Industrial Crops and Products 56 (2014) 175–186

Contents lists available at ScienceDirect

Industrial Crops and Products

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

Optimized extraction of cellulose nanocrystals from pristine and

carded hemp fibres
F. Luzi a , E. Fortunati a,∗ , D. Puglia a , M. Lavorgna c , C. Santulli b , J.M. Kenny a,d , L. Torre a
a
University of Perugia, Civil and Environmental Engineering Department, UdR INSTM, Strada di Pentima 4, 05100 Terni, Italy
b
University of Camerino, School of Architecture and Design, viale della Rimembranza, 63100 Ascoli Piceno, Italy
c
Institute of Polymers, Composites and Biomaterials, National Research Council, P.le Fermi, 1, 80055 Portici, NA, Italy
d
Institute of Polymer Science and Technology, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

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

Article history: The extraction of cellulose nanocrystals (CNC) from Carmagnola hemp fibres has been carried out. Before
Received 8 January 2014 CNC extraction, the effectiveness of two pre-treatment methods, an alkaline chemical and a pectinase
Received in revised form 3 March 2014 enzymatic treatment, applied on the pristine and carded hemp fibres, were compared. Carding allowed
Accepted 8 March 2014
removal of most impurities from the fibres, while it had only a modest effect on their structure. After
Available online 29 March 2014
chemical treatment, hemicellulose was removed, more efficiently in carded hemp, and X-ray diffraction
suggests an increase in the size of cellulose crystallites. Carded hemp fibres, after interaction with pecti-
Keywords:
nase, show the total decomposition of pectin and hemicellulose. On the basis of these results, carded hemp
Natural fibres
Hemp fibres
was selected as start material for CNC extraction and the acid hydrolysis for CNC synthesis was applied
Cellulose nanocrystals on carded hemp after both chemical and enzymatic procedures. The yield of the different hydrolysis
Chemical procedures methods remains approximately at the same level (around 19%): on the other side, a carding procedure,
Enzymatic treatment combined with an alkaline treatment to hemp fibres, represents an optimized process for CNC extraction
by acid hydrolysis.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction total weight, namely 44% alpha-cellulose and 25% hemicellulose

In recent years, the reintroduction of hemp, which was basically
wiped out of the market due to the consequences of antidrug leg-
islation, is taking place in a number of countries, including Italy, as
an effect of the development of hemp varieties with limited con-
tent of the psychotropic agent delta-tetrahydrocannabinol (THC).
However, the value of THC as a medicine for the treatment of mul-
tiple sclerosis suggest separating high THC content varieties for
medical purposes, from low THC content ones, aimed at various
industrial applications (Croxford et al., 2008). These include, among
others, the production of textiles, fatty oils and wood replacement
chips while the conversion of hemp seed oil into biodiesel has been
lately proposed (Li et al., 2010). In the specific case of Italy, one
recently developed very low (less than 0.1%) THC variety is named
Carmagnola, from the place it originates in Piedmont; this variety
already underwent complete chemical analysis, which suggested
that in this variety of hemp polysaccharides form about 70% of
∗ Corresponding author. Tel.: +39 0744492921; fax: +39 0744492950.
E-mail address: elena.fortunati@unipg.it (E. Fortunati).
(Gandolfi et al., 2013).
Of course, the development of a productive system involves
also the presence of waste by-products and in the case of hemp,
this refers particularly to “hemp wool”, usually a bundle of quite
short fibres and hemp shives as the inner wooden core of the
plant. Lower quality fibres no longer than 30–40 cm are used for
the fabrication of hemp wool; when such fibres are mixed with a
binder they form layers used for thermal insulation in buildings
(Collet et al., 2011). Lower quality fibres are usually excluded from
a textile use due to their poor resistance to torque, leading to inef-
fective draping in fabrics and to scarce friction in strands for ropes,
but are nonetheless materials that involve non-negligible labour
and costs in their extraction from the plants. More specifically,
once cut from the plant, hemp stalks are subjected to retting, i.e.,
pectin removal, which is normally achieved by open exposure to
the environment for a few weeks. During retting, the stalks are
turned several times: after retting is completed, they are beaten
and crushed (scutched) for ease separation of the fibres from the
wooden stalk. The obtained fibres are then cleaned and carded to
the desired core content and fineness. In particular, carding is basi-
cally a combing operation aimed at obtaining a sliver, therefore
disentangling fibres and breaking up locks and unorganized clumps

