Biomacromolecules 2010, 11, 1217–1224 1217

Nanostructural Reorganization of Bacterial Cellulose by

Ultrasonic Treatment
Paula C. S. Faria Tischer,*,† Maria Rita Sierakowski,† Harry Westfahl, Jr.,*,‡ and
Cesar Augusto Tischer†
Laboratório de Biopolı́meros, Universidade Federal do Paraná (UFPR), Caixa Postal 19081,
81531-990, Curitiba, Paraná, Brazil, and Laboratório Nacional de Luz Sı́ncrotron (LNLS), CEP 13083-970,
Caixa Postal. 6192, Campinas, SP, Brazil
Received December 4, 2009; Revised Manuscript Received March 16, 2010

In this work, bacterial cellulose was subjected to a high-power ultrasonic treatment for different time intervals.
The morphological analysis, scanning electron microscopy, and atomic force microscopy revealed that this treatment
changed the width and height of the microfibrillar ribbons and roughness of their surface, originating films with
new nanostructures. Differential thermal analysis showed a higher thermal stability for ultrasonicated samples
with a pyrolysis onset temperature of 208 °C for native bacterial cellulose and 250 and 268 °C for the modified
samples. The small-angle X-ray scattering experiments demonstrated that the treatment with ultrasound increased
the thickness of the ribbons, while wide-angle X-ray scattering experiments demonstrated that the average crystallite
dimension and the degree of crystallinity also increased. A model is proposed where the thicker ribbons and
crystallites result from the fusion of neighboring ribbons due to cavitation effects.

Introduction The high crystallinity of bacterial cellulose is the result of a

Cellulose is one of the most important biopolymers and is
widely used based on its availability, biocompatibility, biological
degradability, and sustainable production.1 In particular, bacterial
cellulose is a pure biopolymer, without collateral biogenic
compounds like lignin, hemicelluloses, and pectin, and it is an
attractive biopolymer due to its facile production and purifica-
tion.2 The structure of bacterial cellulose (BC) was described
more than a century ago,3,4 and the material has been extensively
analyzed and characterized.5-9
Bacterial cellulose has been produced from Acetobacter
xylinum by fermentation of different substrates as glucose from
corn syrup,10-12 and more recently, rice bark supplemented with
glucose was used as a substrate to produce cellulose nano-
spheres.13
Cellulose molecules form long chains in polycrystalline
fibrous bundles, which contain crystalline and amorphous
regions, and the degree of crystallinity and crystal dimensions
are dependent on the origin of the cellulose. The biosynthetic
process is the same in all organisms, but there are some
differences in the cellulose synthase complexes that determine
the size and thickness of the cellulose microfibrils, and the great
interest in cellulose macromolecules is due to their crystalline
orientation.14
The microstructures formed by the ultrafine microfibrils of
bacterial cellulose have lengths varying from 1 to 9 µm and
create a dense reticulated structure stabilized by various
hydrogen bonds. These networks show a high index of crystal-
linity (above 60%) and a higher degree of polymerization,
normally around 16.000 or 20.000,15 in comparison with vegetal
cellulose.
* To whom correspondence should be addressed. Phone: +55 41 3361-
3260 (P.C.S.F.); +55 19 3512-1034 (H.W.). E-mail: paula.tischer@pq.cnpq.br
(P.C.S.F.); westfahl@lnls.br (H.W.).
†
Universidade Federal do Paraná (UFPR).
‡
Laboratório Nacional de Luz Sı́ncrotron (LNLS).
10.1021/bm901383a
Published on Web 04/06/2010
high number of inter- and intrahydrogen bonds between adjacent
chains of glucan. These bonds create a regular crystalline
arrangement between the glucan molecules,16 resulting in the
distinct diffraction pattern, swelling, and reactivity of cellulose.
