Cellulose
DOI 10.1007/s10570-014-0330-3
ORIGINAL PAPER
Nanocomposite films based on TEMPO-mediated oxidized
bacterial cellulose and chitosan
Chen Lai • Shujiang Zhang • Xuanchen Chen
Liyuan Sheng
Received: 26 February 2014 / Accepted: 9 June 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Nanobacterial cellulose (BC) and chitosan
(CH) have similar molecular structures. In the present
work, nanocomposite films based on BC and CH were
prepared by stepwise modification instead of by
conventional physical blending. First, surface C6-
carboxylated BC was prepared in a bromide-free
system using 2,2,6,6-tetramethylpyperidine-1-oxyl
(TEMPO) as a catalyst. The carboxylate groups of
oxidised BC could couple to the amine groups of CH.
The composite films were characterised by attenuated
total reflectance Fourier transform infrared spectros-
copy, X-ray photoelectron spectroscopy and Carbon-
13 solid nuclear magnetic resonance 13C NMR. The
results showed that a cross-linking reaction occurred
between TEMPO-mediated oxidised BC and CH.
Even in the absence of cross-linkers, these two
Shujiang Zhang contributed equally to the manuscript and is
also a co-first author.
C. Lai (&) L. Sheng
Shenzhen Key Laboratory of Human Tissue Regeneration
and Repair, Shenzhen Institute, Peking University,
Shenzhen 518057, China
e-mail: laichen1110@msn.com
S. Zhang
The First Affiliated Hospital, Guangzhou Medical
University, Guangzhou 510120, China
X. Chen
Department of Mechanical Engineering, University of
Sheffield, Western Bank Sheffield S10 2TN, UK
•
biopolymers could interact with each other because
of their structural similarity. SEM images and tensile
tests showed that the TEMPO-oxidized BC and CH
composite film prepared at a 0.5:1 ratio was an
exception. The mechanical properties of the composite
films decreased with increasing CH content, passed
through a minimum, and then increased. To explain
this phenomenon, we propose that the hydrogen
bonding in the original BC microstructure plays a
decisive role in the modified nanocomposites. How-
ever, BC/CH composites with excellent properties
could be synthesised at appropriate reactant ratios.
Keywords Nanofibres Bacterial cellulose
Chitosan Hydrogen bond
Introduction
Polysaccharides, cellulose, and chitosan (shown in
Fig. 1) and their derivatives are of increasing interest
as new functional polymeric renewable materials
because of their high abundance and specific proper-
ties. Bacterial cellulose (BC) is a form of cellulose
produced by bacteria, including Gluconacetobacter
(also known as Acetobacter), Acanthamoeba, Achro-
mobactr, and Zoogloea, among others. Acetobacter
xylinum, in particular, generates high yields in com-
mercial bacterial cellulose production. As a cellulosic
polymer, BC is a linear syndiotactic homopolymer
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Fig. 1 Schematic
illustration of the procedure
used to prepare the bacterial
cellulose/chitosan
composites
composed of D-anhydroglucopyranose units (AGU)
that are linked together by b-(1?4)-glycosidic bonds.
Chitosan (CH) is obtained from the partial deacetyla-
tion of chitin, which is the main component of the
exoskeleton of crustaceans and is composed of
2-amino-2-deoxy-D-glucose units linked through b-
(1?4) bonds. It is also crystalline and exhibits
polymorphism, depending on its physical state. BC
has unique mechanical and physical properties due to
its three-dimensional network of microfibres with
diameters of approximately 30 nm. Individual BC
chains aggregate into fibrils, forming ribbons. BC also
has a higher purity, crystallinity, thermal stability
(250–300 °C), biocompatibility, water-holding capac-
ity, tensile strength, and Young’s modulus than
common plant cellulose (Iguchi et al. 2000; Pecoraro
et al. 2008; Yano et al. 2005). CH is a positively
charged molecule and is soluble in dilute acidic
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Cellulose
solutions. Because it is the only polycation in nature
(Davis and Brewster 2004), CH has numerous unique
properties, such as antimicrobial activity (Ali et al.
2010), haemostasis (Hattori et al. 2010), and biode-
gradability by certain human enzymes, especially
lysozyme (Saboktakin et al. 2010). CH can be easily
processed into a membrane, scaffold, sponge, powder,
or hydrogel via different methods. BC and CH exhibit
no immunological reactivity and have been investi-
gated for biomedical applications such as wound
dressings (Archanaa et al. 2013; Lina et al. 2013), drug
carriers (Huang et al. 2013; Shivashankar and Mandal
2013), and bone-repairing scaffolds (Busilacchi et al.
2013; Wang et al. 2013), among others.
Despite their numerous advantages and unique
properties, BC and CH both have drawbacks. For
instance, the BC microstructure may prevent cell
migration into a certain material because the
Cellulose
nanomesh of BC is too dense. Poor degradability and
processability hamper the use of BC in biomedical
applications. The large number of hydroxyl groups on
BC chains results in a strong tendency to form inter-
fibre hydrogen bonds, thereby causing the fibres to
tangle together tightly. The entangled nanofibres lead
to great difficulty in the functionalisation of BC. With
respect to CH, its scaffolds or films are often brittle
and tend to dissolve in vivo, limiting its application in
a weight-bearing area of the human body, such as a
joint or femur. The poor solubility at physiological pH
has been a major obstacle in its development as a
delivery agent or carrier for drugs or proteins in vivo.
The properties of these two polysaccharides are
mutually complementary to some extent. To expand
the applications of these two biopolymers, the prep-
aration of BC composites with CH has recently been
proposed by several researchers. Although the func-
tional groups connected to the second carbon in the
repeating units differ between BC and CH, these two
biopolymers have several structural similarities,
which lead to good interfacial adhesion and strong
interactions between them (Fernandes et al. 2009).
Therefore, a considerable number of studies on the
blending of CH and BC have been reported in the past
few years. Most of them involve dipping BC films or
pellicles into a CH acid solution (Duvey et al. 2005;
Fernandes et al. 2009; Kim et al. 2011) or adding CH
directly to the culture medium during BC biosynthesis
(Ciechańska 2004; Phisalaphong and Jatupaiboon
2008; Yadav et al. 2010). These types of reactions
can proceed smoothly; however, these two biopoly-
mers tend to form composites via physical interactions
instead of chemical bonds. Creating a chemical
reaction between these polymers in the absence of a
catalyst or cross-linkers is extremely difficult. The
hydroxyl groups in BC form hydrogen bonds within
and between the polymer chains, resulting in consid-
erable stiffness of the BC chain and extensive
entanglement of the nanofibres. Therefore, the hydro-
