Article

pubs.acs.org/Biomac

Acidic Deep Eutectic Solvents As Hydrolytic Media for Cellulose

Nanocrystal Production
Juho Antti Sirviö,* Miikka Visanko, and Henrikki Liimatainen
Fibre and Particle Engineering Research Unit, University of Oulu, P.O. Box 4300, Oulu FI-90014, Finland
*
S Supporting Information

ABSTRACT: In this study, a new method to fabricate cellulose nanocrystals (CNCs) based on DES pretreatment of wood
cellulose fibers with choline chloride and organic acids are reported. Oxalic acid (anhydrous and dihydrate), p-toluenesulfonic
acid monohydrate, and levulinic acid were studied as acid components of DESs. DESs were formed at elevated temperatures
(60−100 °C) by combining choline chloride with organic acids and were then used to hydrolyze less ordered amorphous regions
of cellulose. All the DES treatments resulted in degradation of wood fibers into microsized fibers and after mechanically
disintegrating, CNCs were successfully obtained from choline chloride/oxalic acid dihydrate-treated fibers, whereas no liberation
of CNCs was observed with other DESs. The DES-produced CNCs had a width and length of 9−17 and 310−410 nm,
respectively. The crystallinity indexes (CrIs) and carboxylic acid content of the CNCs were 66−71% and 0.20−0.28 mmol/g,
respectively. CNCs exhibited good thermal stabilities (the onset thermal degradation temperatures ranged from 275−293 °C).
The demonstrated acidic DES method exhibits certain advantages over previously reported CNC productions, namely, milder
processing conditions and easily obtainable and relatively inexpensive biodegradable solvents with low toxicity (compared, e.g., to
ILs).

■ INTRODUCTION
Ionic liquids (ILs) can be utilized as an efficient reaction and
treatment media for various biomasses.1,2 ILs contain large
organic cations and inorganic or organic anions having melting
point lower than 100 °C.3 Compared with traditional molecular
solvents, they have several advantages, including high solvent
capacity toward both organic and inorganic materials and
negligible vapor pressure, which reduces the volatile organic
compound (VOC) emissions. Despite their good properties,
ILs have several drawbacks, including synthesis mainly from oil-
based chemicals, toxicity, low biodegradability, low moisture
tolerance, and high cost.4,5
Deep eutectic solvents (DESs) are chemicals that are almost
equivalent to ILs (DESs are even regarded as a subcategory of
ILs according to some sources6) and exhibit similar properties,
such as good solvent capacity and low vapor pressure.7
However, compared with ILs, DESs can be obtained by less
demanding methods by simply heating and mixing two or more
components at an elevated temperature (mainly around 100
°C). On a larger scale, DES can be efficiently synthesized using
the twin screw extrusion process.8
DESs typically consist of a hydrogen bond donor and
acceptor pair. The formation of strong hydrogen bonding and
complexation between the components is assumed to prevent
crystallization, consequently causing the drop in the melting
point and resulting in an eutectic mixture.6 A significant
amount of DESs can be attained directly from low toxic natural
components, such as choline chloride, urea, and glycerol.9
However, the toxicity of DES can be higher compared to its
individual components,10 but the toxicity greatly depends on
the studied organism.11,12 There is even evidence that the
formation of DESs can lower the toxic effect of individual
components (e.g., organic acids). DES systems consisting of
choline chloride and organic acids have been shown to exhibit
certain antimicrobial properties, yet they are well biocompat-
ible, and their toxicity is lower compared to some ILs based on
imidazolium and pyridinium.12 Choline chloride-based DESs
are also readily biodegradable.13 Consequently, these favorable
Received: June 20, 2016
Revised: July 25, 2016

© XXXX American Chemical Society A DOI: 10.1021/acs.biomac.6b00910

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Table 1. Composition of Different DES Systems, Reaction Conditions, Yields, Limiting Viscosity, and Carboxylic Acid Content
before and after Cellulose Treatments
molar reaction time reaction temp yieldd limiting viscosity carboxylic acid content
sample acid ratioa (h) (°C) (%) (dm3/kg) (mmol/g)
b
dissolving 503
pulp
DES1 anhydrous oxalic acid 1:2 2 100 72 104 0.25 ± 0.02
DES2 oxalic acid dihydrate 1:1 2 100 68 113 0.20 ± 0.04
DES3 oxalic acid dihydrate 1:1 4 100 76 104 0.27 ± 0.13
DES4 oxalic acid dihydrate 1:1 6 100 78 104 0.23 ± 0.07
DES5 oxalic acid dihydrate 1:1 2 120 73 100 0.23 ± 0.03
c
DES6 p-toluenesulfonic acid 1:1 2 60 70 108
monohydrate
c
DES7 p-toluenesulfonic acid 1:1 4 60 66 104
monohydrate
c
DES8 levulinic acid 1:2 2 100 88 267
a
Choline chloride/acid. No carboxylic acid groups detected. Carboxylic acid content not measured. Calculated from the mass of fibers after the
b c d

