Rheological behavior of cellulose nanofibers from cassava peel obtained by combination of chemical and physical processes

Carbohydrate Polymers 248 (2020) 116744

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Rheological behavior of cellulose nanofibers from cassava peel obtained by
combination of chemical and physical processes

T

Aline Czaikoski, Rosiane Lopes da Cunha*, Florencia Cecilia Menegalli
Department of Food Engineering, School of Food Engineering, University of Campinas, Campinas, SP CEP 13083-862, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords:
Viscosity
Rheology
Mechanical resistance
Acid hydrolysis
TEMPO-mediated oxidation
Ultra-sonication

This work aimed to produce and characterize cellulose nanofibers obtained from cassava peel with a combination of pre-treatments with acid hydrolysis or TEMPO-mediated oxidation and ultrasonic disintegration. All
nanofibers presented nanometric diameter (5−16 nm) and high negative zeta potential values (around −30
mV). Oscillatory rheology showed a gel-like behavior of the aqueous suspensions of nanofibers (1.0–1.8 % w/w),
indicating their use as reinforcement for nanocomposite or as a thickening agent. Additionally aqueous suspensions of nanofibers obtained by acid hydrolysis presented higher gel strength than those produced by

TEMPO-mediated oxidation. However, ultrasound application increased even more viscoelastic properties. Flow
curves showed that suspensions of nanofibers obtained by acid hydrolysis presented a thixotropy behavior and
viscosity profile with three regions. Therefore our results showed that it is possible to tune mechanical properties
of cellulose nanofibers choosing and modifying chemical and physical process conditions in order to allow a
number of applications.

1. Introduction
Cellulose is the most abundant biopolymer, present in plant fibers,
marine plants, algae, fungi, invertebrates and bacteria (Lavoine,
Desloges, Dufresne, & Bras, 2012; Lima & Borsali, 2004). When cellulose has at least one dimension between 1−100 nm, it is called nanocellulose. The main forms of nanocellulose are nanofibers and nanocrystals, which can be obtained by different chemical, enzymatic and
physical processes. These processes can be used separately or combined
(Kargarzadeh et al., 2018), resulting in particles with varying characteristics.
The production of cellulose nanofibers (CNFs) started around the
1980s from wood fibers using high-pressure homogenization (Turbak,
Snyder, & Sandberg, 1983). Cellulose nanofibers exhibit interesting
properties such as low thermal expansion, high aspect ratio, strengthening effect, good mechanical and optical properties. Due to these
specific characteristics, the cellulose nanofibers have been used in
composites, food packaging, coating additives, aerogels, membranes, as
gas barrier material, fillers, flocculants, Pickering emulsifier, food
thickeners and reinforcement material (Abdul Khalil, Bhat, & Ireana
Yusra, 2012; Abdul Khalil et al., 2014; Choi et al., 2020; Dizge,
Shaulsky, & Karanikola, 2019; Fan et al., 2019; Gao et al., 2018; Kadam
et al., 2019; Liu, Kerry, & Kerry, 2007; Perzon, Jørgensen, & Ulvskov,

⁎

2020; Seo et al., 2020; Tibolla, Czaikoski, Pelissari, Menegalli, & Cunha,
2020; Yousefi, Azad, Mashkour, & Khazaeian, 2018). However, the
technological properties of cellulose nanofibers depend on the raw
material and the treatment used to isolate the fibrils. Many food wastes

have been used for the production of cellulose nanofibers as: corncobs
(Shogren, Peterson, Evans, & Kenar, 2011), carrot juice debris
(Siqueira, Oksman, Tadokoro, & Mathew, 2016), corn stover (Xu,
Krietemeyer, Boddu, Liu, & Liu, 2018), wheat straw (Alemdar & Sain,
2008), soy hulls (Flauzino Neto et al., 2013), sugarcane bagasse (Liu
et al., 2007) and sugar beet pulp (Perzon et al., 2020). However, there
are still several food wastes with the potential to be used in the production of nanofibers. Cassava (Manihot esculenta) is a root crop widely
cultivated in several countries that generates a large amount of waste,
such as peels and residual bagasse during the production of cassava
starch or other food products. Therefore, these residues could be processed to reduce environmental problems and the generation of products with greater added value. In a previous work, cellulose nanofibers
from cassava peel were extracted by acid hydrolysis and characterized
in relation to diameter, aspect ratio, crystallinity among others properties (Leite, Zanon, & Menegalli, 2017), but the rheological behavior of
the dispersions of these nanofibers from the cassava peel has not yet
been evaluated. In addition, cassava peel cellulose nanofibers isolated
with tempo-mediated oxidation have not been observed in the

Corresponding author.
E-mail address: (R.L. da Cunha).

