Ultrasonics Sonochemistry 28 (2016) 136–143

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Fabrication of bacterial cellulose thin films self-assembled from

sonochemically prepared nanofibrils and its characterization
Dimitrios Tsalagkas a, Rastislav Lagaňa b, Ida Poljanšek c, Primož Oven c, Levente Csoka a,⇑
a
University of West Hungary, Institute of Wood Based Materials and Technologies, 9400 Sopron, Hungary
b
Technical University in Zvolen, Faculty of Wood Sciences and Technology, Department of Wood Science, 96053 Zvolen, Slovakia
c
University of Ljubljana, Biotechnical Faculty, Department of Wood Science, 1000 Ljubljana, Slovenia

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

Article history: Bacterial cellulose (BC) film formation could be a critical issue in nanotechnology applications such as
Received 27 March 2015 biomedical or smart materials products. In this research, purified pretreated BC was subjected to high
Received in revised form 8 July 2015 intensity ultrasound (HIUS) and was investigated for the development of BC films. The morphological,
Accepted 14 July 2015
structural and thermal properties of the obtained films were studied by using FE-SEM, AFM, FT-IR,
Available online 14 July 2015
XRD, TGA and DSC characterizations. Results showed that the most favorable purification treatment
was the 0.01 M NaOH at 70 °C for 2 h under continuous stirring. The most suitable ultrasound operating
Keywords:
conditions were found to be, 1 cm distance of ultrasonic probe from the bottom of the beaker, submerged
Microbial cellulose
Ultrasonic irradiation
in cold water bath cooling around 12 ± 2 °C. The power (25 W/cm2), time (30 min), BC concentration
Temperature (0.1% w/w), amplitude (20 lm) and frequency (20 kHz) were maintained constant.
Self-assembled thin films Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Wang and Cheng [16] examined the use of high intensity ultra-
sound to isolate fibrils from four cellulose sources: regenerated cel-
Bacterial cellulose (BC), also known as microbial cellulose, is a lulose (lyocell), pure cellulose fiber, microcrystalline cellulose and
promising natural polymer synthesized by certain bacteria such pulp fiber. Wong et al. [17] investigated the effect of ultrasound
as Gluconacetobacter xylinus. Even though it is chemically identical irradiation time on the depolymerization of plant and bacterial cel-
to plant cellulose, its supramolecular structure and high purity lulose. Tischer et al. [18] subjected BC pellicles to a high power
cellulose content demonstrates unique properties such as high ultrasonic treatment for 15, 30, 60 and 75 min; these were carried
crystallinity (63–71%), high water holding capacity (up to 200 out in an ice bath for tissue engineering applications.
times of its dry mass) and excellent mechanical strength. Young’s The aim of the present study was to examine the effect of two
modulus of BC sheets is in the range of 15–40 GPa, while that of main ultrasound operating conditions, i.e. the effect of temperature
a BC single fibril up to 114 GPa [1,2]. BC binds large amounts of and distance of ultrasonic probe from the bottom of the beaker on
water – up to 99 wt% during its biosynthesis in the aqueous culture morphological, structural and thermal properties of ultrasound
media [3]. Several studies have focused on the utilization of BC as defibrillated BC films. BC was previously pretreated in chemically
reinforcement material, in biomedical applications [4,5] or cellu- mild conditions in order to: (i) maintain its native cellulose I struc-
lose based smart material devices [6,7]. ture, (ii) remove bacterial cell debris and (iii) to emphasize the sub-
The isolation of cellulose nanoparticles without serious degra- sequent ultrasound defibrillation treatment. The overall purpose of
dation, at low costs and using an environmentally friendly method this research was to develop a method of obtaining highly
is constantly being sought. Recently the application of ultrasound crystalline and thermally more stable BC films suitable for energy
assisted extraction of plant polysaccharides [8–10], ultrasound harvesting devices, such as piezoelectric strain sensors.
assisted delignification [11,12], ultrasound assisted size reduction
of cellulose [13] or intensification of enzymatic hydrolysis
[14,15] has gained much interest. 2. Experimental section

