Cellulose (2019) 26:9447–9462

https://doi.org/10.1007/s10570-019-02726-0 (0123456789().,-volV)
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89().,-volV)

ORIGINAL RESEARCH

Choline chloride-lactic acid deep eutectic solvent

for delignification and nanocellulose production of moso
bamboo
Qian Liu . Tao Yuan . Qin-jin Fu . Yuan-yuan Bai . Feng Peng .
Chun-li Yao

Received: 24 April 2019 / Accepted: 3 September 2019 / Published online: 9 September 2019
Ó Springer Nature B.V. 2019

Abstract A facile, green and economical method for Introduction

the high-efficiency utilization and functionalization of
bamboo fiber could significantly improve the devel- Nowadays, developing green energy resources has
opment of biomass industries. Here, we demonstrated become an important task due to the alarming
a deep eutectic solvent, which formed from choline environmental, economic and social issues that are
chloride (ChCl) and lactic acid (LC), as pretreating being caused by massive use of fossil resources.
agent to delignify moso bamboo (Phyllostachys Among others, the industries based on sustainable
pubescens) and produce nanocellulose. The results materials, chemicals and fuels mostly rely on ligno-
showed that most of the lignin was efficiently removed cellulosic biomass (Rabemanolontsoa and Saka 2016).
(94.39%) and 91% of the cellulose was recovered after Plant biomass is an organic substance which is formed
the moso bamboo was pretreated with ChCl–LC at directly or indirectly by photosynthesis in green
120 °C for 3 h with a solid-to-liquid ratio of 1:25. plants. It can be converted into biofuel and platform
Furthermore, nanofibers with widths of 20–80 nm chemicals. In addition, biomass is a kind of abundant,
were successfully prepared from pretreated fibers after widespread, environmentally friendly, and renewable
a simple mechanical process. SEM and AFM images carbon resource (Kumar et al. 2015; Lancefield et al.
showed that they have excellent aspect ratio. In 2017). Bamboo is known as one of the fastest growing
addition, the obtained nanofibers could be used to and highest yielding renewable natural resource in the
fabricate strong films which feature a high tensile world, which with a high growth rate in growing
strength ranged from 163 to 213 MPa. seasons (Ben-zhi et al. 2005; He et al. 2014). Among
the bamboos, moso bamboo (Phyllostachys pubes-
Keywords Pretreatment  Deep eutectic solvent  cens) is an important species in terms of material
Bamboo  Delignification  Nanofibers production which were planted across large swaths of
China. It is widely used in pulping, building and
spinning industries because of its fast growth, excel-
Q. Liu  T. Yuan  Q. Fu  F. Peng  C. Yao (&)
lent strength, and ability to be transformed. Also, it can
Beijing Key Laboratory of Lignocellulosic Chemistry,
Beijing Forestry University, Beijing 100083, China be used to produce bio-ethanol, bio-methane, natural
e-mail: chunliyao2006@163.com food, flavonoids, composite and functional nanoma-
terials (Hu et al. 2017; Huang et al. 2016). Exploitation
Y. Bai
and utilization of moso bamboo can effectively
National Engineering Lab for Pulp and Paper, China
National Pulp and Paper Research Institute Co. Ltd., promote the development of biorefinery industry.
Beijing 100102, China Nevertheless, a common challenge faced with the

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9448 Cellulose (2019) 26:9447–9462