/>Received 5 May 2020; Received in revised form 3 July 2020; Accepted 8 July 2020
Available online 13 July 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 248 (2020) 116744

A. Czaikoski, et al.

with sonication and "wos" to nanofiber without sonication.

literature.
Several authors have studied the rheological behavior of dispersions
of cellulose nanofibers (CNFs) obtained from different chemical and
physical processes, such as high-intensity ultrasonication (Chen et al.,
2013), high pressure homogenization (Shogren et al., 2011) and acid
hydrolysis (Liu et al., 2007; Zhai, Lin, Li, & Yang, 2020). However, the
combination of chemical and physical processes has been more effective in producing nanofibers with enhanced properties. Examples of
these processes are enzymatic hydrolysis/mechanical brillation
(Albornoz-Palma, Betancourt, Mendonỗa, Chinga-Carrasco, & Pereira,
2020), TEMPO-mediated oxidation/ultrasonication or mechanical fibrillation (Benhamou, Dufresne, Magnin, Mortha, & Kaddami, 2014;
Ehman et al., 2020; Souza, Mariano, De Farias, & Bernardes, 2019) and
alkali treatment/high pressure homogenization (Xu et al., 2018). The
rheological properties showed a strong dependence on the type and
process conditions used to obtain the nanofibers, the type of raw material and the concentration of nanofibers in the suspension.
Aqueous suspensions of cellulose or CNF nanofibers generally exhibit gel-like behavior (G’ > G’’), even at low concentrations as 0.125 %
w/w (Bettaieb et al., 2015; Pääkkö et al., 2007). In addition, flow
curves show that CNF suspensions usually present shear thinning and
thixotropic behavior (Bettaieb et al., 2015; Iotti, Gregersen, Moe, &
Lenes, 2011; Naderi, Lindström, & Sundström, 2014). In some cases, the
flow curves exhibit unusual behavior since two shear-thinning regions
with an intermediate viscosity plateau have been observed and this
effect has not yet been even elucidated. Some authors suggest that this
phenomenon occurs due to structural changes of the CNF suspension
during shear flow (Karppinen et al., 2012; Qiao, Chen, Zhang, & Yao,
2016). However some factors such as concentration, ionic strength, pH,
temperature and process conditions can modify the rheological properties of nanofibers suspensions (Chen et al., 2013; Jia et al., 2014;
Naderi et al., 2014).
Therefore, the aim of this work was to analyze the influence of
different combination of physical and chemical processes as hydrolysis
by sulfuric acid, TEMPO-mediated oxidation and high-intensity ultrasound on the properties of cellulose nanofibers from cassava peel.

Nanofibers were characterized by atomic force microscopy (AFM),
transmission electron microscopy (TEM), functional groups from FTIR,
crystallinity index and zeta potential. The rheological behavior of the
CNF suspensions was studied using oscillatory rheology and flow
curves.