xyl groups of BC do not exhibit sufficient reactivity to
react with the amine groups of CH. To avoid these
disadvantages, a more effective composite reaction
between BC and CH has been proposed by Nge et al.
(2010), in which stepwise modification was chosen on
the basis of the carboxylic functionalisation at C6 in
the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)/
NaClO/NaBr system. The reactivity of BC with
carboxylate groups is much greater than that with
hydroxyl groups because the uniformity of accessibil-
ity is enhanced by BC carboxylate. As a consequence,
amidation coupling between CH and TEMPO-medi-
ated oxidised BC (TBC) might occur. In this system,
NaBr was used as an additional catalyst in the reaction
system in which NaClO was used as the primary
oxidant. However, in our research, we observed some
disadvantages of this system, including low repeat-
ability because of the instability of NaClO, which
decomposes under sunlight during short-term storage,
and the side-reaction products of aldehyde groups.
Furthermore, the TBC produced in this system tended
to be yellow and odoriferous. In the present research,
we oxidised BC in the TEMPO/NaClO/NaClO2 sys-
tem that we described in our previous report (Lai et al.
2013b). A large number of carboxylate groups can be
formed with high repeatability and controllability on
the surface of BC nanofibres in a NaBr-free system. In
the present work, we focused on the properties of
nanocomposite films with different TBC/CH ratios
because such films and membranes have wide appli-
cations in the biomedical field, including contact
lenses, topical delivery lidocaine, ventral hernia
patches, and wound dressings. Kim et al. (2011)
reported that the tensile strength and Young’s modulus
of BC–CH composites tends to decrease with increas-
ing CH content. This result is reasonable in that CH is
a brittle material and often decreases the tensile
strength of composites into which it has been incor-
porated. However, in our research, we observed that
the relationship between the decrease in tensile
strength and the CH content was not always linear.
To the best of our knowledge, this phenomenon has
not been previously reported. A new perspective was
taken in this work to provide a theoretical basis for the
synthesis of BC and CH complex films that satisfy
various clinical needs.
Methods
Materials
Bacterial cellulose (BC) pallets (donated by Guangyu
Biotechnology Co., Ltd., China) were stored at 4 °C.
Chitosan (Mw * 389,000 and 92 % deacetylated),
NHS (N-hydroxysuccinimide, AR grade), EDC (1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride, AR grade), TEMPO (AR grade), NaClO
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solution (AR grade, 13.4 % available chlorine), and
NaClO2 (AR grade) were purchased from Sigma–
Aldrich (USA) and were used without further purifi-
cation. The water used in these experiments was
purified by deionising reverse-osmosis water.
Preparation of TEMPO-mediated oxidised
bacterial cellulose (TBC)
TEMPO-oxidised BC (TBC) was prepared via
TEMPO oxidation, in accordance with previously
described methods (Lai et al. 2013b). To obtain TBC
in the TEMPO/NaClO/NaClO2 system, 1 g of BC
suspension in 300 mL of phosphate buffer (0.05 M,
pH 6.86) was used. TEMPO (0.1 mmol/g BC) and
NaClO2 (17 mmol/g BC) were added to the resulting
suspension. NaClO (2 mL) was diluted in 100 mL of
the phosphate buffer solution, and the resulting
mixture was immediately added to the suspension.
The suspension was then sealed and magnetically
stirred at 65 °C for 30 h. The products were washed
four times via centrifugation and then lyophilised for
further analysis.
Preparation of TBC/CH nanocomposites
(CHTBC)
A 10 mg/mL CH solution was prepared by dissolving
CH powder in HCl (0.2 M). The mixture was stirred
overnight at 70 °C, and a clear, viscous, light-yellow
solution was obtained. Freeze-dried TBC (1 g) was
dispersed in 300 mL of deionised water at room
temperature using a homogeniser (IKA T25, Ger-
many) operated at 25,000 rpm. The molar ratio of
CH:EDC:NHS in the present experiment was 1:1:1.
Four CH:TBC weight ratios were investigated: 0.25:1,
0.5:1, 1:1, and 2:1.
First, the NHS was dissolved in 5 mL of distilled
water, which was immediately added to the TBC
suspension. The CH solution was added to the
suspension slowly under intensive agitation. The pH
value of the mixture was adjusted to 5.96–6.2 with
0.5 M NaOH and continuously stirred for 30 min, and
the EDC aqueous solution was subsequently added to
the mixture dropwise. The whole suspension was
intensively stirred for 36 h at room temperature. The
final products were loaded into dialyser bags (pur-
chased from Biotopped; molecular weight cut-off of
8000–14,000) to remove excess EDC, NHS or
123
Cellulose
TEMPO. The dialysis medium was exchanged with
deionised water three times per day until the conduc-
tivity was \2 lm/cm.
For purposes of comparison, we also prepared a
physical blend of CH and TBC without EDC and NHS
by physically mixing the CH solution and the TBC
dispersion. With respect to blending films of CH and
TBC (BCHTBC), previous studies have shown that
maintaining a lower ratio of CH results in higher
tensile strength of composite films. Hence, samples
with a ratio of 0.25:1 (CH:TBC) were identified as a
representative composition. The samples were dia-
lysed and freeze-dried as previously described.
Preparation of the films
One gram each of dried BC, TBC, CHTBC and
BCHTBC was individually dispersed in 100 mL of
distilled water at room temperature using a homoge-
niser (IKA T25, Germany) operated at 25,000 rpm.
The suspension was poured into a Büchner funnel
under vacuum filtration to remove the entrapped air.
After being degassed, the wet membranes were gently
peeled from the PTFE filter paper (2 lm in pore
diameter) and further dried at 35 °C in a vacuum oven
for 24 h under a vacuum of 10 mmHg. The average
thickness of the dried membranes was 0.13 mm.
Characterisation
Solid-state 13C NMR spectra of the films were
recorded at 100 MHz on a Bruker AC-400 spectrom-
eter (Germany). The spectra were obtained according
to the following parameters: spinning rate, 5,000 rpm;
contact time, 1 ms; pulse repetition, 4 s; spectral
width, 36 kHz; and accumulations, 1,036.
X-ray photoelectron spectroscopy (XPS) was
employed to acquire quantitative information on the
chemical species present on the surface of the films.
XPS spectra were recorded with a Kratos AXIS 165
electron spectrometer (Japan) using monochromated
Al Ka X-ray irradiation generated at 350 W. The
standard take-off angle used for analysis was 45°,
which produced a maximum analysis depth in the
range of 3–5 nm. Low-resolution survey spectra were
recorded in 0.5 eV steps with a 187.85 eV analyser
pass energy from 0 to 1,400 eV. High-resolution
carbon (1 s) spectra were recorded in 0.1 eV steps
with an 11.75 eV analyser pass energy from 280 to
Cellulose

