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Cationic nanofibrillar cellulose with high antibacterial properties
Accepted Manuscript Title: Cationic Nanofibrillar Cellulose with High Antibacterial Properties Author: Achraf Chaker Sami Boufi PII: DOI: Reference:
S0144-8617(15)00490-7 http://dx.doi.org/doi:10.1016/j.carbpol.2015.06.003 CARP 9981
To appear in: Received date: Revised date: Accepted date:
21-4-2015 26-5-2015 1-6-2015
Please cite this article as: Chaker, A., and Boufi, S.,Cationic Nanofibrillar Cellulose with High Antibacterial Properties, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4
►Cationic
5
homogenization► the quaternization of the pulp was shown to facilitate the defibrillation
6
processes► the cationic NFC was composed of individual fibrils 4-5 nm in width and length
7
in the micronic scale was ► the inclusion of C-NFC was shown to efficiently enhance the
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antibacterial activity
Research highlights (C-NFC)
cellulose
was
prepared
via
a
high
pressure
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nanofibrillar
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Cationic Nanofibrillar Cellulose with High Antibacterial Properties
10 Achraf Chakera, Sami Boufia*
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University of Sfax, Faculty of Science of Sfax, LMSE, BP 1171-3000 Sfax, Tunisia
ip t
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* Corresponding author: Sami Boufi University of Sfax-Faculty of science BP 1171-3000 Sfax-Tunisia Fax: 216 74 274 437 E-mail [email protected]
21
Abstract
22
Cationic nanofibrillar cellulose (C-NFC) was prepared via a high pressure homogenization
23
using quaternized cellulose fibers with glycidyltrimethylammonium chloride. It has been
24
shown that the quaternization of dried softwood pulp facilitated the defibrillation processes.
25
The effects of the trimethylammonium chloride content on the fibrillation yield, the
26
transparency degree of the gel, the rheological behaviour of the NFC suspension and their
27
electrokinetic properties were investigated. AFM observation showed that the NFC
28
suspension consisted of individualized cellulose I nanofibrils 4-5 nm in width and length in
29
the micronic scale. In addition to their strong reinforcing potential, the inclusion of C-NFC
30
into a polymer matrix was shown to efficiently enhance the antibacterial activity. The
31
reinforcing potential of C-NFC, studied by dynamic mechanical analysis (DMA), was
32
compared to anionic NFC and the difference was explained in terms of the nanofibrils
33
capacities to build up strong networks held by hydrogen bonding.
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Key words: nanofibrillar cellulose, nanocomposites, cationic nanofibrils, antibacterial.
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1. Introduction
36
The production of nanocellulose from natural cellulose resources is undoubtedly one of the
37
most breakthrough in cellulose based materials over the last two decades (Khaliln Bhat,&
38
Yusra 2012, Kalia et al 2014). These bio-nanomaterials combine the advantageous properties
39
of cellulose, namely the broad range of chemical modifications, renewability, and
40
sustainability with the specific attribute of nanosized materials. Among these nanosized
41
fibrils, nanofibrillar cellulose arouses much interest and holds promising applications in a
42
number of technical applications including nanofiller with outstanding reinforcing potential in
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polymers’ matrixes (Boufi, Kaddami & Dufresne 2014, Chaker et al 2014, Besbes, Rei Vilar
44
& Boufi 2011) strength additive of papers (González et al 2011, 2012), in biomedical
45
applications such as drug delivery (Valo et al 2011), implantable scaffolds (Mathew et al
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2011), highly porous cellulose adsorbents (Maatar, Alila & Boufi 2013)
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NFC consists of long, flexible and entangled cellulose nanofibrils formed by alternating
48
crystalline and amorphous domains. It is composed of more or less individualized cellulose
49
microfibrils with lateral dimension in the order of 5 to 50 nm, depending on the production
50
and pretreatment process and length in the micrometer scale.
51
Currently, NFC is produced by disintegration of the cellulosic fibers under a high mechanical
52
shearing action using a high pressure homogenization (HPH), a micro-fluidization and even a
53
conventional high speed blender (Boufi & Gandini 2015, Chaker et al 2014) This intense
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mechanical shear is necessary to overcome the strong interfibrillar hydrogen bonds and
55
break-up the cell walls to progressively release the nanosized microfibrils. In order to
56
facilitate the fibrillation process and reduce the energy demand during the disintegration
57
process, chemical fibers pretreatment is necessary, the most common of which has been
58
recourse to the introduction of charged groups within the fibers. This can be done via a
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TEMPO-mediated oxidation (Saito et al 2006), periodate oxidation (Liimatainen et al 2012),
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carboxymetylation (Wågberg et 2008), sulfatation (Liimatainen et al 2013) and quaternization
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to produce cationized NFC (Liimatainen et al 2014, Olszewska et al 2011).
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In all of these chemical pretreatments, charged groups were introduced to generate repulsive
63
forces between microfibrils that contributed to loosen the microfibrils cohesion held by
64
hydrogen bonding. In fact, the origin of the repulsive forces is the osmotic pressure driven by
65
the difference in the ionic concentration between the interior and the exterior of the fibers.
66
Indeed, as the ionic groups are bounded to the fibrils surface and cannot move out, the
67
solution within the fibers’ pores might be regarded as separated from the external solution by
68
a semipermeable membrane that confines the ionic groups but gives free entry of water into
69
the fibres to reduce the osmotic differential pressure. The resulting fiber swelling contributed
70
to reduce the interfibrillar cohesion and facilitated the cell walls breakdown.
71
Only a limited number of publications have been concerned with cationic nanofibrillar
72
cellulose. The first work has been reported by Olszewska et al (Olszewska et al 2011 ) by
73
disintegration
74
trimethylammonium chloride in isopropanol. Ho et al prepared C-NFC by the conversion of
75
cellulose pulp with chlorocholine chloride in dimethylsulfoxyde (DMSO) followed by a
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mechanical disintegration process (Ho et al 2011) . The C-NFC was used as a matrix to
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embed layered silicates and prepare nanocomposite with improved water vapor barrier
78
properties. It was shown that C-NFC exhibited good interactions with negatively charged
79
layered silicaminerals in composite structures. Pei et al have used C-NFC to prepare
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nanopapers with excellent mechanical properties which was used as an adsorbent for dye
81
removal from aqueous waste streams (Pei et al 2013).
82
In the present work, cationization was used as a pretreatment to facilitate the disintegration of
83
commercial softwood pulp into nanofibrillar cellulose via a high pressure homogenization.
84
Cationic nanofibrillar cellulose (C-NFC) with different contents of trimethylammonium
bleached
sulphite
pulp
cationized
with
N-(2-3-epoxypropyl)
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groups were produced and characterized using different techniques. A correlation between the
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cationic content of the fibers and the efficiency of the nanofibrillation process was
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demonstrated. The effect of the inclusion of C-NFC into a polymer matrix on the antibacterial
88
properties was also studied and compared to negatively charged NFC.
89 90
2. Experimental section
91
2.1. Materials
92
Commercial bleached Eucalyptus pulp (eucalyptus globulus) was used as starting material for
93
the preparation of NFC. The other chemical products were purchased from Sigma Aldrich and
94
used without purification.
95
2.2 Quaternization of cellulose fibers
96 97
The eucalyptus pulp was first soaked in distilled water for 12 h and subjected to a mechanical
98
disintegration using domestic warring blender to obtain a fiber suspension with 5wt %
99
concentration.