http://dx.doi.org/10.1016/j.indcrop.2014.03.006
0926-6690/© 2014 Elsevier B.V. All rights reserved.
176 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
of fibre, finally aligning them unidirectionally. All the subsequent
processes concern the fabrication of textiles: in other words, after
carding, the fibres are ready for operations, such as roving, winding
and weaving, which may involve also the application of treatments
to improve the mechanical performance of the fibres.
It needs to be noted that most of the above operations are basi-
cally tailored on the objective to allow the production of textiles,
which is the highest profile product for hemp fibres. Whenever the
aim of fibre extraction is not the production of a textile yarn, the
question may arise whether the extraction process can be simpli-
fied or not. This has been attempted on hemp retting simplification
by steam explosion, when the purpose was the production of ther-
mal insulation panels. In the case of the present study, aimed at
the production of cellulose nanocrystals (CNC) from low quality
hemp fibres, which are therefore not suitable for textile use, the
question is whether carding would be still necessary for the pur-
pose. Another open question, regarding plant fibre extraction, is
the need to remove non-structural matter, which is mainly formed
by pectin and hemicellulose, to expose the cellulose domains of
the fibres. This is normally carried out using chemical treatments,
the most frequent of which is alkalisation with sodium hydroxide,
which derives its origin from the textile industry practice, start-
ing with cotton fibres (Li et al., 2007). Alkali treatment, though
effective, has proved to be quite aggressive on a number of fibres,
leading to the removal of an excessive amount of material from the
fibres and possibly to their embrittlement. An alternative, which
has been proposed and applied initially on flax fibres (Akin et al.,
2001) and also on other stem extracted fibres, such as hemp and
nettle, is the enzymatic treatment through the use of pectinase to
remove pectin from the fibre. On hemp fibres, on which a number of
research reports are available (Ouajai and Shanks, 2005; Pakarinen
et al., 2012), the removal of pectin showed a strong correlation
with enzymatic hydrolysis, so that the detachment of single fibre
cells within the bast fibre bundle, caused by the partial removal of
pectin, increased the availability of the substrate cell wall surface
area (Pakarinen et al., 2012).
The extraction of CNC from plant fibres is often performed
through acid hydrolysis using sulphuric acid to remove the amor-
phous cellulose and form highly crystalline cellulose. CNC are
typically rigid rod-shaped monocrystalline cellulose domains of
1–100 nm in diameter and from tens to hundreds of nanome-
ters in length, with morphological and structural characteristics,
including entanglement and geometrical dispersion, depending
on the species, cultivar and agronomical factors, such as plant
maturity, characteristics of the soil and fertilisers used. The yield
of the extraction process, defined as the quantity of nanocellu-
lose obtained from a given weight of macrofibre, depends on
both the crystallinity of the specific plant fibre and on the pro-
cedure adopted for extraction. Nanocellulose extraction through
hydrolysis of hemp yarns and use of obtained CNC as coupling
agent to reinforce hemp fibres has been proposed in Dai et al.
(2013).
The method adopted in this research has been applied origi-
nally on sisal fibres by Morán et al. (2008) and already employed
for incorporation of okra bahmia (Abelmoschus esculentus) fibres
in a PVA matrix (Fortunati et al., 2013a), from phormium fibres
(Fortunati et al., 2013b,c) and from Belinka flax fibres (Fortunati
et al., 2013c). The extraction method involves a first chemical treat-
ment leading to the production of holocellulose by the gradual
removal of lignin, while the subsequent sulphuric acid hydroly-
sis process allowed obtaining cellulose nanocrystals in an aqueous
suspension. The same method is applied again in this work, starting
either from carded or non-carded hemp fibres and applying either
chemical or enzymatic treatment on hemp fibres of the Carmagnola
variety, to show the effect of the carding process on the properties
of CNC.
2. Experimental
2.1. Materials
Hemp (Carmagnola) pristine fibres (p-Hemp) were obtained
from Assocanapa in Carmagnola, Piedmont, Italy.
The reagents, toluene, ethanol, acetic acid, sodium chlorite,
sodium bisulphate, sulphuric acid and the buffer solution were
supplied by Sigma–Aldrich.
The enzymes selected for this work were Pectinex® supplied
by Sigma–Aldrich. Pectinex® is prepared using a selected strain of
Aspergillus aculeatus.
2.2. Carding process
A carding treatment was applied to the p-Hemp fibres. Carding is
a mechanical process normally used in the industrial field to disen-
tangle and clean the fibre to produce a continuous sliver suitable for
subsequent processing. The main purpose of this work was to ana-
lyze the effect of carding process on the pre-treatment efficiency
and cellulose nanocrystal extraction yield: chemical, morphologi-
cal and thermal analyses were therefore conducted on pristine and
carded hemp fibres.
The microstructure, dimension and appearance of pristine (p-
Hemp) and carded hemp (c-Hemp) were investigated by means
of field emission scanning electron microscope (FESEM, Supra 25-
Zeiss). The fibres were swollen in distilled water before FESEM
observation. A 1 wt% aqueous solution of fibres was stirred for 4 h
at room temperature. The solution was then subjected to 1 h son-
ication over 2 h in 10 min intervals, in order to loosen up the fibres.
Few drops of the suspension were cast onto silicon substrate, vac-
uum dried for 2 h and gold sputtered before the analysis. FESEM
images of the fibres were analyzed by the NIS-Elements BR (Nikon)
software in order to determine the average diameters of the start
materials. One hundred measurements were performed in order to
obtain a reliable result.
Fourier infrared (FT-IR) spectra of p-Hemp and c-Hemp fibres
were recorded using a Jasco FT-IR 615 spectrometer in the
400–4000 cm−1 range, in transmission mode. The fibres were ana-
lyzed using KBr discs made by using pulverized fibres and dust of
KBr.
Thermogravimetric measurements (TGA) of p-Hemp and c-
Hemp fibres were performed by using a Seiko Exstar 6300 analyzer,
in order to evaluate the effect of the carding process on the thermal
behaviour of hemp fibres. Heating scans from 30 to 600 ◦ C at 10 ◦ C
min−1 in nitrogen atmosphere were performed for each sample.
2.3. Chemical pre-treatment
Pristine hemp fibres and carded hemp fibres were pre-treated
before cellulose nanocrystals extraction. The fibres were washed
with distilled water several times and dried in an oven at 80 ◦ C
for 24 h. Then, they were chopped to an approximate length of
5–10 mm. Finally, a de-waxing step was carried out, boiling in a
mixture of toluene/ethanol (2:1, v/v) for 30 min and dried. Subse-
quently, for cellulose extraction, hemp fibres were firstly treated
with 0.7% (w/v) of sodium chlorite NaClO2 ; the fibres (liquor ratio
1:50) were boiled for 2 h and the solution pH was lowered to
about 4 by means of acetic acid for the bleaching. Then, a treat-
ment with sodium bisulphate solution at 5% (w/v) was carried out
and, at the end of this preliminary chemical process, holocellulose
(␣-cellulose + hemicellulose) was obtained by the gradual removal
