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Industrial Crops & Products

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

Acidic and alkaline deep eutectic solvents in delignification and

nanofibrillation of corn stalk, wheat straw, and rapeseed stem residues
Terhi Suopajärvia,*, Pierfrancesco Riccib, Ville Karvonena, Gianluca Ottolinab,
Henrikki Liimatainena
a
Fiber and Particle Engineering Research Unit, University of Oulu, P.O. Box 4300, FI-90014, Oulu, Finland
b
Institute of Chemistry of Molecular Recognition National Research Council, Via Mario Bianco 9, 20131, Milano, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: In this work, six different deep eutectic solvent (DES) treatments—five acidic (natural organic acid–choline
Biomass chloride) and one alkaline (K2CO3–glycerol)—were used and compared in delignification and nanofibrillation of
Delignification agricultural by-products from wheat straw, corn stalk, and rapeseed stem. The DES treatments were performed at
Lignin 100 °C for 8 h, at 100 °C for 16 h, and at 80 °C for 24 h. The yield of cellulose and lignin fractions was obtained
Nanofibril
gravimetrically, and a more detailed composition of fractions was obtained for the 16 h treatment. All the
Strength properties
samples were further nanofibrillated, and the properties of nanocelluloses and their nanopapers were measured.
Waxes
Acidic lactic acid–choline chloride and alkaline K2CO3–glycerol DESs resulted in the highest delignification
yields (11.8–5.7 wt-%), nanocellulose viscosity (1360–555 Pa s), and crystallinity index (54–38 %), but the
strength properties of nanopapers from alkaline DES treatment (170–132 MPa) were better compared to acidic
DESs (132–24 MPa). A plausible explanation for this difference may be that the alkaline DES also dissolved
waxes and proteins, which can mitigate the adhesion and network formation between the nanofibers. It was also
observed that the separated lignin fractions from acidic and alkaline DES treatments had different characteristics
as determined by FTIR.

1. Introduction
Agricultural by-products such as straws, stalks, and stems, which are
obtained after harvesting and removing the edible products, make up
about half of the yield of agricultural biomass. This means that annually
about 875 megatons of wheat straw, 1000 megatons of corn stover, and
54 megatons of rapeseed residues are produced worldwide (Lal, 2005;
Bentsen et al., 2014). These residues are composed mostly (72–86 wt-
%) from cellulose, hemicellulose, and lignin (Bentsen et al., 2014), all of
which are desired compounds in modern biorefinery platforms. The
minor constituents include waxes, proteins, sugars, salts, and ash (Sin,
2012), which can also potentially be utilized, e.g., in the cosmetics and
food industries.
Currently, some of the cereal residues are used as livestock bedding
and fodder, but agricultural residues mainly return into the soil, in
which they can prevent erosion, increase organic matter content, and
help regulate moisture and temperature as well as promote biota of the
soil (Lal, 2005). Due to the large volumes of agricultural residues pro-
duced, their sustainable use as sources of biomaterials, -chemicals and
energy has also been envisioned as a part of emerging circular and
bioeconomy, depending on the local growing regions and the frequency
of harvesting (Bentsen et al., 2014; Haberl et al., 2011; Monforti et al.,
2015; Hansen et al., 2016; van der Hilst et al., 2018).
The utilization of lignocellulosic biomasses such as agricultural re-
sidues in high-value applications typically requires fractionation or
isolation processes for the separation of its major and minor con-
stituents. For removal of lignin, i.e., the delignification of biomass, al-
kaline, organosolv, ionic liquids, ozonolysis, and many other physico-
chemical or fungal pretreatments have been harnessed (Balat, 2011;
Shirkavand et al., 2016). In recent years, a new class of green chemi-
cals, termed deep eutectic solvents (DES) (Francisco et al., 2012a;
Jablonskỳ et al., 2015; Loow et al., 2017; Lynam et al., 2017; Zhang
et al., 2016; Zulkefli et al., 2017; Tang et al., 2017), has been reported
as an alternative, sustainable approach to fractionating and further
refining the biomass components into various end-products such as
biofuels, furanic derivatives, and other high-value chemicals (Vigier
et al., 2015). DESs have previously been used mainly in the dissolution
of CO2, inorganic salts, organic molecules, and various metal oxides, as
well as in metal recovery in electrochemistry (Zhang et al., 2012), since
they can be easily prepared from low-cost, bulk chemicals by simply

⁎
Corresponding author.
E-mail address: terhi.suopajarvi@oulu.fi (T. Suopajärvi).

https://doi.org/10.1016/j.indcrop.2019.111956
Received 5 September 2019; Received in revised form 30 October 2019; Accepted 6 November 2019
0926-6690/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).