utilization of lignocellulosic materials is the efficiency conversion of furanic derivatives (Das et al. 2016;
of disrupting the close inter-component association Zdanowicz et al. 2016, 2018). However, the develop-
between main constituents (including cellulose, hemi- ment of DESs in biotechnology is still on its infant
celluloses and lignin) of plant cell wall, which hinder stage and the research is not thorough and intensive
the development of industries (Foley and Olabi 2017; yet. Also, the mechanism of biomass pretreatment
Yu et al. 2017). Hence, an efficient method should be using DESs requires more study.
developed to extract different components, especially Recently, a breakthrough was made in the research
the removal of lignin. It is necessary to facilitate the area about utilization of DES in biorefinery indus-
conversion of moso bamboo into high-value and tries—the fabrication of nanocelluloses. Various
biobased products. DESs have been successfully used as alternative green
Pretreatment is an essential process to clear away routes to produce both non-derivatized and anionic
the physical and chemical barriers and improve the nanocelluloses (Chen et al. 2019; Laitinen et al. 2017;
utilization of carbohydrates in lignocellulosic bio- Li et al. 2017; Sirviö et al. 2015). As a case in point,
mass. The common methods include physical (grind- cellulose nanofibrils with widths of 2–5 nm and larger
ing, ball milling, and energy irradiation), nanofibril bundles with widths ranging from 15 to
physicochemical (steam explosion, wet oxidation, 200 nm were successfully achieved after the pretreat-
and auto-hydrolysis), chemical (acid, alkali, organic ment of DES system containing urea and choline
solvents, inorganic salt, and ionic liquid) and some chloride (Sirviö et al. 2015). Alternately, a turbid and
biological methods (Rabemanolontsoa and Saka gel-like cellulose nanofibrils suspension with a mean
2016). Through cleaving various linkages among equivalent spherical diameter of 262 nm was obtained
three components, the aim of all of these methods is from ramie fibers in the study of Yu et al. (2019),
mainly to increase accessibility, porosity and reactiv- which using choline chloride-oxalic acid dehydrate
ity of biomass materials for helping the subsequent and choline chloride-urea as pretreating agents. How-
steps of utilization. However, environmental pollu- ever, most of these research focus on the potential of
tion, poor biodegradability and high production costs urea-based DES systems as pretreatment media for
limit industrial usage of these technologies. Develop- cellulose nanofibril production, and the raw materials
ing new, green and cheap solvents as pretreating of them were almost pulps or with low lignin content
agents becomes the most important subjects, and deep such as bleached kraft birch (Betula pendula) pulp (Li
eutectic solvents (DESs) are the most promising types et al. 2017, 2018), cotton fiber (Ling et al. 2018), waste
of solvents (Willberg-Keyriläinen et al. 2017; board (Suopajarvi et al. 2017) and softwood dissolving
Zdanowicz et al. 2018). cellulose pulp (Sirviö et al. 2019; Sirviö and Visanko
DES is a new type of ionic solvent system firstly 2017). Herein, we reported, for the first time, a facile
discovered by Abbott et al. (2004), attracting growing strategy for fabrication of nanocellulose from moso
interests in academia and industry. It has similar bamboo using a renewable DES based on choline
characteristics with traditional ionic liquids (ILs), but chloride (ChCl) and lactic acid (LC) as a pretreatment
it is much cheaper, readily available, less toxic, media prior to the mechanical disintegration. Further-
biodegradable and environmentally friendly (Espino more, the development of this method would signif-
et al. 2016a, b; Tang et al. 2015). Generally, DES can icantly reduce the environmental problems and the
be synthesized by a halide salt of quaternary ammo- costs of liquid waste disposal which caused by pulping
nium or phosphonium cation, which acts as the and bleaching processes (Lu et al. 2018). Processing of
hydrogen bond acceptor (HBA) and a hydrogen bond the DES to remove lignin of moso bamboo was
donor (HBD) (alcohol, amine, carboxylic acid) at investigated in this paper by single factor and orthog-
suitable temperatures. The melting point of the onal experiments. Related factors including tempera-
mixture is lower than individual components (Loow ture, reaction time and solid-to-liquid ratio were
et al. 2017). Recently, the number of studies related to discussed in terms of delignification. Fibers obtained
properties and applications of DESs have steeply after DES pretreatment were characterized through
increased, as well as the applications of DESs in Scanning Electron Microscopy (SEM), Fourier trans-
biotechnology including phenolic compounds extrac- form infrared (FTIR) spectroscopy, X-ray diffrac-
tion, lignocellulosic biomass delignification, and tometer (XRD) and thermogravimetric (TG) analysis.

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The structure of nanofibers obtained after chemical materials and a certain amount of solvent (solid-to-
and mechanical treatments were analyzed by Atomic liquid mass ratio were 1:15, 1:20 and 1:25). The
Force Microscope (AFM). Furthermore, the mechan- mixture was stirred with a magnetic stirrer at
ical properties of self-standing films produced from 100–120 °C for 2–4 h, respectively using an oil bath
obtained cellulose nanofibrills were measured using a agitation with a rotator. And then, the reaction vessel
universal materialtesting machine. was taken out from the oil bath and added 150 ml
alcohol to wash and recover fibers. Filtrate and solid
were collected, respectively. An excessive amount of
Materials and methods alcohol was used to wash the recovered fibers until the
filtrate was colorless. Then the solid was subjected to
Materials washing with water, filtration and drying to constant
weight. Recovered fibers were named as DES-carbo-
Moso bamboo was obtained from the Gongyi Nature hydrate (DESC). Water was added in the filtrate
Reserve in Huangshan, Anhui Province, China. The (water: filtrate = 9:1), filtration, drying to recover
samples were prepared with 40–60 mesh screens, lignin. Recovered solid was named as DES-lignin
extracted with benzene-ethanol (2:1) solution at 90 °C (DESL). In this section, the reaction time, temperature
for 4 h. Then the materials were dried at 60 °C to and solid-to liquid ratio were sieved by orthogonal
constant weight. The experiment was conducted with designing method. The specific reaction conditions of
deionized water. All chemicals were of reagent grade. different samples were shown in Tables 1 and 2. There
were three parallel samples per specific conditions and
Preparation of ChCl–LC mixtures the data was the average of parallel samples.