One of the methods used to obtain microcrystals of cellulose
is to submit cellulose to controlled acid (H2SO4) hydrolysis
conditions. The hydronium ions can penetrate the cellulose
chains in the amorphous domains, promoting the hydrolytic
cleavage of the glycosidic bonds and releasing individual
crystallites. In this process, sulfate groups are randomly
distributed on the cellulose microfibril surface, inducing a
negative electrostatic layer covering the microfibers.20-22 The
cellulose microcrystals, because of their stiffness, thickness, and
length, are commonly called “whiskers”.17-19
For pursuing applications that require extensively entangled
networks and higher strength, chemically less aggressive hy-
drolysis concepts have been found to maintain a high aspect
ratio of the cellulose I fibrils or fibril aggregates, potentially
allowing strong or even permanent junction points for the
networks. A classic example is provided by omitting the
hydrolysis step and by solely imposing high shearing forces
for disintegration. This process yields a highly entangled
network, which typically consists of elements with a wide size
distribution down to the nanoscale. The resulting material has
been denoted microfibrillated cellulose (MFC), originally in-
troduced by Turback et al.23 and Herrick et al.24
By this method, the cellulose forms a highly entangled
network with a low degree of crystallinity, and some of the
noncrystalline domains remain intact. This method has the same
problems, and the increased energy of tension necessary to cause
the disintegration of fibers requires several steps of treatment.
Recently, a novel route was demonstrated that combines
enzymatic hydrolysis and mechanical shearing to form a long
and highly entangled network of cellulose I elements.25
Another method cited to prepare spherical nanoparticles of
cellulose is based on a hard pretreatment of cellulose: heating
 2010 American Chemical Society
1218 Biomacromolecules, Vol. 11, No. 5, 2010
at 80 °C in 5.0 M NaOH for 3 h, followed by the resuspension
of fibers in DMSO in a water bath at 80 °C for 3 h. Next, the
pretreated fibrils are suspended in an acid solution and sonicated
with heating at 80 °C for 8 h. By this process, nanospheres of
60-570 nm composed of cellulose II are obtained.26
All these methods were realized with vegetal cellulose, and
hard conditions for hydrolysis were used, which caused intense
degradation of the polymer and a consequent reduction in
crystallinity.
The effect of ultrasound in degrading polysaccharide linkages
is well described. Studies with chitosan,27-30 dextran,31-33
agarose and carrageenans,34 xyloglucan,35 cellulose derivatives,
and carboxymethylcellulose36-38 have been described, and in
the majority of cases, the treatment with ultrasound has been
used in acid solutions with relatively low temperature and in
alkaline39 or acidic conditions.40
The energy of ultrasound is transferred to the polymer chains
through a process called cavitation, which is the formation,
growth, and violent collapse of cavities in the water. The energy
provided by cavitation in this so-called sonochemistry is
approximately 10-100 kJ/mol,41 which is within the hydrogen
bond energy scale.42
The main goal of this work was to analyze the effect of
ultrasonic treatment on the reorganization of bacterial cellulose
microfibrils using suitable conditions (aqueous medium, without
acid or heating). Such results are needed to develop an easy
method to obtain films with different nanostructures and
characteristics (porosity, roughness, and crystallinity) and to
develop a process in nanotechnology that permits the formation
of new scaffolds for tissue engineering.
Materials and Methods
Microorganism, Culture Media, and Growth Conditions. The
bacterial strain Acetobacter xylinum ATCC 23769 (reclassified as the
genus Gluconacetobacter), supplied by Foundation André Tosello from
Campinas, São Paulo, was grown in a glucose medium based on the
Hestrin-Schramm’s medium culture.43 All media were autoclaved at
121 °C and 1.02 atm for 20 min.
The inoculum was prepared in a nonagitated 200-mL Erlenmeyer
flask containing about 5 mL of the thawed culture and 40 mL of the
above medium. The flask was incubated at 28 ( 1 °C for 48 h. The
obtained cell suspension was used as the inoculum. The static
fermentation was carried out for 10 days at 28 °C, and the pH was
adjusted to 5.25 with citric acid (5%, w/v).