300 eV, and oxygen (1 s) spectra were recorded in

0.1 eV steps with a 58.7 eV analyser pass energy from
525 to 545 eV. The XPSPEAK 4.1 software was used
to fit Gaussian–Lorentzian curves to the C1s peaks.
ATR-FTIR spectroscopy was performed using a
Nicolet 6700 spectrometer (Thermo, USA) with a
resolution of 4 cm-1; the samples were scanned from
400 to 4,000 cm-1.
Tensile tests were performed under ambient condi-
tions on a universal materials testing machine (Instron,
USA) equipped with a 500 N load cell. Films 50 mm
long, 10 mm wide and 0.13 mm thick were tested at a
cross-head speed of 4 mm/min.
The surface density of film was assessed according
to ATSM D 646-96 using four replicates.
The surface and fracture-sectional images of samples
were recorded using a scanning electron microscope
(SEM, Philips XL-30, Holland). The Transmission
electron microscopy (TEM) images of the samples were
observed under PHILIPS CM300 (Holland) at an
accelerating voltage of 300 kV. The dried samples were
individually dispersed in distilled water at room
temperature using a homogeniser operated at
25,000 rpm. Drops of diluted sample suspensions were
deposited onto carbon-coated electron microscopy grids
and dried at room temperature.

Results

ATR-FTIR and 13C NMR analysis of composite Fig. 2 ATR-FTIR spectra of pure BC, CH, and their
films derivatives

Figure 2 shows the FTIR spectra of the original BC,

CH, and its derivatives. The peaks at 1,157, 1,107,
1,056, and 1,026 cm-1 correspond to C1–O–C4, C2–
O, C3–O, and C6–O stretching vibrations, respec-
tively (Maréchal and Chanzy 2000), and appear in the
spectrum of every sample. The peak at 1,726 cm-1 in
the spectra of the TBC film was assigned to carboxylic
groups, thereby indicating the successful oxidation of
the hydroxyl groups. In the spectra of the covalent
CHTBC, bands were observed at approximately
1,645–1,640 and 1,550 cm-1; these bands are in the
zone related to (–CONH–) and correspond to the C=O
stretching band (amide I) and to the (–NH–) bending
vibration band (amide II), respectively. A substantial
decrease in intensity of the COOH stretching vibration
band at 1,726 cm-1 in the TBC spectra can be
observed in comparison with the spectra of the
CHTBC samples, whereas the CONH peak at
1,645–1,640 cm-1 emerges, thereby confirming the
transformation from COOH to CONH. In the spectra
of the BCHTBC, both the COOH peak and the amide
peak are visible, which suggests that a cross-linking
reaction did not occur between these two polymers
without cross-linkers and that only the incorporation
of CH molecules into the TBC nanostructure occurred
due to their structural similarity (Fernandes et al.
2009). We also observed that the intensity of the
1,430–1,336 cm-1 peaks assigned to CH2 is substan-
tially lower in the CHTBC spectra than in the pure BC
spectra; indeed, these peaks in the CHTBC spectra
more strongly resembled the peaks in the CH pattern.

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13
Fig. 3 Solid-state C NMR spectra of BC and its derivatives (a) and comparison spectra of CHTBC and TBC with partial
enlargement (b)
This result can be attributed to the presence of CH
molecules entangled in the BC microstructure.
NMR spectroscopy, particularly with 13C nuclei,
was performed for the structural analysis of cellulose
and its derivatives. Figure 3a shows that the 13C
spectrum of pure BC consists of six signals, with one
signal assigned to each C atom. The chemical shifts at
104.9 and 88.6 ppm were assigned to C1 and C4,
respectively. The peaks at 75.6–70 ppm correspond to
C2, C3, and C5 and are not easily evaluated because of
their close proximity (Klemm et al. 1998). The carbon
signals at 61.8 ppm, which correspond to the C6
primary hydroxyl groups that are exposed on the
nanofibre surfaces (Viëtor et al. 2002) disappeared
after partial modification occurred at this position. In
contrast, the peak at 64.8 ppm, which corresponds to
the C6 primary hydroxyl group inside the crystalline
fibrils and C2/C3, remained unchanged. These results
may provide evidence of the oxidation of only the
hydroxyl groups of BC on C6 at the surface of the
fibre. No significant change was observed in the
CHTBC 13C NMR patterns, suggesting that the CH
123
Cellulose
content is not significant enough to affect the micro-
structure of CHTBC. Figure 2b presents the evidence