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The fiber suspension was then centrifuged to remove water and solvent
exchanged (twice) with dimethylacetamide (DMAC). Then a known amount of
101
glycidyltrimethylammonium chloride (GMA) was added to the fibers suspension in DMAC
102
(10wt %) and 0.2% of KOH was added as a catalyst. the reaction was carried out at 65°C for
103
8 hours. The reaction mixture was then centrifuged and washed five times with water to
104
completely remove any residual DMAC.
105
2.3 Trimethylammonium chloride content
106
The trimethylammonium chloride content (TMAC) was determined using conductometric
107
titration of chloride ions with AgNO3. The chloride counter ion is precipitated by reaction with
108
Ag+ until Briefly, about (100 mg) of Q-fibers were dispersed in 50 mL of water and titrated
109
with 5 mM silver nitrate (AgNO3). During the titration reaction, the conductivity remained
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roughly constant until all the chloride is precipitated with AgNO3, then the conductivity grow
111
linearly.
112
2.4. Solid state Nuclear magnetic resonance (NMR)
113
Solid-state
114
All spectra were recorded by using a combination of cross-polarization, high power proton
115
decoupling and magic angle spinning (CP/MAS) methods.
116 117
2.5 Fibrillation process
118
NFC was prepared from the delignified pulp by pumping through a GEA Homogenizer
119
processor (NS1001L PANDA 2 K-GEA, Italy). The homogenization was conducted in two
120
steps. Firstly, the fibre suspension at a concentration of 1.5 wt% was passed three times at a
121
pressure of 300 bar (4350 Psi) until the suspension turned into a gel. Then, the fibrillation was
122
pursued by further five passes at a pressure of 600 bar (8700 Psi).
123
2.6. Yield in NFC:
124
A diluted gel (0.2% solid content) was centrifuged at 4500 rpm for 20 min to separate the
125
nanofibrillated material (in supernatant fraction) from non-fibrillated and partially fibrillated
126
fibres which sediment down. The sediment fraction was dried to constant weight at 90°C in a
127
halogen desiccator and the yield of the nanofibrillated fraction was calculated as such:
128
weight of dried sediment 100 Yield % 1 (weight of diluted sample %Sc)
cr
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C NMR experiments were performed on a Bruker AVANCE 400 spectrometer.
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%Sc= solid content of the diluted gel sample
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The results represent the averages of three replications.
132
2.7. Nanocomposites processing:
133
Poly(vinyl alcohol) with 98–99 % hydrolysis degree was used as a matrix to prepare NFC
134
based nanocomposite films. PVA was first dissolved in water under stirring at 90 ° C to
135
obtain aqueous solutions of 10 wt% PVA concentrations. 6 Page 6 of 29
Nanocomposites were prepared via a solution casting method. The desired amount of NFC
137
and additional distilled water (to adjust the total solid content to 5% wt) were added to the
138
PVA solution and the mixture was mechanically stirred at room temperature for 1 h to ensure
139
an effective NFC dispersion. The nanocomposite solutions were degassed, then cast into
140
polystyrene petri dishes. The films were allowed to dry in the air at 23°C and 50% relative
141
humidity before being dried in an oven at 50°C for 4 h. before any mechanical testing, films
142
were stored in standard atmosphere (23 C, 50% relative humidity) for at least 7 days. The
143
nanocomposite films obtained were Translucent to transparent, according to the NFC content
144
with a thickness in the range of 200 to 400 µm.
145 146
2.8. X-ray diffraction analysis:
147
The crystalline degree of cellulose was calculated from an X-ray diffraction profile. X-ray
148
measurements were made on sample sheets cut in small pieces after the air drying of the
149
nanofibres suspension. Cu Kα radiation, generated with a Bruker AXS diffractometer (Bruker
150
AXS, Madison, WI at 30 kV, 100 mA. Sweeps of 5–50°2θ were made with a step size of 0.05
151
s and step time measurement of 10 s. The crystalline index (CrI) was calculated using the
152
diffraction intensities of the crystalline structure and that of the amorphous fraction according
153
to the method of Segal et al. (Segal, Creely, Martin & Conrad, 1959):
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I I CrI % 002 am 100 I 002
154 155
where I002 is the intensity of the crystalline peak at the maximum at 2θ between 22° and 23°
156
for cellulose I, and Iam is the intensity at the minimum at 2θ between 18° and 19° for cellulose
157
I.
158
2.9. Rheological measurements:
159
Rheological measurements were carried out using a controlled speed rotating rheometer
160
(Rheotec RS-30) operated with cone-and-plate geometries (cone angle: 4°, diameter: 3 cm) 7 Page 7 of 29
and a Peltier temperature controller. All the measurements were conducted at a constant
162
temperature of 25 ± 0.2 ◦C.
163
2.10. Antibacterial Activities
164
The antibacterial activity of the films was tested against Gram+ bacteria;Staphylococcus
165
aureus (ATCC 25923), Bacillus cereus (ATCC 14579) and Gram- bacteria (Enterococcus
166
faecalis (ATCC 29212). and (Escherichia coli (ATCC 25922), Salmonella enterica (ATCC
167
13883) and Salmonella typhi (ATCC 14028) using the disc diffusion method on an agar plate
168
with slight modification.
169
The diffusion method was performed using Luria−Bertani (LB) medium solid agar in Petri
170
dishes. The agar slurry on the test specimens was allowed to the gel at room temperature
171
followed by incubation at 37 °C for 18 h. After incubation, films were cut into 15× 15 mm
172
pieces, sterilized in autoclave at 121°C for 20 minutes, and kept in intimate contact with the
173
bacterial agar gel. After incubation during 24h at 37°C, samples were examined visually for
174
growth of bacteria in the area surrounding the film.
175 176
3. Results and discussion
177 178
3.1 Cationization of cellulose fibers
179 180
The cationization of cellulose with glycedyltrimethylammonium chloride is a well known
181
modification approach of cellulose fibers to generate a cationic ammonium site within the
182
cellulose fibers. These latter were incorporated onto cellulose through nucleophilic addition
183
of the hydroxyl groups of cellulose to the epoxy moiety of glycidyltrimethylammonium
184
chloride, as shown in scheme 1.
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185 186 187 8 Page 8 of 29
GTA/fibers molGTA.mol- TMAC content CrI 1
Eucal-P*
-
AGU
(µmol.g-1)
Fibrillation yiel (%)
-
-
C-Eucal-210** 1
1.06
210
C-Eucal-350
0.3
0.21
300
75
C-Eucal-760
0.6
0.64
760
73
C-Eucal-1150
1
1.06
1140
76
75
8 35
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samples
74 86
Table 1: TAMC content in the quaternized softwood pulp according to the amount of used GTA
192
and the fibrillation yield after homogenisation.
an
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*pristine eucalyptus pulp. ** cationization performed without swelling in water
cr
188 189 190 191
In order to limit the hydrolysis of the epoxy ring during the addition reaction, DMAC was
195
used as a solvent instead of water, which is commonly used for this reaction. Moreover, to
196
ensure efficient swelling of the fibers and good accessibility of the reagent to the inner pore,
197
the fibers were first soaked in water for several hours and solvent exchanged with DMAC.
198
Catalytic amount of NaOH was also added to convert alcohol groups of cellulose into more
199
reactive alcoholate groups.