Treatment of Bacterial Cellulose. The bacterial cellulose (BC)
pellicle that formed at the liquid-air interface of the fermentation
medium was removed, rinsed thoroughly with deionized water, and
purified by immersion in an aqueous solution of 0.1 M NaOH for one
day. The films were washed repeatedly with deionized water and then
vacuum-dried at 40 °C. This treatment was controlled to avoid the
mercerization of the BC.
Ultrasonic Process. The pellicles of the BC after vacuum drying at
40 °C were cut with scissors. The cut films (200 mg, immersed in 200
mL Mili-Q water) were shaken vigorously for 15 min. This material
was submitted to ultrasonic treatment for 15, 30, 60, and 75 min using
an ultrasonic processor, Ultrasound SONICS (200 W, 20 kHz). The
ultrasonic treatment was carried out in an ice bath, and the ice was
maintained throughout the entire sonication time. After this process,
the ultrasonicated BC was reconstituted in the form of films and
vacuum-dried at 40 °C. In all of the analysis techniques, the stable
films formed after ultrasonication were used in the solid state as whole
films or cut films.
Small-Angle X-ray Diffraction (SAXS). The SAXS patterns were
used to determine the dimensions of the microfibrils in the cellulose.
SAXS diagrams were recorded on the D02A beamline using a fixed
Tischer et al.
radiation wavelength (λ ) 1.488 Å) at the Brazilian Synchrotron
Light Laboratory (LNLS) also in the transmission geometry. The
samples were thin films, supported in the sample holder and kept
in a 10-2 mbar vacuum. Scattering patterns were recorded during
300 s exposures on a marCCD 165 detector (8 × 8 binning), placed
first at 2507 mm and then at 1484 mm away from the samples.
Calibration of the distances was achieved using a silver Behenate
pattern.44 The SAXS profiles from the samples consisted of isotropic
scattering patterns that were subtracted from the CCD bias and dark-
noise according to a reference.45 The SAXS chamber parasitic
scattering was also recorded (with bias and dark-noise subtraction)
and subtracted from the sample pattern after sample attenuation
correction. The final patterns were normalized by the phase space
volume and azimuthally averaged for a sequence of wave-vectors q
(q ) (4π/λ) sin(θ); 2θ ) scattering angle) ranging from 0.04 to
1.14 nm-1 when the detector was at the longer distance and 0.1 to
2.3 nm-1 for the shorter one.
Wide-Angle X-ray Diffraction (WAXS). The WAXS patterns were
used to determine the relative crystallinity. WAXS diagrams were
recorded on the D03B beamline using a fixed radiation wavelength (λ
) 1.433 Å) at the LNLS in the transmission geometry. The samples
were thin films supported in 1 cm aluminum rings aligned perpendicu-
larly to the beam. Diffraction patterns were recorded during 120 s
exposures on a MarCCD 165 detector (2 × 2 binning), placed 100.0
mm away from the samples. Calibration of the distances was achieved
using a LaB6 pattern. The WAXS profiles from the samples consisted
of circular Debye-Scherrer rings and were subtracted from the CCD
bias and dark-noise according to a reference.45 The air scattering was
also recorded (with bias and dark-noise subtraction) and subtracted from
the sample pattern after sample attenuation correction. The final patterns
were normalized by the phase space volume and azimuthally averaged
for a sequence of wave-vectors q (q ) (4π/λ) sin(θ); 2θ ) scattering
angle) ranging from 1.4 to 28.6 nm-1.
Scanning Electron Microscopy (SEM) Analysis. Scanning electron
microscopy (SEM) was conducted to observe the surface of the bacterial
cellulose films before and after ultrasonic treatment. Samples were
sputter coated with gold and examined using a JEOL JSM-6360LV in
the CEM (Centre of Electron Microscopy) at the UFPR (Universidade
Federal do Parana).
Atomic Force Microscopy (AFM) Analysis. The topography and
surface roughness of the cellulose films were determined using a
Shimadzu FPM-9500J3 operating in air. Small pieces were cut from
each membrane, glued onto metal disks and attached to a magnetic
sample holder located on top of the scanner tube. The membrane surface
was scanned in tapping mode. All of the AFM images were taken at
25 °C.
