Carbohydrate Polymers 143 (2016) 327–335

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

On the polymorphic and morphological changes of cellulose

nanocrystals (CNC-I) upon mercerization and conversion to CNC-II
Ersuo Jin a , Jiaqi Guo b , Fang Yang a , Yangyang Zhu a , Junlong Song a,b,∗ , Yongcan Jin a ,
Orlando J. Rojas b,∗∗
a
Jiangsu Provincial Key Lab. of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, Jiangsu, Peoples Republic of China
b
Bio-based Colloids and Materials, School of Chemical Technology, Aalto University, PO Box 16300, Aalto FIN-00076 Espoo, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Polymorphic and morphological transformations of cellulosic materials are strongly associated to their
Received 8 November 2015 properties and applications, especially in the case of emerging nanocelluloses. Related changes that take
Received in revised form 9 January 2016 place upon treatment of cellulose nanocrystals (CNC) in alkaline conditions are studied here by XRD, TEM,
Accepted 22 January 2016
AFM, and other techniques. The results indicate polymorphic transformation of CNC proceeds gradually
Available online 25 January 2016
in a certain range of alkali concentrations, i.e. from about 8% to 12.5% NaOH. In such transition alkali
concentration, cellulose I and II allomorphs coexists. Such value and range of the transition concentration
Keywords:
is strongly interdependent with the crystallite size of CNCs. In addition, it is distinctively lower than that
Cellulose nanocrystals
CNC
for macroscopic fibers (12–15% NaOH). Transmission electron microscopy and particle sizing reveals that
Mercerization after mercerization CNCs tend to associate. Furthermore, TEMPO-oxidized mercerized CNC reveals the
Polymorphs morphology of individual nanocrystal of the cellulose II type, which is composed of some interconnected
Cellulose I and II granular structures. Overall, this work reveals how the polymorphism and morphology of individual CNC
Crystallinity change in alkali conditions and sheds light onto the polymorphic transition from cellulose I to II.
Crystallite size © 2016 Elsevier Ltd. All rights reserved.

1. Introduction (cellulose I−IV), the structure of which depends on the cellulose

Cellulose, composed of ␤-1,4 linked glucopyranose units, is a
major resource for materials, (bio)chemicals and biofuels (Mosier
et al., 2005; O’Sullivan, 1997; Wyman, Dale, Elander, Holtzapple,
Ladisch, & Lee, 2005). In most cases, it is associated by hydro-
gen bonding and other weak forces to form a semi-crystalline
structure where highly ordered regions (the crystallites) are com-
bined with less ordered (disorder or para-crystalline) domains, also
known as the amorphous phase. The molecular orientation and the
hydrogen-bond network that pack the cellulose chains within the
crystallites can vary widely, giving rise to different polymorphs
Abbreviations: CNC, cellulose nanocrystal; CNC-I, cellulose nanocrystal, allo-
morph of cellulose I; CNC-II, cellulose nanocrystal, allomorph of cellulose II;
CNC-concentration%, cellulose nanocrystals treated under alkaline conditions at
given concentration (%) of NaOH solution.
∗ Corresponding author at: Nanjing Forestry University, Jiangsu Provincial
Key Lab. of Pulp and Paper Science and Technology, Nanjing 210037, China.
Tel.: +86 25 85428163; fax: +86 25 85428689 (J.S.).
∗∗ Corresponding author at: Aalto University (O.J.R.). Tel.: +358 50 5124227;
fax: +358 50 5124227.
E-mail addresses: junlong.song@njfu.edu.cn (J. Song), orlando.rojas@aalto.fi
(O.J. Rojas).
source, method of extraction or treatment. Among them, cellulose
polymorphs I and II are the most widely discussed. In nature, cel-
lulose I is produced into crystalline forms with the Iˇ allomorph
being the most common in plants, while I˛ in algae and bacteria
(O’Sullivan, 1997). Cellulose II is usually obtained from cellulose I
by mercerization (alkali treatment) or regeneration (solubilization
and recrystallization); there are a few reports suggesting the pro-
duction of cellulose II from Gluconacetobacter xylinus (Kuga, Takagi,
& Brown, 1993). Cellulose Iˇ has unit cell dimensions of a = 7.78 Å,
b = 8.20 Å, c = 10.38 Å, ˛ = ˇ = 90◦ , and  = 96.5◦ (Nishiyama, Langan,
& Chanzy, 2002). Cellulose II produced by the mercerization has
unit cell dimensions of a = 8.10 Å, b = 9.08 Å, c = 10.36 Å, ˛ = ˇ = 90◦ ,
and  = 117.3◦ (Gessler et al., 1995; Kobayashi, Kimura, Togawa,
& Wada, 2011; Kolpak & Blackwell, 1976; Langan, Nishiyama, &
Chanzy, 1999, 2001; Raymond, Kvick, & Chanzy, 1995; Sarko &
Muggli, 1974; Stipanovic & Sarko, 1976). There is a general con-
sensus for the parallel chain packing for cellulose I, while the chain
directionality of cellulose II is antiparallel (Lee et al., 2013). A pos-
sible route how antiparallel chains of cellulose II produced from
cellulose I which has parallel chains was proposed by Yamane,
Miyamoto, Hayakawa, and Ueda (2013) Cellulose I and cellulose
II possess different properties and advantages over the other. Com-
pared to cellulose II, cellulose I exhibits much better mechanical

http://dx.doi.org/10.1016/j.carbpol.2016.01.048
0144-8617/© 2016 Elsevier Ltd. All rights reserved.