for amide bond formation. In the enlarged pattern of
CHTBC, a new resonance is detected at 170.1 ppm
that is well separated from the (–COO-) C6 reso-
nance. This peak was attributed to the signal of
amidated C6 carbons (Irwin et al. 1987). The signal at
42.08 ppm corresponds to the N–CH groups of amino-
coupled BC (Fernandes et al. 2013) or to an excess of
unreacted CH. The spectrum of the BCHTBC is
similar to that of TBC, where no split in the COO-
peak was observed, thereby supporting the evidence
that the CH molecules just interposed in the TBC
microstructure instead of chemically binding with
TBC molecules.
Morphological and XPS analyses of the composite
films
Transmission electron microscopy (TEM) results
showed that the individual modified BC nanofibers
with a high aspect ratio were morphologically similar
Cellulose
Fig. 4 TEM images of a pure BC, b TBC and c CHTBC (0.25:1)
to the original BC (Fig. 4), suggesting that BC did not
have any damage during oxidation or crosslink
reaction. However, the groups on the surface can
influence the agglomeration of nanofibers. Lateral
aggregation can be observed in the pure BC (Fig. 4a).
By contrast, better disintegration can be observed at
TBC (Fig. 4b) and CHTBC (Fig. 4c) with less bundles
or aggregates by loosening the adhesion between
nanofibers. A fibre-shaped nanostructure is clearly
visible on the surface of the BC film, as shown in
Fig. 5a, and some nanofibres agglomerate to form
larger clusters, thereby creating a crevice structure on
the surface. After oxidation, the fibre-shaped and
crevice structures were obscured on the surface of the
TBC film (Fig. 5b). However, the network structure
was still visible, and the nanofibres appeared to be
well-dispersed. A high amount of hydrogen bonds
formed by the hydroxyl groups caused the pure BC
nanofibres to entangle or aggregate, thereby leaving
crevices in the microstructure as shown in Fig. 5a. The
regioselective conversion of primary hydroxyl groups
into carboxylate groups after the oxidation treatment
would make it possible to reduce the adhesion between
BC nanofibres by preventing the formation of strong
interfiber hydrogen bonds. Thus, some dispersed and
individual nanofibres could been observed in TBC
microstructure as shown in Fig. 5b. These will
discussed in detail later. When CH was cross-linked
to TBC at a ratio of 0.25:1 (weight ratio), the fibrous
structure could no longer be detected. The smooth
surface of CHTBC, without a noticeable aggregated
formation, is shown in Fig. 5c and suggests that the
CH and TBC are compatible with each other. The
surface of the complex film became coarser with
increasing CH content (0.5:1), and some flocculent
precipitate was visible on the surface, as shown in
Fig. 5d, demonstrating that phase separation occurred
in the CH and TBC reaction system at this reactant
ratio. The film surface tended to become smoother
when the ratio of TBC to CH was increased to 1:1, as
shown in Fig. 5e, although some floccus could still be
observed. However, the surface of the film, which
contained a greater CH concentration, became
smoother, as shown in Fig. 5f. Nonetheless, a low
but detectable amount of precipitate covered the
surface. Thus, the surface of the CHTBC (0.25:1)
composite film was the smoothest among the exam-
ined films. As expected, the SEM image of the
BCHTBC (Fig. 5g) also provides evidence of the good
dispersion of the CH in the TBC membrane, without
obvious aggregates or precipitates.
X-ray photoelectron spectroscopy (XPS) analysis is
the most effective technique for the determination of
surface characteristics. We discussed pure and oxi-
dised BC in our previous paper (Lai et al. 2013a);
therefore, the present research is focused on the
surface properties of CHTBC.
The main spectral regions—C1s, O1s and N1s—of
CHTBC are shown in Fig. 6a. The C1s peak appears at
a binding energy of 288.5 ± 1 eV. The peak at
531.4 ± 1 eV corresponds to the O1s region. The
N1s region contains a very distinctive peak centred at
400.8 eV, which is assigned to the organic nitrogen
atoms in covalent CHTBC. Low but detectable amounts
of surface N were observed in CHTBC (0.25:1) and the
BCHTBC. With increasing CH content, the N peak
became distinct. To acquire more detailed information
about the changes in the types of carbon bonds, the C1s
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 Cellulose