Please cite this article as: Terhi Suopajärvi, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111956
T. Suopajärvi, et al.
Fig. 1. Wheat, corn, and rapeseed raw materials (left), their compositions (middle), and nanofibrillated samples after acidic-DES (lactic acid–choline chloride (5:1))
and alkali-DES (Glycerol–K2CO3 (5:1)) pretreatments (right).
mixing them together with heating or even at ambient temperature
(Tang et al., 2017). The chemicals used in DES can be biodegradable
and sometimes recyclable, which makes them even more attractive in
the sustainable processing of various materials (Sirviö et al., 2015; Yang
et al., 2019).
Previously, DESs formed from organic acids and choline chloride
were found to be able to delignify different biomasses (Francisco et al.,
2012b; Kumar et al., 2016; Loow et al., 2017; Zdanowicz et al., 2018).
These acidic treatments were used, e.g., as a pretreatment for fermen-
tation of sugars into ethanol, since they selectively dissolved lignin but
left cellulose and hemicelluloses intact (Francisco et al., 2012b; Kumar
et al., 2016). The cytotoxicity tests have also shown that these natural
deep eutectic solvents (NADES) do not reveal any cytotoxicity and they
are biodegradable and are considered to be environmental friendly
(Haraźna et al., 2019). However, acidic treatments can decrease the
degree of polymerization of carbohydrates, which can in turn decrease
the material properties of cellulose and hemicelluloses (Mäki-Arvela
et al., 2011). On the other hand, the reports of delignification of bio-
masses by alkaline DES treatments, which can potentially be less
harmful to the properties of carbohydrates, are still rare. It has been
noted that nontoxic alkaline glycerol cooking (without DES formation)
can result in the efficient impregnation of glycerol in the various bio-
masses and provide an effective reaction medium for delignification
(Demirba§, 1998). Moreover, an alkaline DES of glycerol–potassium
carbonate DESs (Ghaedi et al., 2018; Naser et al., 2013) has been re-
ported as potential solvent for industrial applications because of its
viscosity, density, surface tension, refractive index, pH, and con-
ductivity in different temperatures, and it has been reported as a pro-
mising green medium in the pulping of rice straw (Lim et al., 2019). To
the best of our knowledge, a comparison of acidic and alkaline DESs in
delignification and further utilization of biomasses has not been re-
ported previously.
Nanocelluloses, i.e., cellulose particles that typically have a
2
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diameter in nanoscale (< 100 nm) and a length from nanoscale to
several micrometers, are considered to be among the most promising
future materials because of their high strength, light weight, tailorable
surface chemistry, and green origin to enable their use in replacing
plastics (Chin et al., 2018; Nechyporchuk et al., 2016). Typically,
chemical or enzymatic pretreatments are used to loosen the recalci-
trance structure of biomass to unravel elongated and flexible cellulose
nanofibrils (CNF) or shorter and rigid cellulose nanocrystals (CNC) from
biomass (Lee et al., 2014). DESs also offer a potential medium for the
production of nanocelluloses and have been used to form non-
derivatized nanofibrils (Li et al., 2017a; Sirviö et al., 2015; Suopajärvi
et al., 2017), anionic nanofibrils (Selkälä et al., 2016; Sirviö et al.,
2019), and even cellulose nanocrystals (Sirviö et al., 2016).
In this work, six different deep eutectic solvent (DES) treat-
ments—five acidic (natural organic acid – choline chloride) and one
alkaline (K2CO3 – glycerol)—were compared in the delignification and
nanofibrillation of agricultural by-products from wheat straw, corn
stalk, and rapeseed stem. After treatments, the solid fraction, consisting
mainly of carbohydrates (i.e. cellulose and hemicellulose), and the li-
quid fraction, consisting mainly of lignin and hydrolyzed carbohy-
drates, were separated, the composition of the cellulose-rich fractions
was analyzed by gravimetric and analytic methods, and precipitated
lignin was investigated with FTIR. The cellulose-rich fractions were
further nanofibrillated, and properties of nanocelluloses and their na-
nopapers were measured.
2. Materials and methods
2.1. Raw materials and chemicals
The wheat straw (Chile), corn stalk (Argentina), and rapeseed stem
(Argentina) samples were oven-dried to a constant weight and then
milled to a particle size of one millimeter and used as such. The
T. Suopajärvi, et al. Industrial Crops & Products xxx (xxxx) xxxx

Table 1
Composition of DES solutions.
Sample name Acidic-DES Chemical formulas Molar ratio

Monocarboxylic acids
Lac-Ch lactic acid + choline chloride C3H6O3 + C5H14ClNO 2:1
Lac5-Ch lactic acid + choline chloride C3H6O3 + C5H14ClNO 5:1
Lev-Ch levulinic acid + choline chloride C5H8O3 + C5H14ClNO 2:1
Dicarboxylic acids
Malic-Ch malic acid + choline chloride C4H6O5 + C5H14ClNO 1:1
Glu-Ch glutaric acid + choline chloride C5H8O4 + C5H14ClNO 1:1
Alkaline-DES
Gly- K2CO3 Glycerol + K2CO3 C3H8O3 + K2CO3 5:1