2.1. Purification of nata de coco

⇑ Corresponding author. Nata de coco cubes (PT. Cocomas, Indonesia) were washed and
E-mail address: levente.csoka@skk.nyme.hu (L. Csoka). soaked in distilled water (water purification, WP) until the pH was

http://dx.doi.org/10.1016/j.ultsonch.2015.07.010
1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143 137

neutral (pH 5–7) to remove the citric acid and other components of 2.4.2. Atomic force microscopy (AFM)
syrup added for preservation. In order to improve purity of BC, nata AFM experiments were performed using a MultiMode atomic
de coco was further purified by alkaline treatment to remove any force microscopy 8 with a Nanoscope Veeco V controller (Bruker
remaining bacterial cell debris, microorganisms and other soluble Nano Surfaces, Santa Barbara, CA, USA) instrument. Small cut
polysaccharides. After being water purified, the nata de coco cubes pieces of dried BC films were placed on magnetic slides and the
were immersed in 2.5 wt% NaOH (6  103 M) overnight. This pro- scans were obtained in tapping mode using a V-shape Silicon
cess will be hereafter referred as one step purification (OSP). Nitride cantilever. Prior to the measurements, the tip radius and
Another sample was prepared in the same way and successively geometry of the tip were calculated. Two repetitions of imaging
treated with 2.5 wt% NaOCl (3.4  103 M); hereafter referred to (5  5 lm and 1  1 lm) were carried out. These experiments
as two step purification (TSP). OSP and TSP treatments were car- were implemented in an environment with constant relative
ried out by adopting the methodology as reported by Gea et al. humidity and temperature. Width was measured by image analysis
[19]. A third sample was prepared by warming nata de coco in using ImageJ software (ImageJ 1.46, National Institute of Health
0.01 M NaOH at 70 °C for 2 h under continuous stirring; this will (NIH), USA).
be called as 0.01 M NaOH purification.
Subsequently, nata de coco cubes were rinsed under distilled 2.4.3. Fourier Transform Infrared Spectroscopy (FT-IR)
water at room temperature (RT) until the pH of the water became FTIR spectra of the BC films were obtained using a Jasco
neutral. Once neutral pH was reached, BC was mechanically FT/IR6300 equipped with an ATR PRO 470-H spectrometer. A total
ground and homogenized in a 400 W blender for 10 min (medium of 50 cumulative scans were taken per sample with a resolution of
speed, 5 times  2 min with 5 min intervals). Afterwards, blended 4 cm1, in the frequency range of 4000–400 cm1, in absorbance
BC was poured into confined space silicon trays, and dried via sol- mode. ATR correction was applied in each measurement.
vent evaporation in an oven at 50 °C, for two to three days.
2.4.4. X-ray powder diffraction (XRD)
2.2. Ultrasonication of bacterial cellulose films The X-ray diffraction patterns were recorded at room tempera-
ture in the 5–80° 2h angle using an MPD Pro Panalytical diffrac-
After drying, the BC films were cut and were redispersed tometer equipped with an Xcelerator linear detector. Cu-K
(0.1% w/w, immersed in 80 mL distilled water) and subjected to (1.54056A) radiation was used with the 0.016° recording step
further grinding, this time with a hand blender for 20 s, prior to and the 1000 s per step counting time. The samples were pow-
ultrasonication. Sonication was directly applied at low frequency dered before the analysis.
(20 kHz) using an ultrasonic horn (Tesla 150 WS) with a tip diam- XRD peak height method, developed by Segal and coworkers