Fig. 5 SEM images of the surface morphology of different samples: a BC, b TBC, c CHTBC (0.25:1), d CHTBC (0.5:1), e CHTBC
(1:1), f CHTBC (2:1), g BCHTBC

peaks of wide-scan XPS spectra were deconvoluted into
several peaks, as shown in Fig. 6b. The relative
amounts of the different carbon functional groups in
each sample, as obtained from curve fitting of the XPS
spectra, are presented in Fig. 6d. The C1s peaks were fit
by five curves (Kalaskar et al. 2010), indicating that five
types of carbon bonds were present. The C1s1 peak was
attributed to C–C (or C–H) and exhibited the lowest
binding energy of 285 ± .05 eV. The C1s2 peak at
286.0 ± 0.5 eV corresponded to carbon linked to a

123
Cellulose

Fig. 6 Wide-scan XPS spectra of samples prepared under concentrations of different carbon functionalities determined by
various conditions (a), a typical peak fitting of the high- curve fitting of the XPS spectra of the C1s (d) and N1s (e) peaks
resolution wide-scan XPS spectra of C1s (b) and N1s (c),

single nitrogen, C–N. The C1s3 signal at 287.0 ± binding energy at 289.1 ± 0.5 eV represented C1s5
1 eV was assigned to C–O, and the C1s4 signal at and was characterised by the formation of the carbox-
287.5 ± 1 eV was assigned to O–C–O. The highest ylate group, O–C=O. In the present study, the presence