DES treatment vs the mass of the original fibers.

properties have encouraged many researchers to switch their
interest from traditional molecular solvents and ILs to DESs.
Cellulose nanocrystals (CNCs) are stiff, rod-like nanosized
(length generally below a micrometer and width from a few
nanometers to tens of nanometers) cellulosic materials isolated
from natural biomasses, such as plant fibers.14 CNCs have
several unique properties such as lightweight, biodegradability,
stiffness, a large surface area, and are produced from cellulose,
which is renewable and the most abundant biopolymer.15 Due
to the high reinforcement capability, CNCs are widely used in
polymer nanocomposites to improve their properties.16 CNCs
can also be used as a replacement for toxic oil-based chemicals
in mineral flotation, 17 as a stabilizer of oil-in-water
emulsions18,19 and in biomedicine.20 Acidic hydrolysis using
aqueous mineral acids, such as sulfuric,21 hydrochloric,22
hydrobromic,23 and phosphoric acids,24 or polyoxometalates25
has been the most common method to dissolve the amorphous
regions of cellulose to yield CNCs. Several other methods,
mostly based on different oxidation reactions, have also been
applied in CNC production.26−30 Recently, ILs were shown to
act as an efficient swelling and hydrolysis medium to fabricate
CNCs.31−34 DESs have so far been reported to have been used
in pretreatment media for cellulose saccharification35,36 and in
the fabrication of cellulose nanofibers (CNFs;37 by retaining
amorphous cellulose regions intact, allowing the individualiza-
tion of flexible and long nanosized filaments).
In this study, we report the results of DESs obtained from
choline chloride and organic acids as potential acidic hydrolytic
solvents to fabricate CNCs from dissolving pulp. For this
purpose, oxalic (anhydrous and dihydrate), p-toluenesulfonic
monohydrate, and levulinic acids were used as the acidic
components of DESs under different hydrolytic conditions
(temperature and time). The CNCs obtained after DES
pretreatment and mechanical microfluidization were charac-
terized by transmission electron microscopy (TEM), wide-
angle X-ray diffraction (WAXD), diffuse reflectance infrared
Fourier transform spectrum (DRIFT), thermogravimetric
analysis (TGA), and conductive titration.
are discussed elsewhere.38 Choline chloride and p-toluenesulfonic acid
monohydrate were obtained from TCI (Germany), and oxalic acid
(anhydrous and dihydrate) and levulinic acid were purchased from
Sigma-Aldrich (Germany). All the chemicals were used as received,
without further purification.
Hydrolysis of Cellulose Pulp Using Deep Eutectic Solvent.
Molar ratios between choline chloride and acids were 1:2 for
anhydrous oxalic acid39 and levulinic acid40 and 1:1 for oxalic acid
dihydrate41 and p-toluenesulfonic acid monohydrate.42 All the DESs
were produced by heating the mixtures at 100 °C, except in the case of
p-toluenesulfonic acid monohydrate, where a temperature of 60 °C
was used. DES pretreatments of cellulose were performed by adding
1.2 g of dried pulp into 120 g of DES during mixing for the desired
time (2−4 h) at a predetermined temperature (60−120 °C). The
reaction conditions used are presented in Table 1. After mixing, the
reaction mixture was removed from the heating source (oil bath) and
100 mL of deionized water was added. The pretreated pulp
suspensions were filtered and washed with 400 mL of deionized water.
Fabrication of Cellulose Nanocrystals from Deep Eutectic
Solvent-Pretreated Cellulose Fibers. DES-pretreated aqueous
cellulose fiber dispersions (0.5%) were first mixed at 11000 rpm
with an Ultra-Turrax mixer (IKA T25, Germany) at a pH of 7 (the pH
was adjusted using dilute NaOH solution) for 1 min. The fibers were
then disintegrated using a microfluidizer (Microfluidics M-110EH-30,
U.S.A.) to individualize CNCs. Three passes through 400 and 200 μm
chambers at a pressure of 1300 bar and then three passes through 200
and 87 μm chambers at a pressure of 2000 bar were used.
Scanning Electron Microscopy. Scanning electron microscopy
(SEM, Zeiss Zigma HD VP, Germany) images of the freeze-dried
(liquid nitrogen and vacuum drying) samples filtered on a
polycarbonate membrane with a pore size of 0.2 μm were obtained.
The accelerating voltage during imaging was 0.5 kV.
Transmission Electron Microscopy. The morphological features
of the fabricated CNCs were analyzed with a Tecnai G2 Spirit
transmission electron microscope (FEI Europe, Eindhoven, The
Netherlands). Samples were prepared by diluting each sample with
ultrapure water. A small droplet of the dilution was placed on top of a
carbon-coated copper grid. The grid was first coated with polylysine by
applying a small droplet of 0.1% solution of polylysine on the top of
the grid and allowing it to stand for 3 min. The excess polylysine was
removed from the grid by touching the droplet with a corner of a filter
paper. The small droplet of the sample was then placed on the top of