eter of 18 mm immersed in the suspension. HIUS treatment of BC [20], was used to determine the crystallinity index (Cr.I) by the fol-
performed using three levels of temperature; room temperature lowing equation (Eq. (1))
or no water bath (NoW), cold water bath (CW) and ice water bath
I200  Iam
(IW) and two levels of ultrasonic probe distance from the bottom Cr:I ¼ 100 ð1Þ
of a beaker; 1 cm and 4 cm respectively to evaluate the effect of
I200
cavitation active zones, local circulation and ultrasonic intensity where I200 is the peak intensity at the (2 0 0) (2h  22.5°) plane, and
distribution on BC microfibrils. The ultrasonic probe was placed Iam is the minimum intensity (amorphous scatter) at the valley
close to the surface (4 cm distance) and close to the bottom between (2 0 0) and (1 1 0) peaks (2h  18°).
(1 cm distance) of a 100 mL cylindrical beaker. A 7.4 cm distance The interplanar distances of the crystallites (d-spacings) were
(1 wavelength of ultrasound in water) was not possible to be calculated with Bragg’s law,
examined, owing to the height of the beaker. When cold water bath
was used for cooling, the temperature was about 12 ± 2 °C, k ¼ 2d sin h ð2Þ
whereas it was around 5 ± 1 °C when ice bath was used. where k is the wavelength of the X-rays, d is the spacing between
Frequency (20 kHz), amplitude (20 lm), power (25 W/cm2) and the crystal planes in the atomic lattice, and h is the Bragg angle
ultrasonication time (30 min) were kept constant. between the incident ray and the scattering planes [21].
The crystallite sizes at d1, d2 and d3, the three main peaks
2.3. Preparation of bacterial cellulose films respectively, were determined using the Scherrer equation [22]:
0:9k
Resulting ultrasound colloid dispersions were left to stand over- Cr:S: ¼ ð3Þ
night. Thereafter, the liquid supernatant phase (around 40 ml) was
Hhkl cos hhkl
collected from ultrasound treated BC, poured again into silicon where Cr.S. is the crystallite size, k is the wavelength of incident
trays and dried similarly through solvent evaporation, for a second X-rays, Hhkl is the full-width at half-maximum (FWHM) and hhkl is
time. The dried, ultrasound reconstituted BC films were carefully the Bragg angle at the corresponding lattice plane.
removed and stored in plastic bags until further analysis. Due to
the drying method, BC micro/nanofibrils were randomly oriented, 2.4.5. Thermal analysis
which assumes isotropic characteristics for the BC film. Thermal analysis techniques, thermogravimetric analysis (TGA)
and differential scanning calorimetry (DSC) were used to measure
2.4. Characterizations the thermal stability behavior of BC films. Thermogravimetric
(TG) data were acquired between 0 and 500 °C using a Perkin
2.4.1. Field Emission Scanning Electron Microscopy (FE-SEM) Elmer Diamond thermal analyzer under nitrogen purging gas
FE-SEM micrographs were obtained using a Zeiss ULTRA Plus (100 cm3 min1) at a heating rate of 2 °C min1. Differential scan-
(Oberkochen, Germany) instrument at an acceleration voltages of ning calorimetry (DSC) analysis was carried out on a Netsch
1 and 2 kV. The suspensions were filtered through a gilded PC DSC204 instrument under nitrogen purging gas (30 cm3 min1) at
membrane and dried for 1 h at room temperature. All samples a heating/cooling rate of 2 K min1. Temperature and enthalpy
were coated with a highly conductive film of gold by Bal-Tec SCD were calibrated using the melting transition of standard materials
500. (Hg, In, Sn).
138 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143

