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Preparation of Nanocellulose: A Review

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 AATCC Journal of Research

Preparation of Nanocellulose: A Review

By Mohammad Tajul Islam and Mohammad Mahbubul Alam, Politecnico di Torino; and Alessia Patrucco, Alessio Mon-
tarsolo, and Marina Zoccola, Istituto per lo Studio delle Macromolecole
Abstract
The development of nanocellulose has attracted significant interest in the last few decades due to its unique and potentially
useful features. Novel nanocelluloses boost the strongly expanding field of sustainable materials and nanocomposites.
Their potential areas of application include reinforcing agents in nanocomposites, paper, biodegradable films, barriers
for packaging, stabilizing agents in dispersions for technical films and membranes, additives in food, texturing agents in
cosmetics, and medical devices such as wound dressings and bioactive implants. This review organizes current knowledge
on the isolation of microfibrillated and nanofibrillated cellulose from plant sources. Details of the extraction of fibrils from
cellulose are reviewed. In addition, the terms cellulose “microfiber” and cellulose “nanofiber” are formally defined and
distinguished.
Key Terms
Microfibrillated Cellulose, Nanocellulose, Nanocomposites, Nanofibril Isolation

Introduction growing publication activity. The separation of plant fibers

Polymer nanocomposites have been a subject of increas-
ing interest in recent years because of their significantly
enhanced mechanical properties and thermal stability versus
neat polymers or conventional polymer composites.1 Nano-
scale cellulose fiber materials serve as promising candidates
for bio-nanocomposite production due to their abundance,
high strength and stiffness, low weight, and biodegradabil-
ity.2 Isolation, characterization, and search for applications
of novel forms of cellulose—variously termed crystallites,
nanocrystals, whiskers, nanofibrils, and nanofibers—are cur-
rently generating much activity.
Isolated cellulosic materials with one dimension in the
nanometer range are referred to generically as nanocel-
luloses. In a unique manner, these nanocelluloses combine
important cellulose properties such as hydrophilicity, broad
chemical-modification capacity, and the formation of versa-
tile semicrystalline fiber morphologies. These morphologies
have the specific features of nanoscale materials due to their
very large surface area. Cellulose fibrils with widths in the
nanometer range are nature-based materials with unique
and potentially useful features. Most importantly, these
novel nanocelluloses allow new uses of natural cellulose
polymers in the expanding fields of nanocomposites and
sustainable materials. Nanocelluloses have high surface areas
resulting in powerful interactions with surrounding species,
such as water, organic and polymeric compounds, nanopar-
ticles, and living cells.3
So far, a number of automotive components, appliances,
and packaging products are now being manufactured using
thermoplastic and thermoset natural fiber composites.4
Application of cellulose nanofibers in polymer reinforce-
ment is a relatively new research field, although there is
into smaller elementary constituents has typically been a
challenging process with high energy demands, thereby
limiting cellulose nanofiber application development. More
recently, there was a focus on energy-efficient production
methods, whereby fibers are pretreated by various physi-
cal, chemical, and enzymatic methods to decrease energy
consumption. Dufresne, Klemm, Siro, Habibi and Azizi
Samir have written excellent general reviews on the entire
field—from nanocellulose preparation and modification to
application in composites.2,3,5-7 This review specifically cov-
ers various approaches to the preparation of nanocellulosic
materials from plant sources. The focus is on the extraction
and investigation of microfibrillated cellulose (MFC) and
nanocrystalline cellulose (NCC) in particular.
Classification
A classification of nanocellulose material is given in Table I.
Nanocellulose nomenclature has not been used in a com-
pletely uniform manner in the past. Klemm et al.3 have
used the terms MFC, NCC, and BNC, the latter stand-
ing for bacterial nanocellulose. According to them “the
name microfibrillated cellulose (MFC) was coined by the
original investigators and is widely used in the scientific
and commercial literature, whereas NCC and BNC seem
simple and descriptive. Maybe, over time, the nomenclature
‘nanofibrillated cellulose’ will prevail, and the nanocellulose
terminology will become more consistent.”
Nanocellulose
A cellulose fiber consists of bundles of single cellulose fibers,
which have diameters of 25–30 µm. This single cellulose
fiber is made up of bundles of microfibers, which have diam-
eters of 0.1–1 µm. Nanofibers, which have a diameter in the

