Industrial Crops & Products 188 (2022) 115453

Contents lists available at ScienceDirect

Industrial Crops & Products

journal homepage: [Link]/locate/indcrop

Integrated biorefinery of bamboo for fermentable sugars, native-like lignin,

and furfural production by novel deep eutectic solvents treatment
Zongwei Guo a, b, Jianzhen Mao a, Qilin Zhang b, Feng Xu a, *
a
State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
b
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China

A R T I C L E I N F O A B S T R A C T

Keywords: Herein, a novel class of DESs comprising tetramethylammonium chloride/lactic acid (TMAC/LA) and tetrame­
Integrated biorefinery thylammonium bromide/lactic acid (TMAB/LA) was proposed to refine bamboo for cascaded production of
Deep eutectic solvents cellulose residue, native-like lignin, and hemicellulose-derived sugars under a relatively mild condition (120 ◦ C
Enzymatic hydrolysis
for 2 h). The cellulose residue showed an outstanding digestibility with approximately 90 % glucose yield, which
Native-like lignin
Furfural
was ten-fold higher than that of raw bamboo. The efficient conversion was attributed to the open structure and
increased porosity caused by the hemicellulose removal (71.24–86.57 %) and delignification (53.27–73.62 %).
Lignin recovered from the treatment liquor exhibited the β-O-4′ linkages (86.3–89.3 %) characteristic of native
milled wood lignin (MWL). The recovered lignin could be well-suited for phenolic compounds production (yield
> 50 %). After lignin extraction, the residual liquor is a suitable reaction substrate and medium for furfural
production, reaching a furfural yield of 86.9 %. The novel DESs treatment could offer a cascaded biorefinery for
sustainable development.

1. Introduction Lignin is easily removed from lignocellulosic biomass by deep

eutectic solvents (DESs) pretreatment. It has been shown that DESs have
Lignocellulosic biomass such as woody biomass, agricultural and good delignification ability (more than 90 %) from corncob, woody
industrial residues, and dedicated energy crops is considered to the one biomass, and grass, leading to an attractive enzymatic saccharification
of the most abundant renewable feedstocks for transforming to bio- of the pretreated residues (R. Kumar et al., 2020; B. Kumar et al., 2020).
based fuels, commercial chemicals, and functional materials (Ding Besides, the structures and properties of lignin upon DESs treatment
et al., 2012). Bamboo as a bioenergy feedstock is prolific, tolerant to were also studied. One unique feature of DESs lignin is their high purity.
water and nutrients, and positive to environmental conservation (Lin Alvarez-Vasco and coworkers reported that lignin extracted from poplar
et al., 2021). It contains three main components, namely, cellulose, using choline choride/lactic acid showed low molecular weight and high
lignin, and hemicellulose. In the traditional biomass treatment process, purity (95 %) (Alvarez-Vasco et al., 2016). Similarly, Liu et al. reported a
hemicellulose and lignin are removed by efficient treatment to retain low-molecular-weight (913 g/mol) and high-purity lignin (96 %) from
most of cellulose (Yu et al., 2016). Cellulose can be further converted poplar via microwave-assisted choline choride/oxalic acid DES pre­
into high-value chemicals (sugars, hydroxymethylfurfural, and levulinic treatment (Liu et al., 2017). Most investigations showed the regenerated
acid etc.) and biofuels (ethanol and butanol) (Beig et al., 2021; Kang lignin has less β-O-4′ linkages. For example, choline choride/lactic acid
et al., 2018). A typical fate of lignin is being burned for heating and (1:10) were used to pretreatment ecualyptus to extract lignin at 120 ◦ C
electricity. Hemicellulose, often rich in the pretreatment liquor, is for 6 h, but only 4.4 % β-O-4′ linkages in the DES lignin samples (Shen
generally under utilized or discarded. Improved lignin and hemicellu­ et al., 2019). Choline chloride-ethylene glycol-p-toluenesulfonic acid
lose utilization would not only increase the economic viability of bio­ DES lignin exhibited 2.3 % β-O-4′ content, and no peaks of β-5 and β-β
refinery, but can also minimize adverse environmental impacts. (Z. Chen et al., 2019; Y.J. Chen et al., 2019). Alkali lignin was pretreated
Therefore, new methods need to be explored for achieving the complete with lactic acid/choline chloride and oxalic acid/choline chloride at
valorization of the three major biomass components. 120 ◦ C for 6 h resulting in regenerated DES lignin, which exhibited the

* Correspondence to: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China.
E-mail address: xfx315@[Link] (F. Xu).

[Link]
Received 16 April 2022; Received in revised form 5 July 2022; Accepted 29 July 2022
Available online 8 September 2022
0926-6690/© 2022 Published by Elsevier B.V.
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