The eutectic mixture was prepared by combining ChCl Nanofibrillation of cellulose materials
and LC at the molar ratio of 1:9 at 60 °C with a
magnetic stirrer using a water bath until a clear, DESC was diluted with water to a suitable concentra-
homogeneous and colorless solution was obtained. tion and a FSH-2A High speed homogenizer was used
The preparation method is based on literatures (Fran- to refining the mixture for 1 h. Then, water was added
cisco et al. 2012; Hou et al. 2018a, b). again to adjust the concentration of DESC to 1 wt%.
Nanofibers were produced by mechanical disintegra-
Solubility of lignin in the DES tion of 1 wt% DESC solution with a microfluidizer
(Microfluidics M-110EH-30, USA). The sample was
The solubility of pure lignin in the ChCl–LC was named as DES-nanofibers (DES-n).
determined according to previous reports with few
modifications (Francisco et al. 2012; Malaeke et al. Preparation of self-standing films from nanofibers
2018). First, 200 mg of the sample was added to a
glass vial containing 5 g of the DES at constant The DES-n was used to prepare self-standing films for
temperature (80 °C, 100 °C and 120 °C) with stirring. characterization of mechanical properties. First,
The dissolution of lignin was visually checked. If the 0.050 g nanofibers were diluted to 0.1 wt% (corre-
sample was soluble, then consecutive additions of sponding films with a grammage of 40 g/m2) and were
10–50 mg of lignin was added in the reaction system further mixed with a magnetic stirrer to ensure the
until the existence of particles were noticeable. formation of evenly dispersed suspensions. Vacuum
Samples were equilibrated for at least 24 h to check filtration with a glass filter funnel (3.8 cm in diameter)
the dissolution of these particles. All data recorded covered with a polyvinylidene fluoride (PVDF)
were the average values of three parallel samples. membrane (pore size 0.45 lm) was applied for the
fabrication of gel ‘‘cake’’. The gel was placed in two
Chemical pretreatments and optimization PVDF membranes to have a drying process for 9 min
at 93 °C under a vacuum at about a pressure of
Pretreatment of moso bamboo with DES based on 70 mbar.
ChCl–LC was performed with 3.00 g biomass

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Table 1 Factors and levels Level Factor

for orthogonal test of ChCl–
LC pretreatment A: Temperature (°C) B: Reaction time (h) C: Solid-to-liquid ratio

1 100 2 1:15
2 110 3 1:20
3 120 4 1:25

 
Preparation of milled wood lignin (MWL)
Delignification ð%) ¼ 1  m2 c2=m1 c1  100%
The MWL of moso bamboo was prepared according to ð1Þ
previous study (Zhang et al. 2016) and the operations
where m1 is the mass of raw materials, m2 is the mass
were as follows: the ball-milled bamboo powder was
of DESC, c1 is the lignin ratio of raw materials, c2 is
extracted by dioxane (96%) with a solid to liquid ratio
the lignin ratio of DESC.
of 1:20 (g/mL) at room temperature for 24 h with
Cellulose or hemicellulose retention percentage
stirring. This procedure was repeated three times and
was calculated according to the formulas as follows
all the combined filtrate and washing liquid were
(Eq. 2):
concentrated at reduced pressure to obtain the crude
MWL. Water was added in crude MWL to obtain Retention ð%) ¼ m2 ca=m1 cb  100% ð2Þ
suspension and mixed with alcohol (95%). The
where cb is the cellulose (hemicellulose) ratio of raw
mixture was concentrated at reduced pressure until
materials, ca is the cellulose (hemicellulose) ratio of
the volume reached 10 ml and gradually added 6 mol/
DESC.
L HCl solution to adjust the pH to 2.0. Then the MWL
was obtained after subjecting to filtration and drying to
Gel permeation chromatography (GPC) analysis
constant weight.
of DESL
Characterization
For GPC analysis, the DESL (4 mg) was dissolved in
2 ml of tetrahydrofuran (THF). The GPC analysis was
Component analysis
conducted with a high performance liquid chro-
matograph (Knauer LUMTECH/K501, Germany)
Analysis of carbohydrate and lignin in DESC samples
equipped with a refractive index detector and two
were performed according to the standard National
cascaded PL-gel columns (5 lm, 500 Å) at room
Renewable Energy Laboratory (NREL) analytical
temperature with THF as the mobile phase at a rate of
procedure (Sluiter et al. 2008). The material with or
1.0 ml/min. Calibration was performed using
without DES pretreatment were subjected to a two-
polystyrenes with a molecular mass range between
stage sulfuric acid hydrolysis. Subsequently, the
580 Da and 3250 kDa.
carbohydrate content of acid-hydrolyzed filtrate were
determined by high performance liquid chro-
X-ray diffractometry
matograph (HPLC). Acid soluble lignin (ASL) in the
acid-hydrolyzed filtrate was measured using UV–vis
A Bruker D8 ADVANCE X-ray diffraction (Bruker,
spectrophotometer at 320 nm. The acid insoluble
Germany) was used to measure the crystallinity of the
residue was dried and ashed at 105 °C and
DESC pretreated and unpretreated materials. The h–
575 ± 25 °C, respectively, for acid insoluble lignin
2h method was applied to collect the diffraction
(AIL) determination. Weight of lignin in raw and
spectra (Segal et al. 1959). The half cone angle 2h
pretreated materials was calculated as the sum of ASL
range rotated from 5° to 45° at a rate of 2°/min.
and AIL. Removal percentage of lignin was calculated
Crystallinity index (CrI) was calculated as follows
according to the formulas as follows (Eq. 1):
(Eq. 3):