September/October 2014 Vol. 1, No. 5 | 17

 AATCC Journal of Research
Table I.
The Family of Nanocellulose Materialsa
Type of Nanocellulose Synonyms
Microfibrillated Microfibrillated cellulose,
Cellulose nanofibrils and microfibrils,
(MFC) nanofibrillated cellulose
Nanocrystalline Cellulose nanocrystals, crystallites,
Cellulose whiskers,
(NCC) rodlike cellulose
microcrystals
Bacterial Nanocellulose Bacterial cellulose, microbial
(BNC) cellulose,
biocellulose
Reprinted with permission.3 Copyright 2011 John Wiley and Sons.
a
range of 10–70 nm and lengths of thousands of nanometers,
are the constituent of microfibers.8 The cellulose microfiber
has been defined as a fiber consisting of continuous cellulose
chains with negligible lignin and hemicelluloses content
and having a diameter of 0.1–1 µm, with a minimum cor-
responding length of 2–20 µm.9 Traditionally, cellulose
nanofiber has been defined as purely crystalline cellulose
chains having diameters within the range of 5 to 40 nm with
lengths of a few microns.10
Sources
Wood
Mechanical extraction of nanofibers from wood dates back
to the 1980s, when researchers produced MFC from wood
pulp using cyclic mechanical treatment in a high-pressure
homogenizer. The homogenization process resulted in
disintegration of the wood pulp and a material in which the
fibers were opened into their sub-structural microfibrils.
The resulting MFC gels consisted of strongly entangled and
disordered networks of cellulose nanofiber. Bleached kraft
pulp has often been used as the starting material for research
on MFC production.2
Agricultural Crops and By-Products
Wood is certainly the most important industrial source of
cellulosic fibers. Nevertheless, competition from different
sectors, such as the building products, furniture industries,
and the pulp and paper industry, as well as the combus-
tion of wood for energy, makes it challenging to supply all
users with the quantities of wood needed at reasonable cost.
As a result, fibers from crops such as flax, hemp, sisal, and
others, especially from by-products of these different plants,
are likely to become of increasing interest. Other possible
examples of agricultural by-products which might be used
to derive nanocellulose include those obtained from the
cultivation of corn, wheat, rice, sorghum, barley, sugar cane,
18 | Vol. 1, No. 5 September/October 2014
Typical Sources Formation and Average Size
Wood, sugar beet, potato tuber, hemp, Delamination of wood pulp by mechanical pressure before
flax and/or after chemical or enzymatic treatment
diameter: 5–60 nm
length: several micrometers
Wood, cotton, hemp, flax, wheat straw, Acid hydrolysis of cellulose from many sources
mulberry bark, ramie, Avicel, tunicin, diameter: 5–70 nm
cellulose from algae and bacteria length: 100–250 nm (from plant celluloses);
100 nm to several micrometers (from celluloses of tunicates,
algae, bacteria)
Low-molecular weight sugars and Bacterial synthesis
alcohols diameter: 20–100 nm; different types of nanofiber
networks
pineapple, bananas, and coconut crops. Today, these agri-
cultural by-products are either burned or used for low-value
products such as animal feed or in biofuel production. Because
of their renewability, crop residues can be valuable sources of
natural nanofibers.2
Bacterial Cellulose
In addition to its plant origins, cellulose fibers are also secreted
as extracellular structures by certain bacteria belonging to the
genera Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas,
Rhizobium, or Sarcina. The most efficient producer of bacterial
cellulose is Acetobacter xylinum (or Gluconacetobacter xylinus),
a Gram-negative strain of acetic acid producing bacteria.3
Microfibrillated Cellulose (MFC)
MFC production by fibrillation of cellulose fibers into nano-
scale elements requires intensive mechanical treatment.
However, depending upon the raw material and the degree
of processing, chemical treatments may be applied prior to
mechanical fibrillation. These chemical processes are aimed
to produce purified cellulose, such as bleached cellulose pulp,
which can then be further processed. There are also examples
with reduced energy demand in which the isolation of cellu-
lose microfibrils involves enzymatic pre-treatment followed by
mechanical treatments.11
Forcing wood-based cellulose fiber suspensions through
mechanical devices, such as high-pressure homogenizers,
produces microfibrillated cellulose (MFC). Mechanical treat-
ment delaminates fibers and liberates microfibrils that are ~20
nm wide. Pulp is produced from wood by chemical treat-
ment. By using a mixture of sodium hydroxide and sodium
sulfide, so-called kraft pulp (almost pure cellulose fibers) is
obtained. Pulping with salts of sulfurous acid leads to cellulose
named sulfite pulp, which contains more by-products in the
cellulose fibers.
 AATCC Journal of Research