of lignin. The holocellulose was treated with 17.5% (w/v) NaOH
solution, filtered and washed with distilled water. The obtained
 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
cellulose was dried at 60 ◦ C in a vacuum oven until constant weight
(Fortunati et al., 2012a).
2.4. Enzymatic pre-treatment of c-Hemp fibres
Carded hemp fibres were also enzymatically pre-treated. The c-
Hemp were washed with distilled water several times and dried in
an oven at 80 ◦ C for 24 h. Then they were chopped to an approxi-
mate length of 5–10 mm and 5 g of fibres were treated in 100 mL of
deionized water solution containing 800 ␮L solution of Pectinase
Aspergillus (Pectinase concentration 3800 U/mL). The reaction was
conducted for 30 h at 37 ◦ C and pH 3.5–4. Finally, the enzymatically
treated fibres were washed with distilled water several times and
dried in oven at 60 ◦ C for 24 h.
2.4.1. Optimization of the enzymatic procedure
It was initially observed that the enzymatic treatment of c-
Hemp did not result in effective defibrillation. Thus, a standard
sonication process in a sonic bath for 2 h at 80 ◦ C was used to
breakdown fibre bundles prior to enzymatic treatment. Moreover,
a second strategy was applied to increase the efficiency of the enzy-
matic pre-treatment by combining the sonication treatment with
a thermal process. So, carded hemp fibres were boiled in distilled
water for 2 h under magnetic stirring at 150 ◦ C and then sonicated
in a sonic bath for 2 h at 80 ◦ C.
Morphological investigation by means of field emission
scanning electron microscopy and thermal analysis by thermo-
gravimetric measurements (heating scans from 30 to 600 ◦ C at
10 ◦ C/min in nitrogen atmosphere) were conducted on the soni-
cated and sonicated/thermal treated c-Hemp in order to establish
the best preparation method.
Finally, an alkaline treatment, with a 2% (w/v) NaOH solution, at
98 ◦ C for 1.5 h was conducted on the prepared material. The fibres
were washed using distilled water several times, dried in oven at
60 ◦ C for 24 h, and then exposed to the enzymatic pre-treatment.
2.5. Characterization of p-Hemp and c-Hemp treated fibres
The microstructure, dimension and appearance of chemically
and enzymatically pre-treated p-Hemp and c-Hemp fibres, were
investigated by means of field emission scanning electron micro-
scope following the preparation procedure reported in the previous
paragraph. FESEM images of the fibres were analyzed in order to
determine the average diameters after the different pre-treatment
procedures. One hundred measurements were performed on each
sample to obtain a reliable result.
Fourier infrared (FT-IR) spectra of chemically pre-treated fibres
were recorded in transmission mode using KBr discs. Thermo-
gravimetric measurements (TGA) were performed according to the
procedures previously described. The crystallinity of the pristine
and treated hemp fibres was examined by using an Anton Paar
SAXSess diffractometer operating at 40 kV and 50 mA with a CuK␣
radiation source ( equal to 0.1542 nm) and an imaging plate as
detector with 2 ranging from 5 to 40◦ . The fibres were previously
cut as powder and then positioned in a holder cell for powdered
samples. The spectra were collected in transmission mode. The
data were treated first by subtracting the dark current and back-
ground and then normalized for the primary beam intensity. The
crystallinity of the samples as crystalline index was estimated from
diffraction intensity data by adopting the following empirical equa-
tion:
Itot − Iam
Cr = × 100
Itot
where Itot is the diffracted intensity of the main crystalline peak
whereas Iam is the intensity due to the amorphous phase. In
177
practice, for cellulose I the main crystalline band is peaked at 22.5◦
and the amorphous halo is at 19◦ in correspondence to the min-
imum diffracted intensity between the main and the secondary
crystalline peak (i.e. the broad peak at 16◦ ) (Thygesen et al., 2005).
In the case of cellulose II the main peak is at around 21.9◦ whereas
the amorphous halo is at 12.1◦ . There are several methods to get
an estimation of the crystallinity of cellulose materials (Thygesen
et al., 2005). The method proposed in this paper, often referred to as
peak height method has been widely used to compare the relative
changes occurring in biomass subjected to different pretreatment
conditions (Laureano-Perez et al., 2005). The peak full width at half-
maximum, which is used as estimation of the size of the crystals, is
obtained by fitting the peaks with a Lorentz function by using the
deconvolution tool of Origin software.
2.6. Cellulose nanocrystal production and characterization
Cellulose nanocrystal (CNC) suspensions were prepared from
chemically pre-treated p-Hemp and c-Hemp fibres by sulphuric
acid hydrolysis (Fortunati et al., 2012b). Moreover, the acid hydrol-
ysis was also conducted on enzymatic pre-treated c-Hemp and the
results in terms of chemical, morphological and thermal proper-
ties were compared with those offered by nanocrystals extracted
from the chemical pre-treated fibres. For all fibres, the hydrolysis
was carried out with 64% (w/w) sulphuric acid at 45 ◦ C for 30 min
with vigorous stirring. This reaction time was selected to guarantee
the reaction efficiency and avoid crystal degradation. Immediately
after the acid hydrolysis, the suspension was diluted 20 times with
deionized water to quench the reaction. The suspension was cen-
trifuged at 4500 rpm for 20 min to concentrate the cellulose crystals
and to remove the excess of aqueous acid. The resultant precipitate
was rinsed, recentrifuged, and dialyzed against deionized water for
5 days until constant neutral pH was achieved. The suspension was
sonicated repeatedly (Vibracell 75043, 750W, Bioblock Scientific) at
40% output (while cooling in an ice bath) to create cellulose crystals
of colloidal dimensions. The final yield after the hydrolysis process
was calculated as % (of initial weight) of the used pre-treated hemp
fibres.
The microstructure of CNC was investigated by means of a
transmission electron microscopy (TEM, JEOL JEM-1010), with an
accelerating voltage of 100 kV in order to well establish the dimen-
sions of the obtained cellulose nanocrystals. One drop of CNC
aqueous solution was directly placed on the electron microscopic
grids, dried at room temperature and directly observed.
Fourier infrared (FT-IR) spectra of chemically pre-treated fibres
were recorded in transmission mode, while thermogravimetric
measurements were performed using a heating scan from 30 to
600 ◦ C at 10 ◦ C min−1 in nitrogen atmosphere.
The crystallinity of cellulose nanocrystals was determined by
the powder X-ray diffraction method analysis (PXRD) using the
diffractometer of Anton Paar and the operating and data treatment
conditions, previously described.
3. Results and discussion
3.1. Effects of the carding process
The effects of the carding process were investigated by means
of morphological, thermal and chemical analysis. Carding is a
process that represents a compromise between quality improve-
ment (disentanglement and cleaning of fibres) and fibre breakage.
(1) The carding process separates the tufts into individual fibres, and
extracts the impurities and other foreign materials, aligning the
fibres. During this process, the shape and dimension stability of
natural fibres can be profoundly affected. Images of the visual
178 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186