2.3.1. Pre-treatment
Cellulose nanofibers (CNFs) were isolated using the chemical
treatment described by Leite et al. (2017). First, the peel samples were
subjected to alkaline treatment with KOH solution (5 % w/v) in the
proportion of 1:18 (peel samples: KOH solution) at 25 °C and the suspensions were mechanically stirred for 14 h. Then, the wetted samples
were separated by centrifugation (15,345×g /15 °C/15 min). The insoluble material was added in distilled water and centrifuged. This
procedure was carried out until the supernatant color no longer
changed. The remaining insoluble residue was added in distilled water
and the pH adjusted to 5.0 using acetic acid (10 % v/v). Then, the Qchelating treatment with EDTA was performed at 70 °C for 1 h. After
that, a bleaching treatment was conducted at 90 °C for 3 h using hydrogen peroxide (H2O2) (4 % v/v) and three other reagents: NaOH (2 %
v/v), diethylenetriaminepentaacetic acid (DTPA, 0.2 % w/v) and
MgSO4 (3 % w/v). Subsequently this suspension was subjected to a
second alkaline treatment with KOH solution (5 % w/v) at a ratio of
1:5. Between the stages of delignification, the materials were washed
successively with deionized water and centrifuged (15,345×g /15 °C/
15 min). The insoluble material resulting from the last centrifugation
step was added in distilled water, subjected to mechanical agitation and
the pH neutralized with acidic solution (1 % v/v H2SO4). This insoluble
material was subjected to acid hydrolysis (Section 2.3.2) or TEMPOmediated oxidation (Section 2.3.3) before to be submitted to the physical treatment (Section 2.3.4).
2.3.2. Acid hydrolysis
The insoluble material was added in a sulfuric acid solution (30 %
v/v) for 90 min at 60 °C. Subsequently, this mixture was cooled to 40 °C
and subjected to four washes with distilled water. Thereafter, the suspension was diluted with distilled water and neutralized (pH 7.0) with
KOH (5 % w/v). After neutralization, the suspension was centrifuged.

The insoluble material was separated and washed to remove any salts
resulting from the neutralization procedure (Leite et al., 2017).
2.3.3. TEMPO-mediated oxidation
The oxidation process was carried out using the method of Saito,
Kimura, Nishiyama, and Isogai (2007) with some adaptations. The insoluble material (17.07 g corresponding to 1.0 g cellulose, cellulose
content determined according to Sun, Sun, Zhao, and Sun (2004) was
suspended in 100 ml of distilled water with 0.016 g (0.1 mmol) of
TEMPO catalyst (2,2,6,6-tetramethylpiperidin-1-oxyl) and 0.1 g (1
mmol) of sodium bromide (NaBr). Oxidation started with the addition
of 12 % NaClO solution (3.0 mmol/g substrat) to the suspension at
room temperature with stirring at 500 rpm, keeping pH 10 by addition
of 0.5 M NaOH for 25 minutes. Thereafter, the suspension was diluted
with distilled water and neutralized (pH 7.0) with HCl (0.1 M). Subsequently, the insoluble material was washed with distilled water.

2. Material and methods
2.1. Material
Peelings (inner peel and bark) of cassava roots were obtained from
the southeastern region of Campinas - Brazil. All chemicals used were of
analytical grade.
2.2. Raw material preparation

2.3.4. Physical treatment
Half of the suspensions obtained by acid hydrolysis and TEMPOmediated oxidation were subjected to ultrasound treatment. An
Ultrasonic Disruptor/Sonicator (QR 750 W, Ultronique, Brazil) was
used for approximately 20 minutes with a power of 300 W.

First, peelings were properly classified and washed under running
water. Then, they were sanitized with sodium hypochlorite solution
(250 ppm) for 10 min and dried in a forced convection oven at 50 °C for
48 h. Subsequently, the peels were cut and ground in a professional

high-performance blender LT-2.0 Super Skymsen from Metallurgical
Siemsen Ltda. (Santa Catarina, Brazil). The resulting material was
sieved through a 0.15 mm (100-mesh) sieve opening.

2.4. Cellulose nanofibers (CNFs) characterization
Morphology of cellulose nanofibers were evaluated by transmission
electron microscopy (TEM). TEM images were captured with a TEMMSC (JEOL 2100 – Tokyo, Japan) equipped with a LaB6 electron gun,
using an accelerating voltage of 200 kV. In order to determine the
average size of CNFs, 20 measurements of diameter and 40 measurements of length were made in AFM images. AFM images were acquired
on a Microscope Park Systems, model NX-10 (Suwon, Korea) equipped