absence of β-O-4′ bond in the DES lignin (Hong et al., 2020). Thus, the
focus of the work presented here is to study the approaches to preserve
more β-O-4′ linkages in the DESs lignin streams and the catalytic
transformation of DESs lignin to produce aromatic chemicals.
Hemicellulose is the only source of furfural, one of the top twelve
platform chemicals identified by the US Department of Energy (Putten
et al., 2013). Furfural can be obtained by dehydration reaction from
xylose and other pentoses and used as a platform chemical for producing
other chemicals such as 2-methyltetrahydrofuran, tetrahydrofuran, and
levulinic acid (Dong et al., 2015; Gürbüz et al., 2013; Tachibana et al.,
2013). The formation of furfural includes the following steps: (1) The
hydrolysis reaction of converting xylan to xylose; (2) The dehydration
reaction of xylose to form furfural (Luo et al., 2018). The efficient
conversion of xylose and xylan to furfural has been extensively reported.
Similarly, the direct production of furfural from lignocellulosic biomass
has been investigated. For instance, 46 % of furfural yield was obtained
using choline chloride/oxalic acid as catalysts under the optimal con­
dition of 120 ◦ C for 30 min (Bu et al., 2020). When xylan was used the
reaction substrate, high furfural yields of 75 % were obtained with the
Fig. 1. The chemical composition changes of bamboo after DESs pretreatment.
choline chloride/malic acid/water DES under microwave heating for
2.5 min at 150 ◦ C (Morais et al., 2020). However, the downstream
processing of hemicellulose-derived pentoses in the DESs pretreatment 2.2. Pretreatment and fractional separation process
liquor have been rarely investigated. A promising utilization for con­
verting pentoses into furfural is using the DESs pretreatment liquor. The pretreatment trials were performed at 120 ◦ C for 2 h with
Therefore, this offers a potential approach to effective valorization of all magnetic stirring (400 rpm) in a pressure glass tube sealed with a PTEE
major biomass components including cellulose, lignin and resulting in plug. The solid-to-liquid ratio was 1:15, namely, 1.0 g of bamboo and 15
high furfural yield. mL of DESs. After the reaction time was completed, the solid residue and
According to the previous investigation (Lee et al., 2021), lactic acid pretreatment liquor were separated by centrifugation method at 10,000
(LA) as hydrogen bond donors (HBDs) had better pretreatment perfor­ rpm for 10 min. The recovered solid residue (cellulose-enriched pulp)
mance among these monocarboxylic acids. Most of the reported DESs were washed by deionized water and ethanol (2:1) until the filtrate fluid
were prepared using choline chloride (ChCl) or betaine as HBAs was neutral. Then, it was oven-dried at 45 ◦ C for 12 h and stored at
(Alvarez-Vasco et al., 2016). There are very few DESs utilizing tetra­ − 25 ◦ C. The residue containing the original lignin and polysaccharide
methylammonium chloride (TMAC) and tetramethylammonium bro­ could be further digested by cellulase to produce fermentable sugars.
mide (TMAB) as hydrogen bond acceptors (HBAs) and the application on Lignin in the treatment liquor was obtained by precipitation method in
biomass pretreatment is not well reported yet. Additionally, they have neutral water, followed by freeze-dried overnight. Additionally, the
strong phase transfer catalytic ability (Sasson et al., 1996). Based on the neutral water in the treatment liquor was removed via vacuum evapo­
above analysis, TMAB/LA and TMAC/LA DESs integrating solvent and ration at 60 ◦ C until treatment liquor reached the same amount as the
catalyst were synthesized. Specifically, bamboo was fractionated into original solvent volume, and then could be used for furfural production.
three streams: cellulose-enriched pulp, native-like lignin, and
hemicellulose-derived sugars in the treatment liquor. Using bamboo as a 3. Results and discussion
biomass feedstock, the effects of DESs with different mole ratios on the
chemical components, structural changes, enzymatic saccharification of 3.1. Mass recovery and chemical composition
cellulose-enriched pulp, and structural profiling of the regenerated DESs
lignin were systemically evaluated. Subsequently, the pyrolysis of Following the earlier work, the DESs treatment was performed at a
native-like lignin to produce aromatic chemicals was also explored. temperature of 120 ◦ C to achieve a reasonable glucan conversion and
Additionally, the treatment liquor was also collected and tested as a lignin valorization (Guo et al., 2018, 2019a, 2019b). After TMAB/LA
substrate for furfural production. This present study demonstrated a (1:3) pretreatment, the presence of crystallized materials in the sample,
potential integrated biorefinery approach for effective and sustainable indicated an incomplete separation of solid residue and pretreatment
utilization of lignocellulosic biomass. liquor. Thus, the sample was discarded and without further study. The
pretreatment performance was evaluated by analyzing the composition
2. Methods and materials of bamboo. As shown in Fig. 1, the raw bamboo was consisted of 41.05 %
cellulose, 21.64 % xylan, 1.56 % araban, 0.22 % galactan, 27.29 % acid
2.1. Materials insoluble lignin (AIL), and 1.95 % acid soluble lignin (ASL). After
TMAB/LA (1:6) pretreatment, the cellulose content was increased to
Bamboo was collected from Sichuan province, China. The sample 63.37 %, which was attributed to the removal of hemicellulose (77.85
was air-dried, followed by extraction with toluene/ethanol (2:1, v/v) %) and lignin (61.82 %). The lignin content (18.3 %) in the solid residue
and then oven-dried at 45 ◦ C for 12 h. The composition of raw bamboo was comparable to the lignin content (18 %− 19 %) in the oil palm
was cellulose 41.05 %, hemicellulose 23.42 %, and lignin 29.24 %. The empty fruit bunch after ultrasonication and ChCl/urea pretreatment
cellulase was bought from Novozymes (Beijing, China) and its paper (Lee et al., 2021). With increasing molar ratio of LA, the removal ratio of
activity was 100 FPU/mL. Tetramethylammonium chloride (TMAC), lignin and hemicellulose after TMAB/LA pretreatment was enhanced
tetramethylammonium bromide (TMAB), and lactic acid (LA) were gradually. Such increased delignification might be attributed to more
purchased from Sigma Chemical Co., Ltd (USA). All other chemicals active protons donated by lactic acid, which facilitated proton-catalyzed
(analytical grade) were obtained from Beijing Chemical Reagent Co., cleavage of various bonds, such as aryl ether bonds in DESs lignin
Ltd, China. streams, glycosidic bonds in polysaccharide, lignin-carbohydrate link­
ages in biomass. It was also observed that the proportion of LA in
TMAC/LA DESs was positively related to the lignin and hemicellulose

2
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Fig. 2. FE-SEM pictures (*2000) of bamboo before and after DESs treatment, raw bamboo (a), TMAB/LA 1:6 (b), TMAB/LA 1:9 (c), TMAC/LA 1:3 (d), TMAC/LA 1:6
(e), TMAC/LA 1:9 (f).