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 Table 2 Test design and results of orthogonal analysis
Cellulose (2019) 26:9447–9462

Sample A B C Blank Delignification (%) Lignin (%) Cellulose (%) Hemicellulose (%) Cellulose retention (%) Hemicellulose retention (%)

1 1 1 1 1 61.47 15.95 64.82 13.88 92.85 30.38

2 1 2 2 2 72.39 12.33 69.27 15.73 92.02 31.92
3 1 3 3 3 68.37 13.95 66.45 13.72 89.32 28.18
4 2 1 2 3 77.51 10.61 76.69 17.09 96.43 32.83
5 2 2 3 1 89.35 5.54 81.76 12.66 93.14 22.03
6 2 3 1 2 80.26 9.54 74.22 12.46 91.09 23.36
7 3 1 3 2 91.52 4.53 83.88 11.70 93.13 19.85
8 3 2 1 3 87.04 6.65 81.76 12.40 94.57 21.91
9 3 3 2 1 91.42 4.71 84.98 10.85 91.75 17.89
K1 202.24 230.51 228.77 242.24
K2 247.13 248.78 241.32 244.18
K3 269.98 240.05 249.25 232.92
k1 67.41 76.84 76.26 80.75
k2 82.38 82.93 80.44 81.39
k3 89.99 80.02 83.08 77.64
R 22.58 6.09 6.83 3.75
Primary and secondary factors A[C[B
Optimal scheme A3B2C3
9451

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IðcryÞ  IðamÞ according to TAPPI standard test method T230 om-99