Differential Thermal Analysis. This analysis was carried out to
determine the thermal stability of our materials by employing a Mettler
Toledo TGA/SDTA851. Samples were heated from ambient temperature
to 1000 °C in an oxygen current of 50 mL/min at a heating rate of
10 °C/min.
Results and Discussion
SEM Analysis. After ultrasonic treatment for different lengths
of time in an aqueous medium, the cellulose was vacuum-dried
at 40 °C, and the morphology of new films was initially observed
by scanning electron microscopy (SEM). Figure 1a shows the
3-D reticulated structure of the cellulose fibrils with intercon-
nected pores of different sizes, a typical structure of cellulose
produced by A. xylinum in a glucose medium by static
fermentation.16 The bacteria Acetobacter xylinum synthesize
primary nanofibrils with lateral sizes in the range of 7-13 nm,
which aggregate into thin and flat bands or ribbons having
widths of 70-150 nm.46
The SEM micrograph of the native bacterial cellulose showed
bands of microfibrils (ribbons) with widths of 90-140 nm. After
Nanostructural Reorganization of Bacterial Cellulose
Figure 1. Scanning electron micrographs of bacterial cellulose, native (a) and ultrasonicated for 15 (b), 30 (c), 60 (d), and 75 min (e).
the shorter ultrasonication time, we can observe that there is a
reduction in the width of the ribbons and a decrease in the
number/density of pores (Figure. 1b). Apparently, a change in
the morphology causes the surface to become more compact
with fewer pores. Increasing the length of the treatment reduces
the ribbons even further, and the surfaces become more uniform
(Figure 1c-e). The width of the ribbons cannot be determined
by SEM analysis because of their reduced size, so it was
determined by AFM analysis and by SAXS instead.
AFM Analysis. The surface morphology and surface char-
acteristics, such as roughness, are important factors in interfacial
interactions; thus, the surface changes in bacterial cellulose, after
ultrasonic treatment, were determined. The tapping-mode height
images of the ultrasonic cellulose surfaces are shown in Figure
2. The width of the ribbons in the native BC was determined to
be between 110-140 nm, values near those observed by SEM.
In the BC that was ultrasonicated for 15 min, there was,
apparently, a reduction in the width (70-110 nm) and an
increase in the thickness (39-42 nm) of the ribbons; there seems
to be a rather open fibrillar network on the surface of this film.
Biomacromolecules, Vol. 11, No. 5, 2010 1219
Apparently, the ultrasonic treatment changed the microfibrillar
arrangement, leading to a film with a different nanostructure.
From the height obtained with AFM, we observed that after 15
min of ultrasonic processing, there is an increase in the ribbons’
thickness (Table 1). Apparently, increasing the time of ultrasonic
application to 60 min or more caused a reduction of thickness
of the ribbons.
The image presented in Figure 2c shows the surface of the
film that was treated for 60 min with ultrasound, showing
changes in the morphology of the ribbons. The surface of the
BC film that was ultrasonicated for 30 min has similar features
(data not shown). After 75 min, individual ribbons with widths
of 40-70 nm could be observed. The AFM analysis allowed
us to observe that on the surface of the cellulose films that were
ultrasonicated for 30 and 60 min the shape of this nanomaterial
was irregular.
Determining the thickness of the ribbons from AFM is prone
to imprecision due to the arrangement of the ribbons on a flat
surface. The determination of the variation in the thickness and
1220 Biomacromolecules, Vol. 11, No. 5, 2010 Tischer et al.

Figure 2. AFM tapping-mode height images in air of a bacterial cellulose thin film: native (a) and ultrasonicated for 15 (b), 60 (c), and 75 min
(d).

Table 1. Width, Height, and Roughness (rms) of the Native BC

and the Ultrasonicated BC at Different Times, Analyzed by Atomic
Force Microscopy (AFM)a
rms (nm) width (nm) height (nm)
BC 38.5 110.0 25.2
120.0 20.6
140.0 20.4
BC15 37.9 51.0 39.4
64.6 42.3
70.4 40.3
BC60 29.3 30.9 12.6
49.9 12.0
52.9 22.1
BC75 17.3 26.4 22.0
36.2 26.4
39.5 20.0
a
Area: 2 × 2 µm.