2.3. Nanofibers isolation
Cellulose nanofibers were named according to the treatment that
they were submitted, which will be described in the next sections. The
nomenclature is: "CNFs" referring to cellulose nanofibers, "TO" to
TEMPO-mediated oxidation, "HA" to acid hydrolysis, "ws" to nanofiber
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A. Czaikoski, et al.

to those found by Tibolla, Pelissari, Rodrigues, and Menegalli (2017)
for banana peel cellulose nanofibers isolated by enzymatic treatment
with xylanase (1490−1940 nm). However, our results were superior to
those found by Leite et al. (2017), which showed 162–400 nm and
243−296 nm, for cellulose nanofibers from cassava peel and bagasse
obtained by acid hydrolysis, respectively, with an additional centrifugation step.
All treatments generated fibers with a nanometric diameter, but

acid hydrolysis produced cellulose nanofibers with a smaller diameter
than the TEMPO-mediated oxidation. The diameter of the cellulose
nanofibers decreased after being subjected to a sonication process, but
this reduction was more significant for those obtained by the TEMPOmediated oxidation. This effect was also observed by Khawas and Deka
(2016) isolating cellulose nanofibers from banana peel by acid hydrolysis and sonication. Sonication process also produced fibers with
length and diameter more homogeneous than the nanofibers without
sonication, which can be observed from the smaller standard deviation.
This fact may be related to a greater nanofibers fibrillation caused by
ultrasound. In this process waves are produced that cause the cavitation
phenomenon, due to the absorption of ultrasonic energy by the molecules that cause the formation and expansion of microscopic gas bubbles. With the collapse of these bubbles there is local production of heat
and high pressure. These effects facilitate the isolation of nanomaterials, as they break the structural micron-sized fibril into submicron fibrils and then at the nanoscale, producing nanofibers of a more
homogeneous size (Abdul Khalil et al., 2014; Huerta, Silva, Ekaette, ElBialy, & Saldaña, 2020; Wang et al., 2012). Moreover, the aspect ratio
ranged from 242 to 371 for the different nanofibers, ensuring their use
as reinforcement for composites, since the aspect ratio was greater than
100 (Ma, Zeng, Realff, Kumar, & Schiraldi, 2003).
However, the zeta potential of CNFs was not altered after the sonication process, although the method of producing CNFs had an influence on this parameter. The nanofibers obtained by acid hydrolysis
showed a higher negative charge (∼ −49 mV) than those obtained by
catalytic oxidation (∼ −42 mV). A greater negative zeta potential
presented by nanofibers obtained from acid hydrolysis is associated to
the more efficient introduction of sulfate groups on the surfaces of fibers, in comparison to catalytic oxidation that introduces carboxylic
groups. Despite these differences, all nanofibers suspensions presented
electrostatic stability, since the zeta potential was greater than −30 mV
(Everett, 1988).
The nanofibers obtained by acid hydrolysis also showed a higher
crystallinity index (CNFs-HAws = 53.42 % and CNFs-HAwos = 53.47
%) than the nanofibers obtained by catalytic oxidation (CNFs-TOws =
46.67 % and CNFs-TOwos = 46.82 %) (XRD patterns of samples at
Fig. 1.a – Supplementary material). The crystallinity of the material
increased about 8 % after acid hydrolysis, while after catalytic oxidation only 2 % compared to the pretreated material (crystallinity index
of 45 %). A minor increase in crystallinity index after TEMPO-mediated

oxidation can be associated with chemical treatment that only transforms the surface hydroxyls into carboxylate groups, without interfering with the internal conformation of cellulose crystals (Isogai, Saito,
& Fukuzumi, 2011). Acid hydrolysis, on the other hand, acts on the
amorphous fibrils components, lignin and hemicellulose, facilitating
their extraction and, consequently, concentrating the crystalline portions of the material (Alemdar & Sain, 2008). Our results of crystallinity
index were similar to those found by Khawas and Deka (2016), which
observed values between 30.5–63.64 % for cellulose nanofibers extracted from banana peels. The estimate of the amount of cellulose I in
relation to cellulose II for the nanofibers ranged from 1.26 to 1.73
(Table 1). As the values are greater than one, the nanofibers have more
cellulose I than cellulose II. Cellulose I has the best mechanical properties and, therefore, the cellulose nanofibers produced are suitable for
use as a reinforcement material (Mandal & Chakrabarty, 2011).
To assess the chemical structure of CNFs, FTIR spectroscopy analyses were obtained (Fig. 2). The FTIR spectra obtained for the cellulose