Fig. 1. Visual observation (a and b), FESEM images (c and d) and diameter distributions (e and f) of pristine and carded hemp fibres.

observation of the fibres before and after the carding procedure
are reported in Fig. 1a and b, respectively. Before carding, p-Hemp
fibres (Fig. 1a) appear as unorganized clumps, while after the card-
ing process, the fibres result aligned and well individualized due
to the removal of the dust and impurities. Disentangling of neps
(aggregations of small fibres created during fibre processing) is also
noticed in the carding process. The morphological investigation by
FESEM of the fibres, before (Fig. 1c) and after (Fig. 1d) carding, con-
firmed that most of the impurities located at the surface of the
bundles were removed after carding and that the fibres were well
separated. Only a small variation in terms of fibre diameter (Fig. 1e
and f) was observed after carding, with a mean value of 21 ± 5 ␮m
for pristine fibre diameter and of 19 ± 3 ␮m for the carded hemp.
Moreover, a partial removal of dirty material and opening of the
fibres was observed at this stage.
The thermal degradation of the natural fibres is reported in
Fig. 2a. The thermal behaviour of the p-Hemp can be divided into
four stages: moisture evaporation, hemicellulose degradation, cel-
lulose degradation and lignin decomposition (two main peaks due
to moisture evaporation and cellulose degradation are more vis-
ible, while the other two peaks due to hemicellulose and lignin
components can be detected as shoulders of the main peak cen-
tred at nearly 350–360 ◦ C). Moisture is present in the fibre as free
water and bound water. At lower temperature (25–150 ◦ C), free
water uptaken in the fibre pores was removed, while structurally
bound water intimately physisorbed via hydrogen bonds with the
 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
Fig. 2. DTG curves (a), FT-IR spectra (b) and XRD patterns (c) of pristine and carded
hemp fibres.
hydroxyl groups of hemicellulose and lignin, evaporated at higher
temperatures. After the removal of free water, in the temperature
range between 150 and 500 ◦ C, the degradation process begins
in the cellulose, hemicellulose, lignin constituents and the asso-
ciated linked water (Kim and Eom, 2001). In c-Hemp fibres, the
peak attributed to moisture uptake at low temperature (25–150 ◦ C)
seems to be reduced, while a much narrower main derivative curve
(DTG) peak of carded hemp was detected, indicating faster kinet-
ics. Moreover, the shift of this main peak to higher temperature
(360 ◦ C for carded fibres with respect to 354 ◦ C for the pristine ones)
indicates that the improved fineness of the fibre provided higher
specific surface for the thermal decomposition of the cellulosic frac-
tions with acceleration of the thermal degradation kinetics.
179
The chemical analysis of the p-Hemp and c-Hemp was also per-
formed and the results are reported in Fig. 2b. The area between
1800 and 900 cm−1 has many absorption bands due to the various
functional groups present in each component. The bands at around
1740 cm−1 (hemicellulose), 1500 cm−1 (lignin), and 897 cm−1 (cel-
lulose) are typical for the characterization of pristine fibres. The
absorption bands at 1506, 1428, 1250 and 1031 cm−1 arise mostly
from lignin, while the bands around 1373, 1161, 1050 and 897 cm−1
are mainly due to carbohydrates (Gandolfi et al., 2013). When com-
paring with the spectrum of carded hemp fibres, it is possible to
find a slight decrease in the intensity of the peak at 1650 cm−1 , due
to the OH bending of absorbed water (confirming the reduced
water uptake already observed in Fig. 2a) and a reduced inten-
sity for the bands of lignin at 1506 cm−1 and 1250 cm−1 due the
C C aromatic symmetrical stretching and C O stretching of the
lignin component, respectively, due the removal of material by the
carding process on the raw material.
The crystalline structure of the p-Hemp and c-Hemp fibres was
investigated by XRD analysis and the results are reported in Fig. 2c.
In the native state the cellulose shows a semicrystalline structure
with crystalline domains of cellulose I with parallel chains con-
nected side-by-side via hydrogen bonding in flat sheets embedded
in an amorphous phase made by lignin and hemicellulose. Regen-
eration and mercerization of cellulose based materials induce a
transformation from cellulose I structure to cellulose II wherein
the chains are stacked to form corrugated sheets bound together
by a hydrogen bonding network. The XRD pattern of p-Hemp fibre
exhibits four diffraction peaks. The main peak at 22.5◦ , correspond-
ing to (0 0 2) lattice plane, is indicative of the distance between
hydrogen bonded sheets in cellulose I domains. The other three
peaks correspond to the doublet peaks at 14.8◦ and 16.3◦ assigned
to (1 0 1) and (1 0 1) lattice plane and the peak at 34.5◦ which arises
from ordering along the fibre direction and it is sensitive to the
alignment of chains into the fibrils (Besbes et al., 2011). The c-
Hemp fibres exhibit qualitatively the same diffraction pattern of
pristine fibres with a slight increase of the intensity of the peak at
34.5◦ . The crystalline indexes of pristine and carded hemp fibres,
based on the peak height method, are calculated to be 0.83 and
0.85, respectively. These values are comparable to the value of 0.88
determined by Mwaikambo and Ansell (2002) for hemp fibres. The
full width half maximum (FWHM) of the main peak does not change
with the carding process. These data confirm that the carding pro-
cess has only a modest effect on the intrinsic structure of fibres,
while its action is more effective in removing of the impurities on
the fibre surface as confirmed by the other analysis; the increase
of the crystalline index may be ascribed to a partial removal of the
non-cellulosic components from the fibres.
3.2. Chemically treated fibres
Fig. 3 shows the morphological, chemical and thermal charac-
terization of chemically pre-treated p-Hemp and c-Hemp. After
the chemical treatment, both pristine (Fig. 3a) and carded fibres
(Fig. 3b) appear separated into individual micro-sized structures
with a variation with respect to the original dimensions of the
untreated fibres (see Fig. 1c and d for untreated pristine and carded
fibres, respectively). A diameter of 16 ± 5 ␮m was measured for
pre-treated p-Hemp fibres and similar values were detected for the
pre-treated c-Hemp that showed a diameter of 15 ± 4 ␮m. More-
over, in both cases, the fibres appear well individualized and with a
regular, smooth and clean surface, while each elementary filament
shows a compact structure and very long entangled cellulosic fila-
ments. An evident defibrillation process occurred for both hemp
structures studied as a consequence of the lignin and hemicel-
lulose removing effort by the chemical treatments (Kabir et al.,
2013). Moreover, more isolated and individualized fibres were
180 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
Fig. 3. FESEM images (a and b), DTG curves (c), FT-IR spectra (d) and XRD patterns (e) for chemically pre-treated pristine and carded hemp fibres.
evident in the case of carded hemp (Fig. 3b) highlighting the effect
of the mechanical process that, disentangling the fibres, guarantees
a more suitable structure for the subsequent treatment.
This result was confirmed by thermal analysis as reported in
Fig. 3c. For both hemp structures studied, the main degradation
peak was shifted to lower temperatures (309 ◦ C with respect to the
initial 354 ◦ C for pristine fibres and 327 ◦ C with respect to the initial
360 ◦ C for the carded fibres). A higher thermal stability character-
izes the pre-treated c-Hemp fibres, a result which confirmed the
positive influence of the carding procedure. Moreover, a less intense
peak centred at 264 ◦ C attributed to the decomposition of light
fractions (pectin and hemicellulose), was detected for pre-treated
c-Hemp with respect to the pre-treated p-Hemp fibres (Fisher et al.,