removal ratio. At the same molar ratio of LA, the TMAB/LA sample (Varilla-Mazaba et al., 2022), similar to the results by Provost and co­
showed a better performance with lignin removal (73.62 %) and workers (Provost et al., 2022). In conclusion, the above results indicated
hemicellulose removal (86.57 %) than that of TMAC/LA sample lignin that this novel DESs had a unique ability for selective removal of
(60.78 %) and hemicellulose (76.35 %) removal. The better pretreat­ hemicellulose and lignin from bamboo while preserving majority of
ment performance of bromide for ether bond cleavage was also inves­ cellulose.
tigated in acidic ionic liquids (Cronin et al., 2020), whereas
1-H-3-methylimidazolium bromide ([Hmim]Br) demonstrated faster 3.2. Cellulose-enriched pulp
cleavage of veratrylglycerol-b-guaiacyl ether and guaiacyl
glycerol-b-guaiacyl ether model compounds than 1-H-3-methylimidazo­ 3.2.1. SEM analysis
lium chloride ([Hmim]Cl). The results for high delignification were The samples were observed for their morphological characteristics
associated to the stronger nucleophilicity of the bromide anion as by using FE-SEM. As shown in Fig. 2, the raw bamboo exhibited a
compared with to chloride. Bromide had a faster substitution ability of continuous, rough, and rigid surface morphology. However, the surface
the hydroxyl group in lignin to form the corresponding halide inter­ morphologies of treated bamboo samples were altered dramatically
mediate that preceded the ether bond cleavage. Under the optimal (Fig. 2b-f). After TMAC/LA (1:3 and 1:6) DESs pretreatment, the surface
conditions, the delignification ration (73.62 %) was a litter lower than structure became less compact, rough, and some cracks were also
the lignin removal (81.2 %) from sugarcane bagasse after choline observed. When further increasing the molar ratio of LA in the DESs,
chloride (ChCl)/glycerol DES pretreatment at 160 ◦ C for 13.12 h holes and small particle-sized debris could be seen on the surface. After
TMAB/LA (1:6), some holes were also observed as compared with
TMAC/LA pretreatment. Additionally, the surface morphology of the
cellulose-enriched pulp became looser with wider separation of the fi­
bers and more larger holes than that of the DESs treated bamboo, indi­
cating that the TMAB/LA treatment removed more hemicelluloses and
lignin, which was consistent with the chemical composition analysis
(Fig. 1). All these changes of surface morphology resulted in producing
large amounts of reactive sites on the fiber surface, thereby increasing
the accessibility of enzymes and improving the enzymatic digestibility.

3.2.2. FTIR analysis

The functional groups information of cellulose-enriched samples
with different DESs pretreatment was revealed by the FTIR spectra
(1800–800 cm− 1) (Fig. S1). The peak at 1502 cm− 1 was attributed to
aromatic ring stretch vibrations (G) breathing in lignin. The peak
strength of carbohydrate-associated bands at 1733 cm− 1, 1363 cm− 1
and 890 cm− 1 corresponding to the carbonyl functional groups in
hemicellulose, C-H deformation in hemicellulose and cellulose, and C-H
deformation in cellulose, respectively, were reduced, indicating the
cleavage of glycosidic bonds in polysaccharide and lignin-carbohydrate
Fig. 3. Effect of reaction time on enzymatic hydrolysis of untreated and linkages in biomass. Additionally, the intensities of carbohydrates peaks
treated bamboo. decreased to some extent in the DESs treatment process. Especially, the

3
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Table 1
Contents (%) of associated polysaccharides in the regenerated lignin fractions.
Samples Arabinose Galactose Glucose Xylose Total Ash

MWL 0.98 0.02 1.64 2.56 5.20 0.38

TMAB/LA 1:6 0.15 0.02 0.35 0.78 1.30 0.35
TMAB/LA 1:9 0.14 0.02 0.11 0.64 0.91 0.34
TMAC/LA 1:3 0.42 0.03 0.24 1.13 1.82 0.37
TMAC/LA 1:6 0.34 0.04 0.33 1.02 1.73 0.37
TMAC/LA 1:9 0.19 0.06 0.29 0.52 1.06 0.35

decreased intensity of the band at 1729 cm− 1, suggested hemicelluloses

degradation. The decline of the intensities of lignin-associated bands
indicated the partial removal of lignin.