Figure 3. Differential thermal analysis of bacterial cellulose, native

width of the ribbons after ultrasonic treatment is performed by
SAXS analyses in the next section.
The corresponding mean roughness values (rms) were dif-
ferent for all of the films reconstituted after the ultrasonication
process, varying from 38.5 to 17.3 nm (Table 1).
At least two films of the same composition were analyzed at
different areas of the surface. It is difficult to compare the
different roughness values reported in the literature because of
a lack of uniformity in the methods applied to determine surface
roughness; moreover, the size of the scan area is highly variable.
After the ultrasonication process, we can see evidence of
homogeneous profiles and low roughness values due to more
homogeneous substrates, opening the possibility for a future
study of biomolecular adsorption.
and ultrasonicated.
Differential Thermal Analysis. Ciolacu and Popa47 showed
that the lower the crystallinity of the polymer, the lower is its
thermal stability. Differential thermal analysis of native bacterial
cellulose and ultrasonicated samples is shown in Figure 3.
The thermal decomposition pattern of cellulose can be
explained mainly by two different mechanisms.48 The first one
involves the degradation due to the breaking of internal bonds,
dehydration or formation of free radicals, and reactive carbona-
ceous char. The second mechanism involves the cleavage of
secondary bonds and the formation of intermediate products,
Nanostructural Reorganization of Bacterial Cellulose
Figure 4. Guinier plots for native cellulose (a), cellulose treated for
15 min (b), and cellulose treated for 30 min (c).
such as anhydromonosaccharides, which are converted into low
molecular weight polysaccharides and finally carbonized prod-
ucts.49
The results obtained from differential thermal analysis showed
us the onset temperature of the pyrolysis was 208, 250, and
268 °C for the native BC and the BC ultrasonicated for 15 and
30 min, respectively. In addition, after comparing the three
samples, it was observed that the weight losses of the two
degradation events were similar, indicating that the ultrasonic
process did not change the cellulose chemically; apparently, only
structural changes occurred. More data from differential thermal
analysis can be seen in the Supporting Information.
SAXS Analysis. Previous X-ray scattering studies on BC50,51
have already evidenced an ultrastructure composed of nanofibrils
(in the nm range) aggregated into microfibrils (in the 10 nm
range), attached together through interconnecting cellulose
chains, to produce ribbons (in the 100 nm range) that are the
basic units of the 3D network observed in bacterial cellulose.
The BC ribbons are the basic units that contribute most for
the X-ray scattering at small angles. However, due to their
extensive length (L), the full radius of gyration of the ribbons
is way beyond the detection limit of conventional SAXS.
Nevertheless, it is still possible to observe an intermediate
scattering region where a cross sectional radius of gyration,rc,
can be identified and related52 to the thickness a and the width
Biomacromolecules, Vol. 11, No. 5, 2010 1221
Figure 5. SAXS plots from the three samples along with the
extrapolated areas (light dots).
Table 2. Dimensions of the Ribbons Obtained from SAXS
a (nm) b (nm)
BC 7.6 75
BC15 21 86
BC30 15 81
b of the ribbons byrc ) (a2 + b2)/(12). In this region, which in
our experiment belongs to the lowest wave vectors, the cross
sectional radius of gyration is determined53 by the linear
coefficient of the curve ln(qI(q)) × q2 as in Figure 4.
It is clear from Figure 4 that rc increases with ultrasonic
treatment, indicating that one or both cross sectional dimensions
increased with ultrasonic treatment. However, this does not
necessarily imply a thickening of the ribbon. In fact, because
the cross sectional radius of gyration alone cannot determine a
and b independently, we also need to evaluate the Porod cross
sectional area of the ribbons,S ) ab, given by the SAXS curves
as52,53 S ) 2π limqf0(qI(q))/Q. The scattering invariant Q can
be determined by the total area of the scattering curve,53
extrapolated into the Guinier and Porod regions (see Supporting
Information for details of this calculation). From Figure 5, we
can see that the normalized scattering curve, I(q)/Q, is shifted
upward with ultrasonic treatment, indicating that the cross
sectional area of the ribbons has also increased.