123

of C–N bonds is considered to be particularly notewor-
thy. An increase in the CH:TBC ratio from 0.25:1 to
0.5:1 did not lead to substantial changes in the C–N
component. However, a significant increase in C–N was
observed upon the addition of CH beyond this range.
While investigating the BCHTBC samples, we
observed that the C–N content was very low because
CH molecules only interact with TBC through interfa-
cial adhesion in the absence of a cross-linker. CHTBC
contains two nitrogen atoms from different functional
groups: –NH–CO–, which belongs to the covalent
CHTBC, and N?, which belongs to the CH trapped in
the films. The higher-binding-energy N1s2 peak at
401.7 ± 1 eV is assigned to N?, and the N1s1 peak
with a lower binding energy at 399.6 ± 1 eV is
attributed to CO–NH (Ferraria et al. 2010), as shown
in Fig. 6c. The relative amounts of the different
nitrogen functional groups in each sample obtained
from curve fitting of the XPS spectra are presented in
Fig. 6e. With respect to the covalent CHTBC samples,
the –CO–NH– content is much higher than that of N?.
When the CH content was increased, the CO–NH
contribution increased, proceeded through a maximum
(CH:TBC=0.5:1), and then decreased, revealing that
the cross-linking reaction between CH and TBC will
not always proceed with the further addition of CH.
With respect to the BCHTBC, the N? contribution is
much higher than the CO–NH contribution, thereby
demonstrating that the CH molecules are mainly
trapped in the TBC film rather than being cross-linked
with COOH.
Tensile tests
Figure 7a, b show the tensile strength and surface
density of different complex films. The similar trends in
their results suggested that the higher compactness and
uniformity of the films naturally increased the tensile
strength. The tensile strength of the CHTBC (0.25:1)
film was determined to be 42.0 MPa, which is more than
two times greater than the corresponding value for the
pure BC film. The tensile strength of the TBC film was
also considerably higher than that of the BC film. When
the CH:TBC ratio was increased to 0.5:1, the tensile
strength dramatically decreased to 5 MPa. With further
addition of CH, a slight increase in the tensile strength of
the complex film was observed. CHTBC (0.25:1) film
has the highest surface density of 129 mg/cm2. The
surface density of other films ranged from 43 to 84 mg/
123
Cellulose
cm2. The fluctuations of surface density was much
milder than those of tensile strength as shown in Fig. 7b.
It was clear that the difference between CHTBC (0.25:1)
and (0.5:1) films was much greater in the tensile strength
than the surface density. Based on the results, the
difference in tensile strength was not merely a result of
difference in densities. This might have resulted from
the deposition of precipitates (as shown in Fig. 5d) on
the film surface, which may make contribution to the
density. The densities data of the film CHTBC (0.5:1)
and (1:1) could support this deduction. The tensile
strength of CHTBC (1:1) was two times than that of
CHTBC (0.5:1), but the surface density of these two
films appear to be very close. The phase separation (as
shown in Fig. 5d, e), which occurred in the reaction at
this reactant ratio, would lead to precipitate on the film
surface and thus overestimated the surface density.
Figure 8 shows images of the fracture edges after the
tensile tests. The fracture morphology of BC is charac-
terised by a disordered arrangement, with numerous
irregular cracks in the surface (Fig. 8a) and some flakes
pulled out of lamellae. The TBC and CHTBC (0.25:1)
samples exhibited substantial structural differences
(Fig. 8b, c), showing better alignment of the two-
dimensional lamellae throughout their entire cross-
sections. The densely layered structure disappeared in
the CHTBC (0.5:1) film, and some new microcaverns
emerged. When a large excess of CH was added to the
system, a well-ordered microstructure was recon-
structed. As shown in Fig. 8e, f, the microfibre align-
ment appeared again; however, microdefects or
deformations between neighbouring layers were still
observed, suggesting that the interlamellar adhesion was
weaker than that in the cases of the TBC and CHTBC
(0.25:1) samples. We also noted that the BCHTBC film
was more fragile than pure BC but stronger than the
sample of CH. Because of its low fracture strength and
toughness, CH decreases the strength of the blend
composite films. However, the CHTBC (0.5:1) film was
the most fragile of the investigated films. We will
discuss this observation in detail later.
Discussion
The BC/CH composite was synthesised as illustrated
in Fig. 1. The surface carboxylation at the C6 position
was selectively catalysed by TEMPO in the NaClO2/
NaClO system, which we have discussed at length
Cellulose

Fig. 7 Tensile strength (a) and surface density (b) of different samples

Fig. 8 SEM micrographs of the fractured sectional view of different films: a BC, b TBC, c CHTBC (0.25:1), d CHTBC (0.5:1),
e CHTBC (1:1) and f CHTBC (2:1)