3.2.3. X-ray diffraction (XRD) analysis

To investigate the effect of DESs treatment on the cellulose crystal­
line structure, XRD analysis was conducted based on our previous
investigation. As shown in Fig. S2, the typical diffraction peaks of (110), Fig. 4. Weight-average (Mw), number-average (Mn) molecular weights and
(200) and (004) were observed for untreated bamboo. After DESs pre­ polydispersity (Mw/Mn) of the lignin fractions.
treatment, the crystalline structure of cellulose had not changed and
only an enhancement in intensity of diffraction peaks were determined. followed by glucose (0.11–0.35 %), arabinose (0.14–0.42 %), and
The CrI of the raw bamboo was 29.8 %, while CrI of the DESs treated galactose (0.0–0.06 %). Surprisingly, mannose, glucuronic acid, and
bamboo samples slightly increased to 32.7 %− 37.1 %. This enhance­ galacturonic acid were undetectable. The total associated poly­
ment in CrI was attributed to the efficient removal of hemicellulose and saccharides in lignin fractions decreased with increasing molar ratio of
lignin from bamboo, which was consistent with the above compositional LA, indicating that the ether and ester linkages in the plant cell wall of
analysis of bamboo (Fig. 1). bamboo were dramatically cleaved. Notably, the associated poly­
saccharides content (0.91 %− 1.82 %) was significantly lower than the
polysaccharides content in MWL (5.20 %). High lignin purity represents
3.3. Effect of pretreatment conditions on the enzymatic hydrolysis
an important parameter for selecting the optimal operational conditions
for the DESs fractionation (Z. Chen et al., 2019; Y.J. Chen et al., 2019;
To evaluate the effects of DESs treatment on fermentable sugars
Cronin et al., 2020). Most technical lignin samples (kraft lignin and
production, the enzymatic saccharification of the raw and treated
lignosulfonates) contained various amounts of impurities, such as ash,
bamboo was comparatively investigated. As plotted in Fig. 3, the raw
sulfur, and more sugars. Further purification was required prior to
bamboo exhibited a low glucose yield of 10.0 %. This might be related to
biochemical or chemical conversion processes. Sulfur free lignin samples
the cell wall recalcitrance owing to the coating effect of hemicellulose
are extremely versatile in thermosets and resins applications (Huang
and sealing function of lignin for cellulose, hindering the enzymatic
et al., 2019; Llevot et al., 2016). DESs-isolated lignin fractions offer an
hydrolysis (Wang et al., 2018). After TMAB/LA treatment, the enzy­
outstanding alternative to commercial lignin, as they are both sulfur free
matic digestibility was increased to 85.7 % and 91.0 %. This was
and of high purity.
ascribed to the efficient removal of hemicellulose and lignin, resulting in
more accessible surface area and adsorption for enzymes. In the TMA­
3.4.2. Molecular weight distribution
C/LA treatment at 120 ◦ C, the enzymatic digestibility of 82.8 %, 83.1 %,
The Mw, Mn, and polydispersity index (PDI) of all lignin streams
and 85.9 % was slightly lower than that for the TMAB/LA treated
were analyzed by gel permeation chromatography (GPC) (Deuss et al.,
samples at the same molar ratio. This was mainly due to the hemicel­
2015). As given in Fig. 4 and Fig. S4, milled wood lignin (MWL) had a
lulose and lignin contents in the treated samples. Notably, it was found
larger molecular weight (1620 g/mol of Mn and 3850 g/mol of Mw) due
that the bamboo treated with TMAB/LA (1:9) displayed the highest
to less damage to their chemical structures. However, the Mw and Mn of
enzymatic digestibility, as revealed by the chemical component analysis
DESs lignin streams were ranged from 1860 to 2560 g/mol and
(Fig. 1) and morphological features (Fig. 2). At an enzyme loading of
960–1380 g/mol, respectively. This might be ascribed to the cleaving of
15FPU/mL substrate, the TMAB/LA (1:9) had ten-fold higher glucose
aryl ether bonds leading to lignin degradation. Additionally, the mo­
yield than the raw bamboo. Additionally, the enzymatic digestibility of
lecular weight of lignin fractions increased slightly as the acid concen­
TMAB/LA (1:9) sample was higher than that of bamboo treated with
tration was increased, indicating the occurrence of condensation
acetamide/LA (56.44 %), betaine/LA (48.90 %), and choline chlor­
reactions. Furthermore, the DESs lignin samples displayed a relatively
ide/LA (79.07 %) (Ling et al., 2020). It can be concluded that this DESs
low PDI (<2.0) as compared with MWL (2.38), which implied the uni­
treatment had the advantages of short treatment time and high enzy­
form lignin structure.
matic digestibility, indicating a promising and cost-effective approach
for biomass processing and utilization.
3.4.3. 2D HSQC NMR analysis
NMR is a powerful tool to further understand the fundamental
3.4. Lignin characterization structures of lignin streams (Wen et al., 2013; Giummarella et al., 2019).
In the aliphatic-oxygenated region (δC/δH 50–90/2.5–6.0), the
3.4.1. Associated polysaccharides in the lignin streams cross-signals of methoxy groups (δC/δH 55.3/3.61) and side chains in
The native like characteristics of MWL from biomass and was used to β-O-4′ linkages were the most predominant structures in these lignin
clarify the structural characteristics of lignin streams obtained from streams (Table S2 and Fig. 5). The Cβ-Hβ correlations corresponding to
DESs fractionation. In order to detect the relative purity of DESs lignin β-O-4′ substructure linked to G units (Aβ(G)) and S units (Aβ(S)) were
streams, the chemical components and associated polysaccharides con­ distinguished at δC/δH 83.1/4.36 and δC/δH 85.9/4.07, respectively.
tent were also analyzed. As displayed in Table 1, the sugar contents Simultaneously, the signal at δC/δH 61.4/4.12 corresponded to the
ranged from 0.91 % to 1.82 % in the DESs lignin samples. Xylose γ-position of β-O-4′ linkages. The signals corresponding γ-acylated β-O-4′
(49.06–70.33 %) was the dominating sugars in the lignin streams,

4
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Fig. 5. 2D HSQC spectra of lignin fractions and main structures: (A) β-O-4′ linkages; (A′ ) β-O-4′ linkages with acetylated at γ-carbon; (A′′ ) γ-pcoumaroylated β-O-4′
linkages; (B) resinol structures (β-β′ ); (C) phenylcoumaran substructures (β-5′ ); (G) guaiacyl units; (H) p-hydroxyphenyl units; (S) syringyl units; (S′ ) oxidized syringyl
units with a Cα ketone; (FA) ferulate; and (PCA) p-coumarate.

5
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Table 2
Quantification of different linkages in lignin samples.
Samples β-O-4′ β-β′ β-5′ PCA PCA/FA S G H S/G
a b
MWL 54.4 (79.2) 11.6 2.7 38.6 44.6 54.5 39.6 5.9 1.4
TMAB/LA 48.6 (89.3 %) 5.8 -c 51.2 55.9 66.5 27.5 6.0 2.4
TMAC/LA 50.9 (86.3) 8.1 – 53.5 51.5 64.0 33.7 2.3 1.9
a
Results expressed per 100 Ar based on quantitative 2D-HSQC spectra.
b
The percentage of the bond content in the total bonds content.
c
Trace.