■
EXPERIMENTAL SECTION
Materials. Dissolving cellulose pulp (softwood) was obtained as
dry sheets and used as a cellulose material after disintegration in
deionized water. The disintegrated pulp was filtered, washed with
ethanol, and dried at 60 °C for 24 h. The properties of cellulose pulp
the grid. The excess amount of the sample was removed from the grid
by touching the droplet with a corner of a filter paper. Negative
staining of the samples was performed by placing a droplet of uranyl
acetate (2% w/v) on top of each specimen. The excess uranyl acetate
was removed with filter paper, as described above. The grids were
dried at room temperature and analyzed at 100 kV under standard
conditions. Images were captured using a Quemesa CCD camera, and

B DOI: 10.1021/acs.biomac.6b00910
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Figure 1. Schematic flow diagram of CNC production using DES. First step: hydrolysis of wood cellulose fibers in DES at elevated temperature;
Second step: washing and filtrating the DES-treated fibers using water and vacuum filtration; Third step: mechanical disintegration of DES-treated
and washed microfibers in water with microfluidizer.

Figure 2. (a) Dry cellulose pulp before and after DES treatments and washing and (b) examples of SEM images of cellulose pulp and fibers after
DES treatments.

iTEM image analysis software (Olympus Soft Imaging Solutions
GMBH, Munster, Germany) was used to measure the width of the
individual nanocrystals. In total, 60 CNCs of each sample were
measured. The final results were averaged and standard errors were
calculated.
X-ray Diffraction. The crystalline structure of the original pulp
and CNCs was investigated using wide-angle X-ray diffraction
(WAXD). Measurements were conducted on a Rigaku SmartLab 9
kW rotating anode diffractometer (Japan) using a Co Kα radiation (40
kV, 135 mA; λ = 1.79030 nm). Samples were prepared by pressing
tablets of freeze-dried celluloses to a thickness of 1 mm. Scans were
taken over a 2θ (Bragg angle) range from 5−50° at a scanning speed
of 10°/s, using a step of 0.5°. The degree of crystallinity in terms of the
CrI was calculated from the peak intensity of the main crystalline plane
(200) diffraction (I200) at 26.2° and from the peak intensity at 22.0°
associated with the amorphous fraction of cellulose (Iam), according to
eq 1:43
⎛ I − Iam ⎞
CrI = ⎜ 200
⎝ I200 ⎠
⎟ · 100%
(1)
It should be noted that, due to the Co Kα radiation source, the
cellulose peaks have different diffraction angles compared to results
obtained using the Cu Kα radiation source.
Diffuse Reflectance Infrared Fourier Transform Spectrosco-
py. Chemical characterization of raw cellulose and DES-pretreated
C DOI: 10.1021/acs.biomac.6b00910
cellulose was performed using DRIFT. Spectra were collected with a
Bruker Vertex 80v spectrometer (U.S.A.) from freeze-dried samples.
Spectra were obtained in the 600−4000 cm−1 range, and 40 scans were
taken at a resolution of 2 cm−1 from each sample.
Limiting Viscosity. The effect of the DES pretreatment on the
degree of polymerization was estimated using the limiting viscosity,
measured in cupriethylenediamine solution according to the ISO 5351
standard. Samples were freeze-dried prior to the measurements.
Thermogravimetric Analysis. TGA measurements were carried
out with a thermal analyzer Netzsch STA 409 PC (Germany)
apparatus in two different atmospheres: under nitrogen flow and under
air flow (dynamic air), both with a constant rate of 60 mL min−1. Each
measurement was made using approximately 5 mg of the freeze-dried
sample, which was heated from 25−600 °C at a scanning rate of 2 °C/
min−1. The temperature of polymer degradation, Td, was taken as the
temperature at the onset point of the weight loss in the TGA curve
obtained.
■ RESULTS AND DISCUSSION
The fabrication of CNCs is commonly based on the hydrolysis
and dissolution of amorphous regions of cellulose using strong
acids, ILs, or oxidative treatments. Here, four different DES
systems containing choline chloride as a hydrogen bond donor
and organic acids as a hydrogen bond acceptor were studied as
potential new green media for CNC individualization.
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Schematic illustration of the CNC production process is
presented in Figure 1. Table 1 presents the reaction conditions
used and the yields after DES pretreatments. All DES
pretreatments notably decreased the cellulose mass (yield of
66−88%), and the lowest yield was obtained after 2 and 4 h
reaction time with oxalic acid dihydrate and p-toluenesulfonic
acid-based DESs, respectively. However, the yield was seen to
increase slightly with an oxalic acid dihydrate-based system
when reaction time increased. This may be due to the
esterification of the hydroxyl groups of cellulose with oxalic
acid, either by the formation of monoester or cross-linked
diester. In particular, the formation of diester can increase the
yield because it can prevent the dissolution of the hydrolysis
product of cellulose. However, when p-toluenesulfonic acid was
used, the yield decreased when reaction time was increased
from 2−4 h, suggesting that DES systems do not possess
significant reactivity with cellulose.
The fiber structure of the original dissolving pulp was
partially degraded during DES pretreatment (Figure 2a), as
noticed upon visual observation, and powder-like material was
obtained (except in the case of DES8, which remained a fluffy-
