Industrial Crops & Products 191 (2023) 115965

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Microwave-assisted fast co-production of fermentable sugar and

nanocellulose via tunable zinc acetate based deep eutectic
solvents treatments
Ying Xie a, Jinyi Zhao a, Peng Wang a, Zhe Ji b, Zhe Ling a, *, Qiang Yong a
a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037,
China
b
College of Marine Science and Bioengineering, Qingdao University of Science and Technology, Qingdao 266042, China

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

Keywords: Deep eutectic solvent (DES) treatment has been considered as a promising approach to lignocellulose decon­
DES treatment struction and valorization. Nevertheless, some aspects such as high energy consumption and cellulose residue
Bamboo holocellulose loss limited bioconversion efficiency. Herein, neutral DESs based on choline chloride (ChCl) and zinc acetate
Co-production
(ZAC) in different molar ratios were designed for ultra-fast treatment on bamboo holocellulose with the assis­
Enzymatic hydrolysis
Nanocellulose
tance of microwave radiation for the first time. ZAC/ChCl-1 promoted the glucose conversion yield to 67.3%, due
to more hydrophobic sites exposed on crystallite surfaces caused by fibrils swelling and enlarged gaps between
adjacent glucan chains after the treatment. Furthermore, the proposed DES also displayed the ability to exfoliate
cellulose nanofibrils with high aspect ratios from microfibrils at high treatment temperature (120 ◦ C). With a
large amount of negative charge on surface, the nanocellulose maintained satisfying dispersion stability and
homogeneity, which achieved the full use and tunable dual conversion of bamboo cellulose. Thus, the present
study provides a deeper understanding of novel DES pretreatment for biomass conversion and widens the scope
of high-efficiency biorefinery.

1. Introduction
The concerns on environmental changes and the shortage of fossil
energy have increased, augmenting the demand for environmentally
friendly biological resources (Chen et al., 2021a; b, c; Ge et al., 2021).
Biomass wastes such as bamboo or wood processing leftovers can be
used for biorefinery (Li et al., 2021a; b; Yang et al., 2020) or prepared
into carbon-based materials due to their easy availability, low cost,
porosity (Heinze, 2018; Lou et al., 2022; Wang et al., 2023), etc. As the
major component in cell walls of lignocellulose, cellulose is a linear and
high-molecular polymer composed of glucose connected by β-1,
4-glycosidic bonds. The inter- and intra- hydrogen bond network of
glucan chains and van der Waals force formed by the stacking between
chain layers formed a highly-aggregated and condensed two-phase
structure of cellulose (Mohanty et al., 2021; O’Sullivan, 1997). Con­
version of cellulose into glucose is an important procedure in
bio-refining of chemicals, fertilizers, biofuels and other types of bio­
energy (Chen et al., 2022; Chen, 2020; Yu et al., 2020; Zhou et al.,
2022). In addition, cellulose and its derivatives are also important raw
materials in the field of functional materials production (Hu et al.,
2021a; b; Wang et al., 2017). For example, nanocellulose that is
extracted from cellulose microfibrils by chemical or mechanical
methods has been discussed heatedly, due to its advantages in me­
chanical properties and tailorable surface chemistry (Lu et al., 2020).
Although widely distributed and researched, there still appear dif­
ficulties for highly efficient conversion and application of cellulose. The
complex structure, uneven distribution of cellulose in plant cell walls,
and inherent physical barrier formed by lignin-carbohydrate complexes
(LCC) surrounding cellulose, strongly hinder the isolation of cellulose
(Hu et al., 2021a; b). Moreover, the deconstruction of cellulose requires
overcoming complex of highly aggregated cellulose microfibrils
embedded in lignocellulose cell walls, high cellulose crystallinity and
large molecular weight, etc. (Chundawat et al., 2011; Kostag et al.,
2019). Thus, exploration of satisfying pretreatment techniques is
required to alter the aggregated structure of cellulose including thinner
microfibrils and lowered crystallinity for higher conversion efficiency to

* Corresponding author.