3. Results and discussion resulting in solid, bulk films. Similar observations, as in FE-SEM
images were observed in the width of cellulose microfibrils.
3.1. Morphological analysis Although alkali purification treatments were suitable to remove
bacterial debris, they were not appropriate to form suitable films
3.1.1. Field Emission Scanning Electron Microscopy (FE-SEM) for nanocellulose applications. Ultrasound process altered the
FE-SEM images, of dried films after the purification treatments microfibrillar arrangement of BC, leading to new films with differ-
and prior to ultrasonication are shown in Fig. 1. The typical net- ent nanostructure organization, as shown in Fig. 3. Similar films
work structure attributed to native BC was presented in all purified were obtained after drying the supernatant phase of ultrasound
samples. The surface structure of BC is characterized by a 3-D colloid samples at 50 °C, independently of the purification method.
fibrous ultrafine network of well-arranged nanofibrils, stabilized
by the hydrogen bonds existing in cellulose units [23–25]. 3.2. Structural analysis
From Fig. 1a, it can be seen that, WP method was only adequate
to remove the syrups contained within nata de coco package. 3.2.1. Fourier Transform Infrared Spectroscopy (FT-IR)
Bacterial cell debris and other contaminants were slightly removed 0.01 M NaOH treatment, was chosen for further research as the
after the OSP purification treatment, as shown in Fig. 1b. The suc- most favorable purification method. The influence of ultrasound on
cessive treatment with 2.5 wt% NaOCl in TSP caused the removal of 0.01 M NaOH purified BC was first evaluated by FT-IR spec-
a higher amount BC debris (Fig. 1c). However, their existence was troscopy. For a more comprehensive and qualitative analysis and
still visible. From Fig. 1d, it is clear that, BC treated in 0.01 M NaOH investigation of the FT-IR spectra, the FT-IR spectra were divided
at 70 °C for 2 h under continuous stirring, displayed the most ideal in two regions, 4000–2600 cm1 and 1800–800 cm1 (Fig. 4).
results. On their surface there were almost no bacterial skeletons Within the absorbance range of 3600–3000 cm1, a consistent
and only BC nanofibrils had remained. High resolution FE-SEM strong and sharp peak (3340 cm1) typical to cellulose I
images of non-ultrasound water purified BC samples and 0.01 M intramolecular hydrogen bond was observed in all treatments. In
NaOH purified BC samples after ultrasound treatment are shown purified treated samples the CAH stretching band existed in
in Fig. 1e and f. Their mean width was found to be 23.16 and 2900 cm1, which is also assigned to cellulose I. However, after
11.15 nm respectively. ultrasonication the absorbance in this region became broader and
alkaline treated samples displayed two peaks.
The ‘‘fingerprint’’ region 1800–800 cm1 is more complicated.
3.1.2. Atomic force microscopy (AFM) This region contains the largest number of spectral differences,
The surface topography of each BC sample was further charac- which permits the identification of any structural changes within
terized by AFM (Fig. 2). The dense and aggregated typical BC struc- cellulose samples. In all FT-IR spectra, the following characteristic
ture on the dried films, is more apparent by AFM microscopy. bands were observed: 1428 cm1 (CH2 symmetric bending),
Besides, agglomeration of BC microfibrils is also a result of the 1330 cm1 (AOH in plane bending), 1316 cm1 (CH2 wagging at
drying process. Pa’e et al. [26] investigated the effect of different C-6), 1160 cm1 (CAOAC asymmetric stretching), 1111 cm1 (ring
methods on the crystallinity, swelling ability and tensile properties asymmetric stretching), 1058 cm1 (CAO stretching), 1030 cm1
of nata de coco. The surfaces of oven dried BC films were consti- (stretching CAO) and 986 cm1 (CAO valence vibration at C-6).
tuted of numerous randomly oriented and overlapped fibrils pro- This spectra behavior is assigned to native cellulose. The most
ducing an aggregated web structure. During water evaporation, notable changes in cellulose occur at 1430 cm1, 1162 cm1,
the capillary forces among cellulose fibrils increased, their distance 1111 cm1, 986 cm1 and 893 cm1 absorption bands. In these
became much shorter and strong molecular contact was achieved, bands the crystalline cellulose I spectrum differs significantly in