like material similar to raw cellulose) after pretreatment and
drying. Some of the products appeared as slightly greyish
(DES5), whereas the product obtained by DES1 was brown,
indicating the formation of chromophoric cellulose degradation
products that could not be removed during the washing step.
The degradation of the fiber structure of cellulose was
confirmed by SEM (Figure 2b). Oxalic acid and p-
toluenesulfonic acid-based DES pretreatments resulted in the
formation of fragmented cellulose fiber particles, whereas DES8
did not significantly alter the fiber structure of cellulose.
Limiting viscosity was used to indicate the effect of the DES
pretreatment on the degree of polymerization of cellulose
fibers. The original limiting viscosity of the dissolving pulp was
decreased from 503 dm3/kg to approximately 100 dm3/kg
with DES1−7 (Table 1). These results indicate strong cellulose
hydrolysis during the DES pretreatment, which is typical for the
mineral acid hydrolysis of cellulose during CNC production.44
No significant differences between DES pretreatments existed,
except in the case of DES8, where limiting viscosity was almost
three times higher compared to other DES-pretreated fibers.
However, the limiting viscosity of DES8 was still almost half
that of the original pulp. The differences in the limiting
viscosities between DES1−7 and DES8 are most likely caused
by the strong acid characteristic of oxalic acid (pKa of 1.25 and
4.14 in water45) and p-toluenesulfonic acid (pKa of −2.8 in
water46) compared to levulinic acid (pKa of 4.59 in water47).
The esterification of cellulose can take place in the presence
of acids, such as carboxylic acids. The attachment of oxalic acid
to cellulose after DES pretreatment can be seen in the DRIFT
spectra in Figure 3. Compared with the cellulose pulp
spectrum, all the oxalic acid dihydrate DES-pretreated fibers
(DES2−5) exhibited a new band around 1740 cm−1, which is a
superimposed peak of the carbonyl vibrations of ester and
carboxylic acid. The overlapping of the ester and carboxylic acid
carbonyl vibrations is typical of the half-esters of dicarboxylic
acids of cellulose.48 The appearance of carbonyl stretching can
also be seen in the spectrum of anhydrous oxalic acid DES-
pretreated fibers (DES1), where no difference between original
fibers and p-toluenesulfonic (DES6−7) and levulinic acids
(DES8) was observed.
The quantity of carboxylic acid groups on the DES-
pretreated fibers was determined using conductometric
D DOI: 10.1021/acs.biomac.6b00910
Article
Figure 3. DRIFT spectra of cellulose before and after DES
pretreatments. The superimposed peak of the carbonyl vibrations of
ester and carboxylic acid of oxalic acid containing DES-pretreated
fibers is represented by a dashed line.
titration. Titration was performed only for the oxalic acid-
treated fibers, as based on the DRIFT spectra no significant
amount of chemical modification occurred during the DES6−8
treatments. All the oxalic acid (anhydrous and dihydrate) DES-
pretreated fibers exhibited similar carboxylic acid content
(0.20−0.27 mmol/g; Table 1; no detectable amount of the
acid groups was observed in the original cellulose pulp).
Therefore, the results suggest the carboxylic acid content of
DES-pretreated fibers cannot be significantly adjusted by
increasing the temperature or the reaction time. This might
be due to the overesterification of diesters.
All the DES-pretreated fibers were mechanically disintegrated
using a microfluidizer to liberate CNCs. All the samples
underwent similar mechanical treatment expect for DES1,
which blocked the smallest interaction chamber (IXC; 87 μm),
and DES8, which blocked even the largest IXC (200 μm).
Further processing of both specimens was therefore discon-
tinued. Other samples passed through the microfluidizer
without any blocking. However, it was noted that instead of
forming a stable suspension, p-toluenesulfonic acid DES-
pretreated fibers (DES6−7) formed a milky white suspension
in which particle sedimentation occurred almost immediately.
All oxalic acid dihydrate DES-pretreated fibers formed slightly
gel-like suspensions (Figure 4; DES2−5). These suspensions
remained stable at room temperature for several weeks without
any notable sedimentation, excluding DES5, for which minor
sedimentation was observed after two months of free settling at
room temperature.
Due to the sedimentation of DES6 and 7 after disintegration,
only the stable specimens (DES2−5) were studied further.
Based on the TEM images (Figure 5, for higher resolution
images, see Supporting Information), all the oxalic acid
dihydrate DES-pretreated samples (DES2−5) exhibited indi-
vidually occurring short CNCs. Compared with CNCs
obtained at 100 °C, DES5 had a thin, needle-like structure
(average width around 10 nm), whereas DES2−3 had slightly
thicker crystals (average width around 15 nm; Table 2). There
was no significant difference within the specimens in the length
of CNCs; therefore, DES5 had the highest aspect ratio (36)
compared with the others (the aspect ratios of DES2−4 ranged
from 21−28). The width of CNCs obtained after DES
pretreatments were around 2-fold higher than those of the
CNCs typically obtained from sulfuric acid hydrolysis of wood
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Figure 4. Aqueous solutions (0.4%) of DES treated celluloses directly after mechanical disintegration (top row) and after free settling for two
months at room temperature (bottom row).