E-mail address: jjling19@njfu.edu.cn (Z. Ling).

https://doi.org/10.1016/j.indcrop.2022.115965
Received 16 September 2022; Received in revised form 4 November 2022; Accepted 9 November 2022
Available online 21 November 2022
0926-6690/© 2022 Elsevier B.V. All rights reserved.
Y. Xie et al.
~85% (Ling et al., 2019; Zhang, Hara, 2016).
Traditional pretreatment technologies such as hydrothermal pre­
treatment, steam explosion and diluted acid pretreatment have been
widely applied to lignocellulose pretreatment. However, expensive cost,
high energy consumption and high carbon emissions seriously hinder
the process feasibility (Wang and Lee, 2021). Deep eutectic solvent
(DES) is a new type of green pretreatment solvent that has been widely
proved to be a suitable solvent for cellulose pretreatment (Sirviö et al.,
2020). Compared with other chemical pretreatments, DES is economi­
cally low-cost, environmentally friendly, more recyclable, as well as
owning great potential in cleavage of strong hydrogen bonds of cellulose
(Isci et al., 2021). DESs based on choline chloride have shown significant
promise for the enhancement of cellulosic enzymatic hydrolysis (Kumar
et al., 2016; Shen et al., 2019a; b). Meanwhile, similar DESs were also
reported to be used for productions of nanocellulose by breaking
hydrogen bonds and swelling cellulose, recently, which widened the
application of such solvents in lignocellulose conversion research. For
example, Li et al. reported that ChCl-based DES combined with enzy­
matic hydrolysis and high pressure homogenization can obtain stable
LCNF from corncob via (Li et al., 2021a, b). DES (ChCl and anhydrous
citric acid) assisted by high pressure homogenization can produce CNFs
to a width of 20–80 nm from bamboo (Liu et al., 2019). Therefore, it
would be of great interest to explore the intrinsic mechanism of the
production of both products by DES, as well as the convenient process
for the co-production of nanocellulose and fermentable sugars in bio­
refinery systems.
DES pretreatment commonly requires a long treatment time (~2 h)
to improve the bio-conversion performance of lignocellulose (Iurii et al.,
2020). Therefore, lowering energy consumption is also required for the
development of DES pretreatment process. In recent years, microwave
has been shown as an effective approach to reducing pretreatment time
as well as maximizing the ionic character of DESs (Isci et al., 2020).
Recently, our group reported a high enzymatic conversion yield (50%−
60%) of moso bamboo cellulose to glucose after 10 min of ChCl based
DESs treatment with the assistance of microwave radiation (Ling et al.,
2021). Moreover, microwave-assisted deep eutectic solvent pretreat­
ment can fabricate nanocellulose from cotton cellulose with high yields
over 3 min at 80 ℃ (Liu et al., 2017). It is believed that
microwave-assisted DES pretreatment will facilitate the development of
strategies for cellulose deconstruction and nanocellulose isolation. On
the other hand, cellulose could be hydrolyzed to oligosaccharides or
esterified when acid is used as HBD (Zhang et al., 2021). These
by-products can hinder subsequent processing. Thus, methods for the
removal of side reactions of DES will be worth investigating to avoid
their negative impact on bioconversion efficiency and the cost of cel­
lulose. One of the solutions was proved as the selection of neutral DES,
such as metal ions-based DES. Nevertheless, metal ions-based DESs
(Diego, Ramón, 2019; Suopajärvi et al., 2020; Selkala et al., 2016) have
been mainly featured in the studies of activation and conversion of other
chemical compounds. There has been limited literature on such DES
system in lignocellulose pretreatment.
Hence, neutral DESs based on ChCl and zinc acetate as HBA and HBD
were developed for the co-production of fermentable sugars and nano­
cellulose from moso bamboo holocellulose in the present study. The
effects of rapid microwave-assisted pretreatment on moso bamboo
holocellulose were evaluated. In this strategy, the novel DES effectively
changed the crystal structure and microscopic morphology of cellulose
in a short time, favoring its biorefinery. By tuning processing tempera­
tures and ratios of DES, the feasibility of the process to produce nano­
cellulose was further explored. In summary, the proposed fast and new
DES pretreatments on bamboo holocellulose solve the common prob­
lems in biomass pretreatments, such as cellulose residue loss, long pre­
treatment time and limited utilization efficiency. Moreover, it provided
a new strategy for tunable bioconversion of cellulose to fermentable
sugars and applicable nanomaterials.