Finally, combining the data for S and rc, one obtains the cross
sectional dimensions a and b of the ribbons, as presented in
Table 2. The dimensions of the native microfibril bands obtained
here are similar to the ones reported previously;52 however, as
suggested by the SAXS data analysis, the treatment with
ultrasound seems to increase their thickness, resulting in a
modification of the nanostructure of the BC. Note, however,
that this observation does not necessarily imply an increase in
crystallinity because the ribbons are composed of crystalline
microfibrils and amorphous cellulose. The crystallite dimensions
and crystalline fractions of the samples are determined from
the WAXS data analysis in the next section.
WAXS Analysis. The great interest in the mechanical and
medical properties of bacterial cellulose is due to its crystalline
orientation. The physical properties, as well as the accessibility
in the chemical reactions, swelling and adsorption, are strongly
influenced by the crystallinity.54
Due to the linearity of the cellulose backbone, adjacent chains
of cellulose form a framework of water-insoluble aggregates
of varying length and width. These microfibrils consist of both
highly ordered (crystalline) and less ordered (amorphous)
regions. Depending on the source and the conditions used in
cellulose treatment, different degrees of crystallinity can be
obtained.55
1222 Biomacromolecules, Vol. 11, No. 5, 2010
Figure 6. Lorenzian fits to the WAXS reflections in the Debye-Scherrer
mode for the native BC (a), BC treated for 15 min (b), and BC treated
for 30 min (c).
The dimensions of the crystalline regions can also be
obtained from the line widths of the crystalline reflections
using the Scherrer method. Although not accurate, as a result
of neglecting the contributions due to paracrystallinity
defects, this method can be useful for comparing the
crystallite sizes of the three samples. Thus, we fit Lorenzians
to the main crystalline reflections with different positions
and widths, along with a smooth amorphous background, as
presented in Figure 6.
The positions of the crystalline peaks are fixed for the three
samples, using the indexing of the main IR reflections according
to Nishiyama et al.56 Note also that the Miller indices of the
reflections are from a unit cell with the chains aligned along
the a-axis. The widths and heights of the Lorenzian peaks are
fitted simultaneously to the smooth amorphous background,
which we represent by four broad Lorenzians spread along the
entire range of measured wave-vectors and centered at ap-
proximately q ) 0, 7, 12, and 26 nm-1.
The fwhm (full width at half maximum) of the Lorenzian
peaks are determined by the widths of the crystallites in the
direction normal to the Bragg planes given by the Scherrer
relation as follows:
2πK
Dhkl )
∆qhkl
Tischer et al.
Table 3. Crystallinity (xc) and Widths of the Crystallites in the
Directions Normal to the Corresponding Bragg Planes
xc D010 D001 D011 D-411
native 0.44 3.4 10.1 7.5 10.9
BC 15 0.55 6.4 9.6 6.5 19.4
BC 30 0.63 5.4 8.5 6.0 17.6
where K is a constant that depends on the shape of the
crystallites (K ≈ 0.94 for cubic and K ≈ 1.1 for a spherical
shape), and ∆qhkl is the fwhm of the hkl reflection.
Although subtle, the differences in the crystalline structure
of plant and bacterial cellulose have been a subject of great
debate.54 In bacterial cellulose, the major fraction is usually
ascribed to IR crystallinity. For the present data, a Rietveld or
linked atoms least squares (LALS) fit would have to be
performed to determine precisely the relative amounts of IR and
Iβ components.55,56 However, our main concern here is only
the relative amount of crystallinity between the native and the
ultrasonically treated samples. We, therefore, index the reflec-
tions according to IR crystallinity. In Table 3, we present the
average crystallite lengths along selected directions, according
to the Scherrer relation.
