Methodology, Data curation, Investigation, Formal analysis, Writing – original draft Eun-Ju Lee: Methodology, Investi­ gation, Formal analysis Se-Eun Ban: Methodology, Data curation, Formal analysis Jae-Won Lee: Conceptualization, Data curation, Funding acquisition, Writing – review & editing Declaration of competing interest The authors declare no competing financial interest Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No 2021R1A2C100719911) This study was carried out with the support of R&D Program for Forest Science Technology (Project No 2020228C102122-AC01) provided by Korea Forest Service(Korea Forestry Promo­ tion Institute) Appendix A Supplementary data Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2021.118375 References 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(Fenton oxidation or hydrothermal treatment) Treatment induced the degradation of hemi­ cellulose and the amorphous region of cellulose, thereby increasing the crystallinity of the treated biomass. .. biomass (Fenton oxidation or hydrothermal treatment) (Jeong & Lee, 2016) 3.2 Enzymatic hydrolysis of Fenton oxidation- hydrothermal- treated biomass The enzymatic hydrolysis yields of the biomass