linkages substructures were observed in the DESs lignin streams,

implying the acylation of lactic acid. Additionally, resinol (β-β′ , B)
substructures were also obviously found, and the correlated signals of α-,
β-, and γ-positions in the side chain of resinol substructures were
observed at δC/δH 85.3/4.60, 53.6/43.12, and 71.2/3.72 and 4.20,
respectively. Furthermore, the Cα-Hα in the phenylcoumaran (β-5′ , C)
substructures were found in the MWL and TMAC/LA DES lignin, but
barely observed in the TMAB/LA DES lignin. Other small signals asso­
ciated with p-hydroxycinnamyl alcohol end groups (I) were also detec­
ted. Besides, the peaks of carbohydrates (X5 and X4) were found in the
MWL. However, only the X5 signal existed in the TMAB/LA DES lignin,
suggesting that DESs lignin had higher purity. This was consistent with
our previous results on the associated sugars in the DESs lignin streams
(Table 1).
In the aromatic region, the cross-signals from guaiacyl (G), syringyl
(S), and p-hydroxyphenyl (H) lignin units could be clearly distinguished
in these lignin samples. The S units exhibited a significant signal for the
C2,6-H2,6 correlation at δC/δH 104.0/6.55. Additionally, the signals
corresponding to C2,6-H2,6 correlations in Cα-oxidized S units (S′ , δC/δH
Fig. 6. TG curves of lignin samples.
105.5/7.41) were also observed. As compared with MWL, the condensed
structure (S2,6) occurred in the TMAB/LA and TMAC/LA DES lignin
streams, which was attributed to the presence of LA. The G units dis­ facilitated the removal of hemicellulose and lignin from bamboo. In
played different correlations for C2-H2, C5-H5, and C6-H6 at δC/δH 108.4/ short, the isolated lignin fractions preserved its native structure which
6.96, 115.2/6.72–6.72, and 119.4/6.87, respectively. The condensed had a high β-ether content. The obtained high-quality lignin was suitable
structure of G units was hardly detected in these lignin streams, for further pyrolysis to produce aromatic chemicals.
implying S-type units were reacted with other lignin units to form
condensed structures in the DESs fractionation process. Additionally, the 3.4.4. Thermal analysis
signal associated with H unit was distinguished at δC/δH 128.2/7.14. The thermal stabilities of MWL and DESs lignin streams were
Furthermore, the significant signals corresponding to ferulate (FA) and determined by TGA and the thermal decomposition curves at a tem­
p-coumarate (PCA) structures in bamboo were also detected. perature ranging from 50 ◦ C to 600 ◦ C. As illustrated in Fig. 6, most of
The relative abundances of the main lignin interunit linkages (β-O-4′ , the weak C-O linkages in β-O-4′ linkages were cleaved at the initial stage
β-β′ , and β-5′ ), different lignin units (G, S, and H), and basic composi­ of decomposition (≤200 ◦ C) (Pelucchi et al., 2019). When the temper­
tions (PCA/FA and S/G ratio) were semi-quantitatively characterized by ature increased to 220–350 ◦ C (Wen et al., 2013), the cleavage of aryl
the 2D-HSQC spectra. As displayed in Table 2, the content of β-O-4′ ether bond linkages occurred. Afterwards, the increased decomposition
linkages in TAMB/LA and TMAC/LA DES lignin (48.6–50.9/100Ar) rate was closely related with the lignin oxidation, such as the oxidation
were slightly lower MWL (54.4/100Ar), indicating that the β-O-4′ of aliphatic hydroxyl group to carbonylation or carboxylation and chain
linkages was sensitive to the acid condition, which made lignin macro­ dehydrogenation, within a temperature range from 350 ◦ C to 400 ◦ C.
molecule susceptible to depolymerization during the DESs process. As the temperature increases to 400 ◦ C or above, the C–C linkages,
However, the ratio of β-O-4′ linkage in the DESs lignin streams mainly the 5–5 (biphenyl) linkages, and the aromatic rings were
(86.3–89.3 %) was significantly higher than MWL (79.2 %), indicating decomposed releasing CO, CO2, and H2O (Yan et al., 2007). After the
the DESs lignin preserved its native structure. The high β-ether content depletion of elemental oxygen, the residues eventually formed biochar
was dramatically higher than the previous investigations (Guo et al., revealed by a flattened TGA curves at temperature of 500 ◦ C above. At
2019a, 2019b; Shen et al., 2019). It was reported that the preservation of 600 ◦ C, the residual mass was 32.41 %, 33.48 %, and 34.50 % respec­
β-O-4′ linkages was critical for aromatic chemicals production via lignin tively. The weight loss for DESs lignin was very similar with that of
pyrolysis. Moreover, a minor amount of β-β′ (5.8 %− 11.6 %) was also MWL, which indicated that the structural features of DESs lignin sam­
observed in these lignin streams. Additionally, the S/G ratio in lignin ples were close to that of native lignin (MWL).
played a vital role for structural profiling in the DESs fractionation
process. A slightly increased S/G ratio (from 1.4 to 2.4) indicated that 3.4.5. GC-MS analysis of the pyrolysis products
the guaiacyl units were more likely to degradation and recovery than the The products distributions are given in Fig. 7 and Table S3. As
syringyl units. Similar results were also observed by Penín and co­ shown, the pyrolysis products of MWL and DESs lignin streams were
workers (Penín et al., 2020). In the grass lignin, PCA and FA acylate mainly phenolic compounds and aromatic hydrocarbons. The aromatic
arabinoxylans, serve as the bridge between lignin and carbohydrates via hydrocarbons content in MWL (13.56 %) was slightly lower than that in
formation of ether and ester linkages. The increased relative percentages TMAB lignin (20.31 %) and TMAC lignin (20.36 %). There was no dif­
of PCA/FA (from 44.6 % to 55.9 %) indicated the cleavage of the ether ference in the aromatic hydrocarbon contents between the two DESs
bonds between ferulic acid and lignin during DESs pretreatment, which lignin. Aromatic hydrocarbons were mainly composed of toluene, 2,3-

6
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Fig. 8. Effects of reaction time (a), reaction temperature (b), and different
catalysts (c) on furfural yield from the DESs treatment liquid.

structural unit of lignin (p-hydroxyphenyl, guaiacyl, and syringyl). The

Fig. 7. Pyrolysis product distribution of MWL and DESs lignin at 600 ◦ C.
total phenolic compounds yield in TMAB lignin and TMAC lignin were
53.83 % and 52.69 %, respectively. The S-phenols, G-phenols, H-phe­
dihydrobenzofurnan, 1,2,4-trimethoxybenzene, and 3,5-dimethoxyace­ nols, and other phenols were 20.81–22.09 %, 23.88–24.59 %, 6.49–7.40
tophenone. The pyrolysis products of DESs lignin streams were % and 0.60–0.66 %, respectively, which was higher than the yield of
similar, whereas the relative abundance of the peaks slightly varied. As S-phenols (19.18 %), G-phenols (20.01 %), H-phenols (2.40 %), and
expected, significant amount of phenol and phenol derivatives such as 2- other phenols (0.52 %) in MWL. Additionally, the presence of phenolic
methoxy-4-methylphenol, 4-allyl-2,6-dimethoxyphenol, 2-methoxy-4- compounds mainly consisted of 2,4-dimethoxyphenol. This indicated
vinylphenol, syringaldehyde, acetosyringone were observed. Accord­ that more phenolic compounds were obtained by pyrolysis of DESs
ing to the previous investigations, the phenolic compounds could be lignin.
classified into H-phenols, G-phenols, and S-phenols (R. Kumar et al.,
2020; B. Kumar et al., 2020), which derived from three phenylpropane

7
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

Fig. 9. Mass flow for the proposed biorefinery based on 100 kg of bamboo.