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Industrial Crops & Products 191 (2023) 115965
2. Materials and methods
2.1. Materials
Moso bamboo (provided by Yunnan Forestry Center, China) was
milled into 20–80 mesh sample powder. Then the materials are Soxhlet
extraction with a volume ratio of 2:1 acetone-ethanol solution for 6 h to
remove extractives and air-dried overnight.
Zinc acetate (ZAC), ChCl and sodium chlorite were purchased from
Macklin Chemical Company (Shanghai, China). Ethanol, acetic acid and
acetone were purchased from Shanghai Chemical Reagent Company
(Shanghai, China). All the chemicals were analytical grade and used
directly without further purification.
2.2. Delignification
In order to effectively remove lignin, bamboo holocellulose was
prepared by bleaching method. 5 g of samples were treated in 100 ml of
5% (w/w) sodium chlorite solution with stirring at 75 ◦ C for 4 h. During
the process, a certain amount of acetic acid was added to adjust the pH of
the solution to maintain 4.0. After the reaction, the bleached samples
were washed in distilled water to a neutral pH value and air-dried
overnight. Holocellulose is denoted as HC.
2.3. DES synthesis and microwave assisted pretreatment
ZAC and ChCl were mixed at a molar ratio of 1:1 or 1:2, and heated in
an erlenmeyer flask at 80 ◦ C for 2 h. The solution is continuously stirred
by the stirring blade during the heating process. The solvent in which
ZAC and ChCl were mixed at a molar ratio of 1:1 is called ZAC/ChCl-1,
and the other is called ZAC/ChCl-2.
The pretreatment process was carried out in a microwave reactor
(MDS-8 G, Sineo Co. Ltd., Shanghai, China). 5 g of HC powder was added
to a Teflon container and mixed well after adding different 50 ml DESs
separately. The microwave irradiation power was set as 600 W, and the
treatment temperatures were 80 ◦ C, 100 ◦ C, and 120 ◦ C for 3 min
(heating up for 5 min and retention for 3 min). After the treatment was
completed, acetone/water was added for alternate washing, and then
centrifuged to obtain a powder residue. The powders were freeze-dried
and used in various tests to analyze the treatment effect. The sample
powder obtained by the treatment at 120 ◦ C was taken out, dispersed in
deionized water at a solid-liquid ratio of 1:10 (w/w), and subjected to
high-pressure homogenization to obtain nanocellulose. The nano­
cellulose was used for further testing. The materials treated by DES at
the molar ratio of 1:1 at 80 ℃, 100 ℃ and 120 ℃ are respectively noted
as ZAC-1, ZAC-2, and ZAC-3. Correspondingly, the materials treated at
the molar ratio of 1:2 at 80 ℃, 100 ℃ and 120 ℃ are marked as ZAC-4,
ZAC-5, ZAC-6. The process of DES pretreatment is shown in Fig. 1.
2.4. Characterization
2.4.1. Chemical composition analysis
The samples were analyzed for their respective chemical composi­
tions by the National Renewable Energy Laboratory (NREL) method.
Fig. 1. DES pretreatment process with different molar ratios.
Y. Xie et al.
The samples were first hydrolyzed with 72 % sulfuric acid in a constant
temperature water bath at 30 ◦ C for 1 h, and then hydrolyzed with 4 %
dilute sulfuric acid in an autoclave at 121 ◦ C for 1 h. After the reaction,
the filtrate obtained by filtration is put into a liquid chromatograph
(HPLC) for detection. The concentration of monosaccharides is analyzed
by using an Aminex HPX-87 H column (300 × 7.8 mm), a refractive
index detector (column temperature 55 ◦ C, a flow rate of 0.6 ml•min− 1,
0.05 M H2SO4 as the mobile phase), and an Agilent high performance
liquid chromatograph 1200.