Figure 5. TEM images of CNCs obtained from DES-pretreated cellulose fibers: (a) DES2, (b) DES3, (c) DES4, and (d) DES5 (ground circles are
due to the sample preparation, most likely due to the air bubbles trapped in the cellulose solution).

Table 2. Average Dimensions and CrIs for CNCs Obtained

after DES Pretreatment and Mechanical Disintegration
(Error Represents Standard Error)
sample width length aspect ratio CrI
DES2 13.6 ± 1.1 390 ± 25 28 69
DES3 15.7 ± 1.3 337 ± 23 21 69
DES4 13.8 ± 0.7 364 ± 22 26 66
DES5 9.9 ± 0.7 353 ± 16 36 71

pulp, but the lengths of CNCs obtained here were significantly

longer, resulting in similar or even higher aspect ratio values
compared with the traditional acid hydrolysis method.49,50 It
should be noted that no sample fractionation steps were
utilized after mechanical disintegration and therefore a minor
amount of longer cellulose nanofibrils was also observed,
especially with samples (DES2−3) treated in the mildest
conditions. In addition, few thicker (around 200 nm) rod-like
particles were present in each sample. Therefore, it would be
beneficial to perform some final purification (e.g., using
centrifuge) to obtain CNC solutions with higher uniformity.
The X-ray diffraction patterns of the CNCs and original
cellulose pulp are presented in Figure 6. The diffraction
patterns show the characteristic peaks for cellulose I, indicating
no rearrangement to other cellulose allomorphs occurred. The
presence of the diffraction patterns of cellulose I suggest that no
dissolution of cellulose fibers took place during the DES
pretreatment and that hydrolysis was purely heterogeneous in
E DOI: 10.1021/acs.biomac.6b00910
Figure 6. XRD diffraction patterns of original cellulose pulp and
CNCs obtained from DES-pretreated cellulose fibers.
nature. The CrIs of DES2−4 were 66−71% (Table 2), which
are slightly higher compared with original cellulose pulp (66%).
A small increase in the CrI is similar to the hydrochloride acid
hydrolytic treatments reported previously.51 However, the
obtained CrIs are lower compared with CNCs obtained by
acid hydrolysis but are in line with or higher than those
produced by oxidative treatments28,52−54 (it should be noted
that the Segal method used for CrI calculations is not suitable
for the comparison of different types of samples but rather
quantifies changes within a single sample set55). It is plausible
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that crystallinities of DES-pretreated fibers are decreased during
the microfluidizer treatment, as it is well-known that
mechanical treatment can decrease the crystallinity of cellulose
due to strong shear forces during homogenization, which may
loosen the crystalline structure and result in peeling of the
cellulose chains on the crystallites.
The thermal stability of CNCs was studied using TGA
measurements at air and nitrogen atmosphere. It can be seen
from Figure 7 that the onset temperatures of CNCs ranged
Figure 7. TGA curves of original pulp and CNCs prepared after DES
pretreatment (a) in air and (b) in nitrogen.
from 275−293 °C. Even though this is significantly lower
compared to original cellulose pulp (the high thermal stability
of dissolving pulp is most likely due to the high molecular
weight and the absence of a significant amount of hemi-
celluloses), it is still in the range of thermally stable CNCs