3.5. Xylose-enriched pretreatment liquor for furfural production pretreatment liquor resulted in the maximum yield of 86.9 % and 78.6
%, respectively. This might be attributed to the electron configuration of
This novel DESs system fractionated bamboo into three main the cation (Luo et al., 2018). These results indicated the order of the
streams: cellulose-enriched pulp, native-like lignin, and hemicellulose- cation in the catalysts in terms of their furfural production was as fol­
derived sugars in the treatment liquor. We have investigated and dis­ lows: Cu2+>Al3+>Fe3+. As shown in Fig. S4, the reaction mechanism for
cussed the fermentable sugars production and native-like lignin recov­ furfural production in the acidic conditions involved two steps: (1) hy­
ery in the context of an integrated biorefinery. The xylose/xylan- drolysis of hemicellulose in bamboo to xylose in the acidic DESs systems;
enriched treatment liquor can be a suitable substrate for catalytic (2) Xylose was continuously dehydrated to produce furfural. In short,
upgrading. Additionally, the acidic nature of the liquor lends itself as a the above results demonstrated that the DESs pretreatment liquor was
good reaction medium for furfural production. As discussed above, DESs an efficient reaction medium and substrate for the production of plat­
treatment could efficiently remove xylan, leading up to 80 % xylan form chemicals.
removal. Thus, it could be more valuable to valorize the treatment liquor
into furfural while minimizing the generation of waste streams and 3.6. Mass balance
improving the revenue generation in a DESs-based biorefinery.
Due to the higher xylan removal, the pretreatment liquor in the To evaluate the efficiency of the novel DESs treatment, a mass bal­
TMAB/LA (1:9) and TMAC/LA (1:9) was selected as reaction medium ance based on 100 kg of bamboo was shown in Fig. 9. After the inte­
and substrate. Additionally, various catalytic reaction times were first grated process, cellulose was rich in the solid residue and lignin and
explored for optimum furfural production from bamboo pretreatment xylan fractions were rich in the DESs liquid phase, which was easy to
liquor. The investigation was performed at 120 ◦ C for 15–60 min with separate solid and liquid by centrifugation. After separation, the addi­
2 wt. % of AlCl3.6H2O catalyst, and the results are given in Fig. 8(a). tion of deionized water in the liquid phase would lead to the precipi­
Based on the above results, an optimal catalytic reaction time (30 min) tation of 13.35 kg of lignin when bamboo was pretreated by TMAB/LA
was chosen in the following investigation. As shown, the furfural yield in (1:9) at 120 ℃. The deionized water in the liquid fraction was removed
the TMAB/LA and TMAB/LA pretreatment liquor was 44.6 % and 38.5 via vacuum evaporation at 60 ◦ C and then the pretreatment liquor was
% when the reaction was 15 min. Increasing the catalytic reaction time collected and used for furfural production. The use of CuCl2.2 H2O
significantly increased the furfural yield (52.8–66.6 %) in the TMAB/LA catalyst with TMAB/LA pretreatment liquor at 140 ◦ C for 30 min
pretreatment liquor, which was remarkably higher than the furfural resulted in the maximum furfural of 11.69 kg. Additionally, the
yield in the TMAC/LA pretreatment (36.1–45.5 %). It is speculated that maximum yield of fermentable glucose of 38.52 kg. In conclusion, the
the furfural yield was related to the presence of Br-. It should be noted integrated biorefinery offers an effective approach for sustainable uti­
that the highest furfural yield (66.6 %) was obtained at a catalytic re­ lization of lignocellulosic biomass.
action time of 30 min.
Using the reaction time of 30 min, the reaction temperature within in 4. Conclusions
a range of 100 ◦ C, 120 ◦ C, and 140 ◦ C was then optimized for the best
furfural yield. As shown in Fig. 8(b), at 100 ◦ C, there was a relatively low Based on the proposed integrated biorefinery strategy, bamboo was
furfural yield using the TMAB/LA and TMAC/LA pretreatment liquor of refined by DESs at 120 ◦ C for 2 h. The lignin recovered from treated
28.4 % and 21.0 %, respectively. When the reaction temperature was liquor had a high purity (>90 %), low molecular weight (1860–2560 g/
increased from 100 ◦ C to 120 ◦ C, the furfural was increased to 66.6 % mol), low PDI (1.44–1.93), and excellent thermal stability. More
and 45.5 %, respectively. Higher temperature and appropriate reaction importantly, the DESs lignin showed comparable β-O-4′ linkages
times are shown to be beneficial for increasing the furfural yield. The (48.6–50.9 %) as compared with MWL (54.4 %). Pyrolysis of DESs lignin
highest furfural yield in the TMAC/LA pretreatment liquor of 72.6 % was found to result in 52.69–53.83 % of phenolic compounds and
was obtained at 140 ◦ C. However, the furfural yield in the TMAB/LA 20.31–20.36 % of aromatic hydrocarbons. After lignin extraction, under
pretreatment liquor had hardly improved as the temperature increased optimum conditions, the resultant xylose-enriched liquor could be
from 120 ◦ C to 140 ◦ C. Thus, improving the reaction temperature within further converted to a very high furfural yield (86.9 %). Furthermore,
a certain range was beneficial to furfural production. Based on these the enzymatic digestibility of DES treated bamboo biomass was
considerations, the catalytic temperature (140 ◦ C) was chosen for the increased from 10.0 % to 91.0 %. In summary, based on the results
following further investigations. The selection of catalysts (AlCl3.6H2O, presented here, the DES based integrated biorefinery shows great po­
FeCl3.6H2O, and CuCl2.2H2O) were also optimized and the results are tential for complete sustainable valorization of biomass resulting in
shown in Fig. 8(c). The above candidates were chosen from the catalysts value added products including furfural, native-like lignin and cellulose-
widely used in industrial production and laboratory research. Compa­ enriched pulp. This offers tremendous promise for the development of
rable furfural yield (63.4 % and 67.1 %) was observed in the TMAB/LA economically viable and environmentally sustainable nitrated bio­
pretreatment liquor when the catalysts were AlCl3.6H2O and refineries of the future.
FeCl3.6H2O. Similar results were also found TMAC/LA pretreatment li­
quor. The use of CuCl2.2H2O catalyst with TMAB/LA and TMAC/LA