The FTIR analysis was performed by Fourier transform infrared
spectrometer Nicolt iS5. The infrared wavenumber measurement range
was 4000–500 cm− 1, the step distance was 4 cm− 1, and each scan was
20 times.
TGA Q500 analyzer (TA Instruments) was used to test the samples.
During the detection process, it was filled with nitrogen (60 ml), the
injection rate was 10 ◦ C•min− 1, and it was heated to 600 ◦ C.
2.4.2. Enzymatic hydrolysis
The cellulase we selected (Celic CTec2) was purchased from Novo­
zymes, and the enzyme activity of this product is 250 FPU/ml. 20 ml of
5% sample suspension was prepared in a 50 ml Erlenmeyer flask by
adding 1 M citrate buffer, 60 μl tetracycline, and 20 FPU/g dextran
cellulase. The reaction system was placed in a 50 ◦ C water bath for
enzymatic hydrolysis for 72 h.
2.4.3. Scanning electron microscopy (SEM) and X-ray diffraction (XRD)
The samples were analyzed by JOEL JSM-7600 F field emission
scanning electron microscope. The acceleration voltage during the
experiment was 10 kV.
The sample was tested by Shimadzu X-ray diffractometer-6100. The
scanning range is 2θ= 5◦ − 50◦ , the scanning speed is 5◦ /min, the
scanning step is 0.02◦ , the wavelength λ = 1.5419 Å, the voltage is
40 kV, and the current is 40 mA. X-ray diffraction pattern may be
calculated using Maud Rietveld software (French, 2020):
Acrystal
CrI = × 100% (1)
Acrystal + Aamorph
Where CrI is the crystallinity of cellulose, Acrystal is the calculated area of
crystallinity cellulose and Aamorph is the calculated area of amorphous
content.
The crystal size is calculated by the Scherrer formula (Patterson,
1939):
D=
Kλ
(2)
βcosθ
Where K is the Scherrer constant, K= 0.9; β is the half-width of the
diffraction peak; θ is the Bragg diffraction angle; λ is the X-ray wave­
length, which is 1.5419 Å.
Bragg equation (Pope, 1997) was used to calculate the interplanar
spacing:
λ = 2Dsinθ (3)
λ = 1.5419 Å; D represents the distance between molecular chains; θ
represents the angle between the incident ray, the reflected line and the
reflecting crystal plane.
2.4.4. Characterizations of nanocellulose
The obtained nanocellulose suspension was placed in the Malvern
Zetasizer Nano ZS instrument for detection. The instrument can use
dynamic light scattering to measure the particle size and zeta potential
of nanocellulose, so as to judge the stability and particle size of
nanocellulose.
In addition, Asylum Research MFP-3D atomic force microscopy
(AFM) was used to characterize nanocellulose dispersions to obtain
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Industrial Crops & Products 191 (2023) 115965
information about nanocellulose, enabling the possibility of quantitative
calculations. The samples were imaged in ScanAsyst mode and per­
formed using Nanoscope Analysis software.
3. Results and discussion
3.1. Chemical composition analysis
Fig. 2a shows the chemical composition of HC and the pretreated
samples with different DES solvent systems. Raw moso bamboo showed
high contents of cellulose (53.44%) and hemicelluloses that mainly
consist of xylan (22.79%). Pretreatment of ZAC/ChCl-1 and ZAC/ChCl-2
both resulted in an obvious reduction of hemicellulose content to ~18%,
while there is no loss in the content of cellulose during DES pretreat­
ment, similar to the results reported by Pan et al.(Pan et al., 2017). The
partial removal of xylan hemicellulose and cellulose maintenance would
be beneficial for following enzymatic hydrolysis.