compositional analysis (glucan, xylan, lignin, and ash) was performed 2.3. Composition of DES-treated solid fractions
according to the analytical procedure provided by the National
Renewable Energy Laboratory (NREL) (NREL/TP-510-42622 and The compositional analysis of DES-treated solid fractions (glucan,
NREL/TP-510-42618) (Sluiter, 2008; Sluiter et al., 2008) on the raw xylan, lignin, and ash) was performed according to the analytical pro-
biomass previously dried at 105 °C overnight. The analyses were per- cedure provided by the National Renewable Energy Laboratory (NREL)
formed in triplicate. Quantification of monomeric sugars was carried (NREL/TP-510-42622 and NREL/TP-510-42618) (Sluiter, 2008; Sluiter
out by HPLC using the ROA column Phenomenex (USA) at 85 °C and the et al., 2008), after having dried at 105 °C overnight. The analyses were
evaporative light-scattering ELS-2041 detector Jasco (Japan). The mo- performed in triplicate. Quantification of monomeric sugars was carried
bile phase was ultrapure H2O with a flow rate of 0.6 mL min-1. All the out by HPLC using the ROA column Phenomenex (USA) at 85 °C and the
experiments were conducted in triplicate. The raw materials and their evaporative light-scattering ELS-2041 detector Jasco (Japan). The mo-
compositions are shown in Fig. 1. The chemicals used in the preparation bile phase was ultrapure H2O with a flow rate of 0.6 mL min-1. All the
of DES solutions are shown in Table 1. The molar ratio of the hydrogen experiments were conducted in triplicate.
bond donor (HBD) (acids and glycerol) and hydrogen bond acceptor
(HBA) (choline chloride and K2CO3) were chosen according to the lit-
2.4. Nanofibrillation of solid fractions
erature (Francisco et al., 2013, 2012a; Kumar et al., 2016) (acidic DES)
and by knowing that the solubility of lignin compounds increases by
DES-treated wheat, corn, and rapeseed samples were soaked in
increasing the molar ratio of HBD (Soares et al., 2017). However, in the
water overnight at a consistency of 0.5 % and treated with an Ultra
case of glycerol, the viscosity of formed DES limited the used molar
Turrax mixer (IKA T25, Germany) for 20 min at 10,000 rpm before
ratio, which was thus chosen to be 5:1. Choline chloride (99 %) was
nanofibrillation with a microfluidizer (Microfluidics M-110EH-30,
obtained from Algry Quimica, S.L. (Spain) and glycerol (98 %), glutaric
USA). Three passes through a 400 μm chamber without pressure, three
acid (98 %), lactic acid (90 %), levulinic acid (98 %), DL-malic acid (99
passes through 400 and 200 μm chambers at a pressure of 1000 bar, and
%), and ethanol (96 %) from VWR (Finland). K2CO3 (99 %) was ob-
then three passes through 200 and 100 μm chambers at a pressure of
tained from Honeywell (Germany). Sulfuric acid (72 % w/w), CaCO3, D
1500 bar were used to obtain gel-like nanocelluloses suspensions,
(+) glucose and D (+) xylose were used as received and were of
which are shown in Fig. 1.
analytical grade (Merck). For the fabrication of the nanopapers, Dur-
apore polyvinylidene fluoride membranes with pore size of 0.65 μm
were used, and for the preparation of the FESEM samples, track-etched 2.5. Analysis of nanofibrillated samples
Nuclepore membranes with pore size of 0.20 μm from Millipore
(France) were used. Deionized water was used throughout the experi- 2.5.1. Rotational viscosity
ments. The rotational viscosity of nanocellulose suspensions was measured
with the Brookfield DV-II + Pro EXTRA (USA) rotational viscometer
using vane-shaped spindle (V-73). Measurements were performed at
2.2. DES pretreatments constant temperature (20 °C) with the sample consistency at 0.35 %.
Rotational speeds of 10, 20, 50, and 100 rpm and measurement time of
Acidic DES solutions were prepared by mixing choline chloride and 2–5 min were used.
a natural organic acid together, and the alkaline DES solution, by
mixing glycerol and potassium carbonate together, at a chosen molar
2.5.2. X-ray diffraction
ratio (Table 1). Mixtures were heated in an oil bath at 100 °C under
The crystallinity of the CNFs from acidic-DES (Lac5–Ch) and alka-
stirring until a clear solution formed. 3 g of wheat, corn, or rapeseed
line-DES (Gly–K2CO3) treatments were measured from freeze-dried
was added to the suspension with 5 g of water, and the suspension (3
samples, which were pressed into around 1 mm thick tablets. Wide-
wt-%) was allowed to stir at 100 °C for 8 or 16 h or at 80 °C for 24 h. The
angle X-ray diffraction (WAXD) measurements were conducted with a
reaction was stopped with the addition of 100 ml of ethanol, after
Rigaku SmartLab 9 kW rotating anode diffractometer (Japan) by using a
which the warm mixture was filtered and the solid residue washed
Co Kα radiation (40 kV, 135 mA) (λ =1.79030 nm). Scans were taken
twice with warm water, dried at 50 °C for 48 h, and weighed. Around
over a 2θ (Bragg angle) range from 5° to 50° at a scanning speed of 10°/
200 ml of water was added to the ethanol-DES filtrate, and lignin was
s, using a step of 0.5°. The degree of crystallinity in terms of the crys-
allowed to precipitate overnight. The alkaline-DES solution was acid-
tallinity index (CrI) was calculated from the peak intensity of the main
ified with 1 M HCl solution before lignin precipitation. Precipitated
crystalline plane (200) diffraction (I200) at 26.2° and from the peak
lignin was separated from the solution by centrifugation and washed
intensity at 22.0° associated with the amorphous fraction of cellulose
twice with a water-ethanol (9:1) solution. Washed lignin was dried at
(Iam) by using the Segal empirical Eq. (1) (Segal et al., 1959):
50 °C for 48 h and weighed.
I − Iam ⎞
CrI = ⎛ 200
⎜ × 100%
⎟

⎝ I200 ⎠ (1)