CrI ð%) ¼  100% ð3Þ (TAPPI 1999). The DP of samples were calculated as
IðcryÞ
follows (Mazumder et al. 2000; Yu et al. 2019):
where the I(cry) is the intensity of crystalline region
(2h = 22°) and the I(am) is the intensity of amorphous DP0:905 ¼ 0:75ð954 log½g  325Þ ð4Þ
region (2h = 18°).
Scanning electron microscopy (SEM)
Determination of acidic groups
The morphology of DESC was analyzed by a SU8010
The carboxyl content of DESC was analyzed by
scanning electron microscope (HITACHI, Japan)
conductometric titration using a protocol described by
instrument after gold sputtering.
Saito and Isogai (2004).
Atomic force microscope (AFM) test
Thermo-gravimetric (TG) analysis
A BRUKER MULTIMODE 8 atomic force scanning
Thermal gravimetric analyzer (Shimadzu interna-
electron microscope (BRUKER, USA) was used to
tional trade, Shanghai) was used to test the thermal
scan the surface of DES-n. The aqueous 0.02%
stability of the samples. The samples were heated at a
nanofibril suspension was sonicated for 2 h before
temperature increase rate of 20 °C/min under a
use. The samples were prepared by drop-casting the
nitrogen atmosphere. The range of the temperature
suspension (20 mL) onto a freshly cleaved mica
was 40–600 °C.
surface and naturally drying 30 min.
Fourier transform infrared (FTIR) spectroscopy
Results and discussion
FTIR spectra of moso bamboo, DESC and DESL were
analyzed by a Spectrum 100D fourier transform
Optimization of DESs delignification
infrared spectrometry (PerkinElmer, UK). The sam-
ples were pressed uniformly with KBr into pellets for
Here, the DES system containing choline chloride as a
transmission measurements. Sample spectra were
hydrogen bond donor and lactic acid as a hydrogen
scanned from 4000 to 400 cm-1.
bond acceptor was used to remove lignin from moso
bamboo. ChCl is an affordable ammonium salt which
Mechanical properties analysis of films
can be extracted from biomass or synthesized from
fossil resource. It is also an excellent material for
The tensile tests were performed with a universal
feasibility and economic aspects of the process
material testing machine (Zwick D0724587, Switzer-
(Malaeke et al. 2018; Zhang et al. 2012). Lactic acid
land). The films were cut into strips 5 mm wide, and
can be purchased at cheap price (0.56 US$/kg) and
their thicknesses were measured using a micrometer
makes a highly suitable candidate as DES constituent
caliper. Three different locations within the gauge
for long-term biomass processing (Tan et al. 2019).
length were measured to calculate the average thick-
Furthermore, the study of Francisco et al. (2012),
ness of each film. For the tensile tests, a 15 mm gauge
which investigated twenty-eight types of DES system
length was set under a strain rate of 4 mm/min, and
with different molar ratio as solvents for biopolymers
three strips in total were measured for each sample.
(lignin, cellulose and starch), pointed out that lactic
acid–choline chloride mixtures showed no solubility
Degree of polymerization
of cellulose but lignin solubility increased with the
acid ratio. Especially, the solubility of lignin reached
The average degree of polymerization (DP) of DESC
the maximum when the molar ratio of lactic acid:
and nanofibrillated celluloses was evaluated from the
choline chloride was 9:1.
intrinsic viscosity [g], measured with a capillary
viscometer in a cupriethylenediamine solution (1 M)

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The solubility of lignin in ChCl–LC (1:9) at (2015) indicated that the harsh operating conditions
different temperatures were determined by cloud point such as long reaction time and excessive operating
method (Fig. 1). The result indicated that ChCl–LC temperature might lead to thermal degradation of
displayed excellent solubility towards the lignin, sugar content. Therefore, moso bamboo pretreated by
which was in agreement with previous studies, point- ChCl–LC at 100–120 °C for 2–4 h were detailedly
ing that acidic-DESs exhibit excellent dissolution of investigated in this study.
lignin (Francisco et al. 2012; Malaeke et al. 2018). In The orthogonal design method was used to opti-
detail, the solubility of lignin could reach to 13.57% mize the pretreatment conditions. The factors and
w/w at 80 °C in the solvent and the dissolution of levels with temperature, reaction time and solid-to-
lignin increased with the increase of temperature liquid mass ratio as independent variables are shown
(21.84% w/w at 100 °C and 25.54% w/w at 120 °C). A in Table 1. The removal percentage of lignin is taken
possible explanation is that the high temperature as index and the orthogonal test table of 9 (33) is used.
reduce the viscosity of DES to improve the mass The results are shown in Table 2. Ki (i = 1, 2, 3) is the
transfer of the whole system. sum of delignification indexes at the same level of
The cellulose, hemicellulose and lignin contents of each factor, and ki (i = 1, 2, 3) is the average of
raw materials are 44.0%, 28.8% and 26.1%, respec- delignification indexes at the same level of each factor.
tively. At first, the control variable method was used to As shown in Table 2, ChCl and LC based solution
confirm a suitable range of pretreatment temperature at a molar ratio of 1:9 was found to be efficient for
and time (Fig. 2), and the initial range of factors were lignin removal of moso bamboo. It is noted that the
based on literatures (Alvarez-Vasco et al. 2016; Hou removal of lignin could reach 91% when the reaction
et al. 2018a, b; Kumar et al. 2016; Skulcova et al. temperature was 120 °C. The analysis results indicate
2017). As shown in Fig. 2, approximately 23.28% of that temperature is the predominant factor which has
the lignin was removed by ChCl–LC when pretreated an effect on the delignification efficiency of ChCl–LC
at 80 °C for 2 h, and the delignification effect was solvent, followed by solid-to-liquid ratio and reaction
enhanced with the increase of reaction time. Increas- time. More specifically, high reaction temperature and
ing temperature up to 120 °C led to significant jump in solid-to-liquid ratio are beneficial to delignification.
removal percentage of lignin to more than 90%. DES system features a high viscosity at room
However, long term reaction would result in slight temperature compared with ILs and water, which is
decrease of delignification effect when the tempera- attributed to the presence of hydrogen bonds, van der
ture higher than 100 °C. In addition, Loow et al. Waals force, and electrostatic interactions in the