obtained by phosphoric acid hydrolysis24 and significantly
higher than those obtained after sequential periodate oxidation
and reductive amination.53 The highest thermal stability was
found for DES2, whereas there were no significant differences
between the other CNCs. In a nitrogen atmosphere, the onset
of the main degradation temperature was slightly higher
(around 300 °C for all CNCs) compared to the measurement
in air. However, minor degradation (around 10% w) was
observed after 200 °C. The residual mass at 600 °C for CNCs
was over two times higher compared with original cellulose
pulp. The formation of residual char is most likely due to the
dehydration of cellulose by acidic oxalic acid groups.
F DOI: 10.1021/acs.biomac.6b00910
Article
The CNC solutions (0.1% in water) exhibited transmittances
ranging from 45−75% at a wavelength of 800 nm (Figure 8).
Figure 8. UV−vis spectra of DES-pretreated and mechanically
disintegrated CNCs (0.1%).
The transmittance increased when a longer reaction time or a
higher temperature was used in the pretreatment stage,
indicating the higher uniformity of CNCs (presumably due to
the presence of fewer fiber aggregates after more intensive DES
pretreatments). The transmittances in the UV−vis results are
similar to those of the amino-modified CNCs previously
reported by sequential oxidation and reduction treatment28 but
are lower than those of CNCs obtained by periodate oxidation
followed by chlorite oxidation56 or reductive amination with
bisphosphonate containing amine.53 Relatively low trans-
mittance, especially at the lower wavelength, indicates that
some aggregated cellulose particles (particles that might not be
fully disintegrated during the mechanical treatment or
reaggregated CNCs) remained in the solution.
The CNCs are highly promising green nanomaterials for the
emerging bioeconomy to be used in numerous applications.
CNCs themselves have low toxicity at dilute solutions57,58 and
are biodegradable.59 Therefore, in respect of the whole material
production chain, it is important that CNCs can be produced
via environmentally sustainable methods and with few working
hazards. Oxalic acid itself is a moderately toxic chemical and
even though DESs containing oxalic acid has some cytotoxicity
properties,13 low toxicity still indicates that choline chloride and
oxalic acid-based DES is biocompatible and readily biodegrad-
able.12 Therefore, the methodology introduced here can be
seen as one of the most feasible ways to obtain CNCs.
However, there is still room for improvement, and other even
less toxic and cheaper DESs are waiting to be discovered, which
increases the feasibility of producing CNCs.
■
CONCLUSIONS
DES based on choline chloride and oxalic acid dihydrate was
found to be an effective hydrolytic solvent for the production of
CNCs. DESs were used only as a pretreatment media, solvent
was removed by simple filtration and washing steps, and CNCs
were produced mechanically from DES-free fibers. The use of
DES as a pretreatment media thus eliminates cumbersome
separation of CNCs from the reaction mixture. It was found
that even 2 h pretreatment time at 100 °C was suitable for the
fabrication of CNCs with a high aspect ratio, and the width
could be adjusted by increasing the reaction temperature. Due
to the low toxicity, biocompatibility, and biodegradability, DES