Fig. 1. TEM images of dried dispersion of cellulose nanocrystals derived from (a) tunicate (Reprinted with permission.16 Copyright 2008 American Chemi-
cal Society), (b) bacterial (Reprinted with permission.17 Copyright 2004 American Chemical Society), (c) ramie ( Reproduced18 by permission of The Royal
Society of Chemistry), (d) sisal (Reprinted with permission.19 Copyright 2006 Springer), (e) cotton (Reprinted with permission.20 Copyright 1996 American
Chemical Society), and (f) sugar beet (Reprinted with permission.21 Copyright 2004 American Chemical Society).

The major impediment for commercial success has been the
very high energy consumption amounting to over 25,000
kWh per ton in the production of MFC as a result of the
required multiple passes through homogenizers.12 Saito et
al.13 have proposed a new process to obtain MFC based on
the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) radical
reaction and strong mixing. In their study, individualized
MFC was obtained by TEMPO-mediated oxidation at room
temperature and stirring at 500 rpm. At pH 10, optimal
conditions were reached giving cellulose nanofibers of
3–4 nm in width and a few microns in length.5
Nanocrystalline Cellulose (NCC)
NCC is the term often used for cellulose nanocrystals
prepared from natural cellulose by acid hydrolysis. The
nanocrystals formed from wood pulp are shorter and
thinner than the MFC. Also known as whiskers, they
consist of rodlike cellulose crystals with widths and
lengths of 5–70 nm and between 100 nm and several
micrometers, respectively.
The function of the mineral acid hydrolysis is to gener-
ate cellulose nanocrystals by the liberation of crystalline
regions of the semicrystalline cellulosic fibers. This chemi-
cal process starts with the removal of polysaccharides
bound at the fibril surface and is followed by the cleavage
and destruction of the more readily accessible amorphous
regions to liberate rodlike crystalline cellulose sections.
When the appropriate level of glucose-chain depolymer-
ization has been reached, the acidic mixture is diluted
and the residual acids and impurities are fully removed
by repeated centrifugation and extensive dialysis.3 The
structure, properties, and phase-separation behavior of
cellulose-nanocrystal suspensions are strongly dependent on
the type of mineral acid and its concentration, the hydro-
lysis temperature and time, and the intensity of ultrasonic
irradiation used.14 Cellulose sources are variable, and their
degree of crystallinity strongly influences the dimensions
of the liberated crystals. Cellulose nanocrystals show some
dispersibility in aqueous-based mixtures and in organic
solvents with high dielectric constants, such as dimethyl
sulfoxide (DMSO) and ethylene glycol, but tend to aggregate
in highly hydrophobic solutions. Azizi Samir et al.15 freeze-
dried NCC samples and redispersed them by sonication in
N,N-dimethylformamide (DMF).
Transmission electron microscopic (TEM) images of NCCs
are shown in Fig. 1.16-21