123

elsewhere (Lai et al. 2013b). First, the amino groups of
CH were ionised in the acidic solution. The surface
carboxylated BC fibres were then further modified by
amidation coupling using EDC/NHS as a cross-linking
agent, resulting in the formation of amide groups.
The ART-FTIR and 13C NMR results confirmed the
surface modification of BC nanofibres at the C6
position. The signal of the –CONH– group produced
by the amine coupling reaction of TBC and CH was
observed in both the ART-FTIR and 13C NMR spectra.
As evident in the ART-FTIR spectra, NH peaks were
observed both in the case of covalent CHTBC and in
the case of the BCHTBC. However, the disappearance
of the COO- band was accompanied by the appear-
ance of a new –CONH– peak in the spectrum of the
covalent CHTBC sample. The simultaneous existence
of COO- and –NH– was observed in the case of the
BCHTBC sample, suggesting that the cross-linking
reaction between the carboxyl and amino groups did
not occur in the absence of EDC/HMS. The 13C NMR
results are in agreement with the ART-FTIR analysis
results in that the split in the COO- peak in the
spectrum of covalent CHTBC is absent in the spec-
trum of the BCHTBC. These findings confirm that CH
can still interact with TBC through interfacial adhe-
sion even in the absence of a cross-linker because of
their structure similarity. The CH:TBC ratio strongly
affected the film properties, as indicated by observa-
tions of the surface morphologies of the nanocompos-
ite films. The film seemed to become smoother after
carboxylation or amidation or after being blended with
CH. Even in the case of the physically blended films, a
denser network structure was obtained, which suggests
that CH may incorporate with microfibres of BC films
and decrease the pore size (Fig. 5g). However, the
CHTBC (0.5:1) nanocomposite film was an exception
in that numerous sediments settled on its surface, as
shown in Fig. 5d. This phenomenon can be explained
by the occurrence of phase separation at this ratio,
which has also been reported by Shih et al. (2009). We
expected that the presence of excess CH in the
polymer reaction would lead to coprecipitation and
consequently efficient phase separation in the polymer
solution. Under this condition, the surface of the
CHTBC (0.5:1) nanocomposite film became coarse.
However, when additional CH was added, the phase
separation phenomenon was not expected to continue;
it was expected disappear, as shown in Fig. 5e, f. No
theory explaining this phenomenon has been reported,
123
Cellulose
and we therefore attempted to clarify the mechanism
on the basis of hydrogen bonding in the BC network.
The individual nanofibres most often consist of BC
microfibre aggregates rather than smaller individual
nanofibres, as shown in Fig. 9a, c. The hydroxyl
groups in BC exhibit a strong tendency to form inter-
fibril hydrogen bonds, thereby causing the fibres to
entangle tightly, as shown in Fig. 9a. When the surface
hydroxyl groups were oxidised to carboxylate groups,
hydrogen bonding was weakened, leading to the bulk
fibre swelling shown in Fig. 9b. The entangled BC
became looser, and the individual fibres stretched,
improving the contact area between each nanofibre
(Fig. 9d). Well-dispersed TBC nanofibres conse-
quently formed a more uniform and dense film
(Fig. 5b). Cross-linking CH on the fibre further
ruptured surface hydrogen bonding, resulting in the
smoother surface presented in Fig. 5c. However,
phase separation occurred with increased CH content.
Interestingly, CH and TBC reintegrated when the CH
content exceeded a certain value. To theoretically
explain this phenomenon, we provide a schematic
illustration of the diversification of surface charges
and their effects on the BC film structure. As shown in
Fig. 10a, after oxidation, the fibres were unfolded,
with the negative COO- group at the surface C6 site.
Under this condition, the COO- group was highly
accessible to positively charged CH macromolecules.
The cross-linking between carboxyl and amino groups
occurred in the presence of EDC/NHS as the cross-
linker. After the COO- groups at the surface C6
completely transformed to amide groups, the nanofi-
bre surface tended to be electrically neutral. Conse-
quently, the hydrogen bonding between the C2 and C3
hydroxyl groups in the microstructure of BC began to
play a role. Individual fibres interacted via the
enhanced hydrogen bonding and became physically
entangled (Fig. 10b), causing phase separation, which
most likely accounts for the appearance of flocculent
precipitates on the surface in Fig. 5d. With a further
increase in CH content, the situation changed. In the
case of excess CH, the positively charged molecules
could adsorb onto the fibre surface at high density via
electrostatic interactions or van der Waals forces, as
observed in numerous previous studies (Duvey et al.
2005; Fernandes et al. 2009; Kim et al. 2011). Charged
groups on the individual fibre surface repel each other,
inducing extension of the fibre, as illustrated in
Fig. 10c, d. Fully expanded fibres decrease the
Cellulose
Fig. 9 A schematic
diagram of the mechanism
of the increase in the bonded
area between nanofibres
when the hydrogen bonding
changed
entanglement and enhance the contact area between
fibres. Homogenous dispersion of the fibres thus
resulted in the formation of a uniform film, as shown
in Fig. 5e, f. Because the chemical bond between the
positively charged molecules and nanofibre surface
was relatively weak, the homogeneity in the system
was less than that in CHTBC (0.25:1); consequently,
the film surface was coarser.
This mechanism is fully supported by the XPS
analysis. We observed that the C–N component
increased only slightly with increasing proportion of
CH when the ratio was \0.5:1. As described in
Fig. 10, phase separation occurred when the ratio of
CH and TBC increased to 0.5:1. The majority of
precipitates were removed by centrifugation. There-
fore, an increase in the CH content in this range would
not contribute greatly to the C–N component. When
the CH:TBC ratio exceeded 0.5:1, the carboxylate
groups were completely cross-linked with amino
groups; therefore, the excess CH only interacted with
TBC on the surface via electrostatic forces. These
electrostatic forces between CH and TBC were
demonstrated by the BCHTBC sample, which still
exhibited C–N contribution even in the absence of a
cross-linking agent. As a result, the further addition of
CH was expected to cause a significant increase in the
C–N component. The C–N component of CHTBC
(1:1) and CHTBC (2:1) may contain a contribution
from the C–NH and CH macromolecules. The trends
in the carbon and nitrogen XPS analysis results were
similar. N? represents the CH molecule adsorbed by
electrostatic forces onto the TBC nanofibres, whereas
CO–NH is the result of cross-linking. The CO–NH
contribution was the highest in the case of CHTBC
(0.5:1), thereby revealing that the cross-linking of
carboxylate groups was close to completion. Only
123
 Cellulose

Fig. 10 Schematic
illustration of
morphological changes
(swirling or stretching) of
individual nanofibres caused
by surface charges; these
changes explain the phase
separation in the system. a A
surface anionic
carboxylated group
coupling with an ionised
amino group. b When the
amount of surface
carboxylated groups is
reduced, hydrogen bonding
is enhanced and nanofibres
become re-entangled. c The
nanofiber is stretched by
repulsive forces between
groups of like charge.
d Excess ionised amino
groups adsorbed onto the
surface of nanofibres via
hydrogen bonding with OH
groups