Fig. 1 Maximum solubility of lignin in ChCl–LC (1:9) at different temperature

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Fig. 2 Composition of moso bamboo after DES treatment

network. Higher reaction temperature could reduce the DES based on ChCl–LC as a pretreating agent could
viscosity and higher solid-to-liquid ratio increase the efficiently promote high value-added utilization of
contact efficiency between materials, enhancing the moso bamboo biomass.
efficiency of delignification (Smith et al. 2014; Zhang
et al. 2012). In summary, the optimal conditions of FTIR analysis
ChCl–LC solvent pretreat moso bamboo for deligni-
fication are react at 120 °C for 3 h with a solid-to- Figure 4 shows the FTIR spectra of raw material,
liquid ratio of 1:25. The chemical composition of DESC, DESL and milled wood lignin (MWL) of moso
fibers pretreated at optimal conditions was comprised bamboo. DESC and DESL were prepared at optimum
of cellulose (81.4%), hemicellulose (14.8%) and conditions. MWL was prepared according to previous
lignin (3.0%). The delignification ratio of moso study (B. Zhang et al. 2016). Noticeably, the peak at
bamboo pretreated at optimum conditions was 1735 cm-1 is attributed to C=O stretching of the ester
94.39% and more than 91% cellulose was reserved from the acetyl group of hemicellulose. The existence
(Fig. 3). The mechanism of biomass delignification for this peak in spectra of DESC and DESL indicates
using DES is due to their ability to selectively cleave that a large amount of acetyl groups of hemicellulose
the ether bonds between lignin phenylproane units can be reserved after pretreatment. Also the peak may
without affecting the C–C linkages (Alvarez-Vasco be attributed to a superimposed band of the C=O group
et al. 2016). Some researchers pointed that an addition of the ester and the protonated carboxylic acid (Sirviö
of water into the DES improves the accessibility and and Visanko 2017). This finding agrees well with the
digestibility of the biomass by the reduction of previous study in which a new peak around
viscosity (via the weakening of hydrogen bonds 1720 cm-1 for C=O stretching of acid appeared in
between components) (Hou et al. 2017; Kumar et al. the FTIR spectra of lignocellulosic pretreated with
2015). We think the decomposition of ChCl–LC acidic-DES (Tan et al. 2019). Moreover, conducto-
solvent during the pretreatment at a high temperature metric titration was used to determine the quantity of
and the feature of DES (contain 3–7% of water) can carboxylic acid groups on the DESC and the result
greatly facilitate the removal of lignin. However, showed that fibers exhibited 0.30 ± 0.04 mmol/g
because of the complex reaction system involved, carboxylic acid content. The peaks at 1600 cm-1,
more detailed study is required to confirm the mech- 1507 cm-1 and 1460 cm-1 are characteristic peaks of
anism. It is widely known that the aim of biomass lignin. These peaks on the spectrum of DESC are not
pretreatment is breaking the recalcitrant heteropoly- observed, which proves that almost entire lignin of
meric structure of lignocellulose complex and recov- moso bamboo was removed after pretreatment at
ering carbohydrates as much as possible (Lancefield optimum conditions. On the contrary, DESL spectrum
et al. 2017; Sindhu et al. 2016). Therefore, the use of indicates the most intense aromatic skeleton vibration

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Cellulose (2019) 26:9447–9462 9455

Fig. 3 Mass balance during

the pretreatment at 120 °C
with 1:25 solid-to liquid
ratio for 3 h

Fig. 4 The FTIR results of

raw material, DESC, DESL
and MWL

signals at these peaks, which suggests that rich which indicates that the guaiacyl units of moso
aromatic structure existed in the DESL. In addition, bamboo can be largely removed by ChCl–LC solvent.
the peaks at 1324 cm-1 and 1217 cm-1 are assigned The signal at 1033 cm-1 is originated from C–O–C
to syringyl and guaiacyl lignin. The FTIR spectrum of antisymmetric stretching vibration of hemicellulose
DESL has a large absorption peak at 1217 cm-1, and cellulose. In this study, the FTIR spectra of MWL