FTIR spectra showed changes in lignocellulose characteristic peaks
which relate to O-H, C-H, C– – O, C-O bonds stretching (at peak of
3331 cm− 1, 2891 cm− 1, 1734 cm− 1, 1031 cm− 1) (Fig. 2b). After the DES
pretreatment, the characteristic peaks at 3331 cm− 1 almost dis­
appeared. This indicates that the bands between 3500 and 3100 cm− 1
for intramolecular and intermolecular hydrogen bonds have been dis­
rupted by DES (Hinterstoisser and Salmén, 1999). Especially in ZAC-4,
ZAC-5, ZAC-6, the intensities of characteristic peaks at 3331 cm− 1
were much lower than in the others, suggesting the higher capability of
DES ZAC/ChCl-2 on disruption of hydrogen bonds. In addition, both two
types of DES pretreatment showed reduce in absorbance at 1734 cm− 1
as compared with HC. These peaks demonstrate the removal of hemi­
cellulose since they imply the stretching vibration of acetyl groups in
hemicellulose (Thi and Lee, 2019). The phenomenon agrees with the
above compositional analysis. The changes in thermal stability of sam­
ples after DES pretreatment can be analyzed with thermogravimetric
(TGA) and derivative thermogravimetry (DTG) curves (Fig. S1). The
change in DTG curve was attributed to the evaporation of water and the
loss of low molecular weight compounds in samples at 30–100 ℃. As the
temperature increases, the free water is released followed by the
breaking of glycosidic bond and the dehydration of the glucose group.
These reactions will produce primary degradation products such as acids
and alcohols, which will decompose further into CO and CH4 by
breaking carbon-carbon and carbon-oxygen bonds and be aromatized
into charcoal. Simultaneously, hemicellulose polysaccharides also show
dehydration, bond breaking, thermal cracking, polycondensation and
aromatization (Wang et al., 2007). All reactions were revealed by the
mass loss in TGA curves. After pretreatment, the pretreated samples
displayed a lower decomposition temperature of 240–321 ℃ compared
to HC of 338 ℃. Equally, the large weight loss temperature of HC
(254 ℃) was higher than those of others (184–193 ℃). The change in­
dicates that the pretreated samples with the destruction of the cellulose
and the degradation of hemicellulose are more prone to degradation at
lower temperatures. The decreased thermal stability could be ascribed to
the loosened structure of the DESs pretreated samples (Liu et al., 2022).
In addition, the pretreatment which exposed high specific surface area
and surface-enriched active groups facilitate the thermal degradation.
3.2. Morphology and crystal structural analysis
The morphology of the samples after DES pretreatment was deter­
mined by SEM to better understand the changes in cellulose structure
(Fig. 3). The untreated HC had a smooth surface and well-organized
fibrillar structure. After DES pretreatment, the loss of broken fibrils
was observed, indicating that ZAC/ChCl-1 and ZAC/ChCl-2 can both
change the surface morphology of cellulose to different extents, which
was consistent with previous studies (Sun et al., 2022a). The inter- and
intra-hydrogen bonds were broken by DESs, which contributed to more
exposure to microfibrils (Mamilla et al., 2019). In addition, the severity
Y. Xie et al.
Fig. 2. (a) The content of cellulose, hemicellulose and lignin in the samples; (b) FTIR spectra of moso bamboo powders during DES pretreatments.
Fig. 3. SEM observations of HC (a) and ZAC-1 (b), ZAC-2 (c), ZAC-3 (d), ZAC-4 (e), ZAC-5 (f), ZAC-6 (g) DES pretreated moso bamboo powders.
of damage is related to temperature. High temperature is conducive to
more adequate contact between DES and cellulose microfibrils. There­
fore, the surface of the samples treated at higher temperature had
rougher surface, which tied well with previous studies (Isci et al.,
2020b). Furthermore, the larger particle size and surface porosity of the
pretreated samples can be observed clearly. This can be explained as the
swelling of cellulose by DES. Since the dispersion and swelling of cel­
lulose are easily observed in several molten salt hydrates such as Zn
(NO3)2⋅6 H2O (Marcus, 2019), we can speculate that the swelling
behavior of cellulose is attributed to the presence of zinc ions. In
particular, ZAC/ChCl-1 has a higher molar content of zinc ions, which
brought about wider fibrils of cellulose in ZAC-1, ZAC-2, and ZAC-3.