(a) (c) (e)

(b) (d) (f)

Fig. 1. FE-SEM micrographs of (a) WP (1 lm), (b) OSP (1 lm), (c) TSP (1 lm), (d) 0.01 M NaOH purified (1 lm), (e) WP before ultrasound (200 nm) and (f) ultrasonicated
0.01 M NaOH – 1 cm – CW (200 nm) BC samples.
 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143 139

(a) (b) (c)

Fig. 2. AFM images of (a) WP, (b) 0.01 M NaOH purified and (c) ultrasonicated 0.01 M NaOH – 1 cm – CW BC samples.

(a) (b) (c)

Fig. 3. Macroscopic images of (a) WP, (b) 0.01 M NaOH purified and (c) ultrasonicated 0.01 M NaOH – 1 cm – CW BC dried films.
3340

1030
1058
986
1111
1160
2900

1316
1330
1428

(a) 4 (b) 4
[8] [8]

[7] [7]

[6] [6]
Absorbance (a.u.)

Absorbance (a.u.)

[5] [5]
2 2
[4] [4]

[3] [3]
[2] [2]

[1] [1]
0 0
4000 3900 3800 3700 3600 3500 3400 3300 3200 3100 3000 2900 2800 2700 2600 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800
-1 -1
Wavenumbers (cm ) Wavenumbers (cm )
1 1
Fig. 4. Comparative FT-IR spectra of the (a) 3800–2600 cm region and (b) 1800–800 cm region of BC samples: [1] WP no ultrasound, [2] 0.01 M NaOH purification no
ultrasound, [3] 0.01 M NaOH – 1 cm – NoW, [4] 0.01 M NaOH – 1 cm – CW, [5] 0.01 M NaOH – 1 cm – IW, [6] 0.01 M NaOH – 4 cm – NoW, [7] 0.01 M NaOH – 4 cm – CW, [8]
0.01 M NaOH – 4 cm – IW.

relation to cellulose II and amorphous cellulose [27,28]. is closely related to OH region and the intermolecular and/or
Furthermore, there is no indication of the following absorption intramolecular hydrogen bonding network, which sensitively fol-
bands, 1376 and 1278 cm1, assigned mainly to crystalline cellu- lows the physical and structural changes of cellulose during ultra-
lose II and amorphous cellulose [29]. sound treatment [28,33–34]. The results of the infrared
The Lateral Order Index (LOI) and Total Crystallinity Index (TCI), crystallinity ratios (TCI, LOI) and hydrogen bond intensity (HBI)
proposed by Nelson and O’Connor [30,31] and O’Connor [32] are shown in Table 1.
respectively, were used to study the infrared crystallinity changes It should be noted that even though this method is simple and
of cellulose samples. The so-called hydrogen bond intensity (HBI), fast, it provides relative crystallinity index (Cr.I.) values only,
140 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143

Table 1 d3
Determined lateral order (LOI), total crystallinity (TCI) infrared crystallinity indices 90000
and hydrogen bond intensity (HBI) values.
80000
Treatment TCI A1372/A2900 LOI A1430/A898 HBI A3308/A1330 d1
WP – no ultrasound 0.67 0.85 2.97 70000
0.01 M NaOH – no ultrasound 0.73 1.32 3.80 d2

Intensity (a.u.)
0.01 M NaOH – 1 cm – NoW 0.76 1.22 2.81 60000 [4] 0.01 M NaOH - 4 cm - CW
0.01 M NaOH – 1 cm – CW 0.75 1.21 3.35
0.01 M NaOH – 1 cm – IW 0.69 1.23 3.38 50000
[3] 0.01 M NaOH - 1 cm - CW
0.01 M NaOH – 4 cm – NoW 0.62 1.30 3.64
0.01 M NaOH – 4 cm – CW 0.63 1.30 3.89 40000
0.01 M NaOH – 4 cm – IW 0.66 1.40 3.72
30000
[2] 0.01 M NaOH - 1 cm - Now
20000

owing to the fact that the spectrum always contains contributions

10000 [1] 0.01 M NaOH No Ultr.
from both crystalline and amorphous regions [35]. TCI is propor-
tional to the crystallinity degree of cellulose, while LOI is related
10 20 30 40 50 60
to the overall degree of order in cellulose [36]. Poletto et al. report
2 theta (degree)
that cellulose samples with higher TCI, LOI and HBI might demon-
strate higher crystallinity. On the contrary, Carrillo et al. [27] men- Fig. 5. X-ray diffraction patterns of [1] 0.01 M NaOH purification no ultrasound, [2]
tioned that higher crystallinity, in the case of regenerated cellulose, 0.01 M NaOH – 1 cm – NoW, [3] 0.01 M NaOH – 1 cm – CW and [4] 0.01 M NaOH –
was indicated when TCI values were increased and LOI values were 4 cm – CW BC samples.