2002).
The chemical analysis confirmed the results of the thermogravi-
metric analysis. The pre-treated p-Hemp fibres show a peak due to
the residual hemicellulose at 1750 cm−1 than was not so evident
for the carded fibres, underlining the efficiency of carding in the
following alkaline attack. Moreover, for both pre-treated p-Hemp
and c-Hemp, a peak at 1061 cm−1 due to the shift of the origi-
nal 1050 cm−1 related to the xylane and the glycosidic linkages of
hemicellulose, was detected. This shift proves the removal of hemi-
cellulose after treatment, more evident in the case of carded hemp
(Fig. 3d).
The peak at 1373 cm−1 was due to the C OH stretching of
the hydrogen bond intensity of crystalline cellulose. The peak
observed at 897 cm−1 in both pre-treated p-Hemp and c-Hemp
fibres indicates the presence of the glycosidic linkages between
the monosaccharides (Kabir et al., 2013). In Fig. 3e the XRD pat-
terns of chemically pre-treated p-Hemp and c-Hemp fibres are
reported. For p-Hemp, after the alkaline pre-treatment, the main
peaks ascribed to the cellulose I crystalline structure disappear and
a broad asymmetric peak consisting of a doublet at 20◦ and 21.7◦
appears; the intensity of both peaks at around 16◦ and 34.5◦ reduces
whereas a new peak at around 12.1◦ emerges. All these changes
indicate a partial transformation from the cellulose I to the cellu-
lose II structure (Kumar et al., 2010). In fact the presence of the
peak at 21.7◦ along with the presence of other specific peaks at
around 16◦ and 34.5◦ confirm the coexistence of cellulose I domains
embedded in the treated fibres. The same diffraction features can be
observed for c-Hemp fibres treated with alkaline solution. It is well
known that lignin and hemicellulose located between the microfib-
rils, restrict the transformation of cellulose I to cellulose II. With the
NaOH treatment, the lignin and more in general the non-cellulosic
materials, are partially removed, as confirmed by other analysis,
and the crystalline cellulose I can be partially re-arranged in the cel-
lulose II crystalline structure (Abrahama et al., 2011). The intensity
ratio I21.7 /I20 may be used to have a rough estimation of the rela-
tive amount of cellulose I with respect to cellulose II (Mandal and
Chakrabarty, 2011). In this specific case, this ratio appears to be 0.80
for c-Hemp fibres and 0.86 for p-Hemp fibres. This result confirms
that the presence of lignin and hemicellulose hinders the transfor-
mation of cellulose I in II, which probably is controlled by diffusion
of alkaline solution from the external towards the internal part of
the fibres. Finally additional small peaks well evident for pristine
fibre at around 32.3◦ and 33.9◦ do not appear for the carded fibres.
These peaks can be tentatively ascribed to the modifications that
occur during the alkaline treatment in the hemicellulose and lignin
amorphous phases. The crystalline index of cellulose I of p-Hemp
and c-Hemp fibres, based on the peak height method for the peak at
21.7◦ , is calculated to be 0.81 and 0.79, respectively. The FWHM of
the doublet main peaks decrease for the c-Hemp fibres indicating
increased ordering of both the cellulose I and the cellulose II or an
increase in the size of cellulose crystallites.
These results confirm the higher efficiency of the chemical pre-
treatment applied to the c-Hemp suggesting the possibility to use
the mechanical process as a preparation procedure of the fibre in
order to enhance the alkali treatment effect.
 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
Fig. 4. Panel A: Scheme of optimization procedure before the enzymatic treatment. Panel B: FESEM investigation of sonicated and thermal treated/sonicated carded fibres.
3.3. Enzymatically treated c-Hemp fibres
3.3.1. Effect of the sonication and thermal treatments
In order to increase the efficiency of the enzymatic pre-
treatment on the carded fibres, two different procedures were
carried out before the exposition of the micro-fibres to pectinase,
as represented in Fig. 4, panel A. The effect of standard sonication
in a sonication bath at 80 ◦ C (Fig. 4, panel A-a) and the combina-
tion of the sonication with a previous thermal process at 150 ◦ C
(Fig. 4, panel A-b) was investigated by morphological and thermal
analyses.
FESEM investigation (Fig. 4, panel B) confirmed that the ultra-
sonic treatment was powerful to separate the fibres but not
completely successful to remove adhesive substances and non-
cellulosic components from the fibre surface (Kovur et al., 2008). A
preliminary thermal treatment at 150 ◦ C before sonication was able
to eliminate waxes from the surfaces of the fibres, offering a smooth
surface of the c-Hemp after this specific treatment. Moreover, the
thermal characterization by TGA underlines that both procedures,
sonication and sonication plus thermal treatment, do not affect the
thermal degradation of the raw material (data not shown). On the
basis of this result, the combination of thermal and sonication pro-
cess was selected as a good pre-treatment step for c-Hemp fibres
before the enzymatic procedure.
3.3.2. Chemical vs enzymatic treatment of c-Hemp fibres
The results of morphological investigation of the fibres after the
two different treatments, chemical and enzymatic, are reported in
Fig. 5 (panel A). It is possible to observe that for both chemical and
enzymatic treated c-Hemp fibres a change on the original dimen-
sions of the fibres 19 ± 3 ␮m was registered. Specifically, chemically
treated c-Hemp fibres (Fig. 5, panel A-a and b) appear separated
into individual micro fibrils with a mean diameter of 15 ± 2 ␮m,
similar values were detected for the enzymatic carded hemp fibres
181
that showed a mean diameter value of 13 ± 2 ␮m (Fig. 5, panel
A-c and d). As reported by Pakarinen et al. (2012) the refin-
ing of technical fibres produced by steam explosion in response
to pectinase treatment is accomplished by selective removing of
waxy epidermal tissue, adhesive pectins, hemicellulose that bind
fibre bundles among them and to pectin. Moreover, enzymatically
treated c-Hemp fibres show a less rough surface, indicating a more
effective removal of debris and non-cellulosic components from
fibres (Ouajai and Shanks, 2005). From the point of view of thermal
behaviour, DTG of c-Hemp fibres after interaction with pectinase
in comparison with chemically pre-treated c-Hemp (Fig. 5, panel
B-a) reveals a quite different profile due to the total decompo-
sition of pectin and hemicellulose fractions that are still visible
in the case of chemical pre-treatment. The strong chemical pre-
treatment to which the carded fibres are subjected decreased the
thermal stability of the fibres, while this effect was not visible
in the case of enzyme treated c-Hemp fibres. As reported by Li
and Pickering (2008), the initial decomposition temperatures of
the fibres, for the two different treatments, are completely differ-
ent: the chemically pre-treated c-Hemp fibres started to degrade
at about 196 ◦ C, while this value increased to 286 ◦ C for the enzy-
matic treated c-Hemp fibres. These results indicate an increase in
the thermal stability of the treated fibres due to the reduced amount
of non-cellulosic material present in the fibre and the presence of
high crystalline cellulosic components. This was probably because
more non-cellulosic material was removed during the treatments
and a high degree of structural order was expected. This revealed
a relationship between the structure and the thermal degradation
of cellulose. A greater crystalline structure would require a higher
degradation temperature (Kim et al., 2010; Ouajai and Shanks,
2005). The XRD diffraction patterns for carded fibres exposed to
enzymatic and chemical treatments (Fig. 5, panel B-b) confirm the
result of the thermal analysis. The c-Hemp fibres, subjected to the
enzymatic treatment, exhibit the diffraction features characteristic
182 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186