Fiber swelling and removal of the association of hemicellulose may
improve the accessibility of cellulose towards detection reagent (Gos­
sett, 1985).
DES pretreatment also altered the cellulose crystal structure revealed
by the XRD determination (Chundawat et al., 2011). Three diffraction
peaks at around 2θ = 14.9◦ , 16.7◦ and 22.5◦ of XRD patterns are
assigned to (1 − 1 0), (1 1 0) and (2 0 0) lattice planes of crystalline
cellulose Iβ (French, 2014) (Fig. 4a). The XRD patterns showed that,
limited impacts on the allomorphs of cellulose were caused by
ZAC/ChCl-1 and ZAC/ChCl-2 DES. The information of cellulose crystal
structures calculated by Maud Rietveld analysis is shown in Fig. 4b.
After DES pretreatment, the crystallinities of ZAC-1, ZAC-2, ZAC-4,
ZAC-5 were 73.4%− 77.5%, which was higher than that of HC
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Industrial Crops & Products 191 (2023) 115965
(66.7%). The removal of amorphous components such as hemicellulose
from biomass contributed to higher crystallinity (Chen et al., 2021a; b,
c). On the other hand, the preferential degradation of the amorphous
region of cellulose by DES also resulted in increased crystallinity. It is
notable that the crystallinity of ZAC-3 and ZAC-6 decreased despite the
removal of amorphous components, indicating the possible cellulose
decrystallization at higher treatment temperatures. Combined with FTIR
spectra, it can be seen that the decrease in crystallinity is due to the
breaking of the complex inter- and intra-molecular hydrogen bonds of
cellulose by solvents ZAC/ChCl-1 and ZAC/ChCl-2. Focusing on the
hydrophobic (2 0 0) plane of cellulose that is regarded as the cellulase
enzyme adsorption plane (Ding, 2012), the d-spacing generally
increased, but the crystallite sizes showed different trends owing to
different ratios of ZAC and ChCl in DES pretreatment (Fig. 4c and
Fig. 4d). The crystallite sizes and d-spacing values are in line with earlier
reports (Ling et al., 2021b). The increase in d-spacing is dependent on
the weak interplay between intramolecular hydrogen bonding. In
particular, more zinc ions in ZAC/ChCl-1 worked on the surface of the
cellulose chains and attracted polar hydroxyl groups, which led to
swelling as revealed in SEM observations (Burger et al., 2020). The in­
crease in the distance among the adjacent lattice planes will facilitate
the contact of cellulose and cellulose enzymes. Meanwhile, the smaller
crystallite size of ZAC-1, ZAC-2, and ZAC- 3 may be due to some release
of outer glucan chains on the lattice planes, despite the larger d-spacings
in presence of higher zinc ions concentration. Nevertheless, the
Y. Xie et al.
Fig. 4. (a) XRD patterns of moso bamboo holocellulose before and after the DES pretreatments.; (b) Crystallinity (c) d-Spacings and (d) crystallite sizes perpendicular
to (2 0 0) lattice plane for samples.
crystallite size of ZAC-5 and ZAC-6 increased due to their weakened
hydrogen bonding, which tends to make the chains move at high
temperatures.
3.3. Enzymatic hydrolysis
Digestibility of the pretreated bamboo holocellulose was proposed
by enzymatic hydrolysis (Fig. 5). HC showed low enzymatic hydrolysis
yield of 50.30%, which was promoted after DES pretreatment. The
glucose yield of pretreated samples after 72 h hydrolysis was
Fig. 5. Enzymatic hydrolysis yield of samples.
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Industrial Crops & Products 191 (2023) 115965
63.8–67.3%. It demonstrated that the pretreatment performance of
ZAC/ChCl-1 and ZAC/ChCl-2 is obviously superior to other neutral DES
pretreatments, such as ChCl/urea or ChCl/glycerol (Hu et al., 2020).