328 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
properties, while the first offer benefits in terms of functionality
(Diddens, Murphy, Krisch, & Muller, 2008; Wang, Li, Yano & Abe,
2014).
Cellulose II, as indicated before, can be produced by alkali treat-
ment of cellulose I or by regeneration from cellulose solution;
however, cellulose II can be obtained from native cellulose through
other approaches as well. For example, Ago, Endo, and Hirotsu
(2004) demonstrated that a mechano-chemical treatment of native
cellulose with a given amount of water (∼30 wt% as present in the
cellulose solid state) via ball milling for several hours, caused the
crystalline transformation to cellulose II polymorph, even though
the crystallinity index of cellulose II was quite low, ranging from 30
to 37%. Sèbe, Ham-Pichavant, Ibarboure, Koffi, and Tingaut (2012)
used a combination of extended treatment time (>50 min) and high
final acid concentration (>64% H2 SO4 ) to obtain cellulose nanocrys-
tals with some cellulose II. They also found that pure cellulose II
nanowhiskers are obtained only in a narrow range of conditions
(66% H2 SO4 /60 min addition). Martins et al. (2015) also produced
cellulose II crystals through sulfuric hydrolysis (under a concentra-
tion of acid not reported). In principle, sulfuric acid is a swelling
agent for cellulose and therefore recrystallization of cellulose may
have occurred upon subsequent removal of the swelling agent or
during the hydrolysis process. (Sèbe et al., 2012).
Cellulose nanocrystals (CNC) display a high specific strength,
modulus and aspect ratio which are main reasons for their effect in
improving the mechanical properties of synthetic polymers at low
loading levels (Habibi, Lucia, & Rojas, 2010). Benefits derived from
the utilization of CNCs are being reported in fields of material sci-
ence, electronics, catalysis and biomedicine (Gatenholm & Klemm,
2010). In most cases, CNCs are obtained by sulfuric acid hydrolysis
of native cellulose, which results in purified CNCs. The properties of
the nanocrystals (e.g., shape, length, and diameter) depend on the
source of the cellulose and the degradation process (e.g., time and
temperature for acid hydrolysis, high-pressure homogenization
conditions, etc.). Thus, the properties of CNC-based materials are
closely related to CNC pretreatment method and resultant nanopar-
ticle morphology (Liu, Song, Anderson, Chang, & Hua, 2012; Lu &
Hsieh, 2010).
Despite the fact that Mercer introduced the “mercerization”
transformation in 1850, the unraveling of its mechanism remains
a question of intense debate. As for the macroscopic state of cel-
lulose, i.e. cellulosic fibers, the general agreement indicates that
at room temperature the polymorphic transformation starts at an
alkali concentration of 12.5% and is completed at a concentration
of 17.5% (Halonen, Larsson, & Iversen, 2013; Purz & Fink, 2003;
Revol, Dietrich, & Goring, 2011). However, if the temperature or
other conditions are changed, the polymorphic transformation may
occur at different alkali concentrations. For instance, the transi-
tion occurs at 14–15% NaOH concentration for the mercerization
of jute fibers at 85 ◦ C and 4 h (Yu et al., 2014). Polymorphic trans-
formation from cellulose I to cellulose II for cotton linter by alkali
pretreatment and urea additive was investigated by Gupta, Uniyal,
and Naithani (2013). Their results showed a sudden polymorphic
transformation at a concentration of 15 wt% NaOH, regardless of
the presence of urea. In two-phase solvent systems, such as the
mercerization experiments conducted with four different solution
systems (ethanol/water, acetone, dimethyl sulphoxide and xylene),
structural changes of cellulose crystallites depended primarily on
the distribution and solubility of sodium hydroxide in the sol-
vent. However, there was a critical concentration in the hydrophilic
phase that must exceed 7–8% NaOH before cellulose I undergoes
complete crystal change to cellulose II (Mansikkamaki, Lahtinen
and Rissanen, 2005).
The understanding of the polymorphic transformation of cel-
lulose in the nanoscale, i.e. nanocellulose crystals, has found some
controversies in the literature. Li, Ding, Li, and Jiang (2002) reported
Fig. 1. Schematic diagram illustrating the general process to obtaining individual
cellulose nanocrystals with dominant polymorphs I or II.
that CNCs obtained by sulfuric acid hydrolysis undergo complete
polymorphic transformation by reaction with 1% NaOH solution in
3 s. Liu and Hu (2008) treated bamboo nanofibers with 12% NaOH
solution for 20 min to generate cellulose II with some residual cel-
lulose I while full conversion to cellulose II was achieved at 16%
NaOH concentration. Abe and Yano (2011) treated wood cellulose
nanofibers and full conversion of cellulose I into II was accom-
plished in 15% NaOH solution. Interestingly, the reported values
of NaOH concentration for mercerization of nanocellulose are very
close to those needed for polymorphic transformation of macro-
scopic fibers.