Fig. 5. Panel A: FESEM investigations of chemically (a and c) and enzymatically (b and d) pre-treated carded fibres. Panel B: DTG curves (a), XRD patterns (b) and FT-IR spectra
(c) for chemically and enzymatically pre-treated carded hemp fibres.

of cellulose I crystalline domains. For comparison, the XRD pattern
of c-Hemp fibres submitted to chemical treatment is also reported,
displaying the characteristic features of cellulose II formed from
cellulose I. The XRD results show that the enzymatic treatment does
not affect significantly the crystalline index of c-Hemp fibres, which
results to be equal to 0.84, whereas the peak at 34.5◦ increases
significantly after the treatment.
The results of the chemical analysis (Fig. 5, panel B-c) of the c-
Hemp fibres after the enzymatic treatment confirm the results of
the thermal and structural investigations. Even if the spectrum of
the c-Hemp fibre is in general similar to that of the enzyme treated
hemp, some changes can be detected. In fact, an increase in inten-
sity of the 897 cm−1 band, assigned to the C O C stretching of the
␤-(1→4)-glycosidic linkage in cellulose and a decrease in intensity
of the 1637 cm−1 band attributed to bending mode of absorbed
water, confirm the effects of fibre cleaning due to the enzyme action
and the major exposition of the cellulosic fraction. Moreover, other
peaks corresponding to cellulose (1316 cm−1 and 1370 cm−1 ) show
higher intensity, and the peaks at 1160 and 1129 cm−1 assigned to
the C O stretching vibration show relatively higher intensity after
the enzyme treatment (Li and Pickering, 2008; Ouajai and Shanks,
2005).
3.4. Cellulose nanocrystals extracted from chemically pre-treated
p-Hemp and c-Hemp
After the fibre treatment, the obtained micro-fibres were
hydrolyzed with a sulphuric acid process in order to produce
 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
Fig. 6. TEM images (a and b), DTG curves (c), FT-IR spectra (d) of cellulose nanocrystals prepared from pristine and carded chemically pre-treated hemp fibres.
cellulose nanocrystals (CNC). CNC suspensions were prepared from
pristine and carded chemically pre-treated fibres. The resulting cel-
lulose nanocrystal aqueous suspensions were approximately 0.6%
(w/w) and the hydrolysis yield was ca. 19%. This value is close to
the lower limit estimated for an optimized hydrolysis process and
no particular differences in the reaction yield values were detected
related to the two starting materials.
Fig. 6a and b shows the typical needle-like structure of CNC.
TEM images evidence the effectiveness of the acid hydrolysis
treatment, confirming that the aqueous suspensions contained cel-
lulose nanocrystals consisting mostly of individual crystals. TEM
images show that most of the cellulose nanocrystals obtained from
the hydrolysis of both pristine and carded chemically pre-treated
fibres are characterized by an acicular structure typical of cellulose
nanocrystals or whiskers. No particular differences in the shape and
dimensions of CNC extracted from the two fibre typologies occurred
since the dimensions of the nanocrystals obtained in both cases
ranged from 100 to 200 nm in length and around 15 nm in width
(Fortunati et al., 2012b).
The results of the thermal degradation of CNC structures per-
formed on both carded and pristine pre-treated fibres are reported
in Fig. 6c. It has been reported (Wang et al., 2007) that in the two-
step pyrolysis for acid-treated nanocellulose crystals, the first peak
can be attributed to acid sulphate groups on the crystal surface.
Since the extent of the first pyrolysis process depends on the actual
number of sulphate groups on the crystal surface, acid treated
183
crystals obtained from pristine pretreated fibres seems to facili-
tate the formation of sulphate groups on the surface of the CNC
and resulted less thermally stable if compared with CNC from pre-
treated c-Hemp fibres. Moreover, while in the case of pre-treated
c-Hemp fibres there is no variation in the main registered peak
temperature (327 ◦ C) and only one main peak was detected in
the degradation profile of CNC, in the case of pre-treated p-Hemp
fibres two well-separated pyrolysis processes in the DTG curves are
observed. From the comparison of the decomposition temperature
related to the main degradation peak (354 ◦ C with respect to the
initial 309 ◦ C for pristine fibres and 327 ◦ C for the carded fibres),
it can be observed that the degradation of the cellulose molecules
was initiated at lower temperatures in the case of carded fibres
with respect to pre-treated p-Hemp fibres, likely due to the weaker
interaction of OH groups in cellulose that require less energy to
start the thermal degradation process. It has been observed that
higher crystallite size cellulose have also higher thermal stability.
Kim et al. (2010) studied different types of cellulose and reported an
increase of thermal stability promoted by a higher crystallite size.
Therefore, the different thermal stability of CNC from pre-treated p-
Hemp and c-Hemp fibres could be probably due to different amount
of hydrogen bonds between cellulose chains that can lead to differ-
ent ordered and packed cellulose regions; this can probably modify
the thermal decomposition temperature of cellulose (Kim et al.,
2010; Ouajai and Shanks, 2005). Fig. 6d shows that the fingerprint
region of the spectra of CNC obtained from pre-treated p-Hemp and
184 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186

c-Hemp fibres revealed common and easily identifiable bands as,

for example, adsorbed water in cellulose (1637 cm−1 ), and bands
at 1428, 1373, 1339 and 1317 cm−1 attributed to CH2 symmetric
bending, CH bending, in-plane OH bending, CH2 rocking vibration,
respectively (Chen et al., 2010). Moreover, no difference can be
detected in the two spectra for CNC samples. Furthermore, the
signals at 1162, 1111, 1061, 1033, 897 cm−1 that are assigned to
asymmetric C O C bridge stretching, anhydroglucose ring asym-
metric stretching, C O stretching, in-plane C H deformation and
C H deformation of cellulose, respectively (Corgié et al., 2011) did
not change in the two different spectra.
Fig. 7 reports the XRD patterns of cellulose nanocrystals pre-
pared from p-Hemp and c-Hemp chemically pre-treated fibres.
The cellulose nanocrystals obtained by both p-Hemp and c-Hemp
chemically pre-treated hemp fibres show the characteristic diffrac-
tion features of cellulose II crystalline domains. However, the
presence of small peaks at around 34.5◦ and a broad halo around
14◦ –16◦ together with the peak at 21.7◦ confirm that cellulose I
domains are still present. From the intensity ratio I21.7 /I20 which is Fig. 7. XRD patterns of cellulose nanocrystals prepared from pristine and carded
chemically pre-treated hemp fibres.
equal to 0.88 and 0.83 for p-Hemp and c-Hemp fibres respectively,
it is inferred that the carding process allows a higher conversion of
cellulose I in cellulose II justifying also the decreased thermal sta- contrast, the FWHM of both the cellulose I and II doublet peaks do
bility observed in DTG curves related to the presence of a less pure not change with respect to the pristine CNC, indicating similar size
cellulosic material (Ouajai and Shanks, 2005). These results confirm of the cellulose I and II crystallites.
that the carding process allows a higher conversion of cellulose I in
cellulose II. The crystalline index, based on the peak height method 3.5. CNC from enzymatically pre-treated c-Hemp
for the peak at 21.7◦ , is equal to 0.87 and 0.90 for CNC from p-
Hemp and c-Hemp pre-treated fibres, respectively. This high value The c-Hemp fibres, after the enzymatic treatment, were
could be due to the removal of amorphous non-cellulosic or amor- hydrolyzed with the same sulphuric acid process conducted for
phous cellulosic compounds during the hydrolysis procedure. In the chemically pre-treated c-Hemp fibres, in order to produce