According to above studies, weak intermolecular hydrogen bonding and
swelling can make a difference in the exposure of cellulose chains (Ling
et al., 2020; Zhang et al., 2014). Moreover, larger gaps between the
abutting hydrophobic planes were also beneficial to decreasing steric
hindrance for cellulase to contact cellulose surface (Shen et al., 2019a;
b). These phenomena provide more adsorption sites for cellulases for
rapid and efficient hydrolysis of cellulose (Shishir, Chundawat, 2020a;
b, c; Zhu and Pan, 2022a; b). Furthermore, the decrystallization capa­
bility of DES ZAC/ChCl-1 and ZAC/ChCl-2 produced more amorphous
regions of cellulose which can be hydrolyzed more easily by cellulase
(Nisha, 2018). Although lower crystallinity of cellulose favors enzymatic
saccharification, no further increased glucose yield appeared during
enzymatic hydrolysis at 120 ◦ C. It was hypothesized that, 100 ◦ C may be
the optimal temperature for the pretreatment of ZAC/ChCl-1 and
ZAC/ChCl-2, which was in consistent with the previous report that ac­
etate helps separate the cellulose chains and optimum treatment tem­
perature is relatively low (Sun et al., 2022b). Besides, due to more
microfibrils swelling, higher surface explosion and smaller crystallites,
ZAC/ChCl-1 DES is more suitable for optimizing enzymatic saccharifi­
cation as a pretreatment solvent.
3.4. Factors affecting enzymatic hydrolysis
Factors that have effects on enzymatic conversion yields vary from
different samples and the pretreatment methods. In this study, ZAC/
ChCl-1 has better performance, such as high yields of glucose, more
microfibrils swelling, higher surface explosion and smaller crystallites.
Therefore, taking representative samples pretreated by ZAC/ChCl-1
solvent as examples, the correlation between d-spacing or xylan
Y. Xie et al. Industrial Crops & Products 191 (2023) 115965

content of HC, ZAC-1, ZAC-2, ZAC-3 and the enzymatic hydrolysis yield Table 1
was fitted by non-linear (R2 = 96.9%) and linear (R2 =96.7%) function Zeta potential and particle size of nanocellulose.
respectively (Fig. 6). It can be seen that the d-spacing of the hydrophobic Aspect ratio Average Length (nm) Zeta Potential (mV)
plane of cellulose is positively correlated with the enzymatic hydrolysis
ZAC-3 60 483.87 ± 58.71 -18.83 ± 2.58
performance while the xylan content is opposite. Other studies reported ZAC-6 87 413.43 ± 29.04 -12.63 ± 0.64
similar results (Shimizu et al., 2020). As the physical blockage of
enzymatic hydrolysis of cellulose, hemicellulose has negative effects on
cellulose accessibility (Zhu and Pan, 2022a; b). Therefore, the removal proved in FTIR may be responsible for the nanofibrillation of cellulose
of xylan is a meaningful measure in enhancing inherent substrate after mechanical homogenization. In summary, this stable and uniform
reactivity. As mentioned above, the increased d-spacing perpendicular aqueous CNFs dispersion can be conducive to the preparation of func­
to (2 0 0) plane can play a role in a higher hydrolysis degree of cellulose, tional materials.