September/October 2014 Vol. 1, No. 5 | 19

 AATCC Journal of Research
Bacterial Nanocellulose (BNC)
Cellulose biosynthesis takes place not only in plants, but
also in bacteria such as Acetobacter, Acanthamoeba, and
Achromobacter spp., algae such as Valonia, Chaetamorpha
spp., and fungi. By selecting the substrates, cultivation
conditions, various additives, and finally the bacterial strain,
it is possible to control the molar mass, the molar mass
distribution, and the supramolecular structure. Thus, it is
possible to control important cellulose properties, and also
the course of biosynthesis (e.g., kinetics, yield, and other
metabolic products).
Materials also known as bacterial cellulose, microbial cel-
lulose, or bio-cellulose are formed by aerobic bacteria, such
as acetic acid bacteria of the genus Gluconacetobacter, as a
pure component of their biofilms. These bacteria are wide-
spread in nature where the fermentation of sugars and plant
carbohydrates takes place. In contrast to MFC and NCC
materials isolated from cellulose sources, BNC is formed as
a polymer and nanomaterial by biotechnological assembly
processes from low-molecular-weight carbon sources, such
as D-glucose. Although identical to cellulose of plant origin
in terms of molecular formula, bacterial cellulose is quite
different. The degree of polymerization (DP) is very high,
with DP values of 2000–8000. Crystallinity is also high, with
values of 60%–90%. Bacterial cellulose is characterized by its
high purity (with no accompanying substances like hemicel-
luloses, lignin, or pectin) and by an extremely high water
content of 90% or greater.22
In contrast to existing methods for obtaining nanocellulose
through mechanical or chemo-mechanical processes, BNC
is produced by bacteria through cellulose biosynthesis and
the building up of microfibril bundles.23 Bacterial cellulose
biosynthesis from low-molecular weight sugars or other
carbon sources via uridine diphosphate glucose has been
elucidated in detail. The formed cellulose chains are excreted
into the aqueous culture medium as fibers with diameters
in the nanometer range. Further approaches include the
continuous harvesting of cellulosic filaments, pulps, and
fibers. Other examples are the linear conveyor reactor and
the rotary disk reactor, developed by Bungay and Serafica.24
These approaches lead to rather non-uniform BNC mate-
rial owing to the bundling and aggregation of thin layers
or filaments.25
Cellulose Nanofiber Isolation
Cellulose nanofibers in the cell wall impart the high tensile
strength of plant fibers. It is essential to extract them from
the cell wall to physically measure the properties of the
nanoscopic fibrous component, which may risk substantive
chemical or mechanical damage. Then, analysis of these iso-
lated nanofibers for their reinforcing potential in composite
manufacturing can be performed.
20 | Vol. 1, No. 5 September/October 2014
Chemical Treatment
Cellulose nanocrystals are generated by the liberation of
crystalline regions of the semicrystalline cellulosic fibers
by hydrolysis with mineral acids. This chemical process
starts with the removal of polysaccharides bound at the
fibril surface and is followed by cleavage and destruction of
the more readily accessible amorphous regions to liberate
rodlike crystalline cellulose sections.3 To facilitate crystal-
line region liberation, several pre- treatments are extensively
used for the removal of everything other than cellulose, such
as hemicelluloses, lignin, fat, wax, and pectins surrounding
the cellulose structure.
Alkali Pretreatment
Alkali pretreatment removes a certain amount of lignin,
wax, and oils covering the external surface of the fiber cell
wall. This treatment disrupts the lignin structure and helps
to separate the structural linkages between lignin and carbo-
hydrates.26–29 Sodium hydroxide (17%–18%) is used for that
purpose—a very similar treatment to cotton mercerization.
Purification by mild alkali treatment results in the solubili-
zation of lignin and remaining pectins and hemicelluloses.
Alkali extraction needs to be carefully controlled to avoid
undesirable cellulose degradation and to ensure that hydro-
lysis occurs only at the fiber surface so that intact nanofibers
can be extracted.26,30
Oxidative Pre-Treatment
Application of TEMPO radicals as an oxidative pretreatment
before mechanical treatment was primarily introduced by
Isogai et al.13 Solving the aggregation problem arising from
the presence of native cellulose -OH groups, TEMPO-medi-
ated oxidation is a promising surface modification method
by which carboxylate and aldehyde functional groups can be
introduced into solid native celluloses under aqueous and
mild conditions.31,32 Oxidation occurred only at the surface
of the microfibrils, which became negatively charged. This
negative charge resulted in repulsion of the nanofibers, thus
easing fibrillation.
Enzymatic Pre-Treatment
In nature, cellulose is not degraded by a single enzyme, but
by a set of cellulases. These can be categorized as A- and
B- type cellulases, termed cellobiohydrolases, and C- and
D-type cellulases, or endoglucanases. Cellobiohydrolases can
attack highly crystalline cellulose, whereas endoglucanases
generally require some disorder in the structure to degrade
cellulose. Cellobiohydrolases and endoglucanases show
strong synergistic effects to facilitate MFC disintegration.
The MFC produced from enzymatically pretreated cellulosic
wood fibers showed a more favorable structure, having a
higher average molar mass and a larger aspect ratio, than
nanofibers produced by subjecting pulp fiber to strong acid
hydrolysis.11