with Si Nano sensor probes manufactured with a constant spring of 42
N.m−1. The resonance frequency was about 320 kHz and the acquired
images were treated with the software GWYDDION version 2.4 to obtain the mean diameter and length of the nanofibers.
The zeta potential was determined using the Zetasizer model Nano
ZS from Malvern Instruments Ltd. (United Kingdom, U.K) at a detection
angle of 173°. Nine measurements of zeta potential were performed for
each sample at room temperature (25 °C). The crystallinity index was
determined from X-ray diffraction (XRD) patterns registered on a
D5005 diffractometer equipped with a graphite monochromator and a
CuKα source (λ =0.154 nm) at 40 kV and 30 mA. The crystallinity
index (IC) was calculated from the ratio of intensity of the crystalline
peak to the intensity of diffraction of the non-crystalline material
(Segal, Creely, Martin, & Conrad, 1959). An estimate of the ratio of type
I cellulose to type II cellulose was also obtained, according to Mandal
and Chakrabarty (2011). This ratio was calculated using the peak intensity at 21.7° over the peak intensity at 20°. Functional groups were
analyzed by Fourier transform infrared (FTIR) spectroscopy accomplished on the Fourier transform infrared spectrometer (JASCO FTIR6100, Japan) in the infrared region from 4000 to 600 cm−1 (Vicentini,
Dupuy, Leitzelman, Cereda, & Sobral, 2005).
2.5. Rheological characterization

Nanofiber suspensions (1.0 %, 1.4 % and 1.8 % w/w) were prepared
in distilled water and their rheological behavior was studied using a
stress-controlled rheometer MCR 301 (Anton Paar, Austria) equipped
with cone and plate geometry (6 cm diameter, cone truncation of 0.208
mm and 2°). After being placed on the rheometer plate, the CNFs suspensions were allowed to rest for 3 min in order to minimize the shear
history imposed by loading. All the measurements were carried out at
25 °C. Flow curves were obtained by up-down-up steps program with
shear rate ranging from 0 to 300 s−1. Apparent viscosity values were
evaluated at 100 s−1 since this shear rate is associated to chewing
(Whitcomb, Gutowski, & Howland, 1980) and other process conditions,
such as agitation and flow in pipes. In addition thixotropy degree was
estimated from the area between the up and down curves, in order to
compare how much the material microstructure was changed with the
shear stress (Barnes, 1997).
Viscoelastic properties were evaluated from oscillatory rheology.
First, strain sweeps of 0.1–10 % at constant angular frequency of 1 Hz
were performed to define the linear viscoelastic range. After that, frequency sweeps were done in the range of 0.01–10 Hz with a strain
within the linear viscoelastic range.
2.6. Statistical analysis
Results were evaluated by analysis of variance (ANOVA) and the
Tukey test. The significance level was 5 %.
3. Results and discussion
3.1. Characterization of cellulose nanofibers
3.1.1. Length, diameter, zeta potential, crystallinity index and functional
groups
Length distribution of the cellulose nanofibers is shown in Fig. 1 and
Table 1 summarizes the properties of the cellulose nanofibers obtained
from cassava peels after acid hydrolysis and TEMPO-mediated oxidation with and without sonication. Nanofibers obtained by TEMPOmediated oxidation without sonication showed the widest distribution
range. These nanofibers (CNFs-TOwos) also had the biggest length and
diameter (Table 1), with the highest number of nanofibers obtained in

the length range of 2500−3500 nm. In contrast, the other samples of
cellulose nanofibers showed the highest percentage of length distribution in the range of 1500−2500 nm. These lengths were slightly higher
3


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A. Czaikoski, et al.