3
T. Suopajärvi, et al.
2.5.3. TEM and FESEM imaging
An energy filtered transmission electron microscope (EFTEM), LEO
912 OMEGA (Germany), was used to observe the morphology of the
produced nanofibrils. TEM samples were prepared by dropping the
diluted nanocellulose suspension on a carbon-coated copper grid.
Uranyl acetate (2 % w/v) was used in the negative staining of the
samples, after which the samples were dried at room temperature. The
TEM images were captured with a Quemesa CCD camera (Japan), using
100 kV as an accelerating voltage. The widths of individual nanofibrils
were measured from images with ImageJ processing software. As a
pretreatment to FESEM imaging, nanofibrillated samples were filtered
on the polycarbonate membrane (pore size of 0.2 μm), rapidly frozen in
liquid nitrogen, and freeze-dried in a vacuum overnight. Samples were
sputter-coated with platinum and imaged using an accelerating voltage
of 5 kV and a working distance of 5 mm.
2.6. Preparation and characterization of nanopapers from nanofibrillated
samples
Nanopapers were produced by filtering 300 mg (abs.) nanofi-
brillated samples on a polyvinylidene fluoride membrane (pore size
0.65 μm). After filtration, the moist nanopaper was covered with an-
other membrane, placed between two blotting boards, and dried with a
Rapid-Köthen sheet dryer (Karl Schröder KG, Germany) at 94 °C under
900 mbar vacuum for 10 min. The samples were stored at standard
conditions of 23 °C and 50 % relative humidity. After preconditioning in
the standard environment for at least 48 h, the sample thickness was
measured in five different locations using a precision thickness gauge
(Hanatek FT3, UK), and the results were averaged. The nanopaper was
cut into 6 strips with a width of 5 mm prior to the strength measure-
ments. The tensile tests were performed with a Zwick D0724587
(Switzerland) universal material testing machine equipped with a
100 N load cell. For the tensile tests, the gauge length was set to 40 mm,
and six replicates of each sample were tested at a strain rate of 4 mm
min-1 using a pre-strain of 0.1 N at the standard conditions of 23 °C
temperature and 50 % relative humidity.
2.7. Analysis of lignin
2.7.1. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFT)
DRIFT was used for chemical characterization of the separated
lignin fractions. Spectra were collected with a Bruker Vertex 80v
spectrometer (USA) for precipitated and dried lignin samples. For each
sample, 40 scans were taken at a resolution of 2 cm-1, and spectra were
obtained in the 400–4000 cm-1 range.
3. Results and discussion
3.1. Characteristics of DES-treated biomasses
Five acidic and one alkaline DES treatments were used to delignify
wheat straw, corn stalk, and rapeseed stem samples. The samples in-
itially contained 19–24 wt-% lignin, 27–40 wt-% cellulose, and 11–18
wt-% hemicelluloses. The gravimetric yield of lignin from DES treat-
ment varied from 1.0 wt-% to 9.5 wt-% for wheat, 0.8–8.5 wt-% for
corn, and 0.8–11.8 wt-% for rapeseed stem (Fig. 2a–c). The results show
that acidic Lac5–Ch resulted in the highest lignin yield among the dif-
ferent DESs with all biomasses, and the 16 -h reaction at 100 °C was the
optimum condition for lignin removal with Lac5–Ch (8.5 wt-% for
wheat, 9.5 wt-% for corn, and 11.8 wt-% for rapeseed) and other acidic
DES treatments. However, it was noticed that the Lac-Ch and Lac5-Ch
DESs evaporated slightly during long treatment times at 100 °C. It has
been noted before that the characteristics of HBDs affect notably the
thermal stability of DESs and they decompose or volatilize first in the
DES. (Chen et al., 2018; Haraźna et al., 2019). Evaporation was not
observed with other acids nor with glycerol. Despite, the evaporation
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the Lac DESs had the highest delignification efficiency. The lignin yield
with the alkaline DES treatment was from 0.8 to 5.7 wt-% and was
comparable to acidic DESs with wheat and rapeseed and lower with
corn. However, the lignin content measured from solid fractions
(Fig. 2d–f) was generally only slightly lower with Gly-K2CO3s, thus
indicating similar delignification efficiency to that of Lac–Ch and
Lac5–Ch. This result may be attributed to incomplete precipitation of
lignin from alkaline DES, which is seen as a low lignin recovery.
The gravimetric yield of the cellulose-rich fraction varied from 44.9
wt-% to 70.1 wt-% for wheat, 32.2–53.6 wt-% for corn, and 35.9–57.8
wt-% for rapeseed stem. Both lactic acid DESs gave the lowest yield for
cellulose-rich fractions for wheat (45.5 wt-% with Lac–Ch and 44.9 wt-
% with Lac5–Ch) and corn (32.2 wt-% [Lac–Ch] and 33.3 wt-%
[Lac5–Ch]) samples treated for 16 h at 100 °C, but for rapeseed stem the
lowest yield was obtained with Gly–K2CO3 DES (35.9 wt-%) at the same
conditions. The highest yield in the cellulose-rich fraction was obtained
with Lev–Ch samples treated 24 h at 80 °C with all raw materials. On
the other hand, the samples having the highest yield in the cellulose-
rich fraction had the lowest cellulose content and highest content of
lignin and others fraction, thus also showing the poorest delignification
performance. The total yield of the cellulose-rich fraction and the lignin
fraction varied from 35 to 70 wt-%, indicating that notable amounts of
compounds like hemicelluloses, ash, and waxes dissolved in the DES
solutions and did not precipitate out of the DES-ethanol/water solu-
tions.
The compositional analyses of original samples and cellulose-rich
solid fractions after 16 -h reactions at 100 °C in DES are presented in
Fig. 2d–f. The carbohydrates are presented as cellulose- and hemi-
cellulose-derived glucans and hemicellulose-derived xylans. The ash,
lignin, and other compounds (waxes and proteins) are also presented.
The cellulose and hemicellulose contents varied from 62.0 wt-% to 72.3
wt-% for wheat, 53.2–70.7 wt-% for corn, and 50.3–70.7 wt-% for ra-
peseed stem, while residual lignin contents ranged from 12.1–19 wt-%
for wheat, 16.3–26.8 wt-% for corn, and 16.6–22.7 wt-% for rapeseed
stem. The highest cellulose contents were reached with acidic Lac–Ch
and Lac5–Ch treatments (wheat and corn) as well as with alkaline Gly-
K2CO3 reaction (rapeseed). In contrast with acidic DES treatments, the
alkaline Gly–K2CO3 DES did not decrease the hemicellulose content of
the biomasses. Overall, the higher solubility of hemicelluloses com-
pared to cellulose is related to shorter chain length (100∼200 with a
degree of polymerization) of hemicelluloses compared to that of cel-
lulose (10,000–14,000) (Xiao et al., 2001), diff ;erent ring structures