200
Evidence of the occurrence of the addition reaction was confirmed by 13C CP/MAS (Figure 1)
201
showing in addition to the cellulose backbone between 60 and 110 ppm, the typical signal of
202
the (CH3)3N+ moiety appearing around 55 ppm. The resonances of the cellulose skeleton
203
appeared at 105 (C-1), 88 (crystalline C-4), 84 (amorphous C-4), 75, 72 (C-2, C-3, C-5), and
204
64 (C-6) ppm. Based on the peaks integration, the content of the trimethylammonium chloride
205
is determined with accuracy and found to be in good agreement with conductometric titration
206
of chloride ions using AgNO3. By changing the ratio of glycedyltrimethylammonium
207
chloride/cellulose fibers, three samples of cationized fibers with various trimethylammonium
208
contents
209
given in Table 1. It is worth noting that, when the reaction was performed in DMAC without
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were prepared. The content of TMAC according to the GMC/Cellulose ratio is
9 Page 9 of 29
swelling the fibers in water, the cationic content of the fibers was about five fold lower than
211
that obtained with the swelling treatment (C-NFC-210) (see Table 1). This difference might
212
be explained by the lower accessibility of the hydroxyl groups within the cellulose fibers.
213
Indeed, delignified cellulose fibers exhibit a multiscale hierarchical structure and are a porous
214
material which can absorb water and other molecular components. This internal porosity,
215
created during the lignin removal, opens the cell wall and allows the fibers to expanse in
216
water, making them accessible to a wide range of molecules with different size (Thompson,
217
Chen & Grethlein 1992). Since cationization is presently adopted to facilitate the fibrillation
218
process, it is crucial that the reaction occurred inside the fibers on the surface of the accessible
219
microfibrils.
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C ellu lose M icrof ib ril
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C2,3,5
O
CH2
CH
CH2
N(CH3)3+
OH
C1
C6
C4 N(CH3)3+
220 221
Figure 1 : CP-MAS 13C NMR spectra of quaternized fibers with TMAC content of 1140
222
µmol.g-1. (C1 to C6 stands for carbones of the glucosic unit of cellulose) 10 Page 10 of 29
223 Analysis of the crystalline structure of the fibers by X-ray diffraction, revealed no evolution
225
of the cellulose I polymorph, nor in the crystallinity degree after the cationization treatment
226
(see supplementary material), which indicates that the cationization reaction occurred mainly
227
on the surfaces of the cellulose microbrils. All the samples showed a typical diffraction
228
pattern of cellulose I, with the typical diffraction peaks at 2 16, 22.6°. The CrI remained
229
roughly constant around 75% irrespective of the TMAC content.
230 231
3.2 Nanofibrillation behaviour of the cationized fibers
232
To investigate how the cationic content affected the fibrillation behavior of the fibers, a high
233
pressure homogenization was progressively carried out, starting with several passes at 300 bar
234
until the suspension turned into a gel. Then, it was pursued at 600 bar to further promote the
235
breakdown of the cell walls. During the disintegration process, samples were withdrawn at
236
regular passes and the NFC suspension was analyzed in terms of its nanofibrillation yield,
237
transparency and rheological behavior.
238
A strong dependence of the fibrillation yield with the TMAC content of the fibers was noted
239
(Table 1, Figure 2 supplementary material), with a huge enhancement in the fibrillation
240
efficiency as the TMAC content increased. The fibrillation yield reached about 35, 70 and
241
85% as the cationicity grew from 350, to 750 and 1160 µmol.g-1, respectively. In the absence
242
of any pretreatment and adopting the same disintegration mode, the fibers failed to be
243
converted into NFC and multiple clogging occurred during high pressure homogenization.
244
This phenomenon was not observed in the presence of cationized treated fibers.
245
How to explain the strong enhancement of the nanofibrillation efficiency with the TMAC
246
content? In fact, cellulose is a hierarchical material formed of elementary fibrils aggregated
247
into larger microfibrils 5–50 nm in diameter and several microns in length, sticking together
248
in a spiral manner within a multiple-layer-composite to form the cellulose fibers. Upon
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immersion in water, the fibers underwent expansion as a result of water diffusion inside the
250
fibers microspores. Then, after solvent exchange and addition of GMA, the accessible
251
microfibrils, namely those in the amorphous domain, were cationized with GMA, resulting in
252
the generation of quaternary trimethylammonium groups bounded on the surface of
253
microfibrils. The presence of these ionic groups within the cell wall resulted in an osmotic
254
swelling of the fibers that reduced the hydrogen bonding among the microfibrils by increasing
255
the gap between neighbouring fibrils as schematically illustrated in scheme 1. The
256
proportionality between the osmotic pressure and the content of ionic groups
257
Posm RT (
258
and V is the liquid volume of the fibre wall (m3/g) accounted for the enhancement in the
259
fibrillation yield as the content in TMC is going up.
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cr
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249
260
Ac ce p
OH OH
te
OH OH
d
M
an
N ion ) ( Posm = where Posm (N/m2), Nion is the moles of charged groups (moles/g), V
OH O CH2 CH CH2 N(CH3)3+ OH
Cl-
OH
Microfibril
Elementary fibril
261 262
P >P + osm+ ext +
+ +
+
+ +
Electrostatic repulsion
+
+
+
Osmotic pressure n Posm c RT V
263 264
Scheme 1: schematic illustration of how the fibers quaternization enhanced the
265
nanofibrillation process.
266 12 Page 12 of 29
The transparency degree of the C-NFC after the homogenization process was shown to be
268
greatly affected by the TMAC content of the fibers. As shown in Figure 2, at a TMAC content
269
of 350 µmol.g-1, the C-NFC gel is quite opaque and turned to a translucid gel as the TMAC
270
increased over 750 µmol.g-1. This change in the optical transparency is further highlighted by
271
the transmittance measurement of a diluted suspension of C-NFC. The transmittance level for
272
C-NFC-350 did not exceed 30% and grow to 65-70% and 72-80% for C-NFC-750 and C-
273
NFC-1160 respectively, which is indicative of the low fraction of micro-sized particles
274
responsible for light scattering. The evolution in the optical transparency is explained by the
275
change in the fibrillation yield and the dispersion of the cellulose nanofibrils in water. The
276
enhancement in the transmittance with increasing content of ionic groups is indicative of a
277
decrease in the amount of non-fibrillated and partially fibrillated fractions responsible for a
278
light-scattering phenomenon. In addition, the increase in the TMAC groups is expected to
279
enhance the colloidal stability of the cellulose nanofibrils through electrostatic stabilization
280
brought by the presence of cationic groups on the surface of the nanofibrils. This stabilization
281
prevents the likelihood tendency of nanosized fibrils to self-aggregate through hydrogen
282
interaction. The aggregation risk of nanosized particles is another phenomenon likely to affect
283
the transparency degree of the suspension.
284
The transmittance spectra of C-NFC also showed also a strong wavelength-dependence with
285
lower transmittance at shorter wavelength, namely with TMA content of 750 and1160
286
µmol.g-1. This phenomenon is explained by the dependence of scattering intensity to the
287
fourth power of wavelength ( I 1 / 4 ) for nanosized particles with square section lower than
288
50 nm.
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A 1140
300
760
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760
300
1140
cr
290 90 80
1140 µmol.g-1
us
B
760 µmol.g-1
an
60 50
300 µmol.g-1
40
M
Transmittance (%)
70
30
291
500
600
700
800
Wavelength (nm)
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0 400
te
10
d
20
292
Figure 2. (A) Visual aspect of Q-NFC gel at a solid content of 1 wt% with different TMAC
293
amount, and (B) Visible transmittance spectra of the Q-NFC suspension for different TMAC
294
content after 5 passes at 300 bar and 5 additional passes at 600 bar
295 296
The AFM images of C-NFC samples deposited on wafer substrate and dried at room
297
temperature are shown in Figure 3. these observations were relative to C-NFC samples with a
298
TMAC content of 750 and 1160 %mol.g-1 and produced via high pressure homogenization
299
following the same pressure sequence (5 passes at 300 bar followed by 5 additional passes at
300
600 Bar). Both samples showed a network of entangled individual nanofibrils having typical
301
width of 20–40 nm and lengths in the micrometer range. The length evaluation of the fibril is 14 Page 14 of 29
qualitative as the long fibrils formed an entangled network. Besides, it is hard to evaluate the
303
entire length of the fibrils accurately.