decreased.
In this study, it had been attempt to investigate the possibility
Table 2
of empirically estimating the crystallinity of BC relying on LOI, XRD d-spacings, peak intensities, crystallite sizes at 2h angles and Cr.I. of 0.01 M
TCI and HBI values. In accordance with the results, a combination NaOH BC under the investigated ultrasound operating conditions.
of higher TCI and lower LOI indices was selected for comparison
Treatment d spacings (nm) Cr. size (nm) Cr.I (%)
among ultrasound treated 0.01 M NaOH purified BC samples, as
d1 d2 d3 d1 d2 d3
reported in Carillo et al. The other option, i.e. the combination of
higher TCI and LOI values was preferred among the different BC 0.01 M NaOH – No ultrasound 0.60 0.52 0.39 5.45 5.47 5.52 77.78
purification treatments. 0.01 M NaOH – 1 cm – NoW 0.60 0.53 0.39 4.88 5.43 6.17 80.46
0.01 M NaOH – 1 cm – CW 0.60 0.52 0.39 4.71 5.19 5.81 77.62
The following ultrasound operating conditions of 0.01 M NaOH 0.01 M NaOH – 4 cm – CW 0.60 0.53 0.39 5.55 5.57 5.62 81.70
purified BC samples were chosen for further characterizations with
XRD, TGA and DSC techniques: (i) 1 cm distance of the ultrasonic
probe – no water bath (1 cm – NoW), (ii) 1 cm distance of the ultra-
sonic probe – cold water bath (1 cm – CW) and (iii) 4 cm distance investigated the properties of acid and mechanically treated,
of the ultrasonic probe – cold water bath (4 cm – CW). No ultrason- spray-dried BC obtained from nata de coco. Nevertheless, the crys-
icated 0.01 M NaOH purified BC samples were also examined as  0 (d1 spacing), 1 1 0 (d2 spacing) and 2 0 0 (d3 spac-
tallite sizes in 1 1
control. ing) lattice planes displayed shifted values, influenced by the
XRD results showed that in water purified and the highly crys- different ultrasound operating conditions. These results indicate
talline 0.01 M NaOH purified BC, Cr.I. was almost identical. This that ultrasound treatment resulted in changes to the crystalline
could be a possible explanation of obtaining similar TCI and LOI shape of BC microfibrils.
values after ultrasonication. In contrary, there were observed
changes in these values among purification treatments and in
ultrasonicated OSP and TSP bacterial cellulose samples. Cr.I was
3.3. Thermal analysis
notably increased after application of ultrasound in OSP and TSP
bacterial cellulose.
3.3.1. Thermogravimetric analysis (TGA)
The thermogravimetric (TGA) and derivative thermogravimetric
3.2.2. X-ray diffraction analysis (XRD) (dTGA) curves of ultrasound treated 0.01 M NaOH alkaline-purified
The X-ray diffraction patterns of ultrasound-treated BC BC samples have been shown in Fig. 6.
obtained from 0.01 M NaOH purified nata de coco are presented Thermal degradation of BC includes depolymerisation, dehydra-
in Fig. 5. Diffractograms displayed three main characteristic peaks tion and decomposition of glycosic units, and subsequent oxidation
assigned to cellulose I in X-ray diffractions, and these are located at leading to the formation of a charred residue. To examine the
approximately 2h = 14°, 16° and 22° Corresponding to 1 1  0 (d1 thermal degradation behavior, the onset thermal degradation
spacing), 1 1 0 (d2 spacing) and 2 0 0 (d3 spacing) crystallographic temperature (Td) was taken at 10% weight loss. The thermal
planes respectively. The diffractogram peak located at around 2h decomposition of cellulose known as maximum degradation tem-
= 35°, corresponds to 0 4 0 crystallographic plane [37,38]. The perature (Tdmax), was determined from dTGA curves.
X-ray diffractograms, showed no change in peak intensities located The degradation patterns in terms of shape and temperature
at the crystallographic plane reflections, indicating no transforma- seems to be quite similar for ultrasound-treated samples. All sam-
tion of cellulose I into cellulose II allomorph due to ultrasonication. ples showed a slight weight loss at low temperatures (<100 °C) cor-
d-spacing values, crystallite sizes of the three characteristic planes, responding to evaporation of absorbed water. The Td temperature,
as well as their crystallinity index (Cr.I) calculated from X-ray were observed at 285 °C, 309 °C, 316 °C and 312 °C for 0.01 M
diffractograms are given in Table 2. NaOH purified, 0.01 M NaOH – 1 cm – NoW, 0.01 M NaOH – 1 cm
Results demonstrated no changes in the interplanar distances – CW, 0.01 M NaOH – 4 cm – CW BC samples respectively. The
(d-spacings) among untreated and ultrasound treated BC samples. dTGA peak accounting for Tdmax temperature of cellulose, were
d-spacing values are in accordance with Amin et al. [39] who observed at around 368 °C, 376 °C, 374 °C and 374 °C respectively.
 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143 141