8
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

CRediT authorship contribution statement Gürbüz, E.I., Gallo, J.M.R., Alonso, D.M., Wettstein, S.G., Lim, W.Y., Dumesic, J.A., 2013.
Conversion of hemicellulose into furfural using solid acid catalysts in
γ-valerolactone. Angew. Chem. Int. Ed. 52, 1270–1274. [Link]
Zongwei Guo: Experiments performing, Writing e review & editing, anie.201207334.
Jianzhen Mao: Experimental data acquisition, Methodology, Qilin Hong, S., Shen, X.J., Pang, B., Xue, Z., Cao, X.F., Wen, J.L., Sun, Z.H., Su, S.L., Yuan, T.Q.,
Zhang: Writing e review & editing, Feng Xu: Supervision, Project Sun, R.C., 2020. In-depth interpretation of the structural changes of lignin and
formation of diketones during acidic deep eutectic solvent pretreatment. Green.
administration. Chem. 22, 1851–1858. [Link]
Huang, K.F., Ma, S.Q., Wang, S., Li, Q., Wu, Z.J., Liu, J.C., Liu, R., Zhu, J., 2019.
Sustainable valorization of lignin with levulinic acid and its application in polyimine
Declaration of Competing Interest thermosets. Green. Chem. 21, 4964–4970. [Link]
Kang, S.M., Fu, J.X., Zhang, G., 2018. From lignocellulosic biomass to levulinic acid: a
review on acid-catalyzed hydrolysis. Renew. Sust. Energy Rev. 94, 340–362. https://
The authors declare that they have no known competing financial [Link]/10.1016/[Link].2018.06.016.
interests or personal relationships that could have appeared to influence Kumar, B., Bhardwaj, N., Agrawal, K., Chaturvedi, V., Verma, P., 2020. Current
the work reported in this paper. perspective on pretreatment technologies using lignocellulosic biomass: an emerging
biorefinery concept. Fuel Process. Technol. 199, 106244 [Link]
fuproc.2019.106244.
Data availability Kumar, R., Strezov, V., Weldekidan, H., He, J., Singh, S., Kan, T., Dastjerdi, B., 2020.
Lignocellulose biomass pyrolysis for bio-oil production: a review of biomass pre-
treatment methods for production of drop-in fuels. Renew. Sust. Energy Rev. 123,
No data was used for the research described in the article. 109763 [Link]
Lee, K.M., Hong, J.Y., Tey, W.Y., 2021. Combination of ultrasonication and deep eutectic
solvent in pretreatment of lignocellulosic biomass for enhanced enzymatic
Acknowledgements
saccharification. Cellulose 28, 1513–1526. [Link]
03598-5.
We gratefully acknowledged the National Key Research and Devel­ Lin, W.Q., Yang, J.L., Zheng, Y.Y., Huang, C.X., Yong, Q., 2021. Understanding the
opment Program of China (2019YFB1503804) and Programme of effects of different residual lignin fractions in acid-pretreated bamboo residues on its
enzymatic digestibility. Biotechnol. Biofuels 14 (1), 143. [Link]
Introducing Talents of Discipline to Universities (Project 111, B21022). s13068-021-01994-y.
Ling, Z., Guo, Z.W., Huang, C.X., Yao, L., Xu, F., 2020. Deconstruction of oriented
crystalline cellulose by novel levulinic acid based deep eutectic solvents
Appendix A. Supporting information
pretreatment for improved enzymatic accessibility. Bioresour. Technol. 305, 123025
[Link]
Supplementary data associated with this article can be found in the Liu, Y.Z., Chen, W.S., Xia, Q.Q., Guo, B.T., Wang, Q.W., Liu, S.X., Liu, Y.X., Li, J., Yu, H.
P., 2017. Efficient cleavage of lignin-carbohydrate complexes and ultrafast
online version at doi:10.1016/[Link].2022.115453.
extraction of lignin oligomers from wood biomass by microwave-assisted treatment
with deep eutectic solvent. ChemSusChem 10, 1692–1700. [Link]
References cssc.201601795.
Llevot, A., Grau, E., Carlotti, S., Grelier, S., Cramail, H., 2016. From lignin-derived
aromatic compounds to novel biobased polymers. Macromol. Papid. Comm. 37 (1),
Alvarez-Vasco, C., Ma, R.S., Quintero, M., Guo, M., Geleynse, S., Ramasamy, K.K.,
9–28. [Link]
Wolcott, M., Zhang, X., 2016. Unique low-molecular-weight lignin with high purity
Luo, Y.P., Li, Z., Liu, X.F., Liu, X., Fan, J.J., Clark, J.H., Hu, C.W., 2018. The production
extracted from wood by deep eutectic solvents (DES): a source of lignin for
of furfural directly from hemicellulose in lignocellulosic biomass: a review. Catal.
valorization. Green. Chem. 18, 5133–5141. [Link]
Today 319, 14–24. [Link]
Beig, B., Riaz, M., Naqvi, S.R., Hassan, M., Zheng, Z.F., Karimi, K., Pugazhendhi, A.,
Morais, E.S., Freire, M.G., Freire, C., Coutinho, J., Silvestre, A., 2020. Enhanced
Atabani, A.E., Chi, N.T.L., 2021. Current challenges and innovative developments in
conversion of Xylan into furfural using acidic deep eutectic solvents with dual
pretreatment of lignocellulosic residues for biofuel production: a review. Fuel 287,
solvent and catalyst behavior. ChemsusChem 13 (4), 784–790. [Link]
119670. [Link]
10.1002/cssc.201902848.
Bu, C.Y., Yan, Y.X., Zou, L.H., Ouyang, Z.J., Zheng, Z.J., Ouyang, J., 2020.
Pelucchi, M., Cavallotti, C., Cuoci, A., Faravelli, T., Frassoldati, A., Ranzi, E., 2019.
Comprehensive utilization of corncob for furfuryl alcohol production by chemo-
Detailed kinetics of substituted phenolic species in pyrolysis bio-oils. React. Chem.