So far, investigations dealing with mercerization of cellulose
have only been focused on whole cellulose fibers, i.e., organized
assemblies of microfibrils. Very few investigations have dealt with
the properties of cellulose I and II nanocrystals (Yue et al., 2012).
Thus, it is necessary to conduct a systematic study on the trans-
formation of individual CNCs, especially their polymorphic and
morphological changes. Our results show that there are effects
induced by the nano-scale size of the CNCs, which do not hold
in the case of macroscopic fibers. The polymorphic transforma-
tion of CNCs upon mercerization proceeds gradually, starting below
8% NaOH concentration and is complete above 12.5% NaOH. This
transition range also depends on the initial cellulose source. CNCs
produced by sulfuric acid hydrolysis contain negatively-charged
groups (anionic sulfate half-ester groups) that can be removed
during mercerization under alkaline conditions. Additionally, indi-
vidual CNCs are difficult to observe (TEM, AFM) but appear to form
aggregates. Thus, moderate TEMPO-oxidation of the mercerized
cellulose crystals (schematic illustration in Fig. 1) is required for
better dispersion and observation.
2. Experimental
2.1. Materials
Two microcrystalline cellulose grades were used in this study,
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China),
thereafter referred to as MCC1 and, AvicelTM PH-101, provided
by Sigma-Aldrich Co., Ltd (Shanghai, China), referred to as MCC2.
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 99% (GC) purity
was purchased from Jiana Chemical Co., Ltd (Changzhou, China).
Sodium hypochlorite, sodium hydroxide, sulfuric acid and other
reagents were analytical grade and purchased from Nanjing Chem-
ical Reagent Co., Ltd (Nanjing, China).
 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
2.2. Preparation of cellulose nanocrystals (CNCs)
The procedure for preparation of CNCs is described elsewhere
(Beck-Candanedo, Roman, & Gray, 2005; Fu, He, Jin, Cheng, & Song,
2012; Song, Fu, Cheng, & Jin, 2014; Viet, Beck-Candanedo, & Gray,
2007). Briefly, MCC powder (10 g) was hydrolyzed with sulfuric
acid (250 mL, 64 wt%) at 45 ◦ C for 1 h under continuous stirring.
The hydrolysis was quenched by adding a large amount of water
(∼10 times the volume of the acid solution used). The system was
centrifuged (12,000 rpm) at 4 ◦ C for 10 min and the supernatant
was decanted. This procedure for acid removal was repeated three
times until a white suspension was obtained. Then the suspen-
sion was dialyzed with cellulose acetate membrane tubes (MWCO
14000) against deionized water, for about 5–7 days, until a constant
pH was reached. A portion of the CNC aqueous dispersion (there-
after referred to as CNC-I) was stored in a bottle and the other was
freeze-dried.
2.3. Mercerization of CNC-I
10 mL of NaOH solutions (1.0, 2.0, 5.0, 8.0, 10, 12.5, 15 and 17.5%
concentration) were prepared initially. Then 100 mg precisely
weighted CNC was added into the alkali solution under constant
stirring for 30 min at ambient temperature. Upon completion of the
reaction, the dispersion was neutralized with 5% sulfuric acid solu-
tion. The respective mercerized CNC dispersion was dialyzed with
cellulose acetate membrane tubes (MWCO14000) against deion-
ized water for 3–5 days, until a constant conductivity was reached.
The obtained samples, herein indicated by a numeral representing
the NaOH concentration used during mercerization (e.g. CNC-10%),
were freeze-dried and stored in a desiccator. For comparison, the
other MCC sample, MCC1, was mercerized with 17.5% NaOH for 24 h
at first, and then subjected to sulfuric acid hydrolysis as described in
the previous section (a shorter time was used, 20 min). This sample
is thereafter denoted as CNC-II.
2.4. TEMPO-mediated oxidation of mercerized CNCs
Negative charges on CNC-I are reduced to some degree after
treatment with alkali, due to hydrolysis of ether bonds (Lokanathan,
Uddin, Rojas, & Laine, 2014). As a consequence, CNCs tend to
agglomerate after mercerization, limiting the possibility of detailed
studies, for example, via imaging. Therefore mercerized CNCs were
subjected to TEMPO-mediated oxidation, which adds negative
charges to the surface and improves colloidal stabilization. The
TEMPO-mediated oxidation followed the method described else-
where (Habibi, Chanzy, & Vignon, 2006) except that more moderate
conditions were used. Briefly, the CNC-17.5% sample (50 mL dis-
persion) was first sonicated for 5 min. Then TEMPO (14.75 mg,
0.094 mmol) and NaBr (162 mg, 1.57 mmol) were added to the dis-
persion. NaClO solution (0.4 mL, 1.24 M) was added slowly and the
pH of the mixture was maintained at 10 by adding 0.5 M NaOH.
The oxidation was terminated after 30 min by adding methanol
(1 mL) and the pH was adjusted to 7 using 0.5 M HCl solution. The
aqueous dispersion was then introduced in cellulose acetate mem-
brane tubes (MWCO14000) for dialysis against deionized water.