304
The fibril size, estimated from the height of the fibrils directly attached to the wafer, was
305
found to be between 3 and 6 nm for the individual non-aggregated fibrils. The NFC produced
306
from fibers with a TMAC content of 1160 µmol.g-1 seems to exhibit lower square section than
307
that with lower TMAC content. The difference between the thickness of the fibrils and their
308
width is likely to be due to the dimensions and geometry effect of the AFM tip that led to
309
surface feature broadening. This latter effect became obvious when the surface features have
310
the same or smaller size than the AFM tip. Under this condition, the tip will not be able to
311
correctly draw the profile contours giving rise to the so called convolution effects with artifact
312
appearance results, and the height should give more reliable measurement of NFC width.
313
From the AFM analysis, it can be confirmed that cationized celluloses were efficiently
314
homogenized to individual nanofibrils having lateral dimensions lower than 8 nm. Further
315
confirmation of the nanoscale dimension of C-NFC was given by FE-SEM observation
316
(Figure 4) with a larger area view of C-NFC, and where the partially fibrillated material with
317
width within 100 nm was clearly shown.
319
cr
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A
4.2 nm
3.5 nm
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cr
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2.5 nm
320
an
321
6.4 nm 4 nm
322 323
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B
324
Figure 3: AFM height images of Q-NFC with TMAC content of (A) 1140 and (B) 760 µmol.g-
325
1, and the corresponding height profile analysis (the arrow marks the points the points used
326
for the measurements of the height profile
327
16 Page 16 of 29
90 nm 10 nm
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Partially fibrillated material
Figure 4 : FE-SEM images of C-NFC produced from quaternized fibers with TMAC content of 350 µmol.g-1. (C-NFC-350)
333
3.3 Colloidal properties of C-NFC
334 335
The colloidal stability and the rheological behavior of NFC in water are highly dependant on
336
the zeta-potential of the cellulose nanofibrils. The electrokinetic properties and the rheological
337
behavior of C-NFC were studied according to the TMAC content. The evolution of the zeta-
338
potential versus pH for the C-NFC suspension is shown in Figure 5. For the different contents
339
in TMAC, the nanofibrils exhibited a positive ζ-potential over the whole range of pH.
340
However, the magnitude of the ζ-potential is shifted to higher value as the TMAC content
341
increased and showed a pH dependence between pH 5 and 11. The former effect is expected
342
since a higher content in TAMC led to an increase in the surface charge density of the fibrils.
343
The value of ζ-potential exceeding +30 mV for C-NFC with a TMA content of 760 and 1160
344
µmol.g-1 accounts for the good dispersion stability of the NFC suspension in water as attested
345
by the individualization of the nanofibrils in AFM observations and good transparency of the
346
C-NFC suspension, even at a solid content over 1wt.% .
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328 329 330 331 332
17 Page 17 of 29
40 1140 µmol.g
-1
30
760 µmol.g
-1
ip t
25 20 300 µmol.g
-1
cr
Zeta-potential (mV)
35
10 2
3
4
5
6
7 pH
348
8
9
10
11
an
347
us
15
Figure 5: Change in the zeta-potential versus pH of Q-NFC with different TMAC content.
M
349
d
1000
te
Ac ce p
Viscosity (Pa.s)
100
1140 µmol.g-1
10
760 µmol.g-1
1
300 µmol.g-1
0.1
0
2
4
6
8
10
12
Shear rate (s-1)
350 351
Figure 6. Viscosity as a function of shear rate of the Q-NFC gel at 1% solid content with
352
different TMAC content.
353
NFC suspension is known to exhibit a gel-like aspect as the solid content exceeds a critical
354
threshold in the range of 0.1-5 wt%, depending on the origin and the morphological features 18 Page 18 of 29
of the cellulose nanofibrils. The evolution of the viscosity vs. shear rate of C-NFC at different
356
TMAC contents is shown in Figure 6. All of the C-NFC suspensions exhibited a strong shear
357
thinning behavior, namely when the TMAC content exceeds 750 µmol.g-1, as attested by the
358
intense decrease in the viscosity as the shear rate increases. For instance, the viscosity of the
359
C-NFC gel with a cationicity of 1160 µmol.g-1, falls down from 725 to about 35 Pa.s, as the
360
shear rate goes from 0.4 to 5 s-1. This behaviour, typical of most NFC suspensions in water, is
361
explained by the hydrodynamic properties of charged cellulose nanofibrils with a high aspect
362
ratio. Indeed, , the NFCs flexibility along with their strong tendency to self interact through
363
hydrogen bonding led to the formation of coiled network formed by entangled cellulose
364
fibrils. Upon the application of a shear rate the network is progressively broken down, the
365
elementary nanofibrils start to be oriented in the shear direction leading to their
366
disentanglement. Consequently, the flow resistance is lowered and the viscosity is decreased.
367
Once in rest, the nanofibrils network is reformed and the viscosity rebuilt again. The shift in
368
the viscosity toward higher value as the cationic content increases is mainly due to the
369
enhancement in the fibrillation extent- as the TMAC content exceeded 700 µmol.g-1.
370 371
3.4. Antibacterial properties.
372 373
To evaluate the antibacterial activity of the C-NFC when incorporated in a polymer matrix,
374
the diffusion method using Gram+ and Gram- bacteria was adopted. Pure PVA matrix and
375
nanocomposite films prepared from negatively charged A-NFC and PVA matrix was used as
376
a reference for the sake of comparison.
377
Figure 7 presents the picture of the diffusion assay resulting from pure PVA films, C-NFC-
378
PVA and A-NFC-PVA nancomposite films with 3 and 7% nanofiller loading. Pure PVA film
379
did not show any inhibitory effects on .all the bacterial tested. The same tendency was
380
observed for nanocomposite films prepared with A-NFC, with colonies growing of BC and E-
Ac ce p
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19 Page 19 of 29
colis growing on the agar plates as well as on the surface of the A-NFC-PVA films. On the
382
other hand, for all nanocomposite films prepared with C-NFC, bacteria increased on the agar
383
plates around the films, but not on top or under the films, and no trace of colonisation of the
384
film was observed. In the presence of Staphylococcus aureus and Salmonella enterica, better
385
antibacterial activity was observed as attested by the appearance of inhibition zone around the
386
film. The absence of a clear and large inhibition zone is probably due to the immobilization
387
of antibacterial agent (C-NFC) on the polymer matrix restricting the diffusion of the
388
antibacterial agent to areas close to the specimen. The antibacterial activity of C-NFC-PVA
389
seems to be independent on the NFC loading over the range between 3 to 10wt. %. These
390
results clearly demonstrate the trivial role of C-NFC in preventing bacteria proliferation when
391
incorporation into a polymer matrix at a content as low as 4wt%. The efficient antibacterial
392
activity of C-NFC against a board range of bacteria is likely due to the presence of quaternary
393
ammonium groups on the surface of the C-NFC. These groups are known to be one of the
394
most useful antiseptics and disinfectants used in wide range of applications in cosmetic
395
products, clinical purposes, textiles and active packaging. The mechanism proposed to
396
account for these strong antibacterial properties is the interaction with the lipids and proteins
397
that compose the cytoplasmic membrane damaging the outer layer of bacterial disturbing their
398
metabolism (Denyer 1995).