and hydroxyl configurations, which give the amorphous nature, and a
more branched structure for hemicelluloses (Wyman et al., 2004).
Therefore, they are generally more reactive than cellulose and can be
selectively removed from cellulosic substrates. However, the degree of
branching and substitution and composition of hemicelluloses can
differ depending on their natural resources (Kishani et al., 2018). The
mechanism of chemical hydrolysis is based on the catalytic cleavage of
glycosidic bonds in polysaccharides, in which acid acts as a catalyst
(Mäki-Arvela et al., 2011). The acid hydrolysis rate of hemicelluloses
varies depending on their structure, and both random scission and se-
lective scission mechanisms of the side chain have been reported (Mäki-
Arvela et al., 2011). Previously, the solubility of pure lignin, cellulose,
and hemicellulose was studied in choline chloride–lactic acid DES, and
it was found that only lignin was selectively dissolved into the DES
(Kumar et al., 2016). However, our results indicate that all the tested
acidic treatments remove hemicellulose as well as lignin, and in the
case of Glu-Ch and Malic–Ch DESs (Fig. 2d–f), more hemicelluloses
were removed than lignin.
The ash content in the wheat and corn samples was clearly lower
after alkaline Gly-K2CO3 treatment than with the acidic treatments
(0.9–2.5 % vs. 4.5–7.8 %), indicating high solubility of inorganic
compounds in alkaline DES. However, a higher content of inorganics
was noted with the rapeseed. The ash in the corn and wheat consisted
mainly of silicates (Adebisi et al., 2017; Kraszkiewicz et al., 2017; Li
T. Suopajärvi, et al.
Fig. 2. The yield of solid and lignin fractions after different DES treatments for (a) wheat, (b) corn, and (c) rapeseed, and compositions of solid fractions of (d) wheat,
(e) corn, and (f) rapeseed samples without treatment and after DES treatments at 100 °C for 16 h.
et al., 2014), but in the rapeseed stem the primary inorganics are cal-
cium (CaO); potassium (K2O), phosphorus (P2O5), and magnesium
(MgO) oxides; and metals such as Fe, Mo, Mn, Cu, Zn, and Co
(Kraszkiewicz et al., 2017; Paleckienė et al., 2012), which have a poor
solubility in alkaline media. The remaining part of the solid fractions
(others in the diagrams Fig. 2d–f) consists of waxes and proteins and
was not analyzed further. Those compounds are more soluble in alka-
line conditions than in acidic conditions. Thus, Gly–K2CO3 samples
have a clearly lower fraction of these compounds than acidic ones,
especially for the rapeseed sample.
3.2. Nanocelluloses from DES-treated biomasses
Solid fractions from DES treatments were further nanofibrillated
using a microfluidizer to test their feasibility in nanocellulose produc-
tion. All specimens were successfully disintegrated into viscous gels
except those from rapeseed treated with Glu–Ch and Malic–Ch DESs for
24 h at 80 °C. Based on the compositional analysis (Fig. 2f), these
samples also had the highest lignin content. In acidic DESs second
carboxylic group in the HBD may lower the solubility of lignin (Soares
et al., 2017), which explains the poor delignification properties of Glu-
Ch and Malic-Ch. Also, the longer alkyl chain length has reported to
increase the solubility of lignin (Soares et al., 2017), which seems to
correlate with the performance of dicarboxylic acids since Glu-Ch dis-
solves more lignin than Malic-Ch. However, monocarboxylic acids be-
haved differently and Lac-Ch dissolved more lignin than Lev-Ch. In
addition both Lac-Ch and Malic-Ch have one hydroxyl group in addition
to carboxylic group. Therefore, it seems that the solubility of the lignin
increases when the polarity of the HBD of acids decreases as has been
noticed before (Haraźna et al., 2019; Soares et al., 2017). Lignin is
known to hinder the individualization of nanofibrils, because it makes
the lignocellulose structure more stiff, affects the surface charge, and
reduces water uptake and swelling (Lê et al., 2018). The lignin removal
was promoted by the higher temperature during DES treatment
5
Industrial Crops & Products xxx (xxxx) xxxx
(Fig. 2a–c); i.e., more efficient delignification was observed at 100 °C
than at 80 °C, despite the longer treatment time. The same trend was
also noted during the nanofibrillation; the samples from DES treatment
at 100 °C for 16 h were nanofibrillated most easily without clogging of
the microfluidizer, while the samples treated at 80 °C for 24 h were
most difficult to disintegrate into homogenous nanocellulose suspen-
sions because of clogging of the microfluidizer. However, harsh acidic
conditions were observed to darken the samples, which can clearly be
seen especially with rapeseed samples (Fig. 1). This darkening was
probably because of the coaling of the cellulose. Lignin may also de-
grade to acid-soluble form in acidic conditions and further depoly-
merize to smaller Mw products, which may condense or repolymerize
into the insoluble residual lignin during long reactions (Mäki-Arvela
et al., 2011). In addition, a high content of calcium ions, which are
present especially in the rapeseed stem samples, increases the pre-
cipitation of lignin onto the fibers (Gellerstedt and Al-Dajani, 2003) and
can cause a dark color in the acidic DES-treated cellulose-rich fractions.
Based on solid fraction analysis (highest cellulose content) and on
visual evaluation of the quality of nanocellulose samples, the specimens
from acidic Lac5–Ch and alkaline Gly–K2CO3 DES treatments were se-
lected for further analysis. Rotational viscosity was used to indicate the
homogeneity, dimensions, and aggregation of the samples, which are
shown in Fig. 3. Low share rate viscosities are reported to correlate
especially with the morphology of the cellulose particles (Besbes et al.,
2011; Iotti et al., 2011), and thinner and longer nanofibrils possess
higher viscosities in the given concentration (Lasseuguette et al., 2008).
Acidic-DES samples treated at 100 °C for 16 h (Fig. 3) had the highest
viscosities, even though viscosities varied significantly between the raw
materials. The highest viscosity was obtained with corn, and the lowest,
with the rapeseed sample. The differences in pH of DES treatments may
affect the viscosities, since acidic treatments leave the samples in pro-
tonated form, which increases the viscosity of the nanocellulose sus-
pension by decreasing the interfibrillar charge repulsions. The highest
differences in viscosities with alkaline treatment are seen in the
T. Suopajärvi, et al. Industrial Crops & Products xxx (xxxx) xxxx