The obtained sample, after freeze–drying, is referred thereafter as
CNC-17.5%-TEMPO.
2.5. X-ray diffraction
X-ray diffraction measurements were carried out by using a
Goniometer Ultima IV (Rigaku Co. Ltd., Japan) X-ray diffractome-
ter equipped with CuK␣ radiation (0.15406 nm) generated at 40 kV
and 30 mA at room temperature. Vacuum dried cellulose powder
samples were placed on quartz substrates that have no background
329
signal. X-ray diffraction data were collected from 2 = 5–55◦ in steps
of 0.02◦ ; and were further analyzed using the MDI Jade 6.5 software
(Materials Data, Inc.). XRD patterns were smoothed and baseline-
corrected by a software-generated parabolic curve. Curve-fitting
was performed to identify individual peaks and to calculate the
crystallinity and crystallite size. The Segal method (Creely, Segal, &
Ziifle, 1956) uses the ratio of the peak height at the 2 = 18◦ position,
which is assumed to result from the contribution of amorphous
regions of cellulose, to that of plane 200. This method is usually
applied to cellulose I but not for cellulose II. Here, we used the
areas of the crystalline and amorphous regions in the XRD spectra.
Therefore, the crystallinity based on the ratio of crystalline region’s
area to the total area was employed for convenient comparison.
The Scherrer’s equation was used for estimating the crystallite size
(Mittal, Katahira, Himmel, & Johnson, 2011):
k
ˇ=
 cos 
where  is the wavelength of the incident X-ray (0.15406 nm),  is
the Bragg angles corresponding to the planes, ˇ is the full-width
at half maximum (FWHM) of the X-ray peak corresponding to the
planes;  is the X-ray crystallite size, and k is a constant of 0.89.
2.6. CNC imaging and sizing
A drop of aqueous CNC dispersion in water (∼0.01%w/v) was
deposited on a carbon-coated electron microscope grid and then
negatively stained with phosphotungstic acid. A piece of tissue
paper was used to absorb excess water and then allowed to dry
in air. The sample on the grids was imaged with a JEM-2100
UHR (JEOL, Japan) transmission electron microscope operated at
an accelerating voltage of 200 kV.
AFM observation was performed using Multimode Nanoscope
V controller (Bruker Corporation, USA) in air at room temperature
(23 ◦ C). A drop with respective CNC dispersion was placed on a sil-
icon wafer and spin-dried at a speed of 3000 rev/min using a spin
coater (WS-650-23, Laurell Technology Co. Ltd., USA). Images from
AFM were recorded in tapping mode by using silicon cantilevers.
The commercial AFM probes had a spring constant of 20–80 N/m
and a resonance frequency of 300–340 kHz.
The relative size of mercerized CNC aggregates as well as CNC-
II was determined by dynamic light scattering using a Malvern
Zetasizer NanoS90 at a 90◦ scattering angle.
3. Results and discussion
3.1. Untreated cellulose nanocrystals
Two untreated cellulose nanocrystal samples (CNC-I1 and
CNC-I2) were obtained by sulfuric acid hydrolysis from two micro-
crystalline cellulose sources, MCC1 and MCC2, respectively. In
Fig. 2a, both XRD patterns of CNC-I1 and CNC-I2 exhibit a sharp
high peak at 2 = 22.6◦ and two overlapping, weaker diffraction
peaks at 2 = 14.7◦ and 2 = 16.4◦ , which are assigned to the typ-
ical pattern of cellulose I (French, 2014; French & Cintrón, 2013).
The d-spacings of these planes calculated by Bragg equation are
0.39 nm, 0.60 nm and 0.54 nm, respectively. These parameters are
consistent with those reported in the literature (Ishikawa, Okano,
Sugiyama, Ishikawa, & Okano, 1997; Sèbe et al., 2012). However,
compared to the diffraction bands of CNC-I1, the pattern for CNC-
I2 is smoother and overlaps more markedly. Two diffraction peaks
at 14.7◦ and 16.4◦ merge into one broad band and the small peak at
2 = 20.1 (plane 012) is integrated into the most pronounced peak
of plane 200. The crystallinity index (CrI) of CNC-I1 and CNC-I2 are
quite similar, 77.9% and 77.5%, respectively.
330 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
Fig. 2. X-ray diffraction patterns of CNC-I1 and CNC-I2 (a) and a representative TEM image of CNC-I1 (b) (the images for CNC-I2 are very similar, not included here).
The morphology of CNC-I1 and CNC-I2 as observed by TEM
is quite similar (Fig. 2b). The crystals show a needle-like shape,
100–300 nm in length and ca. 10 nm in width, consistent with other
values reported in the literature for MCC-derived CNCs (Habibi
et al., 2010).
3.2. CNC polymorphic transformation from cellulose I into
cellulose II and size-induced polymorphism
A systematic evaluation of the process by which cellulose I
nanocrystals evolve into cellulose II crystals was undertaken and
the role of size-induced polymorphic transformation upon mercer-
ization was assessed. To this end, the two CNC samples (CNC-I1 and
CNC-I2) were treated with NaOH solutions of given concentrations,
from 1% to 17.5%, for 30 min and at room temperature. The X-ray
diffraction patterns of CNC-I1 and CNC-I2 after alkali treatment are
shown in Fig. 3a and b, respectively.