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399
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381
20 Page 20 of 29
399 a
C-NFC
b
c
1 401 4 3
4
403
405 406
Bacillus cereus
C-NFC Staphylococcus aureus
C-NFC
A-NFC
d
an
408 409 410 Escherichia coli
M
Bacillus cereus
412 413
418
A-NFC
te
d
Figure 7. The antibacterial activity of control NFC-PVA nanocomposite film prepared from quationized NFC C-NFC (a, b and c) and from anionic A-NFC (d, e) tested against Gram- and Gram+ bacteria: (1) neat PVA matrix, (2) 3% NFC, (3) 5% NFC and (4) 7% NFC.
Ac ce p
414 415 416 417
C-NFC Salmonella enterica
e
407
411
3
3
us
404
4
ip t
402
1
1
cr
400
C-NFC
419
3.5 Reinforcing potential
420
In addition to the antibacterial effect imparted by the inclusion of C-NFC into a polymer
421
matrix, a reinforcing effect is also expected by the addition of NFC. To investigate the
422
reinforcing potential of the C-NFC, nanocomposite films based on PVA matrix and different
423
nanofiller loadings were prepared by solvent-casting and were analysed by DMA. For the
424
sake of comparison, similar films were also prepared using nanofibrillar cellulose bearing
425
carboxylic groups (A-NFC). The temperature dependence of the storage tensile modulus, E΄ ,
426
as a function of temperature at 1 Hz for PVA-NFC (C-NFC and A-NFC) nanocomposite with
427
different NFC are reported in the supplementary material. 21 Page 21 of 29
30
(A)
At 15% A-NFC 27-fold matrix
25
E'c/E'm.100
20
ip t
15 10
cr
At 15% Q-NFC 11 fold matrix
5
0
1
2
3
4
5
6
7
us
0 8
9 10 11 12 13 14 15 16
429 (B)
an
NFC content (wt%)
428
Cationized Cellulose nanofibril
OH
OH
OH CH2
CH2 CH O
OH
N+
H3C OH CH3
OH
OH
CH3 N+ H3C OH CH3
OH
d
OH
OH OH CH CH3 CH2 CH2 O
M
OH
OH
te
Cationized Cellulose nanofibril
430 431
OH
Ac ce p
OH
OH
OH
OH
OH
OH
OH
OH
PVA matrix
(C)
Carboxylated Cellulose nanofibril
OH
OH
O
OH
HO
C
O
OH
OH
OH
C
OH
OH O
OH
OH
HO
C
O
OH
OH C
OH
OH
Carboxylated Cellulose nanofibril
OH
OH
COOH
OH
OH
OH OH
432 433
OH OH
PVA matrix
22 Page 22 of 29
Figure 8. (A) The relative storage modulus versus NFC content at 70 °C for nanocomposite films prepared from C-NFC and A-NFC and schematic illustration of the hydrogen potential interaction among (B) C-NFC, and(C) A-NFC, when embedded into a polymer matrix.
438
At a temperature lower than the glass transition, the storage modulus undergoes only a slight
439
enhancement with respect to the unfilled matrix. For instance, at 15% NFC E΄ is about two
440
fold higher. Besides, at 20°C the nanocomposites containing 15 wt% C-NFC and A-NFC
441
exhibit a storage modulus around 1.2 and 1.4 GPa, which represents a 2.8 and 3.3-fold
442
enhancement, respectively, over that of the neat matrix (0.42 GPa). This behavior, common to
443
nanocomposite materials based on nanosized cellulose particles, is explained by the low
444
difference between the modulus of the glassy matrix (close to 1 GPa) and that of the
445
nanofiller network (being in the range of 10–15 GPa).
446
Above the glass transition, however, the storage modulus is more sensitive to the presence of
447
the nanofiller and increased significantly with NFC addition, which is in line with the well-
448
known reinforcing effect of nanocellulose based nanofiller.(Boufi 2014)
449
The stiffening effect imparted by the addition of NFC can be expressed by the evolution of
450
the relative modulus Er
451
and unfilled matrix respectively measured in the rubbery region taken here at 70°C), versus
452
the NFC content (Figure 8A). The continuous rise in the relative modulus clearly confirms the
453
reinforcing potential of the NFC. However, the extent of the reinforcing potential is different
454
from cationic to anionic NFC. At the same nanofiller loading, the modulus enhancement is
455
lower for C-NFC than that observed for A-NFC. This is namely observed when the NFC
456
content exceeded 2%. For instance, at 15 wt %, the modulus is about 27 and 11 times higher
457
for samples prepared with A-NFC and C-NFC respectively.
458
To explain the lower reinforcing potential of C-NFC compared to that of A-NFC, it is worth
459
noting that the unusual enhancement of the stiffness and strength in the nanocellulose
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an
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434 435 436 437
Ac ce p
E 'c (with Ec and Em are the storage modulus of the nanocomposite E 'm
23 Page 23 of 29
reinforced nanocomposites arouse mainly from the setup of an entangled interconnected
461
network held-up by strong hydrogen bonding. The strength of the NFC network is dependant
462
on the aspect ratio of the cellulose nanofibrils and their capacity to interact through hydrogen
463
bonding. Presently, we infer that the presence of a relatively high surface density of appended
464
2-hydroxypropyltrimethylammonium chloride moiety reduced the potential of hydrogen
465
bonding among neighbouring nanofibrils and weakened the network cohesion. This can be
466
deduced from the following analysis. Indeed, assuming that the square section of the
467
nanofibrils is 5 nm and that the average values between cellulose chains within a transversal
468
cross section ranges from 0.54 to 0.61 nm, then for a content of 1100 µmol.g-1, the surface
469
substitution degree should be DSS=DS/ratio of surface chains=0.19/0.4=0.47 . This means
470
that
471
hydroxypropyltrimethylammonium
472
trimethylammonium group to share hydrogen bonding with hydroxyl groups, the interaction
473
among entangled cellulose nanofibrils network is expected to weaken due to the shielding
474
effect imparted by the trimethylammonium group. For A-NFC, the presence of carboxylic
475
groups on the surface of the nanofibrils will, on the contrary, favour hydrogen interaction
476
among NFC. A schematic illustration of this phenomenon is given in Figure 8Aand B.
477
Though, the production of C-NFC via the quaternization of cellulose fibers as a pretreatment
478
was already reported in several paper as noted in the introduction (Olszewska et al 2011, Ho
479
et al 2011 and Pei et al 2013), the main finding of the present work is twofold; correlate the
480
fibrillation efficiency with the content of cationic groups, and highlight the great potential of
481
C-NFC to enhance the antibacterial resistance of the material when included as additive into a
482
polymer matrix. The potential usefulness of C-NFC gel as wound healing might be also
483
considered. It follows that the quaterization of NFC as both a pretreatment and method for
each
two
anhydroglucosic
M
of
chloride
units
moiety.
is
Given
linked the
to lack
the
2-
of
the
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24 Page 24 of 29
surface modification open the way for usefulness of C-NFC in biomedical application. This
485
work is under investigation.
486
4. Conclusion
487
Cationized NFC from eucalyptus pulps was prepared by cationization of the cellulose fibers
488
with GTMAC in DMF and mechanical disintegration using a high pressure homogenization.
489
What can be concluded from the present study is that:
cr
490
ip t
484
1- The cationization of the bleached cellulose fibers is an efficient approach to facilitate the breakup of the cell wall and the release of cellulose nanofibrils.
us
491
2- The fibrillation efficiency is meaningfully enhanced as the TMAC content exceeded
493
700 µmol.g-1. Over this content, thick transparent gel was obtained with good
494
colloidal stability for long-term storage.
