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

Carbohydrate Polymers 270 (2021) 118375

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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.

1. 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: (J.-W. Lee).
/>Received 19 April 2021; Received in revised form 5 June 2021; Accepted 23 June 2021
Available online 26 June 2021
0144-8617/© 2021 The Authors.
Published by Elsevier Ltd.
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S.-Y. Jeong et al.

Carbohydrate Polymers 270 (2021) 118375

Fig. 1. 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. 2. 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
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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 5 mM H2SO4 as the mobile phase
at a flow rate of 0.6 mL/min for 55 min.

Table 1
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 5 mM H2SO4
as the aqueous mobile phase at a flow rate of 0.6 mL/min for 55 min. 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/
min for 55 min.
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 6 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 1 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 4 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 1 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 1 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.
2. 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 1 L Erlen­
meyer flask. The pH of the mixture was adjusted to 3 using 1 M NaOH
and the reaction was conducted at 50 ◦ C for 1 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 min (Jeong & Lee, 2020). The treated
biomass and hydrolysate were then separated by filtration and stored at
4 ◦ 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
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Carbohydrate Polymers 270 (2021) 118375

Fig. 3. 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− 1 at a resolution
of 4 cm− 1 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 4 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 4 h at room temperature with stir­
ring. The pH of reaction mixture was adjusted to 2 with 1 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 2 s, respectively. Additionally, the 1JC-H was 150 Hz.
Prior to Fourier transformation, the data matrices were filled from 0 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,

3. 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

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Carbohydrate Polymers 270 (2021) 118375

Fig. 4. 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. 1. 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
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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 2
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 1.
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. 2.
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
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Carbohydrate Polymers 270 (2021) 118375

Fig. 5. 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. 3. 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. 4 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
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Carbohydrate Polymers 270 (2021) 118375

Fig. 6. 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 1 and
Fig. 3.
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,
8


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Carbohydrate Polymers 270 (2021) 118375

Fig. 7. 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. 7 and 8 show the linkage types in the LCC1 and LCC3 of yellow
poplar and larch, as linkages were not detected in LCC2. Phenyl
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Carbohydrate Polymers 270 (2021) 118375

Fig. 8. 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. 7 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.
4. 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.

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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.
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