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and DESL were similar and the number-average bamboo after being pretreated with ChCl–LC at
molecular weight of DESL obtained at optimum optimal conditions. There are peaks at 22° and 18°
conditions was 2.06 9 103 g/mol. The DESL recov- (both untreated and treated fibers), typical for cellulose
ered from the pretreatment waste has a potential for I, showing that ChCl–LC pretreatment did not alter the
the application as a high-value chemical precursor. fundamental crystal structure of cellulose. However,
the crystallinity of the material was increased from
DP, XRD and TG analysis 49.20 to 60.43% which may be attributed to the
removal of non-crystalline components, such as
The change in the length of the cellulose polymer hemicellulose and lignin (Xu et al. 2016). The increase
chains was studied by measuring the degree of of crystallinity suggests the function of ChCl–LC
polymerization (DP). The result showed that the DP pretreatment, which retains the cellulose of bamboo
of DESC was 1376, showing approximately 2% and manages to separate cellulose from lignin and
reduction compared to previous studies in which the hemicellulose, can facilitate the utilization of
DP value of original bamboo fibers was 1400 (Cao carbohydrate.
et al. 2014; Ma et al. 2013). This finding indicated that The thermal behavior of untreated and delignified
the using of ChCl–LC was able to swell and loosen the (at optimal conditions) moso bamboo was explored by
fiber macroscopic structural integrity without thermo-gravimetric analysis (TGA) in an N2 atmo-
notable degradation of the molecular cellulose chain sphere. The results of differential thermogravimetry
structure. Moreover, The DP values displayed a analysis (DTG) and TG are shown in Fig. 6. The major
significant decrease from 1376 (DESC) to 904 weight loss of fibers with or without pretreatment
(DES-n) after mechanical treatments, which suggested takes place between 280 °C (330 °C for DESC) and
that the molecular chains of cellulose had been broken 360 °C. In this stage, the degradation rate of DESC
by high cutting force of this disintegration method. was 69.61% and of raw materials was 58.14%. The
The XRD analysis (Fig. 5) was used to determine DTG curves show that the maximum degradation rate
the alteration in cellulose crystallinity of moso of fibers increased from 331 to 352 °C after ChCl–LC

Fig. 5 X-ray diffraction

analysis of raw materials
and DESC

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Cellulose (2019) 26:9447–9462 9457

Fig. 6 Thermo-gravimetric
analysis of raw material and
DESC

pretreatment. Thermal decomposition of biomass DESC prepared at optimal conditions. It showed the
frequently presents two distinct peaks at the temper- existence of characteristic graminae cells and that the
ature range of 200–400 °C in the DTG curves, surfaces of pretreated fibers were smooth in Fig. 7b.
referring to the decomposition of hemicellulose and As shown in the magnified SEM image, there is a large
cellulose (Marcilla et al. 2013; Yiin et al. 2018). The amount of longitudinal cracks on the surface of
DTG curve of DESC exists only one peak in the individual fibers, indicating that the DES pretreatment
temperature range of 300–400 °C indicates large released the tight connection of bamboo fibers along
amount of hemicellulose in moso bamboo was its growth direction by removing hemicellulose and
dissolved during the DES pretreatment. DES pre- lignin.
treated fibers exhibited better initial and maximum
degradation temperature than original fibers, indicat- Cellulose nanofibrillation and characterization
ing the enhanced thermal stability of moso bamboo
fibers. The alteration in thermal decomposition tem- Recently, the DES used in biomass pretreatment has
perature was most likely due to the removal of increasingly been reported, but only a few studies
hemicellulose and the decrease of chemical linkages focused on the use of DES for nanocellulose produc-
after pretreated with DES. tion and functionalization have been published (Sirviö
and Visanko 2017; Zdanowicz et al. 2018). In this
SEM analysis work, larger nanofibril bundles with widths ranging
from 20 to 80 nm were observed after mechanical
The picture of bamboo fibers pretreated with ChCl– disintegration with a microfluidizer (Fig. 7a). The
LC at optimal conditions was shown in Fig. 7a. The surface structure of DES-n was observed via AFM test
pretreated fibers distributed in water can form a (Fig. 7c). The images clearly show network structure
homogeneous supernatant. Furthermore, the color of of DES-n consisting of nanofibril bundles and indi-
the products became slightly darken which may be vidual nanofibrils. The AFM images evidently indi-
attributed to the generation of chromophoric groups cate that the pretreatment of ChCl–LC was not enough
during DES pretreatment. The SEM observations were to enable the liberation of evenly sized nanofibers. The
conducted to understand the surface morphologies of width profiles (Fig. 8a) of the individual nanofibers

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Fig. 7 Morphology study of raw and pretreated fibers (DESC and DES-n); a pictures of raw materials, DESC and DES-n. b SEM
surface images of DESC. c AFM topography images of DES-n