The XRD patterns of CNC-I1 upon treatment with dilute alkali,
e.g. 1, 2, 5, and 8% NaOH solutions display similar peaks as those
of untreated CNC and indicate the main contribution of cellulose
I (French, 2014). However, for the sample treated by 8% NaOH,
a small bump at the position of 2 = 12.0 starts emerging. When
the alkali concentration is increased to 10%, two additional distinc-
tive diffraction peaks appear at 2 = 12.0 and 2 = 20.0, which are
assigned to cellulose II (French, 2014). This observation hints to
the fact that at concentration of ca. 10% NaOH, both polymorphs,
cellulose I and cellulose II, coexist. When the alkali concentration
is increased to 12.5%, three peaks from cellulose II dominate but
Fig. 3. XRD patterns after mercerization (30 min, room temperature) of CNC-I1 (a) and CNC-I2 (b) under given concentrations of NaOH solution, from 1 to 17.5%.
two noticeable peaks at 2 = 14.7 and 16.4, assigned to cellulose I,
still remain. Samples subjected to NaOH treatment at concentra-
tions above 12.5% (i.e. 15 and 17.5%) indicate similar XRD patterns
and display only the characteristic profile of cellulose II, with three
peaks positioned at 2 = 12.0, 20.0 and 21.8o .
The alkali-treated CNC-I2 samples produce similar trends as
those discussed before for CNC-I1, except that the transition con-
centration is shifted from 10% to 8% (CNC-I1).
Crystallinity index, d-spacing and crystallize size for each
plane were determined via Jade code. The d-spacings of the
(1 −1 0), (1 1 0) and (2 0 0) planes for cellulose I are 6.01 ± 0.06 Å,
5.35 ± 0.05 Å and 3.92 ± 0.02 Å, respectively. While these values are
shifted to 7.41 ± 0.06 Å, 4.43 ± 0.03 Å and 4.08 ± 0.04 Å for cellulose
II. The crystallite size for the (1 −1 0) and (1 1 0) planes of cellulose I,
and (1 1 0) and (2 0 0) for cellulose II varied extensively due to over-
lap. However the values of the average crystallize size of the most
prominent peak(s), i.e., 2 = 22.6◦ for cellulose I and 2 = 20.0 and
21.8◦ for cellulose II, can be considered accurate. The crystallinity
indices and average crystallite sizes of CNC-I1 and CNC-I2 after
treatment with alkali solutions of given concentrations are listed
in Tables 1 and 2, respectively.
In the case of CNC-I1, CrI continuously decreases with the
increase of alkali concentration used in the treatment. The frac-
tion of cellulose I and cellulose II can be estimated by the area of
fitted curves. At 10% NaOH concentration, the fraction of Cellulose
I allomorph is 72.8%, indicating the presence of 27.2% cellulose II
allomorph. In the case of 8% NaOH, cellulose I dominates (84.0%)
and only a small portion (16.0%) of cellulose II allomorph is
 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335 331

Table 1
Crystallinity indices and crystallite size of CNC-I1 after alkali treatment for 0.5 h.

NaOH Conc. (%) CrI (%) Crystallite size (nm)

Cellulose I Cellulose II

MCC1 83.7 6.9 –

CNC-I1 77.9 6.6 –
1 76.5 6.8 –
2 75.9 6.8 –
5 73.8 6.8 –
8 71.4 6.5 5.2
10 66.6 6.3 5.3
12.5 68.2 5.1 5.5
15 68.2 – 5.5
17.5 56.7 – 5.5

Table 2
Crystallinity indices and crystallite size of CNC-I2 after alkali treatment for 0.5 h.
Fig. 4. XRD patterns of CNC-I1 treated with alkali at the transition concentration
NaOH Conc. (%) CrI (%) Average crystallite size (nm) (10%) for given periods of time.
Cellulose I Cellulose II

MCC2 69.9 4.4 – is slightly higher than that of the produced CNCs. This is explained
CNC-I2 77.5 4.1 – by the fact that in the production of CNC some molecules located
1 78.9 3.9 –
in the periphery of the crystals react with sulfuric acid and become
2 81.2 3.9 –
5 73.1 3.9 – dissolved. The reason why two different MCC sources have differ-
8 72.1 2.8 4.5 ent crystallite sizes may be due to the difference in fiber source and
10 70.8 – 5.0 treatments applied in their production. This issue needs further
12.5 67.7 – 4.8
elucidation.
15 59.2 – 5.0
17.5 55.3 – 4.9
3.3. Time for CNC polymorphic transformation

identified. For 12.5% NaOH, cellulose II dominates (82.4%) and a
small portion (17.6%) of cellulose I allomorph is identified. These
observations hint that the polymorphic transformation proceeds
gradually, starting from below 8% NaOH and ending at a NaOH
concentration above 12.5%.