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pretreatment can be seen as an environmentally friendly
method for CNC production.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.6b00910.
TEM images of CNCs with different magnitutes (PDF).
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: +358294482424. Fax: +358 855 323 27. E-mail: juho.
sirvio@oulu.fi.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The following people are gratefully acknowledged for their
contribution to this work: Ms. Vilma Haarala for her
contribution in the experimental part of the study, Dr. Ilkka
Miinalainen for his help with the TEM and SEM images, Mr.
Tommi Kokkonen for TG analysis, and Mr. Sami Saukko for
his guidance with WAXD analysis. The facilities at the Center
of Microscopy and Nanotechnology at the University of Oulu
were utilized in this research.
■
REFERENCES
(1) Brandt, A.; Gräsvik, J.; Hallett, J. P.; Welton, T. Green Chem.
2013, 15, 550−583.
(2) de Oliveira, H. F. N.; Farès, C.; Rinaldi, R. Chem. Sci. 2015, 6,
5215−5224.
(3) Petkovic, M.; Seddon, K. R.; Rebelo, L. P. N.; Pereira, C. S. Chem.
Soc. Rev. 2011, 40, 1383−1403.
(4) Egorova, K. S.; Ananikov, V. P. ChemSusChem 2014, 7, 336−360.
(5) Liwarska-Bizukojc, E.; Maton, C.; Stevens, C. V. Biodegradation
2015, 26, 453−463.
(6) Hammond, O. S.; Bowron, D. T.; Edler, K. Green Chem. 2016,
18, 2736−2744.
(7) Smith, E. L.; Abbott, A. P.; Ryder, K. S. Chem. Rev. 2014, 114,
11060−11082.
(8) Crawford, D. E.; Wright, L. A.; James, S. L.; Abbott, A. P. Chem.
Commun. 2016, 52, 4215−4218.
(9) Dai, Y.; van Spronsen, J.; Witkamp, G.-J.; Verpoorte, R.; Choi, Y.
H. Anal. Chim. Acta 2013, 766, 61−68.
(10) de Morais, P.; Gonçalves, F.; Coutinho, J. A. P.; Ventura, S. P.
M. ACS Sustainable Chem. Eng. 2015, 3, 3398−3404.
(11) Juneidi, I.; Hayyan, M.; Hashim, M. A. RSC Adv. 2015, 5,
83636−83647.
(12) Zhao, B.-Y.; Xu, P.; Yang, F.-X.; Wu, H.; Zong, M.-H.; Lou, W.-
Y. ACS Sustainable Chem. Eng. 2015, 3, 2746−2755.
(13) Radošević, K.; Cvjetko Bubalo, M.; Gaurina Srček, V.; Grgas, D.;
Landeka Dragičević, T.; Radojčić Redovniković, I. Ecotoxicol. Environ.
Saf. 2015, 112, 46−53.
(14) Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors,
M.; Gray, D.; Dorris, A. Angew. Chem., Int. Ed. 2011, 50, 5438−5466.
(15) Habibi, Y. Chem. Soc. Rev. 2014, 43, 1519−1542.
(16) Mariano, M.; El Kissi, N.; Dufresne, A. J. Polym. Sci., Part B:
Polym. Phys. 2014, 52, 791−806.
(17) Laitinen, O.; Kemppainen, K.; Ä mmälä, A.; Antti Sirviö, J.;
Liimatainen, H.; Niinimäki, J. Ind. Eng. Chem. Res. 2014, 53, 20092−
20098.
(18) Zoppe, J. O.; Venditti, R. A.; Rojas, O. J. J. Colloid Interface Sci.
2012, 369, 202−209.
G DOI: 10.1021/acs.biomac.6b00910
Article
(19) Ojala, J.; Sirviö, J. A.; Liimatainen, H. Chem. Eng. J. 2016, 288,
312−320.
(20) Lin, N.; Dufresne, A. Eur. Polym. J. 2014, 59, 302−325.
(21) Bondeson, D.; Mathew, A.; Oksman, K. Cellulose 2006, 13,
171−180.
(22) Yu, H.; Qin, Z.; Liang, B.; Liu, N.; Zhou, Z.; Chen, L. J. Mater.
Chem. A 2013, 1, 3938−3944.
(23) Sadeghifar, H.; Filpponen, I.; Clarke, S. P.; Brougham, D. F.;
Argyropoulos, D. S. J. Mater. Sci. 2011, 46, 7344−7355.
(24) Camarero Espinosa, S.; Kuhnt, T.; Foster, E. J.; Weder, C.
Biomacromolecules 2013, 14, 1223−1230.
(25) Liu, Y.; Wang, H.; Yu, G.; Yu, Q.; Li, B.; Mu, X. Carbohydr.
Polym. 2014, 110, 415−422.
(26) Qin, Z.-Y.; Tong, G.-L.; Frank Chin, Y. C.; Zhou, J.-C.