ionic electrostatic forces occurred with the further
addition of CH, which determined the increase in the
N? contribution.
The mechanical test results can be explained by the
‘‘Page’’ equation published in 1969 to describe the
tensile strength of paper sheets (Batchelor and
Kibblewhite 2007). The Page equation is given as
follows:
1 9 12A qW
¼ þ ð1Þ
T 8Z p l b RBA
where T is the tensile strength (kNm/kg), Z is the zero-
span tensile index (kNm/kg), p is the fibre perimeter
(m), l is the fibre length (m), b is the fibre–fibre bond
strength (N/m2), A is the average fibre cross-sectional
area (m2), qW is the density of the fibre (kg/m3), and
RBA is the relative bonded area, which describes the
fraction of the fibre surface bonded to other fibres.
According to the Page equation, the tensile strength is
dependent on both the fibre strength and the bonding that
occurs between fibres. Under the conditions of our
experiment, BC modification only occurred on the fibre
surface, whereas the inner microstructure remained
unchanged. Fibre strength was therefore unchanged
before and after modification. On the basis of previously
published research, the incorporation of a polymer with
weaker mechanical properties into BC often causes a
decrease in tensile strength (Chen et al. 2013; Trovatti
et al. 2012). As expected, BCHTBC exhibited a lower
tensile strength than pure BC. However, the CHTBC with

123
Cellulose
a 0.25:1 ratio exhibited greatest strength. However, the
strength of CHTBC with a 0.5:1 ratio decreased dramat-
ically, but slowly increased with the further addition of
CH. Such behaviour was difficult to reconcile with
previously published results. On the basis of the previ-
ously discussed assumption that the change of hydrogen
bonding in the BC network determines the nanofibres
aggregation or dispersion, we can explain this discrep-
ancy. As described in Fig. 9, swollen fibre bundles are
more conformable and their bonded area is increased.
Decreased hydrogen bonding can increase the RBA. If
the other parameters in Eq. (1) constant, an increase in the
RBA leads to an enhancement of the tensile strength. As
discussed in the film morphology section, conversion of
the primary hydroxyl groups at the C6 site to carboxyl or
amide groups would enable the adhesion between
nanofibres to loosen by preventing the formation of
strong inter-fibre hydrogen bonding. The presence of a
homogenous dispersion of nanofibres increased the RBA;
thus, greater tensile strength was observed in the TBC and
CHTBC (0.25:1) films. Given the previously discussed
changes in the hydrogen bonding of individual nanofi-
bres, an increase in the CH/TBC ratio to 0.5:1 led to
aggregation and phase separation. The RBA decreased
due to the fibre swirl, thereby resulting in a dramatic
decrease in tensile strength. However, when the CH
content was increased, the repulsion force produced by
like charges on the nanofibre surface caused fibre
stretching, and the RBA increased. Therefore, the tensile
strength of the film exhibited an upward trend. The
fracture section images of nanocomposite films provided
additional evidence supporting the proposed mechanism.
The densely packed morphology of TBC and CHTBC
(0.25:1) suggested good material homogeneity and strong
interlayer binding. As expected, the further addition of
CH led to phase separation accompanied by serious
delamination of the layers, as shown in Fig. 8d. However,
the presence of excess CH led to the recovery of the
homogeneity of the system, and the fracture edge again
appeared to be ordered.
Conclusion
Chitosan (CH) and BC nanocomposite films were
synthesised in the present research using a more effective
amine coupling reaction instead of the conventional
impregnation or physical blending methods. Although
numerous researchers have demonstrated that the
mechanical properties of such composites are expected
to be superior to their unmodified counterparts, the
composites of CH and BC did not behave as predicted in
our research. The CH:TBC ratio in the reaction appears to
strongly influence the properties of the films. The
predominant feature of the BC microstructure is the
existence of a large amount of hydrogen bonding.
Therefore, we proposed a mechanism to explain the
phenomenon observed in the present research. Inter-fibre
hydrogen bonds caused the nanofibres to tangle together
tightly, thereby decreasing the accessibility and reactivity
of the hydroxyl groups of BC. Surface carboxylation
resulted in a decrease in the number of strong inter-fibre
hydrogen bonds and enabled amine coupling with CH.
When the carboxylate groups on the surface of the
nanofibres had completely reacted with the amino groups
of CH, the hydrogen bonds produced by the C2 and C3
hydroxyl groups again dominated the microstructure in
the crystal and again caused swirling and aggregation of
the nanofibres. This swirling and aggregation could
explain the phase separation in the CHTBC (0.5:1)
sample. After all, the molecular structure of CH and BC
are highly similar, and they exhibit good interfacial
adhesion and strong interactions. Therefore, when an
excess of CH was added to the reaction system, the
ionised CH would adhere to the nanofibres. The like
charges on the surface led to the stretching of individual
nanofibres, thereby increasing the RBA. The tensile
strength and homogeneity of the nanocomposite films
subsequently increased. Therefore, tensile strength
depends on the single fiber strength but mostly on degree
of bonding between fibres. However, the use of an
appropriate CH:BC ratio in the synthesis reaction can
yield composite films with excellent properties, thereby
providing a promising method for functionalising BC and
CH to satisfy technical needs in a large variety of
applications.
Acknowledgments This research was supported by the Major
State Basic Research Development Program (973 Project No.
2012CB933600).
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