This gradual transition is also evidenced in the average crys-
tallize size. The average crystallize size for MCC1 is 6.9 nm. This
size is reduced to 6.6 nm for CNC-I1. When CNC-I1 is treated in
dilute alkali, the size remains similar. When the alkali concentration
increased to 8%, the average crystallize size for cellulose I crys-
tallites are reduced slightly, to 6.5 nm. When the concentrations
increased to 10% and 12.5%, the average crystallize size for cellu-
lose I crystallites is reduced further to 6.3 and 5.1 nm, respectively.
In the meantime, the average size of cellulose II crystallites grows
from 5.2 nm to 5.5 nm. At an alkali concentration above 12.5%, the
average size of cellulose II crystallites doesn’t change further.
The CrI variation upon pretreatment of CNC-I2 with alkali fol-
lows the same trend as that for CNC-I1, i.e. CrI continuously
decreases with the increase in alkali concentration used in the
treatment. At 8% NaOH, the CNC mixture comprises 68.0% Cellu-
lose I and 32.0% Cellulose II crystallites. The fraction of cellulose II
is higher than that in the case of CNC-I1 for treatment with the same
alkali concentration. Furthermore, negligible amounts of cellulose
I or II are found at 5% and 10% NaOH.
In terms of the average crystallite size of CNC-I2, it also fol-
lows the same trend as that of CNC-I1, i.e. the average crystallite
size for cellulose I gradually reduces at the transition alkali con-
centration, while the average crystallite size for cellulose II grows.
A very interesting is that the average crystallite size of cellulose
I in CNC-I2, 4.0 nm, is much smaller than that of CNC-I1, 6.8 nm.
The difference in crystallize size may be one of the reasons why the
polymorphic transformation for CNC-I2 takes place at a lower alkali
concentration and is completed within a narrow range. The differ-
ence in crystallize size for both CNC samples can be traced back to
the MCC sources. The crystallite size in MCC1 is 6.9 nm, while that
in MCC2 is only 4.4 nm. The crystallite size for both MCC samples
The XRD pattern of CNC pretreated at the transition NaOH con-
centration includes all the peaks of cellulose I and cellulose II. This
facilitates determination of the extent of completion of the poly-
morphic transformation, i.e., via monitoring the change in XRD
patterns with the pretreatment time. As such, Fig. 4 includes XRD
patterns of CNC-I treated with 10% NaOH during given times, up to
24 h. In all cases, from 0.5 h and up to 24 h treatment, the same dis-
tinguishable features in the XRD profiles are observed, indicating
that an extensive treatment is not conducive of further polymor-
phic transformation. The polymorphic transformation occurs quite
rapidly, in less than 30 min, regardless of the concentration of alkali
used. This result is in agreement with the conclusion of Liu and Hu
(2008).
3.4. Morphological changes of cellulose nanocrystals during
polymorphic transformation
An AFM image of CNC-I1 after treatment with 10% NaOH is
shown in Fig. 5a. Needle-like shapes are observed, with ca. 10 nm
in width and 100–200 nm in length. Negligible differences in size
and morphology are observed if compared to the untreated CNC-I.
CNC-I1 pretreated with 17.5% NaOH resulted in important mor-
phological changes as observed by AFM and TEM (Fig. 5b and c),
respectively. Round-shaped agglomerates, about 200 nm in size,
composed of granules (size of around 30 nm), can be observed in
Fig. 5b; no individual crystals are identified, even if the dispersion
was diluted before drying and imaging. Such agglomerates can be
clearly seen under TEM (Fig. 5c) as uniform spherical aggregates
of ca. 150–200 nm. The size of granules for mercerized CNC is also
consistent with that of regenerated cellulose (Yamane et al., 2013).
Untreated CNCs produced by sulfuric acid hydrolysis carry neg-
atively charged groups and are dispersed easily in aqueous medium
(Habibi et al., 2010). The ester bonds of negatively charged sulfate-
half ester groups break down, at least partially, upon treatment in
an alkaline solution (Lokanathan et al., 2014). Therefore, the surface
charge of CNCs is reduced and facilitates their agglomeration. This
332 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
Fig. 5. AFM image of CNC-I1 upon treatment at the critical concentration of alkali, 10% (a) and corresponding images (AFM, b and TEM, C) upon CNC-I1 treatment at higher
concentration (17.5% NaOH).
Fig. 6. Size of CNC and corresponding agglomerates as determined by light scatter-
ing in aqueous dispersions of nanocrystals (CNC-I1) before and after treatment at
given alkali concentrations. The image of CNC-II is also included.
tendency is clearly demonstrated in Fig. 6 for size measurements
that were carried out in aqueous dispersion via light scattering.
Bearing in mind that the size recorded can only be taken as a
relative value and cannot be compared with that determined by
TEM, it is still interesting to note similar observations: the diame-
ter of untreated CNC-I1 and that after treatment with 10% NaOH
solution is somewhat similar. For CNC-17.5%, the size shown
indicates large aggregates. As a comparison, the size of CNC-II
(hydrolysis product of mercerized MCC), is shown to be 20-30 nm
(see Fig. 7a), which is in agreement with the granule size in AFM
observations for CNC-17.5% (Fig. 5b).