BioResources 2011, 6 (2), 1136−1146.
(27) Leung, A. C. W.; Hrapovic, S.; Lam, E.; Liu, Y.; Male, K. B.;
Mahmoud, K. A.; Luong, J. H. T. Small 2011, 7, 302−305.
(28) Visanko, M.; Liimatainen, H.; Sirviö, J. A.; Heiskanen, J. P.;
Niinimäki, J.; Hormi, O. Biomacromolecules 2014, 15, 2769−2775.
(29) Tejado, A.; Alam, M. N.; Antal, M.; Yang, H.; van de Ven, T. G.
M. Cellulose 2012, 19, 831−842.
(30) Sirviö, J. A.; Visanko, M. T.; Heiskanen, J. P.; Liimatainen, H. J.
Mater. Chem. A 2016, 4, 6368−6375.
(31) Mao, J.; Heck, B.; Reiter, G.; Laborie, M.-P. Carbohydr. Polym.
2015, 117, 443−451.
(32) Mao, J.; Osorio-Madrazo, A.; Laborie, M.-P. Cellulose 2013, 20,
1829−1840.
(33) Man, Z.; Muhammad, N.; Sarwono, A.; Bustam, M. A.; Vignesh
Kumar, M.; Rafiq, S. J. Polym. Environ. 2011, 19, 726−731.
(34) Tan, X. Y.; Abd Hamid, S. B.; Lai, C. W. Biomass Bioenergy
2015, 81, 584−591.
(35) Xia, S.; Baker, G. A.; Li, H.; Ravula, S.; Zhao, H. RSC Adv. 2014,
4, 10586−10596.
(36) Kumar, A. K.; Parikh, B. S.; Pravakar, M. Environ. Sci. Pollut. Res.
2016, 23, 1−11.
(37) Sirviö, J. A.; Visanko, M.; Liimatainen, H. Green Chem. 2015, 17,
3401−3406.
(38) Sirvio, J.; Hyvakko, U.; Liimatainen, H.; Niinimaki, J.; Hormi, O.
Carbohydr. Polym. 2011, 83, 1293−1297.
(39) Habibi, E.; Ghanemi, K.; Fallah-Mehrjardi, M.; Dadolahi-
Sohrab, A. Anal. Chim. Acta 2013, 762, 61−67.
(40) Maugeri, Z.; de María, P. D. RSC Adv. 2012, 2, 421−425.
(41) Jablonský, M.; Škulcová, A.; Kamenská, L.; Vrška, M.; Šíma, J.
BioResources 2015, 10, 8039−8047.
(42) Assanosi, A. A.; Farah, M. M.; Wood, J.; Al-Duri, B. RSC Adv.
2014, 4, 39359−39364.
(43) Segal, L.; Creely, J. J.; Martin, A. E.; Conrad, C. M. Text. Res. J.
1959, 29, 786−794.
(44) Sun, B.; Zhang, M.; Hou, Q.; Liu, R.; Wu, T.; Si, C. Cellulose
2016, 23, 439−450.
(45) Knowles, C. F.; Hodgkinson, A. Analyst 1972, 97, 474−481.
(46) Guthrie, J. P. Can. J. Chem. 1978, 56, 2342−2354.
(47) Kopetzki, D.; Antonietti, M. Green Chem. 2010, 12, 656−660.
(48) Li, W. Y.; Jin, A. X.; Liu, C. F.; Sun, R. C.; Zhang, A. P.;
Kennedy, J. F. Carbohydr. Polym. 2009, 78, 389−395.
(49) Sacui, I. A.; Nieuwendaal, R. C.; Burnett, D. J.; Stranick, S. J.;
Jorfi, M.; Weder, C.; Foster, E. J.; Olsson, R. T.; Gilman, J. W. ACS
Appl. Mater. Interfaces 2014, 6, 6127−6138.
(50) Beck-Candanedo, S.; Roman, M.; Gray, D. G. Biomacromolecules
2005, 6, 1048−1054.
(51) Palme, A.; Theliander, H.; Brelid, H. Carbohydr. Polym. 2016,
136, 1281−1287.
(52) Yang, H.; van de Ven, T. G. M. Cellulose 2016, 23, 1−11.
(53) Sirviö, J. A.; Hasa, T.; Ahola, J.; Liimatainen, H.; Niinimäki, J.;
Hormi, O. Carbohydr. Polym. 2015, 133, 524−532.
(54) Sirviö, J. A.; Visanko, M.; Laitinen, O.; Ä mmälä, A.; Liimatainen,
H. Carbohydr. Polym. 2016, 136, 581−587.
(55) Ahvenainen, P.; Kontro, I.; Svedström, K. Cellulose 2016, 23,
1073−1086.
Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules Article

(56) Sirviö, J. A.; Honkaniemi, S.; Visanko, M.; Liimatainen, H. ACS

Appl. Mater. Interfaces 2015, 7, 19691−19699.
(57) Hanif, Z.; Ahmed, F. R.; Shin, S. W.; Kim, Y.-K.; Um, S. H.
Colloids Surf., B 2014, 119, 162−165.
(58) Du, L.; Arnholt, K.; Ripp, S.; Sayler, G.; Wang, S.; Liang, C.;
Wang, J.; Zhuang, J. Ecotoxicology 2015, 24, 2049−2053.
(59) Kümmerer, K.; Menz, J.; Schubert, T.; Thielemans, W.
Chemosphere 2011, 82, 1387−1392.

H DOI: 10.1021/acs.biomac.6b00910
Biomacromolecules XXXX, XXX, XXX−XXX