Fig. 8. TEM image of CNC obtained from carded fibre after enzymatic treatment (a), DTG curves (b), FT-IR spectra (c) of cellulose nanocrystals prepared from chemically or
enzymatically pre-treated hemp fibres and XRD pattern for CNC crystals obtained from carded fibre after enzymatic treatment (d).
 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
cellulose nanocrystals and to compare the effects of chemically
and enzymatic treatments on the CNC extraction process. The
resulting concentration of cellulose nanocrystal aqueous suspen-
sion in this case was approximately 0.5% (w/w) and the hydrolysis
yield was very similar to the previous results discussed for the
other procedure (ca. 19%), underlining that no particular differ-
ences are revealed in the reaction efficiency due to the different
pre-treatment procedures of the fibres.
The morphological investigation (TEM, Fig. 8a) underlines that
CNC extracted from c-Hemp after the enzymatic procedure show
the same shape and dimensions just discussed for the CNC
extracted from the alkaline treated fibres.
The results of the thermal analysis of CNC extracted from
enzymatically pre-treated fibres in comparison with chemically
pre-treated fibres confirmed that no substantial differences can be
detected in the DTG profiles of the two cellulosic structure (Fig. 8b).
On the other hand, the thermal stability of CNC from enzymati-
cally treated fibres seems to be affected (the temperature of the
main peak shifted from 362 ◦ C to 314 ◦ C) when compared with the
results of the TGA analysis of reference fibres (see Fig. 5, panel B-
a). As reported by Henriksson et al. (2007) the pre-treatment of
fibres with a very low enzyme concentration (0.02%) preserved the
molecular weight and length of cellulose nanofibres, but it was also
able to attack the amorphous regions of cellulose chains, making it
is easier to separate the cellulose as nanosized structures by sub-
sequent acid attack. According to this behaviour, it is reasonable to
conclude that enzymatically pre-treated fibres could be more easily
attacked by the acid and consequently would reduce the thermal
stability of the produced CNC structures.
FT-IR spectra (Fig. 8c) for the same systems showed several
common characteristic peaks and prominent changes. The peak at
1637 cm−1 can be attributed to the O H bending of the absorbed
water while the vibration peak detected at 1375 cm−1 is related to
the bending vibration of the C H and C O bonds in the polysaccha-
ride aromatic rings. Moreover, the peak observed at 1061 cm−1 is
due to the C O C pyranose ring skeletal vibration. Another signifi-
cant band, which is more evident in the case of CNC from chemically
pre-treated carded fibres, is the one at 897 cm−1 , which corre-
sponds to glycosidic C H deformation, with a ring vibration
contribution and O H bending, characteristic of the ␤-glycosidic
linkage between the anhydroglucose units in cellulose (Alemdar
and Sain, 2008) and could be related to a different chemical struc-
ture of the two CNC samples.
Fig. 8d shows the XRD pattern of the CNC crystals obtained
from carded fibres after the enzymatic treatment. The CNC crys-
tals extracted from c-Hemp fibre pre-treated with the enzymatic
process show the diffraction features typical of cellulose I crystal
domains. The crystal index is equal to 0.9 indicating a high extent
of crystalline domains. However, the enzymatic pre-treatment
resulted in major changes in X-ray diffraction patterns of carded
hemp fibres. The typical cellulose pattern, already observed for
CNC extracted from chemically pre-treated fibres, shows addi-
tional well-defined peaks at 29.4◦ , 35.8◦ and 39.8◦ . These diffraction
peaks, which correspond to very short atomic interactions, may be
tentatively ascribed to the formation of new crystalline domains
likely originated from the enzymatic degradation of cellulose I or
amorphous cellulose (Henriksson et al., 2007). In fact, the enzy-
matic treatment might depolymerize the cellulose macromolecules
with the formation of shorter chains able to form denser crystalline
domains. A similar behaviour was observed during the enzymatic
degradation of pectin (Gnanasambandam and Proctor, 1999).
4. Conclusions
The extraction of cellulose nanocrystals has been successfully
carried out from hemp fibres of the Carmagnola cultivar. Pristine
185
and carded fibres were pre-treated by both an alkaline chemical
procedure and an enzymatic treatment through the action of pecti-
nase, and the effects of the two procedures on the thermal, chemical
and structural properties of the obtained fibres were compared.
Carding allowed removal of most impurities from the fibres and
thermal analysis confirmed that the improved fineness of the fibre
provided higher specific surface for the thermal decomposition of
the cellulosic fractions, while carding has only a modest effect on
fibre structure. After chemical treatment, hemicellulose is mainly
removed, more evidently in the case of c-Hemp. X-ray diffraction
data suggest that an increased ordering of cellulose I and cellulose
II structures results in a likely increase in the size of cellulose crys-
tallites. Carded hemp fibres after interaction with pectinase show
the total decomposition of pectin and hemicellulose fractions, as an
effect of the higher efficiency of the chemical pre-treatment. The
enzymatic treatment does not affect the crystallinity of c-Hemp
fibres, whilst increases significantly the alignment of the cellulose
chains in the fibre direction.
Cellulose nanocrystals were extracted by acid hydrolysis from
c-Hemp after both chemical and enzymatic procedure. TEM anal-
ysis showed that most cellulose nanocrystals, obtained from the
hydrolysis of both chemically pre-treated p-Hemp and c-Hemp
fibres, were characterized by an acicular structure typical of cel-
lulose nanocrystals and in both the cases CNC had the dimensions
ranging from 100 to 200 nm in length and 15 nm in width. The yield
of the different CNC extractions remains approximately at the same
level (around 19%) and no substantial differences can be detected
in the thermogravimetric profiles of the two cellulosic structures.
However, the thermal stability of CNC obtained from enzymatically
treated fibres seemed to be rather compromised, as the main degra-
dation peak decreased from 362 to 314 ◦ C while the enzymatic
pre-treatment resulted in major changes in X-ray diffraction pat-
terns of carded hemp fibres. For these reasons, a carding procedure
combined with an alkaline treatment to hemp fibres represents the
optimized process for the CNC extraction by acid hydrolysis.
This research evidences the success of the applied extrac-