enzymatic sequential catalysis in a biphasic system. Bioresour. Technol. 319, 124156
Eng. 4, 490–506. [Link]
[Link]
Penín, L., López, M., Santos, V., Alonso, J.L., Parajo, J.C., 2020. Technologies for
Chen, Y.J., Zhang, L.L., Yu, J., Lu, Y.Z., Jiang, B., 2019. High-purity lignin isolated from
Eucalyptus wood processing in the scope of biorefineries: a comprehensive review.
poplar wood meal through dissolving treatment with deep eutectic solvents. R. Soc.
Bioresour. Technol. 311, 123528 [Link]
Open. Sci. 6 (1), 181757 [Link]
Provost, V., Dumarcay, S., Ziegler-Devin, I., Boltoeva, M., Trébouet, D., Villain-
Chen, Z., Jacoby, W.A., Wan, C.X., 2019. Ternary deep eutectic solvents for effective
Gambier, M., 2022. Deep eutectic solvent pretreatment of biomass: influence of
biomass deconstruction at high solids and low enzyme loadings. Bioresour. Technol.
hydrogen bond donor and temperature on lignin extraction with high β-O-4 content.
279, 281–286. [Link]
Bioresour. Technol. 349, 126837 [Link]
Cronin, D.J., Chen, X.W., Moghaddam, L., Zhang, X., 2020. Deep eutectic solvent
Putten, R.V., Van, D., Jong, E.D., Rasrendra, C.B., Heeres, H.J., Vries, J.D., 2013.
extraction of high-purity lignin from a corn stover hydrolysate. ChemSusChem 13
Hydroxymethylfurfural, a versatile platform chemical made from renewable
(17), 4678–4690. [Link]
resources. Chem. Rev. 113 (3), 1499–1597. [Link]
Deuss, P.J., Scott, M., Tran, F., Westwood, N.J., Vries, J., Barta, K., 2015. Aromatic
Sasson, Y., Negussie, S., Royz, M., Mushkin, N., 1996. Tetramethylammonium chloride as
monomers by in situ conversion of reactive intermediates in the acid-catalyzed
a selective and robust phase transfer catalyst in a solid-liquid halex reaction: the role
depolymerization of lignin. J. Am. Chem. Soc. 137 (23), 7456–7467. [Link]
of water. Chem. Commun. 297 (3), 1–2. [Link]
org/10.1021/jacs.5b03693.
Shen, X.J., Wen, J.L., Mei, Q.Q., Xue, C., Sun, D., Yuan, T.Q., Sun, R.C., 2019. Facile
Ding, S.Y., Liu, Y.S., Zeng, Y.N., Himmel, M.E., Baker, J.O., Bayer, E.A., 2012. How does
fractionation of lignocelluloses by biomass-derived deep eutectic solvent (DES)
plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science
pretreatment for cellulose enzymatic hydrolysis and lignin valorization. Green.
338 (6110), 1055–1060. [Link]
Chem. 21 (2), 275–283. [Link]
Dong, F., Zhu, Y.L., Guoqiang, Ding, G.Q., Cui, J.L., Li, X.Q., Li, Y.W., 2015. One-step
Tachibana, Y., Yamahata, M., Kasuya, K., 2013. Synthesis and characterization of a
conversion of furfural into 2-methyltetrahydrofuran under mild conditions.
renewable polyester containing oxabicyclic dicarboxylate derived from furfural.
ChemSusChem 8 (9), 1534–1537. [Link]
Green. Chem. 15 (5), 1318–1325. [Link]
Giummarella, N., Lindén, P., Areskogh, D., Lawoko, M., 2019. Fractional profiling of
Varilla-Mazaba, A., Raggazo-Sanchez, J.A., Calderon-Santoyo, M., Gomez-Rodríguez, J.,
kraft lignin structure: unravelling insights on lignin reaction mechanisms. ACS
Aguilar-Uscanga, M.G., 2022. Optimization of lignin extraction by response surface
Sustain. Chem. Eng. 8, 1112–1120. [Link]
methodology from sugarcane bagasse using deep eutectic solvents (DES). Ind. Crop.
acssuschemeng.9b06027.
Prod. 184, 115040 [Link]
Guo, Z.W., Ling, Z., Wang, C., Zhang, X.M., Xu, F., 2018. Integration of facile deep
Wang, W., Tan, X.S., Yu, Q., Wang, Q., Qi, W., Zhuang, X.S., Wang, Z.M., Yuan, Z.H.,
eutectic solvents pretreatment for enhanced enzymatic hydrolysis and lignin
2018. Effect of stepwise lignin removal on the enzymatic hydrolysis and cellulase
valorization from industrial xylose residue. Bioresour. Technol. 265, 334–339.
adsorption. Ind. Crop. Prod. 122, 16–22. [Link]
[Link]
indcrop.2018.05.053.
Guo, Z.W., Zhang, Q.L., You, T.T., Ji, Z., Xu, F., 2019. Heteropoly acids enhanced neutral
Wen, J.L., Xue, B.L., Sun, S.L., Sun, R.C., 2013. Quantitative structural characterization
deep eutectic solvent pretreatment for enzymatic hydrolysis and ethanol
and thermal properties of birch lignins after auto-catalyzed organosolv pretreatment
fermentation of Miscanthus x giganteus under mild conditions. Bioresour. Technol.
293, 122036 [Link]
Guo, Z.W., Zhang, Q.L., You, T.T., Zhang, X., Xu, F., Wu, Y.Y., 2019. Short-time deep
eutectic solvent pretreatment for enhanced enzymatic saccharification and lignin
valorization. Green. Chem. 21, 3099–3108. [Link]

9
Z. Guo et al. Industrial Crops & Products 188 (2022) 115453

and enzymatic hydrolysis. J. Chem. Technol. Biot. 88 (9), 1663–1671. [Link] Yu, Q., Zhuang, X.S., Wang, W., Qi, W., Wang, Q., Tan, X.S., Kong, X.Y., Yuan, Z.H.,
org/10.1002/jctb.4017. 2016. Hemicellulose and lignin removal to improve the enzymatic digestibility and
Yang, H.P., Yan, R., Chen, H.P., Lee, D.H., Zheng, C.G., 2007. Characteristics of ethanol production. Biomass. Bioenergy 94, 105–109. [Link]
hemicellulose, cellulose and lignin pyrolysis. Fuel 86 (12–13), 1781–1788. https:// biombioe.2016.08.005.
[Link]/10.1016/[Link].2006.12.013.

10