Fig. 1. Length distribution of CNFs produced from different chemical and physical treatments.

nanofibers, cassava peel and pre-treated material exhibited a wide band
in the region of 3500 cm−1 at 3200 cm−1 corresponding to the free
vibration of the OHe stretches of the OH groups of the cellulose molecules. In addition, the spectra showed the CeH stretch characteristic
of hemicellulose and cellulose around 2895 cm−1 (Khalil, Ismail,
Rozman, & Ahmad, 2001). All nanofibers showed a peak located at
1030 cm−1 that is associated with COe elongation, characteristic of the
presence of cellulose.
The FTIR absorption peak at 1430 cm−1 corresponds to the vibration of the CH2 bonds, attributed to the cellulose "crystallinity band".
The band at 890 cm−1 is attributed to the C-O-C stretching vibration of
β-cell (1 → 4) glycosidic bonds, which is considered to be an "amorphous band" (Shankar & Rhim, 2016). All the nanofibers presented
peaks in these bands, demonstrating the presence of amorphous and
crystalline celluloses. The peaks 2464 cm−1, 1509 cm−1 and 1601
cm−1 are characteristic of the existence of aromatic rings and CHe
bonds, indicating the presence of lignin (Liu, Wang, Zheng, Luo, & Cen,
2008). The two peaks close to the nanofibers obtained in this study, at
1614 cm−1 and 1409 cm−1, showed that the processes used were not
enough to remove all existing fractions of lignin. However, these peaks
were less evident that in cassava peel and pre-treated material.

Fig. 2. FTIR spectra of the cassava peel, the pre-treated material and cellulose
nanofibers obtained from acid hydrolysis without (CNFs-HAwos) and with
(CNFs-HAws) sonication, TEMPO-mediated oxidation without (CNFs-TOwos)
and with (CNFs-TOws) sonication.

pictures revealed fibers with a wide size distribution both in diameter
and in length. Sonicated nanofibers were more separated than nanofibers without this process showing some fibers with intact bundles. In
addition, acid hydrolysis produced more dispersed (separated) nanofibers than the samples obtained by TEMPO-mediated oxidation.
Furthermore, it is possible to observe the presence of cellulose nanospheres after the ultrasound process. As cellulose hydrolysis generally begins in the superficial amorphous region and subsequently

3.2. Microscopy observations
Fig. 3 shows the morphological characteristics of CNFs. TEM

Table 1
Characteristics of the cellulose nanofibers obtained from acid hydrolysis without (CNFs-HAwos) and with (CNFs-HAws) sonication; TEMPO-mediated oxidation
without (CNFs-TOwos) and with (CNFs-TOws) sonication.
Sample

Mean Length (nm)

Mean Diameter (nm)

Aspect ratio (L/D)

Zeta potential (mV)

Cellulose ratio (I/II)*

CNFs-HAwos
CNFs-HAws

CNFs-TOwos
CNFs-TOws

2527 ± 991bc
1857 ± 835c
3867 ± 1597a
2750 ± 1499b

8 ± 6.2b
5 ± 2.1b
16 ± 14.0a
8 ± 4.3b

316
371
242
344

−49.65 ± 1.6a
−49.33 ± 4.3a
−41.81 ± 4.2b
−42.22 ± 1.9b

1,73
1,39
1,26
1,33

a,b,c,

Different superscripts letters in the same column indicate a statistically significant difference (p < 0.05).
* Cellulose I in relation cellulose II.
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A. Czaikoski, et al.

Fig. 3. Transmission electron microscopy (TEM) images for CNFs (scale bar =100 nm).

penetrates the internal amorphous region, the pretreatment together
with acid hydrolysis or TEMPO-mediated oxidation must have caused
the superficial hydrolysis that favored the ultrasonic treatment to penetrate the amorphous inner region, facilitating the formation of
smaller cellulose fragments, such as cellulose nanospheres (Neng,
Enyong, & Rongshi, 2008).