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Fig. 8 Width and height distributions for cellulose nanofibrils; a width distributions. b Height distributions

were evaluated and the results show that the width of ranged from 4.11 to 5.38 9 106 N m/g, which are
nanofibers were mainly distributed in 20–60 nm much higher than films produced by urea-based DESs
(82%). In addition, the heights of nanofibers were pretreated nanofibers reported previously (the range of
mainly distributed in 1–5 nm (Fig. 8b). Nanofibrils tensile strength was 135–189 MPa and of tensile
produced in this method shows a longer length (the strength index was approximately
length of nanofibrill was more than 4 lm which shown 2.36–3.31 9 106 N m/g) (Li et al. 2017; Sirviö and
in Fig. 7c) and higher DP value (904) compared with Visanko 2017). In addition, the elastic modulus ranged
that of nanocelluse prepared from pulps by DES from 9.0 to 11.0 GPa is also higher than those films
treatments (Laitinen et al. 2017; Ling et al. 2018; Yu (6–7 GPa). The high strength properties of films in this
et al. 2019). Furthermore, based on these measured study can be attributed to the cross-linking in DES-n
lengths and widths, the DES-n aspect ratios mainly fall with excellent aspect ratio and high DP value (904)
in the range of 67–101 which was higher than (Ho et al. 2014; Yu et al. 2019). Also nanofibers with
nanofibers prepared by other DESs (Ling et al. 2018; 0.26 mmol/g carboxyl contents could be combined
Sirvio et al. 2016). Nearly 0.26 mmol/g carboxylic frequently in drying process to make films compact.
acid content in DES-n was conducive to the stability of Herein, we demonstrate the potential of ChCl–LC
prepared nanacelluse emulsions. pretreatments on moso bamboo for delignification and
Structure analysis indicated that the DES treatment nanofibrillation. The good performance of ChCl–LC
can loosen the structure of the cellulose cell wall so in bamboo pretreatment, the low cost, easily available
that the fibrils began unraveling from the surface of the materials (bamboo and lactic acid), and the green and
cellulose fibers (Suopajarvi et al. 2017). In this study, facile approach indicate that the application of ChCl–
nearly 94% of lignin (which as an adhesive between LC on bamboo pretreatment shows an attractive
fibers in biomass) was removed by the ChCl–LC prospective in the conversion and high-value utiliza-
pretreatment, resulting in the main interaction tion of bamboo and in turn promoting the development
between fibers by inter- and intra-hydrogen bonds. of biorefinery industry.
Hence, the high impact, high-shear, and cutting force
of mechanical treatment could effectively disperse the
fibers to nano-sized through fibers colliding with each Conclusions
other and the wear-resistant surfaces of the chambers.
The self-standing films produced by DES-n exhib- An easily obtained DES based on ChCl and LC was an
ited excellent mechanical properties (Fig. 9). In more efficient solvent for moso bamboo delignification.
detail, the films exhibited a tensile strength ranged More than 94% lignin was removed when pretreated at
from 163 to 213 MPa, and tensile strength index 120 °C for 3 h with a solid-to-liquid ratio of 1:25.

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9460 Cellulose (2019) 26:9447–9462

Fig. 9 Stress and stain

curves of self-standing films
(40 g/m2) produced by
DES-n

DESC prepared at optimal conditions was further application. J For Res 16(2):143–147. https://doi.org/10.
nanofibrillated to a width of 20–80 nm with a 1007/bf02857909
Cao S, Ma X, Lin L, Huang F, Huang L, Chen L (2014) Mor-
microfluidizer. Excellent aspect ratio and high DP phological and chemical characterization of green bamboo
value confer the self-standing films prepared by DES- (Dendrocalamopsis oldhami (Munro) Keng f.) for dis-
n with excellent mechanical properties (tensile solving pulp production. BioResources 9(3):4528–4539
strength reached 213 MPa). Consequently, the Chen L, Yu Q, Wang Q, Wang W, Qi W, Zhuang X, Wang Z,
Yuan Z (2019) A novel deep eutectic solvent from lignin-
ChCl–LC pretreatment appears to be a promising derived acids for improving the enzymatic digestibility of
route for exploitation and utilization of moso bamboo. herbal residues from cellulose. Cellulose
26(3):1947–1959. https://doi.org/10.1007/s10570-018-
Acknowledgment Thanks to National Key R&D Program of 2190-8
China (2017YFD0600804). Das AK, Sharma M, Mondal D, Prasad K (2016) Deep eutectic
solvents as efficient solvent system for the extraction of
kappa-carrageenan from Kappaphycus alvarezii. Carbo-
hydr Polym 136:930–935. https://doi.org/10.1016/j.
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