Since CNCs loss their charges after mercerization and tend to
agglomerate, TEMPO-mediated oxidation was carried out in order
to facilitate dispersion and to observe the morphology of individual
particles. Oxidation strength was carefully adjusted by the molar
ratio of NaClO/equivalent anhydroglucose unit (AGU). In a conven-
tional TEMPO-mediated oxidation, this value is about 0.5, which is
too severe for the intended purpose. After several tests, it was found
that NaClO/AGU = 0.15 is the minimum necessary to promote col-
loidal stabilization of the CNCs, i.e., by oxidation of some of its C6
hydroxyl groups. A TEM image of CNC-17.5%-TEMPO is shown in
Fig. 7b. The morphology of individual mercerized cellulose crystal
indicates interconnected spherical features and bended kidney-like
granules. The size of these granules is consistent with that observed
in AFM (Figs. 5b and 7a), ca. 30–40 nm.
To the best of our knowledge, this is the first report on the
morphology of individual CNC with cellulose II polymorph, espe-
cially obtained from the mercerization. Sèbe et al. (2012) have
imaged individual cellulose nanocrystals with cellulose II allo-
morph obtained from acid regeneration. Compared with that of
cellulose I nanowhiskers, their morphology (AFM images) showed
a ribbon-like shape with rounded tips, with a smaller size but
 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
Fig. 7. AFM image of CNC-II (a) and TEM image of CNC-17.5%-TEMPO (b).
expanded width. The morphology shown in AFM images demon-
strate that such ribbons were composed of several granules. Hence
the morphology includes several interconnected granules shared
by both individual nanocellulose samples, regardless the treat-
ment process used, either by concentrated acid or mercerization;
they undergo swelling and recrystallization. Yue et al. (2012) have
reported rod-like crystals for cellulose II having similar widths but
reduced length, compared with pristine cellulose I crystals. How-
ever these authors obtained cellulose II crystals from mercerized
macroscopic fibers, via sulfuric acid hydrolysis and followed by
high-pressure homogenization. This means that the polymorphic
transformation occurred at the macroscopic state and the mor-
phological changes may have been restricted by the surrounding
crystals and were not able to relax, which is in contrast to the
present observations.
The XRD patterns of CNC-17.5%-TEMPO, and CNC-II are shown
in Fig. 8. The XRD pattern for CNC-17.5% is added for comparison.
They all include the characteristic peaks of cellulose II but with
different intensity. After TEMPO-mediated oxidation, mercerized
CNCs still retain their crystalline structure. The crystallinity indices
and crystallite size of CNC-17.5%-TEMPO and CNC-II are given in
Table 3. Since CNC-II contains mainly the crystalline component
(upon removal of amorphous regions), its crystallinity, 75.4%, is
close to the original CNC samples, and higher than that of mercer-
ized CNC (CNC-17.5%), 56.7%. This value is much higher than that
of TEMPO-oxidized sample (CNC-17.5%-TEMPO), 37.6%. The aver-
age crystallite size of CNC-II, 5.1 nm, is slightly smaller than those
Fig. 8. XRD patterns for cellulose nanocrystals with polymorph of cellulose II.
333
of mercerized CNCs, 5.5 nm. While the average crystallite size of
TEMPO-oxidized sample (CNC-17.5%-TEMPO), is only 4.2 nm. This
observation indicates that the crystallite size of CNC-II and CNC-
17.5%-TEMPO is reduced in their production.
All the above observations provide some evidence and shed
some light to better understanding how the polymorphic trans-
formation of cellulose takes place. For a regular cellulose I crystal,
with a width of several nanometers and a length of hundreds
nanometers, and at a NaOH concentration above the critical value,
hydroxide ions start to penetrate into the crystals. As hydroxide
ions diffuse within the crystallite and swells it, the cellulose chains
rearrange their orientation as hydroxide ions are removed from the
lattice of the cellulose matrix. This process takes place as suggested
by Yamane et al. (2013) who indicated that some six ring conform-
ers of glucose form hair-pin turns and therefore cellulose chains
fold and pack antiparallel forming a more thermodynamically sta-
ble state, allomorph cellulose II. As such, the cellulose II crystallites
formed have much shorter length, ca. 30–40 nm (see TEM images),
if compared with their original state, cellulose I, 100–300 nm. Our
results also demonstrate that one cellulose chain may be involved
in the formation of several cellulose II crystallites. The “bridge”
connecting granules provides this evidence. While images of indi-
vidual CNC composed of the cellulose II polymorph can be obtained,
the process by which cellulose I evolves into cellulose II, and the
variables that influence this process, remain unresolved subjects.
Computational simulation and related approaches may shed more
light on these issues.
Overall, the results obtained suggest that: (1) cellulose I allo-
morph cannot be converted under dilute alkali treatment (<5%
NaOH); (2) polymorphic transformation proceeds gradually, in the
alkali transition concentration range. In the transition state, both
allomorphs, cellulose I and II, coexist in the cellulose nanocrystals.