3.3. Rheological properties
3.3.1. Oscillatory rheology
The viscoelastic properties of CNF suspensions prepared with different methods are illustrated in Fig. 4. All CNF suspensions exhibited
gel-like properties with G’ > G’’, but nanofibers treated by acid hydrolysis showed G' greater than those obtained by TEMPO-mediated
oxidation. In addition, cellulose nanofibers obtained by acid hydrolysis
presented moduli with less frequency dependence than those obtained
by catalytic oxidation. These results can be, at least partly, related to
the greater negative charge observed for CNFs obtained by acid hydrolysis. A higher negative charge favors repulsive forces between the
nanofibers, causing a better dispersion to entrap water molecules in the

Fig. 4. Storage (G’) and loss (G’’) moduli of suspensions with 1.4 % (w/w) CNFs
as a function of frequency for: Cellulose nanofibers obtained from acid hydrolysis without ( ) and with ( ) sonication; TEMPO-mediated oxidation without (
) and with (♦) sonication. Filled symbols correspond to G’ and open symbols

correspond to G’’.

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A. Czaikoski, et al.

Fig. 5. Shear stress as a function of shear rate
for suspensions with 1.4 % (w/w) CNFs: (○)
First sweep (up); ( ) second sweep (down) and
(x) third sweep (up).

from the difference between the first and second/third flow curves. The
sonication process increased the degree of thixotropy of the nanofibers,
which could be associated with greater separation of the nanofibers, as
can been seen on the TEM images (Fig. 3). This separation increases the
aspect ratio of these fibers and consequently increases the interaction
between the nanofibers and the degree of thixotropy (Barnes, 1997).
Although the viscosity of CNFs suspensions obtained by acid hydrolysis decreased to shear rate up to 20 s−1, the viscosity became almost constant, forming a Newtonian plateau, between 20 s−1 and 40
s−1 (Fig. 6) and the viscosity decreased again (shear-thinning behaviour) above 40 s−1. However, the nanofibers obtained by catalytic
oxidation presented only shear-thinning behavior within the shear rate
range. A similar behavior of suspensions of CNFs obtained by acid hydrolysis was reported in other studies. Iotti et al. (2011) found a similar
viscosity behavior for microfibrillated cellulose, which was attributed
to production of another structure by shear leading to the formation of
the Newtonian plateau. However Karppinen et al. (2012), working with
suspensions of microfibrillated cellulose and using images captured in a
transparent outer cylinder in concentric cylinders, verified a shear-induced phase separation in the range of shear rate of the Newtonian
plateau. Bettaieb et al. (2015) attributed this behavior to the slippage of

the suspension over the sensor, even using used rough surfaces on the
measuring sensor.
Slippage effects on the wall of rheometer sensors occurs because the
dispersed phase separates from the dispersant of the suspension, leaving
a liquid layer formation that shows low viscosity near the sensor and
cause a lubrication or slipping effect. The characteristics that usually
lead to slipping effects in the flow are: existence of large particles with
high aspect ratio; use of smooth walls on measuring sensors and low
speeds/flow rates; walls and particles carrying electrostatic charges and
electrically conductive continuous phase (Barnes, 1995). The formation
of the Newtonian plateau in the suspensions of nanofibers produced by
acid hydrolysis may have occurred because they present several characteristics that can lead to the occurrence of the slippage. Nanofibers
isolated by TEMPO-mediated oxidation may not have this effect due to
the lower zeta potential or lower quantity of electrostatic charges and

vicinity of the fibers and increase the elastic character of the suspensions (Benhamou et al., 2014; Li et al., 2015). Another factor that may
have influenced this behavior more significantly is the difference in
aspect ratio of the different nanofibers. Materials that show a high aspect ratio tend to flocculate or form interlacings/entanglements among
them. These entanglements between the fibers cause a certain restriction of movement with the flow and, therefore, induce a solid type
behavior (Sato & Cunha, 2012). The sonication process also contributed
to the increase in storage moduli for CNFs suspensions. Mishra, Manent,
Chabot, and Daneault (2011) report that the sonication treatment
causes a greater separation of the nanofibers, which is corroborated in
our results (Fig. 3). This more pronounced fibrillation caused by the
ultrasound treatment increased the surface area of the fibers, facilitating the physical interactions between nanofibers, promoting entanglements between them and increasing their gel strength.