Fig. 3. Top left: Rotational viscosities of aqueous nanofibril suspensions (0.35 %) for (a) wheat, (b) corn, and (c) rapeseed samples from acidic (Lac5–Ch) (solid line)
and alkaline (Gly–K2CO3) (dotted line) DES treatments at the temperature of 100 °C for 8 h and 16 h and at the temperature of 80 °C for 24 h. Top right: FESEM
images of nanofibrils after DES treatment at 100 °C for 16 h (Scale bar 200 nm). Bottom: TEM images of nanofibrils after DES treatment at 100 °C for 16 h (Scale bar
200 nm).

rapeseed stem samples, which also had largest differences in the yield treatments showed the typical elongated structure of nanofibrils and
of lignin and cellulose-rich fractions (Fig. 2c). their continuous network. The average width of nanofibrils ranged from
TEM images (Fig. 3) of nanofibrils from Glu–Ch and Lac–Ch 3 to 12 nm for all raw materials. FESEM imaging (Fig. 3) confirmed that

6
T. Suopajärvi, et al. Industrial Crops & Products xxx (xxxx) xxxx

Fig. 4. X-ray diffractograms and the crystallinity indexes of cellulose-rich fractions after acidic (Lac5–Ch) and alkaline (Gly–K2CO3) DES treatments for (a) wheat, (b)
corn, and (c) rapeseed samples.

Fig. 5. Tested visual appearance of nanopapers (left),tensile strength (top row) of (a) wheat, (b) corn, and (c) rapeseed nanopapers, and force-strain curves (bottom
row) of the (d) wheat, (e) corn, and (f) rapeseed nanopapers prepared from the nanofibrillated samples after DES treatments of 16 h at 100 °C.

Table 2 Table 3
Tensile strength, strain, Young’s modulus, and thickness of the nanopapers Tensile strength, strain, Young’s modulus, and thickness of the nanopapers
prepared from the samples after DES treatments of 16 h at 100 °C. prepared from the samples after DES treatments of 8 h at 100 °C.
Sample Tensile strength Strain [%] Young’s Thickness of the Sample Tensile strength Strain [%] Young’s Thickness of the
[MPa] modulus [GPa] film [μm] [MPa] modulus [GPa] film [μm]

Wheat Wheat
Glu-Ch 62 ± 6 2.1 ± 0.5 4.4 77.8 Glu–Ch 69 ± 2 2.6 ± 0.3 4.6 76.9
Gly-K2CO3 132 ± 7 4.9 ± 0.5 5.8 60.1 Gly–K2CO3 117 ± 9 4.0 ± 0.5 6.1 63.9
Lac-Ch 114 ± 9 4.8 ± 0.9 5.5 64.3 Lac–Ch 111 ± 8 4.2 ± 0.7 5.3 63.1
Lac5-Ch 132 ± 6 4.8 ± 0.5 6.6 55.5 Lac5–Ch 127 ± 7 4.8 ± 0.6 5.5 59.0
Lev-Ch 52 ± 4 2.0 ± 0.4 3.8 74.0 Lev–Ch 45 ± 3 1.8 ± 0.2 3.7 74.7
Malic-Ch 57 ± 2 2.9 ± 0.3 4.0 71.9 Malic–Ch 35 ± 3 1.4 ± 0.1 3.1 87.0
Corn Corn
Glu-Ch 50 ± 3 1.6 ± 0.4 4.1 60.3 Glu–Ch 56 ± 4 1.8 ± 0.3 4.3 81.6
Gly-K2CO3 163 ± 11 4.9 ± 0.6 7.6 89.1 Gly–K2CO3 143 ± 12 3.7 ± 0.7 7.6 65.9
Lac-Ch 99 ± 9 4.8 ± 0.5 5.4 71.4 Lac–Ch 92 ± 9 3.6 ± 0.9 5.2 73.5
Lac5-Ch 95 ± 3 3.6 ± 0.7 4.9 75.3 Lac5–Ch 95 ± 4 3.3 ± 0.6 5.3 71.5
Lev-Ch 68 ± 4 2.3 ± 0.2 4.9 77.4 Lev–Ch 65 ± 2 2.0 ± 0.2 4.8 75.9
Malic-Ch 52 ± 4 2.4 ± 0.3 3.3 91.0 Malic–Ch 54 ± 8 1.5 ± 0.3 4.9 79.5
Rapeseed Rapeseed
Glu-Ch 80 ± 6 1.8 ± 0.3 6.0 68.7 Glu–Ch 82 ± 5 1.7 ± 0.1 6.4 67.4
Gly-K2CO3 161 ± 11 4.2 ± 0.7 7.5 53.2 Gly–K2CO3 160 ± 4 4.0 ± 0.3 8.3 53.8
Lac-Ch 90 ± 5 1.9 ± 0.1 5.9 60.4 Lac–Ch 80 ± 6 1.7 ± 0.2 5.5 63.2
Lac5-Ch 86 ± 4 1.5 ± 0.1 6.2 62.4 Lac5–Ch 103 ± 6 1.7 ± 0.2 6.9 58.1
Lev-Ch 70 ± 6 1.7 ± 0.2 5.2 65.4 Lev–Ch 71 ± 4 1.6 ± 0.2 5.6 60.5
Malic-Ch 57 ± 4 1.2 ± 0.1 5.5 81.7 Malic–Ch 67 ± 6 1.3 ± 0.2 6.4 70.8