M
495
an
492
3- AFM observation confirmed the nanosized scale of C-NFC with a square section in the range of 5 nm.
d
496
4- The inclusion of C-NFC within a polymer matrix strongly enhanced the antibacterial
498
activity of the nanocomposite films by imparting a reinforcing effect. These
499
nanocomposites films could be useful in active packaging material for food
501 502
Ac ce p
500
te
497
preservation. These results clearly suggested the great potential of cationic NFC as reinforcement additive for polymer with effective antibacterial activity.
503
References
504
Besbes I., Rei Vilar M., & Boufi S.(2011) Nanofibrillated cellulose from alfa, eucalyptus and
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pine fibres: preparation, characteristics and reinforcing potential. Carbohyd. Polym. 86, 1198-
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1206.
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Boufi S. Chapter 14 in . Biomass and Bioenergy. Khalid Rehman Hakeem,Mohammad
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Jawaid,Umer Rashid. Springer 2014, pp 267-305.
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Boufi S., Kaddami H.,& Dufresne A. (2014) Mechanical performance and transparency of
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nanocellulose reinforced polymer nanocomposites. Macromol. Mater. Eng. 299, 560–568
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Boufi S.,& Gandini A.. Triticale crop residue: a cheap material for high performance
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nanofibrillated cellulose, RSC Adv., 2015, 5, 3141–3151
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Chaker A., Mutjé P., Rei Vilar M.,& Boufi S. (2014) Agriculture crop residues as a source for
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the production of nanofibrillated cellulose with low energy demand, Cellulose 21, 4247–4259
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Chaker A., Mutje P., Vilaseca F.,& Boufi S. (2014) Reinforcing potential of nanofibrillated
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cellulose from nonwoody plants. Polym. Compos. 34, 1999-2007.
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Denyer, S.P. (1995) Mechanisms of action of antibacterial biocides. Int. Biodeterior.
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Biodegradation 36, 227–245
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González I., Boufi S., Pèlach M. A., Alcalà M., Vilaseca F.,& Mutjé P. (2012)Nanofibrillated
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cellulose as paper additive in bleached hardwood pulps. Bioresources 7, 5167-5180.
527 528
González I., Vilaseca F., Alcalá M., Pèlach M., Boufi S.,& Mutjé P. (2013) Effect of the
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combination of biobeating and NFC on the physico-mechanical properties of paper. Cellulose,
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20 (3), 1425-1435.
531 532
Ho T. T. T., Zimmermann T., Hauert R.,& Caseri W.,(2011) Preparation and characterization
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of cationic nanofibrillated cellulose from etherification and high-shear disintegration
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processes, Cellulose 18, 1391–1406
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Kalia S., Boufi S., Celli A., & Kango S. (2014) Nanofibrillated cellulose: surface
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modification and potential applications. Colloid. Polym. Sci. 292, 5–31
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Khalil H.P.S.A, Bhat A.H,& Yusra A.F.I,(2012) Green composites from sustainable cellulose
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nanofibrils: A review, Carbohydrate Polymers 87 (2), 963-979
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Liimatainen H., Suopajärvia T., Sirviöa J., Hormib O.,& Niinimäki J. (2014) Fabrication of
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cationic cellulosic nanofibrils through aqueousquaternization pretreatment and their use in
544
colloid aggregation, Carbohydrate Polymers 103, 187– 192
545
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Liimatainen H., Visanko M., J Sirvio¨ J., Hormi O.,& Niinima¨ki J., (2013) Sulfonated
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cellulose nanofibrils obtained from wood pulp through regioselective oxidative bisulfite pre-
548
treatment, Cellulose 20,741–749
549 Liimatainen H., Visanko M., Sirvio¨ J., Hormi O., Niinima¨ki J (2012) Enhancement of the
551
nanofibrillation of wood cellulose through sequential periodate–chlorite oxidation.
552
Biomacromolecules 13, 1592–1597
553 554
Maatar W., Alila S.,& Boufi S. (2013) Cellulose based organogel as an adsorbent for
555
dissolved organic compounds. Ind Crop Prod 49, 33–42
556 557
Mathew A.P, Oksman K., Pierron D.,& Harmand M.F. (2012) Fibrous cellulose
558
nanocomposite scaffolds prepared by partial dissolution for potential use as ligament or
559
tendon substitutes. Carbohydr Polym 87:2291–2298
560
Olszewska A., Eronen P., Johansson L.S., Malho J.M., Ankerfors M., Lindstrom T.,
561
Ruokolainen J., Laine J.,& Osterberg M. (2011) The behaviour of cationic NanoFibrillar
562
Cellulose in aqueous media, Cellulose 18, 1213–1226
563 564
Pei A., Butchosa N., Berglund L.A., Zhou Q.(2013) Surface quaternized cellulose nanofibrils
565
with high water absorbency and adsorption capacity for anionic dyes, Soft Matter, 9, 2047–
566
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Saito, T., Nishiyama, Y., Putaux, J. L., Vignon, M., & Isogai, A. (2006). Homogeneous
568
suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of
569
native cellulose. Biomacromolecules, 7, 1687–1691.
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571
Thompson D. N., Chen H-C,& Grethlein H. E., (1992) Comparison of Pretreatment Methods
572
on the Basis of Available Surface Area Bioresource Technology 39, 155-163
573 574
Valo H., Kovalainen M., Laaksonen P, Hakkinen M, Auriola S, Peltonen L, Linder M,
575
Jarvinen K, Hirvonen J,& Laaksonen T (2011) Immobilization of protein-coated drug
576
nanoparticles in nanofibrillar cellulose matrices-enhanced stability and release. J Control
577
Release 156:390–397
578
Wågberg L, Decher G, Norgren M, Lindstro¨m T, Ankerfors M,& Axna¨s K (2008) The
579
build-up of polyelectrolyte multilayers of microfibrillated cellulose and cationic
580
polyelectrolytes. 27 Page 27 of 29
581
Langmuir 24:784–795
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582
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583
Figure captions
584 Figure 1 : CP-MAS 13C NMR spectra of quaternized fibers with TMAC content of 1140
586
µmol.g-1. (C1 to C6 stands for carbones of the glucosic unit of cellulose)
587
Scheme 1: schematic illustration of how the fibers quaternization enhanced the
588
nanofibrillation process.
589
Figure 2. (A) Visual aspect of Q-NFC gel at a solid content of 1 wt% with different TMAC
590
amount, and (B) Visible transmittance spectra of the Q-NFC suspension for different TMAC
591
content after 5 passes at 300 bar and 5 additional passes at 600 bar
592
Figure 3: AFM height images of Q-NFC with TMAC content of (A) 1140 and (B) 760
593
µmol.g-1, and the corresponding height profile analysis (the arrow marks the points the points
594
used for the measurements of the height profile
595
Figure 4 : Change in the zeta-potential versus pH of Q-NFC with different TMAC content.
596
Figure 5. Viscosity as a function of shear rate of the Q-NFC gel at 1% solid content with
597
different TMAC content.