The ratio of each fraction depends only on the alkali concentra-
tion used in the treatment. Below this transition, cellulose I clearly
dominates the system while above the transition concentration,
cellulose II dominates. (3) If compared to the polymorphic trans-
formation of macroscopic fibers, where the polymorph conversion
only occurs when alkali concentration is above 12.5%, the critical
Table 3
Crystallinity indices and crystallite size of CNC-17.5%-TEMPO and CNC-II.
Sample Polymorph CrI (%) Average crystallite size (nm)
CNC-17.5%-TEMPO II 37.6 4.2
CNC-II II 75.4 5.1
334 E. Jin et al. / Carbohydrate Polymers 143 (2016) 327–335
transition concentration for CNCs is reduced, to about 8% NaOH.
This indicates a size-induced polymorphic transformation, which
is strongly interconnected with the CNC crystallite size. However,
this effect is not as pronounced as that claimed by Li et al. (2002)
who reported complete conversion upon treatment with 1% NaOH
solution for three seconds. This may be attributed to the con-
centrated alkali that remains after mercerization during drying,
which facilitates the polymorphic transformation. At the transition
NaOH solution concentration of 8–12.5%, and for the polymorphic
transformation of the studied CNCs, it is hypothesized that the
hydrated hydroxide ions start to penetrate the cellulose crystals
and undergo partial reaction with cellulose. At alkali concentra-
tions above the transition concentration, hydroxide ions penetrate
extensively within the crystals, producing CNC swelling. Upon ion
removal during dialysis, the cellulose chains recrystallize into cellu-
lose II, which is more stable thermodynamically. As a consequence,
the respective XRD patterns display the contribution of cellulose II
allomorph while the characteristic peaks of native cellulose I are
absent. Treatment with alkali at a concentration below the transi-
tion value provides conditions that are not sufficient for hydroxide
ions to penetrate into the cellulose I crystals and therefore no poly-
morphic transformation takes place, even if the size of the particle
is in the nano-scale (Abe & Yano, 2011; Dinand, Vignon, Chanzy,
& Heux, 2002; Fengel, Jakob, & Strobel, 1995). Based on the dis-
cussion of Liu and Hu (2008), the size of the hydroxide ions vary
with the alkali concentrations. At a low concentration, hydrox-
ide ions are fully hydrated and their size may prevent them from
penetrating and disrupting the cellulose lattice. However, as the
NaOH concentration increases, the amount of free water available
for hydrating the hydroxide ions is limited. As such, the charac-
teristic size of hydrated hydroxide ions are relatively smaller and
more easily penetrate into the cellulose lattices. When their size is
reduced to dimensions small enough to penetrate into the cellulose
lattices, the critical alkali concentration required for polymorphic
transformation is identified. At the NaOH transition concentration,
some crystals of cellulose I are converted into cellulose II. The ratio
of cellulose I to II is a function of alkali concentration used. There
is indication that the polymorphic transformation of CNCs is actu-
ally a dynamic process. Above the NaOH transition concentration,
hydroxide ions penetrate easily into all pristine crystals and convert
them into crystals of cellulose II polymorph.
In terms of the penetration of hydrated hydroxide ions, as for
CNC particles of inherently large surface area, the penetration is
facilitated. This the reason for size-induced polymorphic transfor-
mation. In regards to the CNC sample with a small crystallite size,
hydroxide ions penetrate easily into the matrix and therefore shift
the concentration required for polymorphic transformation to a
lower value and within a narrower range.
4. Conclusions
The polymorphic and morphological changes of cellulose
nanocrystals were systematically investigated by treating CNCs
under various concentrations of NaOH (1, 2, 5, 8, 10, 12.5, 15 and
17.5%). It is found that a transition range of alkali concentration
exist for polymorphic transformation of cellulose and the mercer-
ization process is completed in less than 30 min.
The transition NaOH concentration is found to be in the range
of 8–12.5%, which strongly correlates with the CNC crystallite size.
Within this transition concentration range, both allomorphs of cel-
lulose I and cellulose II coexists while below the polymorph of
cellulose I dominates. Above this concentration range, cellulose I
converts to cellulose II completely. The polymorphic transforma-
tion proceeds gradually, which is evidenced by the gradual decrease
in crystallite size of cellulose I and the growth of cellulose II.
The nano-size induced polymorphic transformation plays a role in
reducing the critical concentration required for allomorph conver-
sion. Finally, morphological changes (TEM and AFM) of individual
cellulose crystal during polymorphic transformation illustrate that
CNC comprising the Cellulose II polymorph exhibits interconnected
granules. This indicates that single cellulose chains may be involved
in Cellulose II the formation of Cellulose II crystals.
Acknowledgements
This project was financially supported by Special Fund for
Forestry Scientific Research in the Public Interest (201404510),
National Natural Science Foundation of China (Nos. 31270613,
31200454), Qing-Lan Project, Jiangsu Co-Innovation Center for Effi-
cient Processing and Utilization of Forest Resource (NJFU) and the
Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions. JS acknowledges funding support from China
Scholarship Council provided for his research mission in Aalto Uni-
versity. This work was also partially supported by the Academy of
Finland Centres of Excellence Programme (2014–2019) “Molecular
Engineering of Biosynthetic Hybrid Materials Research” (HYBER).
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