tion procedures and the possibility to obtain a filler with high
potential characteristics to be employed in the production of bio-
nanocomposites with enhanced properties.
Acknowledgments
The authors gratefully acknowledge Assocanapa for supply-
ing carded and non-carded hemp fibres. The Authors gratefully
acknowledge Prof. Juan López Martínez (Instituto de Tecnología
de Materiales, Universitat Politècnica de València, Spain) and Dr.
Marina P. Arrieta for TEM examinations.
References
Abrahama, E., Deepaa, B., Pothana, L.A., Jacobc, M., Thomasb, S., Cvelbard, U., Anand-
jiwalac, R., 2011. Extraction of nanocellulose fibrils from lignocellulosic fibres:
a novel approach. Carbohyd. Polym. 86, 1468–1475.
Akin, D.E., Foulk, J.A., Dodd, R.B., McAlister III, D.D., 2001. Enzyme-retting of flax and
characterization of processed fibers. J. Biotechnol. 89, 193–203.
Alemdar, A., Sain, M., 2008. Biocomposites from wheat straw nanofibres: morphol-
ogy, thermal and mechanical properties. Compos. Sci. Technol. 68, 557–565.
Besbes, I., Vilar, M.R., Boufi, S., 2011. Nanofibrillated cellulose from TEMPO oxidized
eucalyptus fibres: effect of the carboxyl content. Carbohyd. Polym. 84, 975–983.
Chen, H., Ferrari, C., Angiuli, M., Yao, J., Raspi, C., Bramanti, E., 2010. Qualitative
and quantitative analysis of wood samples by Fourier transform infrared spec-
troscopy and multivariate analysis. Carbohydr. Polym. 82, 772–778.
Collet, F., Achchaq, F., Djellab, K., Marmoret, L., Beji, H., 2011. Water vapor proper-
ties of two hemp wools manufactured with different treatments. Constr. Build.
Mater. 25, 1079–1085.
Corgié, S.C., Smith, H.M., Walker, L.P., 2011. Enzymatic transformations of cel-
lulose assessed by quantitative high-throughput Fourier transform infrared
spectroscopy (QHT-FTIR). Biotechnol. Bioeng. 108, 1509–1520.
Croxford, J.L., Pryce, G., Jackson, S.J., Ledent, C., Giovannoni, G., Pertwee, R.G.,
Yamamura, T., Baker, D., 2008. Cannabinoid-mediated neuroprotection, not
186 F. Luzi et al. / Industrial Crops and Products 56 (2014) 175–186
immunosuppression, may be more relevant in multiple sclerosis. J. Neuroim-
munol. 193, 120–129.
Dai, D., Fan, M., Collins, P., 2013. Fabrication of nanocelluloses from hemp fibers
and their application for the reinforcement of hemp fibers. Ind. Crop Prod. 44,
192–199.
Fisher, T., Hajaligol, M., Waymack, B., Kellogg, D., 2002. Pyrolysis behavior and kinet-
ics of biomass derived materials. J. Anal. Appl. Pyrolysis 62, 331–349.
Fortunati, E., Puglia, D., Monti, M., Peponi, L., Santulli, C., Kenny, J.M., Torre, L., 2012a.
Extraction of cellulose nanocrystals from Phormium tenax fibres. J. Polym. Envi-
ron. 21, 319–328.
Fortunati, E., Armentano, I., Zhou, Q., Iannoni, A., Saino, E., Visai, L., Berglund,
L.A., Kenny, J.M., 2012b. Multifunctional bionanocomposite films of poly(lactic
acid), cellulose nanocrystals and silver nanoparticles. Carbohyd. Polym. 87,
1596–1605.
Fortunati, E., Puglia, D., Monti, M., Santulli, C., Maniruzzaman, M., Kenny, J.M., 2013a.
Cellulose nanocrystals extracted from okra fibres in PVA nanocomposites. J.
Appl. Polym. Sci. 128, 3220–3230.
Fortunati, E., Puglia, D., Monti, M., Peponi, L., Santulli, C., Kenny, J.M., Torre, L., 2013b.
Extraction of cellulose nanocrystals from phormium tenax fibres. J. Polym. Envi-
ron. 21, 319–328.
Fortunati, E., Puglia, D., Luzi, F., Santulli, C., Torre, L., Kenny, J.M., 2013c. Binary PVA
bio-nanocomposites containing cellulose nanocrystals extracted from different
natural sources: Part I. Carbohyd. Polym. 97, 825–836.
Gandolfi, S., Ottolina, G., Riva, S., Pedrocchi Fantoni, G., Patel, I., 2013. Complete
chemical analysis of carmagnola hemp hurds and structural features of its com-
ponents. Bioresources 8, 2641–2656.
Gnanasambandam, R., Proctor, A., 1999. Preparation of soy hull pectin. Food Chem.
65, 461–467.
Henriksson, M., Henriksson, G., Berglund, L.A., Lindström, T., 2007. An environ-
mentally friendly method for enzyme-assisted preparation of microfibrillated
cellulose (MFC) nanofibers. Eur. Polym. J. 43, 3434–3441.
Kabir, M.M., Wang, H., Lau, K.T., Cardona, F., 2013. Effects of chemical treatments on
hemp fibre structure. Appl. Surf. Sci. 276, 13–23.
Kim, U.J., Eom, S.H., Wada, M., 2010. Thermal decomposition of native cellulose:
influence on crystallite size. Polym. Degrad. Stab. 95, 778–781.
Kim, H.J., Eom, Y.G., 2001. Thermogravimetric analysis of rice husk flour for a
new raw material of lignocellulosic fiber thermoplastic polymer composites.
J. Korean Wood Sci. Technol. 20, 59–67.
Kovur, S.K., Schenzel, K.C., Grimm, E., Diepenbrock, W., 2008. Characterization of
refined hemp fibers using nirftraman micro spectroscopy and environmental
scanning electron microscopy. Bioresources 3, 1081–1091.
Kumar, S., Gupta, R., Lee, Y.Y., Gupta, R.B., 2010. Cellulose pretreatment in subcritical
water: effect of temperature on molecular structure and enzymatic reactivity.
Bioresour. Technol. 101, 1337–1347.
Laureano-Perez, L., Teymouri, F., Alizadeh, H., Dale, B., 2005. Understanding factors
that limit enzymatic hydrolysis of biomass. Appl. Biochem. Biotechnol. 121–124,
1081–1099.
Li, S.Y., Stuart, J.D., Li, Y., Parnas, R.S., 2010. The feasibility of converting Cannabis
sativa L. oil into biodiesel. Bioresour. Technol. 101, 8457–8460.
Li, X., Tabil, L.G., Panigrahia, S., 2007. Chemical treatments of natural fiber for
use in natural fiber-reinforced composites: a review. J. Polym. Environ. 15,
25–33.
Li, Y., Pickering, K.L., 2008. Hemp fibre reinforced composites using chelator and
enzyme treatments. Compos. Sci. Technol. 68, 3293–3298.
Mandal, A., Chakrabarty, D., 2011. Isolation of nanocellulose from waste sugarcane
bagasse (SCB) and its characterization. Carbohyd. Polym. 86, 1291–1299.
Morán, J.I., Alvarez, V.A., Cyras, V.P., Vázquez, A., 2008. Extraction of cellulose and
preparation of nanocellulose from sisal fibers. Cellulose 15, 149–159.
Mwaikambo, L.Y., Ansell, M.P., 2002. Chemical modification of hemp, sisal, jute, and
kapok fibers by alkalization. J. Appl. Polym. Sci. 84, 2222–2234.
Ouajai, S., Shanks, R.A., 2005. Morphology and structure of hemp fibre after bioscour-
ing. Macromol. Biosci. 5, 124–134.
Pakarinen, A., Zhang, J., Brock, T., Maijala, P., Viikari, L., 2012. Enzymatic accessibility
of fiber hemp is enhanced by enzymatic or chemical removal of pectin. Bioresour.
Technol. 107, 275–281.
Thygesen, A., Oddershede, J., Lilholt, H., Thomsen, A.B., Stahl, K., 2005. On the deter-
mination of crystallinity and cellulose content in plant fibres. Cellulose 12,
563–576.
Wang, N., Ding, E., Cheng, R., 2007. Thermal degradation behaviors of spherical
cellulose nanocrystals with sulfate groups. Polymer 48, 3486–3493.