7
T. Suopajärvi, et al. Industrial Crops & Products xxx (xxxx) xxxx

Table 4 3.3. Characteristics of nanopapers from DES-treated nanofibrils

Tensile strength, strain, Young’s modulus, and thickness of the nanopapers
prepared from the samples after DES treatments of 24 h at 80 °C. Flexible and opaque nanopapers (Fig. 5) were successfully prepared
Sample Tensile strength Strain [%] Young’s Thickness of the using a vacuum filtration method from all DES-treated nanofibrils ex-
[MPa] modulus [GPa] film [μm] cept those from rapeseed treated with Glu–Ch and Malic–Ch DESs for
24 h at 80 °C, which we were not able to nanofibrillate. Tensile
Wheat
strengths of all nanopapers and force-strain curves of nanopapers after
Glu–Ch 55 ± 4 2.2 ± 0.2 3.8 77.4
Gly–K2CO3 101 ± 7 3.8 ± 0.3 5.2 71.3 DES treatment for 16 h at 100 °C are shown in Fig. 5. In Table 2 are
Lac–Ch 84 ± 4 2.7 ± 0.1 5.6 64.2 listed the tensile strength, strain, Young’s modulus, and thickness of the
Lac5–Ch 75 ± 2 2.7 ± 0.2 4.4 66.1 prepared nanopapers for wheat, corn, and rapeseed stem samples after
Lev–Ch 24 ± 4 1.1 ± 0.2 2.4 89
16 -h reaction at 100 °C. In the Table 3 are listed the same parameters
Malic–Ch 39 ± 2 1.9 ± 0.2 3.3 75.8
Corn
after 8 h at 100 °C and in the Table 4 after 24 h at 80 °C. The highest
Glu–Ch 46 ± 5 4.8 ± 0.2 4.0 92.5 strengths at break (132 MPa, 163 MPa, and 170 MPa for wheat, corn,
Gly–K2CO3 146 ± 6 6.0 ± 0.4 7.7 64.3 and rapeseed stem, respectively), strain (4.9 %, 4.9 %, and 6.0 %, re-
Lac–Ch 71 ± 5 5.3 ± 0.5 3.9 76.0 spectively), and Young´s modulus (6.1 GPa, 7.7 GPa, and 8.6 GPa, re-
Lac5–Ch 74 ± 6 5.8 ± 0.3 4.9 73.9
spectively) were obtained with the nanopapers prepared from alkaline
Lev–Ch 46 ± 4 4.0 ± 0.2 4.1 81.2
Malic–Ch 40 ± 6 5.6 ± 0.2 3.8 90.7 DES-treated nanofibrils for all biomasses, while acidic DES treatments
Rapeseed showed notably lower values (132–24 MPa, 4.8–1.1 %, and 6.6–2.4 GPa
Gly–K2CO3 170 ± 17 4.2 ± 0.9 8.6 54.3 for wheat; 99–39 MPa, 5.8–1.5 %, and 5.4–3.3 GPa for corn; and
Lac–Ch 67 ± 7 1.4 ± 0.2 5.5 62.0
102–57 MPa, 1.8–1.1 %, and 6.9–5.1 GPa for rapeseed stem; see Tables
Lac5–Ch 72 ± 6 1.4 ± 0.2 6.2 60.6
Lev–Ch 77 ± 6 1.7 ± 0.2 5.1 61.0
2–4. Acidic treatments of wood chips in hemicellulose extraction prior
to pulping have been previously reported to reduce pulp strength
properties even with dilute acid conditions (0.3 % (v/v)) (Vena et al.,
all the samples consisted of individual nanofibrils but also contained 2015). For example, the tensile index of handsheets prepared from
larger nanofibril bundles and irregular aggregates. The average dia- wood pretreated with diluted H2SO4 was reduced by about 15 % (Vena
meter of individual nanofibrils was from 4 to 6 nm, which is similar to et al., 2015). However, with the wheat samples, differences in the
ranges in nanofibrillated wheat (Ma et al., 2018), corn(Xu et al., 2018), strength of nanopapers between alkaline Gly–K2CO3 and acidic Lac–Ch
and rapeseed (Svärd et al., 2019) samples in previous studies. and Lac5–Ch samples were small (Fig. 5a and Table 2). The only ex-
X-ray diffractograms (Fig. 4) indicated that the crystallinity of cel- ception is after a 24 -h reaction at 80 °C (Table 4), in which case the
lulose in all nanofibrillated samples remained in cellulose I allomorph, tensile strength and strain are clearly higher with Gly–K2CO3 (101 MPa
with all DES treatments showing the characteristic peaks at 2θ of 18, and 3.8 %) than with lactic acid samples (84–75 MPa and 2.7 %). For
26.2, and 40 without the existence of other peaks associated with cel- the wheat samples, the amount of waxes and proteins (others fraction)
lulose. The peak at 2θ value of 35 in the Gly–K2CO3 16 h corn and (Fig. 2d) was also small with acidic treatments compared to the corn
rapeseed samples (Fig. 4b and c) is CaCO3 and originates from the raw and rapeseed samples (Fig. 2e and f). Presumably, the residual waxes
materials, according the fingerprint library of the measuring device. reduced the interfibrillar adhesion and mechanical properties of na-
The Segal-crystallinity indexes (CrI) calculated from the diffractograms nopapers. It can also be seen from Fig. 5f that the strain of all acidic DES
varied from 38 to 54 % for wheat, 42–52 % for corn, and 44–52 % for nanopapers is low in rapeseed samples whereas Lac–Ch and Lac5–Ch
rapeseed stem. Overall, the differences in CrI values of acidic and al- resulted in high strain values with wheat and corn samples (Fig.5d and
kaline DES treatments were small, and no systematic trend in the role of e and Table 2). Those samples also had the highest cellulose content
treatment temperature and time was detected. The reason for this and lowest lignin content of all the samples. Thus, lignin can make the
finding could be the fact that the CrI values reflect the changes in nanofibrils stiffer and result in more fragile nanopapers (Ma et al.,
cellulose crystallinity but are also affected by the content of lignin and 2018).
hemicelluloses because they cause extra X-ray scattering (Agarwal The strength properties of alkaline Gly–K2CO3 nanopapers, e.g., for
et al., 2013). Lignin and hemicelluloses particularly affect the 2θ region the corn samples, were high (tensile strength 163 MPa and Young’s
of 18 − 25° by broadening the cellulose [002] peak and increasing the modulus 7.6 GPa) (Table 2) and remarkably higher than recently pub-
underlying background (Agarwal et al., 2017, 2013; Carrillo et al., lished results for corn nanopapers obtained from alkaline sodium hy-
2018). Thus, the differences in lignin and hemicellulose content of DES- droxide treatment, which also included very intense mechanical treat-
treated samples is presumed to increase the variance in CrI values. ment of 30 passes with a high pressure homogenizer (tensile strength