598 599 600 601 602 603 604 605 606
Figure 6. The antibacterial activity of control NFC-PVA nanocomposite film prepared from C-NFC (a, b and c) and from A-NFCtested against Gram- and Gram+ bacteria: (1) neat PVA matrix, (2) 3% NFC, (3) 5% NFC and (4) 7% NFC.
te
d
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an
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585
Ac ce p
Figure 7. the relative storage modulus versus NFC content at 70 °C for nanocomposite films prepared from Q-NFC and A-NFC, and schematic illustration of the hydrogen potential interaction among (A) C-NFC, and A-NFC, when embedded into a polymer matrix
29 Page 29 of 29
S0144-8617(15)00490-7 http://dx.doi.org/doi:10.1016/j.carbpol.2015.06.003 CARP 9981
To appear in: Received date: Revised date: Accepted date:
21-4-2015 26-5-2015 1-6-2015
Please cite this article as: Chaker, A., and Boufi, S.,Cationic Nanofibrillar Cellulose with High Antibacterial Properties, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4
►Cationic
5
homogenization► the quaternization of the pulp was shown to facilitate the defibrillation
6
processes► the cationic NFC was composed of individual fibrils 4-5 nm in width and length
7
in the micronic scale was ► the inclusion of C-NFC was shown to efficiently enhance the
8
antibacterial activity
Research highlights (C-NFC)
cellulose
was
prepared
via
a
high
pressure
ip t
nanofibrillar
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9
Cationic Nanofibrillar Cellulose with High Antibacterial Properties
10 Achraf Chakera, Sami Boufia*
11 a
University of Sfax, Faculty of Science of Sfax, LMSE, BP 1171-3000 Sfax, Tunisia
ip t
12 13
* Corresponding author: Sami Boufi University of Sfax-Faculty of science BP 1171-3000 Sfax-Tunisia Fax: 216 74 274 437 E-mail [email protected]
21
Abstract
22
Cationic nanofibrillar cellulose (C-NFC) was prepared via a high pressure homogenization
23
using quaternized cellulose fibers with glycidyltrimethylammonium chloride. It has been
24
shown that the quaternization of dried softwood pulp facilitated the defibrillation processes.
25
The effects of the trimethylammonium chloride content on the fibrillation yield, the
26
transparency degree of the gel, the rheological behaviour of the NFC suspension and their
27
electrokinetic properties were investigated. AFM observation showed that the NFC
28
suspension consisted of individualized cellulose I nanofibrils 4-5 nm in width and length in
29
the micronic scale. In addition to their strong reinforcing potential, the inclusion of C-NFC
30
into a polymer matrix was shown to efficiently enhance the antibacterial activity. The
31
reinforcing potential of C-NFC, studied by dynamic mechanical analysis (DMA), was
32
compared to anionic NFC and the difference was explained in terms of the nanofibrils
33
capacities to build up strong networks held by hydrogen bonding.
34
Key words: nanofibrillar cellulose, nanocomposites, cationic nanofibrils, antibacterial.
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1. Introduction
36
The production of nanocellulose from natural cellulose resources is undoubtedly one of the
37
most breakthrough in cellulose based materials over the last two decades (Khaliln Bhat,&
38
Yusra 2012, Kalia et al 2014). These bio-nanomaterials combine the advantageous properties
39
of cellulose, namely the broad range of chemical modifications, renewability, and
40
sustainability with the specific attribute of nanosized materials. Among these nanosized
41
fibrils, nanofibrillar cellulose arouses much interest and holds promising applications in a
42
number of technical applications including nanofiller with outstanding reinforcing potential in
43
polymers’ matrixes (Boufi, Kaddami & Dufresne 2014, Chaker et al 2014, Besbes, Rei Vilar
44
& Boufi 2011) strength additive of papers (González et al 2011, 2012), in biomedical
45
applications such as drug delivery (Valo et al 2011), implantable scaffolds (Mathew et al
46
2011), highly porous cellulose adsorbents (Maatar, Alila & Boufi 2013)
47
NFC consists of long, flexible and entangled cellulose nanofibrils formed by alternating
48
crystalline and amorphous domains. It is composed of more or less individualized cellulose
49
microfibrils with lateral dimension in the order of 5 to 50 nm, depending on the production
50
and pretreatment process and length in the micrometer scale.
51
Currently, NFC is produced by disintegration of the cellulosic fibers under a high mechanical
52
shearing action using a high pressure homogenization (HPH), a micro-fluidization and even a
53
conventional high speed blender (Boufi & Gandini 2015, Chaker et al 2014) This intense
54
mechanical shear is necessary to overcome the strong interfibrillar hydrogen bonds and
55
break-up the cell walls to progressively release the nanosized microfibrils. In order to
56
facilitate the fibrillation process and reduce the energy demand during the disintegration
57
process, chemical fibers pretreatment is necessary, the most common of which has been
58
recourse to the introduction of charged groups within the fibers. This can be done via a
59
TEMPO-mediated oxidation (Saito et al 2006), periodate oxidation (Liimatainen et al 2012),
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3 Page 3 of 29
carboxymetylation (Wågberg et 2008), sulfatation (Liimatainen et al 2013) and quaternization
61
to produce cationized NFC (Liimatainen et al 2014, Olszewska et al 2011).
62
In all of these chemical pretreatments, charged groups were introduced to generate repulsive
63
forces between microfibrils that contributed to loosen the microfibrils cohesion held by
64
hydrogen bonding. In fact, the origin of the repulsive forces is the osmotic pressure driven by
65
the difference in the ionic concentration between the interior and the exterior of the fibers.
66
Indeed, as the ionic groups are bounded to the fibrils surface and cannot move out, the
67
solution within the fibers’ pores might be regarded as separated from the external solution by
68
a semipermeable membrane that confines the ionic groups but gives free entry of water into
69
the fibres to reduce the osmotic differential pressure. The resulting fiber swelling contributed
70
to reduce the interfibrillar cohesion and facilitated the cell walls breakdown.
71
Only a limited number of publications have been concerned with cationic nanofibrillar
72
cellulose. The first work has been reported by Olszewska et al (Olszewska et al 2011 ) by
73
disintegration
74
trimethylammonium chloride in isopropanol. Ho et al prepared C-NFC by the conversion of
75
cellulose pulp with chlorocholine chloride in dimethylsulfoxyde (DMSO) followed by a
76
mechanical disintegration process (Ho et al 2011) . The C-NFC was used as a matrix to
77
embed layered silicates and prepare nanocomposite with improved water vapor barrier
78
properties. It was shown that C-NFC exhibited good interactions with negatively charged
79
layered silicaminerals in composite structures. Pei et al have used C-NFC to prepare
80
nanopapers with excellent mechanical properties which was used as an adsorbent for dye
81
removal from aqueous waste streams (Pei et al 2013).
82
In the present work, cationization was used as a pretreatment to facilitate the disintegration of
83
commercial softwood pulp into nanofibrillar cellulose via a high pressure homogenization.
84
Cationic nanofibrillar cellulose (C-NFC) with different contents of trimethylammonium
bleached
sulphite
pulp
cationized
with
N-(2-3-epoxypropyl)
Ac ce p
te
of
d
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an
us
cr
ip t
60
4 Page 4 of 29
groups were produced and characterized using different techniques. A correlation between the
86
cationic content of the fibers and the efficiency of the nanofibrillation process was
87
demonstrated. The effect of the inclusion of C-NFC into a polymer matrix on the antibacterial
88
properties was also studied and compared to negatively charged NFC.
89 90
2. Experimental section
91
2.1. Materials
92
Commercial bleached Eucalyptus pulp (eucalyptus globulus) was used as starting material for
93
the preparation of NFC. The other chemical products were purchased from Sigma Aldrich and
94
used without purification.