as was found in previous studies (Imai et al., 2020). Similarly, the
decrease of steric hindrance and the increase of cellulase adsorption sites 4. Conclusions
help for better adsorption of cellulase on cellulose. The treatment tem­
perature of 100 ◦ C remarkably enlarged the d-spacing, while the higher In summary, DESs based on ChCl as HBA and ZAC as HBD were
temperature did not bring about an increase of the glucose yield. This successfully prepared and high co-production yields of glucose and
means that the morphological structure of cellulose may be transformed nanocellulose were achieved. ZAC/ChCl significantly promoted enzy­
at 120 ◦ C, which is worth exploring. matic hydrolysis of moso bamboo holocellulose (from 50.3 % to 67.3 %)
in 3 min with microwave radiation assistance. Deconstruction of
hydrogen bonding connections, varied morphological and crystal
3.5. Co-production of nanocellulose
structures of cellulose were revealed, leading to the satisfying biocon­
version performance. This rapid and treatment technique for industrial
Both DES treatment and microwave treatment are emerging tech­
biorefinery showed practical significance and socio-economic owing to
nologies for the preparation of nanocellulose. Hence, we attempted to
drastic reduction in pretreatment time and high-yield sugars. In addition
extract nanocellulose from pretreated samples after high-pressure ho­
to operating costs, this neutral DES also effectively avoids acid corrosion
mogenization. It turned out that, nano-sized cellulose fibrils from ZAC-3,
of the equipment, contributing to environmental sustainability. More­
ZAC-6 appeared and dispersed homogeneously in the water. Due to the
over, both ZAC/ChCl-1 and ZAC/ChCl-2 DES can exfoliate CNFs with
degradation of xylan by DES, the very small amount of xylan on the
high aspect ratio and stability from the surface of fibrils at the treatment
surface of the sample are worth ignoring (Ang et al., 2020). Nano­
temperature of 120 ◦ C. The properties make CNF promising for appli­
fibrilllation of treated samples may cause solid loss during separation as
cations in film and hydrogel. From these results, ZAC/ChCl appeared to
well as lower crystallinity, which corresponds to the above results. As
be an excellent route for co-production of fermentable sugars and CNF.
well, it could also be responsible for the slightly decreased enzymatic
The present ZAC/ChCl DESs are proved to be excellent pretreatment
conversion yields for samples treated at 120 ◦ C. We can find that
agents to produce high-value-added products from renewable moso
nanocelluloses with high aspect ratios were by both ZAC/ChCl-1 and
bamboo feedstock.
ZAC/ChCl-2 DES (Table 1). Therefore, we defined the product as cel­
lulose nanofibrils (CNFs). As proposed in Fig. 7 and Table 1, the heights
CRediT authorship contribution statement
of CNFs in ZAC-3 and ZAC-6 were 8.01 nm and 4.75 nm and their
lengths were 483.87 nm and 413.43 nm, respectively. The aspect ratios
Xie Ying: Conceptualization, Data curation, Formal analysis,
of CNFs are 60–87, which is higher than DES-prepared nanocelluloses
Writing – review & editing. Jinyi Zhao: Methodology. Peng Wang:
reported before (Ma et al., 2019). In a study by Wang et al., CNFs pre­
Formal analysis. Zhe Ling: Funding acquisition, Supervision, Writing –
pared by ChCl/OA (1:1) in 5 h with the assistance of ball milling has an
review & editing. Qiang Yong: Supervision, Project administration.
average width of 14.29 nm. Thus, the shorter preparation time for
large-sized CNFs which were separated from ZAC-3 and ZAC-6 has
higher application potential. As illustrated in Table 1, the zeta potentials Declaration of Competing Interest
of ZAC-3 and ZAC-6 were − 13.83 mV and − 12.63 mV, indicating good
electrostatic repulsion and stability of the CNFs (Chen et al. (2021a); b, The authors declare that they have no known competing financial
c. The strong hydrogen bonding disruption at high temperatures as interests or personal relationships that could have appeared to influence

Fig. 6. (a) The correlation between d-spacing or xylan content and enzymatic hydrolysis yield of ZAC-1, ZAC-2, ZAC-3 and original sample; (b) the correlation
between xylan content and enzymatic hydrolysis yield of ZAC-1, ZAC-2, ZAC-3 and original sample.

6
Y. Xie et al.
Fig. 7. (a) is the AFM images and average height calculations of nanocellulose obtained in ZAC-3; (b) is the AFM images and height calculations of nanocellulose
obtained in ZAC-6.
the work reported in this paper.
Data Availability
No data was used for the research described in the article.
Acknowledgements
The authors acknowledge the funding support from the National
Natural Science Foundation for Youth (32001270) and Natural Science
Foundation of Jiangsu Province for Youth (BK20200797). This work was
also supported by State Key Laboratory of Pulp and Paper Engineering
(Project Number 202115).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.indcrop.2022.115965.
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