Fig. 6. Appearance of alkaline and acidic lignins (left) and DRIFT spectra (right) of wheat and corn acidic-DES and alkaline-DES lignins.

8
T. Suopajärvi, et al.
was 76.4 MPa and Young’s modulus 2.2 GPa) (Xu et al., 2018). In ad-
dition, lower tensile strength values have been reported for nanopapers
of sodium hydroxide-anhtraquinone pulped and chlorine dioxide and
hydrogen peroxide bleached rapeseed stem and organosolv-treated
wheat (114 MPa (Yousefi et al., 2013) and 42.3 MPa (Barbash et al.,
2017) vs. 57–170 MPa and 24–132 MPa for rapeseed and wheat, re-
spectively) (Tables 2–4).
3.4. Lignin from DES treatments
Lignin was separated from all DES-treated wheat, corn, and rape-
seed stem samples. Fig. 6 shows the visual appearance of Lac–Ch and
Gly–K2CO3 lignins and DRIFT spectra of wheat and corn samples in
different reaction conditions. The amounts of rapeseed lignin were too
low to be analyzed. The color of the acidic-DES lignin was black-brown,
and the lignin particles were quite small compared with the alkaline-
DES lignin, which had a lighter brown color. All lignins show a broad
band around 3400 cm-1 attributed to the hydroxyl groups in phenolic
and aliphatic structures and bands at 2930 cm-1 and 2860 cm-1 arising
from CH stretching in aromatic methoxyl groups and in methyl and
methylene groups of side chains (Kang et al., 2012; Kumar et al., 2016;
Strassberger et al., 2015; Xu et al., 2006). Bands at 1515–1500 cm-1 and
1460 cm-1 are associated with aromatic ring and CeH deformation,
respectively (Kang et al., 2012; Xu et al., 2006). The peaks at 1750 cm-1
(unconjugated carbonyl/carboxyl stretching (Kang et al., 2012; Xu
et al., 2006)), 1370 cm-1 (phenolic OH and aliphatic CeH in methyl
groups (Kang et al., 2012; Li et al., 2017b)), and 1140 cm-1 (C]O
stretching/guaiacyl lignin) for Lac5–Ch lignin are more visible than the
Gly–K2CO3 lignin peaks for both wheat and corn. In addition, the peak
at 1260–1280 cm-1, which is characteristic for the guaiacyl unit ring
(Kang et al., 2012), is broader in the Gly–K2CO3 lignin than in the Lac5.
Thus, there seem to be differences between the alkaline- and acidic-DES
lignin. However, the spectral region below 1400 cm-1 is difficult to
analyze, since most bands have contributions from various vibration
modes and are specific to the different monolignol units (Boeriu et al.,
2004).
4. Conclusions
The performance of acidic lactic acid–choline chloride and alkaline
K2CO3–glycerol DESs in delignification of wheat straw, corn stalk, and
rapeseed stem was good, and the nanofibrillation of these delignified
cellulose-rich fractions was possible without other pretreatments. The
average width of prepared individual nanofibrils was 4–6 nm with all
raw materials. Both acidic lactic acid–choline chloride and alkaline
K2CO3–glycerol nanocellulose samples had the highest viscosity
(1360–555 Pa s) and crystallinity properties (54–38 %), but the nano-
papers from alkaline DES treatment had better tensile strength prop-
erties and Young´s modulus (170–132 MPa and 8.6–5.2 GPa) compared
with nanopapers from acidic lactic acid–choline chloride DESs
(132–67 MPa and 6.9–3.9 GPa). The separated lignin fractions also
differed between acidic and alkaline DES treatments visually and ac-
cording to the FTIR results.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests.
Acknowledgements
The authors acknowledge the financial support of the ERANet-LAC
and Academy of Finland, project: Towards a novel and sustainable
9
Industrial Crops & Products xxx (xxxx) xxxx
biorefinery concept based on green technologies for main commercial
grain crop residues (BIOCODE) (No. 311974). Marcin Selent is grate-
fully acknowledged for his contribution to the XRD measurements. The
facilities of the Center of Microscopy and Nanotechnology of the
University of Oulu were utilized in this research.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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