Acid Hydrolysis
Treatment of cellulosic, starch, or hemicellulosic materials
using acid solutions to break down the polysaccharides to
simple sugars is known as acid hydrolysis. Lignocellulosic
fibers such as flax contain 20% to 40% hemicelluloses,
which are heteropolysaccharides consisting mainly of pen-
toses and hexoses. Acid hydrolysis can yield these sugars as
monomers. Being more amorphous compared to cellulose,
hemicelluloses are more prone to oxidation and degrada-
tion reactions. Hydrolysis of glycosidic bonds is possible in
both acid and alkaline medium, but much faster hydrolysis
occurs at lower pH. Hydrochloric and sulfuric acid are
two choices for acid hydrolysis to produce nanocrystals.
Hydrochloric acid gives almost neutral nanocrystals with
limited dispersibility in water whereas sulfuric acid gives
more stable product over a wide range of pH values.33
Reaction time is an important parameter to be considered
during hydrolysis (e.g., long reaction time results in com-
plete digestion of cellulosic hemp fiber). Insufficiently short
reaction times will only yield large un-dispersible fibers
and aggregates.
Mechanical Treatment
A purely mechanical process can also produce refined,
fine fibrils several micrometers long and between 50 to
1000 nm in diameter. Hepworth and Bruce suggested that
nanofibers, even without their extraction from the cell wall,
could be used for their reinforcing ability. In their work,
fragments of cell wall are extracted from vegetable paren-
chyma tissue and pressed with PVA to form a composite
sheet.34 As compared to purely mechanical methods,
cellulose nanofibers from primary and secondary cell
walls can be extracted by mechanical treatments followed
by chemical treatments (chemo-mechanical treatments)
without degrading the cellulose. A chemo-mechanical
process can yield even finer fibrils of cellulose with diam-
eters ranging between 5 to 50 nm. The main objective of
natural fiber chemical treatments is the selective removal of
non-cellulosic compounds.
Purely mechanical methods include the following techniques.
Refining and High-Pressure Homogenization
MFC is now available as a commercial product from
various companies and other organizations (e.g., Daicel,
Japan; Rettenmaier, Germany; or Innventia AB, Sweden).2
MFC manufacturing is now generally based on mechanical
treatment consisting of refining and high-pressure homog-
enizing process steps. The dilute fiber suspension is forced
through a gap between rotor and stator disks by using a
disk refiner. These disks have surfaces fitted with bars and
grooves against which the fibers are subjected to repeated
cyclic stresses. This mechanical treatment brings about
irreversible changes in the fibers, increasing their bonding
potential by modification of their morphology and size.23
AATCC Journal of Research
Grinding
Some researchers have attempted to use modified com-
mercial grinders with specially designed disks to fibrillate
cellulose fibers. In such equipment, the cellulose slurry is
passed between a static grind stone and a rotating grind
stone revolving at a speed of about 1500 rpm. The fibrillation
mechanism of grinder treatment results from shearing forces
generated by the grinding stones. This brakes down the cell
wall structure consisting of nanofibers in a multi-layered
structure and hydrogen bonds. As a result, nanosized fibers
are individualized from the pulp.2
Cryocrushing
Cryocrushing is a method for producing nanofibers in
which fibers are frozen using liquid nitrogen followed
by the application of high shear forces.35 The objective of
cryocrushing is to form ice crystals within the cell wall.
When high impact forces are applied to the frozen fibers,
ice crystals inside the fibers exert pressure on the cell walls,
causing them to rupture and thereby liberating microfi-