3.3.2. Flow properties
Figs. 5 and 6 show the flow curves and viscosity behavior, respectively, of the different nanofibers suspensions obtained by acid hydrolysis and by TEMPO-mediated oxidation, with and without sonication.
These nanofibers suspensions presented a hysteresis loop, indicating a
thixotropic behavior. The degree of thixotropy of these suspensions was

calculated from the area between the first and the third curve (steady
state) of shear stress-shear rate (Fig. 5) and the results are shown in
Fig. 7. Nanofibers obtained by catalytic oxidation presented a smaller
hysteresis area (CNFs-TOws =14.56 Pa.s−1; CNFs-TOwos =4.39
Pa.s−1) than the nanofibers treated by acid hydrolysis (CNFs-HAws
=143.95 Pa.s−1; CNFs-HAwos =72.28 Pa.s−1), corroborating a
weaker and less complex network as observed in viscoelastic properties.
Thixotropic behavior is common in dispersions that exhibit flocculated,
entangled or aligned fibers. Due to the presence of the interweaving of
the fibers, they present greater restriction to the alignment with the
flow and, consequently, show higher viscosity (Fig. 6). As shear increases, these entanglements are broken. This structural change causes
the fibers to separate, facilitating their alignment with the flow, which
causes a decrease in viscosity (Fig. 6). This behavior can be observed
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A. Czaikoski, et al.

Fig. 6. Viscosity of suspensions with 1.4 % (w/w) CNFs as a function of shear rate: (○) first sweep (up); ( ) second sweep (down) and (x) third sweep (up).

entanglement between nanofibers, as higher concentrations of nanofibers increase the interaction between them and form stronger network
structures (Iotti et al., 2011). As observed in Figs. 5 and 6, higher
pseudoplasticity was observed in nanofibers treated with acid

also smaller aspect ratio.
Fig. 7 shows the dependence of the storage moduli, viscosity and
degree of thixotropy with the increase in the concentration of nanofibers. The increase in rheological properties was also related to the

Fig. 7. Degree of thixotropy, apparent viscosity at 100 s−1 (ƞ) and storage moduli (G’) at
1 Hz of the suspensions of cellulose nanofibers
obtained from acid hydrolysis without (CNFsHAwos) and with (CNFs-HAws) sonication;
TEMPO-mediated oxidation without (CNFsTOwos) and with (CNFs-TOws) sonication at
different concentrations (n = 3).

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A. Czaikoski, et al.

hydrolysis and after ultrasound treatment, demonstrating again that
these systems showed more complex rheological behavior and, consequently, network structure.

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4. Conclusion
Our results demonstrated that the agricultural residue of cassava
peel has great potential for the production of cellulose nanofibers. The
chemical methods employed, such as alkaline pre-treatment, acid hydrolysis and TEMPO-mediated oxidation, were efficient for the isolation
of cellulose nanofibers with diameters between 5 and 16 nm. Acid
treatment was more efficient to introduce negative charge on surface
and to produce nanofibers with greater aspect ratios. These characteristics facilitated the formation of entanglements between the nanofibers, increasing the complexity of the structural behavior and, consequently, presenting suspensions with higher gel strength than the
nanofibers obtained by TEMPO-mediated oxidation. The sonication
process on the nanofibers also interfered in the gel strength of their
suspensions, since this process also induced an increase of aspect ratio.
Nanofibers suspensions that presented higher gel strength also showed
higher degree of thixotropy, pseudoplasticity and viscosity, showing
that the rheological behavior was essential to identify the better method
to produce cellulose nanofibers with potential features as a strengthening of polymeric matrix.
CRediT authorship contribution statement
Aline Czaikoski: Conceptualization, Formal analysis, Investigation,

Methodology, Validation, Writing - original draft. Rosiane Lopes da
Cunha: Conceptualization, Writing - original draft. Florencia Cecilia
Menegalli: Conceptualization, Funding acquisition, Writing - original
draft.
Acknowledgements
This study was financed in part by the Coordenaỗóo de
Aperfeiỗoamento de Pessoal de Nớvel Superior Brasil (CAPES) - (2952/
2011) and the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) (131520/2015-6 and 307168/2016-6). The authors would also like to acknowledge the Brazilian Nanotechnology
National Laboratory (LNNano) for allocation of the equipments.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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