As we can see, due to the ultrasonic treatment, the average
crystallite dimension increases in the (010) direction but shrinks
in the other directions. These changes in crystalline dimensions
are compatible with the increase in the ribbon dimensions
obtained from the SAXS analysis.
The crystallinity of the native bacterial cellulose and the
samples ultrasonicated for 15 and 30 min was calculated from
the invariant integral of the crystalline and total scattering
contributions as
∫ Icr(q)q2dq
xc )
∫ I(q)q2dq
where Icr(q) is the contribution to the scattering curve due to
only the crystalline peaks and I(q) is the full (amorphous +
crystalline) WAXS curve.
The crystallinity of these samples, native and ultrasonically
treated for 15 and 30 min, is 0.44, 0.55, and 0.63, respectively.
Although this method is not as reliable for obtaining the
crystallinity57 as the Rietveld method, the relative increase in
xc is clear and appears to be due to the conversion of the
amorphous halo background directly into the (010) facets of
the new crystallites.
The transformation of amorphous cellulose into cellulose I
is expected because polymerization and crystallization are
coupled in Acetobacter xylinum. It has been shown by Benziman
et al.58 that by preventing crystallization using Calcofluor, the
amorphous cellulose is formed as bundles of parallel glucan
chains that crystallize into cellulose IR after drying. This suggests
that, in normal biogenic conditions, the amorphous parts from
bacterial cellulose are cell-directed into parallel bundles ready
to crystallize into cellulose I. The activation energy required
for such a process is provided by cavitation from the ultrasonic
treatment. This process occurs primarily in the amorphous
regions, where water can more effectively penetrate.
It is clear that part of the amorphous background correspond-
ing to the broad features around q ) 7 and 12 nm-1 (the blue
curve in Figure 6), which may come from the intermicrofibrillar
cellulose chains, is being converted into crystallites. However,
if the conversion of amorphous material into crystalline material
Nanostructural Reorganization of Bacterial Cellulose
Figure 7. Scheme illustrating the changes after ultrasonic treatment
and the creation of new crystallites.
occurred in each ribbon independently, we would expect a
decrease in their thickness rather than an increase because the
density of crystalline cellulose is higher than that of amorphous
cellulose.
Therefore, we propose that the thicker crystallites result from
the fusion of neighboring ribbons, as shown in Figure 7.
Indeed, it is expected that the surface of the ribbons will be
much more susceptible to the effects of cavitation than the
crystalline core. This scenario supports the conclusions obtained
from SEM, AFM, SAXS, and WAXS.
Conclusions
In the present work, we have demonstrated that ultrasonic
processing in mild conditions was effective in changing the
microfibrillar structure of bacterial cellulose. The ultrasound
energy is transferred through shearing and cavitation to the
glucan chains, promoting the conversion of amorphous material
into crystalline material. The energy scale of the cavitation
processes is within the hydrogen bond energy scale and occurs
primarily in the amorphous regions because these are the regions
where water can more effectively penetrate. Moreover, due to
the intrinsic nature of polymerization and crystallization pro-
moted by Acetobacter xylinum, the crystalline material formed
is of type I and not of type II, as would be expected by random
crystallization of the amorphous phase. A scheme illustrating
this effect is depicted in Figure 7. The shape of these crystallites
changed, and this process created films with higher crystallinity
and lower surface roughness.
Acknowledgment. We thank CNPq for a postdoctoral
fellowship (P.C.S.F.T.), CAPES/Brazil for financial support,
FINEP for financial support for the use of electronic microscopy
equipment (CEM), Prof. Paulo Cesar Camargo for the use of
AFM microscopy, and LAMIR (Laboratório de Análises de
Minerais e Rochas) for Differential Thermal Analysis. We also
Biomacromolecules, Vol. 11, No. 5, 2010 1223
thank Mateus B. Cardoso for insightful discussions and help
with Figure 7.
Supporting Information Available. Details of the SAXS
data analysis and additional data obtained by differential thermal
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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