brils.26 Bhatnagar and Sain30 obtained nanofibers with an
estimated diameter of 5–80 nm by applying cryocrushing
of chemically treated flax, hemp, and rutabaga fibers. The
cryocrushed fibers may then be dispersed uniformly into a
water suspension using a disintegrator before high-pressure
fibrillation. Cryocrushing combined with a high-pressure
fibrillation process was used also by Wang and Sain26,27
for isolation of nanofibers with diameters in the range of
50–100 nm from soybean stock.
Sonication
Ultrasound is a part of the sound spectrum in the range of
20 KHz to 10 MHz generated by a transducer that converts
mechanical or electrical energy into high-frequency acousti-
cal energy. High-intensity ultrasonication (HIUS) waves can
produce a very strong mechanical oscillating power due to
cavitation, which is a physical phenomenon that includes
the formation, expansion, and implosion of microscopic gas
bubbles when the molecules in a liquid absorb ultrasonic
energy. Within the cavitation bubble and the immediate sur-
rounding area, violent shock waves are produced, resulting
in temperatures up to 5000 °C and pressures of greater than
500 atm at implosion sites. Ultrasonic radiation is hence
used in many processes, including emulsification, catalysis,
homogenization, disaggregation, scission, and dispersion.36
Isolation of fibrils from several cellulose resources (e.g.,
regenerated cellulose fiber, pure cellulose fiber, MCC, and
pulp fiber) can be performed using HIUS energy in a bath
process. A mixture of microscale and nanoscale fibrils can be
obtained. The temperature of the fiber suspension increased
at a faster rate when the power was higher—the higher the
power, the better the fibrillation. The temperature of the
water suspension could reach up to 91 °C without water
cooling. The higher the temperature, the better the cellulose
September/October 2014 Vol. 1, No. 5 | 21
 AATCC Journal of Research
Fig. 2. Methods of production of nanocelluloses from macroscopic cellulose fibers (Reprinted with permission.38 Copyright 2007 American
Chemical Society).
fibrillation, whereas the longer the raw fiber, the lower the
fibrillation. The cellulose concentration of the suspension
depends on the dimensions of the cellulose fiber, resulting
in lower concentrations when the fiber was longer. A larger
distance from the HIUS probe tip to the beaker bottom was
not beneficial to fibrillation.37 Fig. 2 summarizes nanocellu-
lose production mechanisms.38
Conclusion
Growing research efforts have recently been reported regard-
ing the formation and use of nanocelluloses, particularly
in the last seven years. It was confirmed that MFC, which
exhibits gel-like properties, can be obtained by a purely
mechanical process such as high-pressure homogenizer. A
22 | Vol. 1, No. 5 September/October 2014
second type of nanocellulose with much smaller dimensions,
NCC, requires chemical treatment such as acid hydrolysis to
remove amorphous section of partially crystalline cellulose.
NCC shows liquid crystalline properties. Another nanocel-
lulose variant, BNC, unlike the previous two categories,
is prepared from low molecular weight resources, such
as sugar, with the help of acetic acid bacteria of the genus
Gluconacetobacter. The shape, structure of the nanofiber
network, and composite formation can be controlled by in
situ bio-fabrication of BNC.
The rapidly advancing state of knowledge in all three
categories of the nanocellulose family, especially NCC,
makes a review focusing on preparation imperative. The
potential of NCC research and development is very high.
 AATCC Journal of Research

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DOI: 10.14504/ajr.1.5.3 September/October 2014 Vol. 1, No. 5 | 23

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