100 0.0

-0.2
Weight of the sample (%) (a) -0.4 (b)

Rate of weight loss [% (oC)]

80
-0.6

-0.8
60
-1.0

-1.2
40
-1.4

-1.6
0.1 M NaOH pur. no ultrasound
20 0.01 M NaOH No Ultr. 0.1 M NaOH pur.1 cm no water bath
-1.8
0.01 M NaOH - 1 cm - NoW 0.1 M NaOH pur. 1 cm cold water bath
0.01 M NaOH - 1 cm - CW -2.0 0.1 M NaOH pur. 4 cm cold water bath
0.01 M NaOH - 4 cm - CW
0 -2.2
0 100 200 300 400 500 0 100 200 300 400 500
Temperature (oC) Temperature (oC)

Fig. 6. (a) TGA and (b) dTGA curves of 0.01 M NaOH purified BC under the examined ultrasound operating conditions.

The onset temperature and the maximum degradation temper- 3.4. Effect of alkaline treatment
ature of simply 0.01 M NaOH purified BC were both lower than that
of the ultrasound treated. The improved thermal stability of ultra- Even though BC is free from hemicelluloses and lignin contam-
sonicated BC films is useful for potential medical and energy har- inants, the ferment broth contains other impurities such as BC deb-
vesting applications. ris and the remaining culture medium. The most suitable
purification methods for the removal of the mentioned impurities
are distilled water, sodium hydroxide and sodium hypoclorite
solutions, or organic acids like acetic acids [40,41].
3.3.2. Differential Scanning Calorimetry (DSC)
The most common purification BC method is in aqueous solu-
DSC thermograms of 0.01 M NaOH alkaline-treated BC samples
tions of 0.1 M NaOH. However, in this study it was found that
obtained after the considered ultrasound operating conditions are
0.01 M NaOH at 70 °C for 2 h under continuous stirring was cap-
shown in Fig. 7.
able enough of removing BC debris. Warm temperature conditions
The thermal behavior presented in all samples is related to
at 70 °C allowed better alkali penetration into the inner parts of
monotropic solid-to-solid transition, which is an endothermic
nata de coco cubes and BC aggregated mirofibrils than by carrying
reaction and frequently occurs in organic compounds, like cellu-
out at ambient temperatures. It is well known that proteins and
lose. This kind of feature was possible to be observed because of
nucleic acids can be hydrolyzed with alkaline solutions at warm
the low DSC heating rate (2 K min1) and indicated the slow
temperatures.
transition and rearrangement of amorphous regions to a stable
Alkaline treatments, even at these low concentrations, can
crystalline phase. A possible explanation is given by Tischer et al.
cause a weak to very low swelling on BC samples [42,43]. It is
[18]. They claim that ultrasound energy transferred through shear-
assumed, that swelling increased the contact surface area between
ing and cavitation phenomena to the glucan chains, promoted the
solid and liquid phases and assisted in accelerating the mass trans-
conversion of amorphous material into crystalline material.
fer and shear forces produced during ultrasound, causing friction of
BC microfibrils, thus crystallite size changes took place.

3.5. Effect of ultrasound operating conditions

1.5
Ultrasound function, although seems to be simple, actually is
1.0 quite complex. Ultrasonication is influenced by many operating
Heat flow (mW/mg)

parameters and even slight changes could attribute to significant

0.5 variations in ultrasound efficiency. Some parameters, such as
geometry of ultrasonic horn, operating frequency, ultrasound
energy distribution are not easy to change or to control.
0.0
For this study, a frequency of 20 kHz was considered as the opti-
0.01 M NaOH No Ultr. mum ultrasound frequency since it was found to be effective for
-0.5
0.01 M NaOH - 1 cm - NoW extraction of plant contents [9]. The intensity of ultrasound, which
0.01 M NaOH - 1 cm - CW is proportional to the amplitude of vibration [44] was kept con-
-1.0 0.01 M NaOH - 4 cm - CW stant at 20 lm. Water due to its susceptible action to formation
of hydroxyl radicals during cavitation was used as solvent.
-1.5 On the contrary, other parameters can be investigated in order
0 20 40 60 80 100 120 140 160 of achieving the most suitable ultrasound conditions. Considering
the reaction of a cavitational process, bulk operating conditions
Temperature (oC)
such as time, ultrasonic power, distance of ultrasonic probe and
Fig. 7. DSC curves of 0.01 M NaOH purified BC under the investigated ultrasound temperature are crucial factors, which often interact with each
operating conditions. other.
142 D. Tsalagkas et al. / Ultrasonics Sonochemistry 28 (2016) 136–143

It was found that as ultrasonication time was increased, faster Cellulose-based energy harvesting devices, such as piezoelectric
cellulose degradation rate was observed and crystallinity of cellu- sensors, are an emerging field of research. Further investigations in
lose was also decreased [13]. Moreover, ultrasonic power influ- alignment of BC fibrils, cellulose reactivity, piezoelectric film
ences the number of cavitation bubbles formed, their lifetime dimensions and fabrication of piezoelectric cantilever, modeling
and the generated cavitation intensity. On account of preliminary of ultrasonication and piezoelectric effect should be considered.
research the selected operating time and power were 30 min and However, the developed method resulted in the formation of self
around 25 W/cm2 respectively, to attain defibrillation and libera- assembled, highly crystalline thin films, which could be useful in
tion of nanofibrils, but which simultaneously avoids the possible nanotechnology applications where the utilization of such films
cellulose decomposition. is important.
Acoustic cavitation phenomenon is a function of vapor pressure Our results demonstrated that ultrasonication was an advanta-
and temperature. High solvent temperatures are more desirable to geous technique of isolating cellulose nanofibrils, while concur-
disrupt strong interaction forces such as van der Waals, hydrogen rently enhanced their crystallinity. This mild way of mechanical
bonds and dipole attraction between the solute molecules and treatment facilitated the separation of inherent aggregated, puri-
solute-matrix. In contrast at lower temperatures depending on fied BC microfibrils, altered the size and shape of BC crystallites
ultrasound intensity, vapor pressure is less intense, a large number and introduced a new reformed homogeneous, well-redispersed
of bubbles collapse more violently with smaller radius, higher film. Variations in TCI, LOI and HBI index values imply that ultra-
internal temperature is reached and cavitation activity become sound influenced the hydrophilic behavior and cellulose surface
stronger [44]. accessibility and reactivity of the obtained BC films.
The decreasing bath temperature influenced the temperature of
BC aqueous dispersions, so the ultrasound intensity and cavitation
efficiency as well. In the absence of water cooling, rapidly high Acknowledgements
temperature was obtained into the BC aqueous colloid biphasic
system. As a result, isolation of BC micro and nanofibrils could be Dimitrios Tsalagkas is grateful to the Balassi Institute and the
amplified, yet ultrasound energy conversion efficiency was Hungarian Scholarship Board (HSB) for their financial support dur-
promptly reduced. In ice water bath, liquid dispersion attained ing his PhD studies. This research – as a part of the Talentum
the lowest temperature. Likelihood, acquired temperature was Project for Science and Student Talent Fostering at WHU,
not sufficient enough of breaking van der Waals and hydrogen TÁMOP-4. 2. 2. B – 15/1/KONV-2015-0005 – was sponsored by
bond forces of BC microfibrils. It is believed that moderate temper- the EU/European Social Foundation. The financial support is grate-
ature conditions demonstrated in cold water bath treatment is the fully acknowledged.
most favorable option.
Ultrasound intensity and the pressure field are not uniformly
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