95
2.2 Quaternization of cellulose fibers
96 97
The eucalyptus pulp was first soaked in distilled water for 12 h and subjected to a mechanical
98
disintegration using domestic warring blender to obtain a fiber suspension with 5wt %
99
concentration.
d
M
an
us
cr
ip t
85
te
The fiber suspension was then centrifuged to remove water and solvent
exchanged (twice) with dimethylacetamide (DMAC). Then a known amount of
101
glycidyltrimethylammonium chloride (GMA) was added to the fibers suspension in DMAC
102
(10wt %) and 0.2% of KOH was added as a catalyst. the reaction was carried out at 65°C for
103
8 hours. The reaction mixture was then centrifuged and washed five times with water to
104
completely remove any residual DMAC.
105
2.3 Trimethylammonium chloride content
106
The trimethylammonium chloride content (TMAC) was determined using conductometric
107
titration of chloride ions with AgNO3. The chloride counter ion is precipitated by reaction with
108
Ag+ until Briefly, about (100 mg) of Q-fibers were dispersed in 50 mL of water and titrated
109
with 5 mM silver nitrate (AgNO3). During the titration reaction, the conductivity remained
Ac ce p
100
5 Page 5 of 29
110
roughly constant until all the chloride is precipitated with AgNO3, then the conductivity grow
111
linearly.
112
2.4. Solid state Nuclear magnetic resonance (NMR)
113
Solid-state
114
All spectra were recorded by using a combination of cross-polarization, high power proton
115
decoupling and magic angle spinning (CP/MAS) methods.
116 117
2.5 Fibrillation process
118
NFC was prepared from the delignified pulp by pumping through a GEA Homogenizer
119
processor (NS1001L PANDA 2 K-GEA, Italy). The homogenization was conducted in two
120
steps. Firstly, the fibre suspension at a concentration of 1.5 wt% was passed three times at a
121
pressure of 300 bar (4350 Psi) until the suspension turned into a gel. Then, the fibrillation was
122
pursued by further five passes at a pressure of 600 bar (8700 Psi).
123
2.6. Yield in NFC:
124
A diluted gel (0.2% solid content) was centrifuged at 4500 rpm for 20 min to separate the
125
nanofibrillated material (in supernatant fraction) from non-fibrillated and partially fibrillated
126
fibres which sediment down. The sediment fraction was dried to constant weight at 90°C in a
127
halogen desiccator and the yield of the nanofibrillated fraction was calculated as such:
128
weight of dried sediment 100 Yield % 1 (weight of diluted sample %Sc)
cr
ip t
C NMR experiments were performed on a Bruker AVANCE 400 spectrometer.
Ac ce p
te
d
M
an
us
13
129 130
%Sc= solid content of the diluted gel sample
131
The results represent the averages of three replications.
132
2.7. Nanocomposites processing:
133
Poly(vinyl alcohol) with 98–99 % hydrolysis degree was used as a matrix to prepare NFC
134
based nanocomposite films. PVA was first dissolved in water under stirring at 90 ° C to
135
obtain aqueous solutions of 10 wt% PVA concentrations. 6 Page 6 of 29
Nanocomposites were prepared via a solution casting method. The desired amount of NFC
137
and additional distilled water (to adjust the total solid content to 5% wt) were added to the
138
PVA solution and the mixture was mechanically stirred at room temperature for 1 h to ensure
139
an effective NFC dispersion. The nanocomposite solutions were degassed, then cast into
140
polystyrene petri dishes. The films were allowed to dry in the air at 23°C and 50% relative
141
humidity before being dried in an oven at 50°C for 4 h. before any mechanical testing, films
142
were stored in standard atmosphere (23 C, 50% relative humidity) for at least 7 days. The
143
nanocomposite films obtained were Translucent to transparent, according to the NFC content
144
with a thickness in the range of 200 to 400 µm.
145 146
2.8. X-ray diffraction analysis:
147
The crystalline degree of cellulose was calculated from an X-ray diffraction profile. X-ray
148
measurements were made on sample sheets cut in small pieces after the air drying of the
149
nanofibres suspension. Cu Kα radiation, generated with a Bruker AXS diffractometer (Bruker
150
AXS, Madison, WI at 30 kV, 100 mA. Sweeps of 5–50°2θ were made with a step size of 0.05
151
s and step time measurement of 10 s. The crystalline index (CrI) was calculated using the
152
diffraction intensities of the crystalline structure and that of the amorphous fraction according
153
to the method of Segal et al. (Segal, Creely, Martin & Conrad, 1959):
Ac ce p
te
d
M
an
us
cr
ip t
136
I I CrI % 002 am 100 I 002
154 155
where I002 is the intensity of the crystalline peak at the maximum at 2θ between 22° and 23°
156
for cellulose I, and Iam is the intensity at the minimum at 2θ between 18° and 19° for cellulose
157
I.
158
2.9. Rheological measurements:
159
Rheological measurements were carried out using a controlled speed rotating rheometer
160
(Rheotec RS-30) operated with cone-and-plate geometries (cone angle: 4°, diameter: 3 cm) 7 Page 7 of 29
and a Peltier temperature controller. All the measurements were conducted at a constant
162
temperature of 25 ± 0.2 ◦C.
163
2.10. Antibacterial Activities
164
The antibacterial activity of the films was tested against Gram+ bacteria;Staphylococcus
165
aureus (ATCC 25923), Bacillus cereus (ATCC 14579) and Gram- bacteria (Enterococcus
166
faecalis (ATCC 29212). and (Escherichia coli (ATCC 25922), Salmonella enterica (ATCC
167
13883) and Salmonella typhi (ATCC 14028) using the disc diffusion method on an agar plate
168
with slight modification.
169
The diffusion method was performed using Luria−Bertani (LB) medium solid agar in Petri
170
dishes. The agar slurry on the test specimens was allowed to the gel at room temperature
171
followed by incubation at 37 °C for 18 h. After incubation, films were cut into 15× 15 mm
172
pieces, sterilized in autoclave at 121°C for 20 minutes, and kept in intimate contact with the
173
bacterial agar gel. After incubation during 24h at 37°C, samples were examined visually for
174
growth of bacteria in the area surrounding the film.
175 176
3. Results and discussion
177 178
3.1 Cationization of cellulose fibers
179 180
The cationization of cellulose with glycedyltrimethylammonium chloride is a well known
181
modification approach of cellulose fibers to generate a cationic ammonium site within the
182
cellulose fibers. These latter were incorporated onto cellulose through nucleophilic addition
183
of the hydroxyl groups of cellulose to the epoxy moiety of glycidyltrimethylammonium
184
chloride, as shown in scheme 1.
Ac ce p
te
d
M
an
us
cr
ip t
161
185 186 187 8 Page 8 of 29
GTA/fibers molGTA.mol- TMAC content CrI 1
Eucal-P*
-
AGU
(µmol.g-1)
Fibrillation yiel (%)
-
-
C-Eucal-210** 1
1.06
210
C-Eucal-350
0.3
0.21
300
75
C-Eucal-760
0.6
0.64
760
73
C-Eucal-1150
1
1.06
1140
76
75
8 35
ip t
samples
74 86
Table 1: TAMC content in the quaternized softwood pulp according to the amount of used GTA
192
and the fibrillation yield after homogenisation.
an
us
*pristine eucalyptus pulp. ** cationization performed without swelling in water
cr
188 189 190 191
In order to limit the hydrolysis of the epoxy ring during the addition reaction, DMAC was
195
used as a solvent instead of water, which is commonly used for this reaction. Moreover, to
196
ensure efficient swelling of the fibers and good accessibility of the reagent to the inner pore,
197
the fibers were first soaked in water for several hours and solvent exchanged with DMAC.
198
Catalytic amount of NaOH was also added to convert alcohol groups of cellulose into more
199
reactive alcoholate groups.
200
Evidence of the occurrence of the addition reaction was confirmed by 13C CP/MAS (Figure 1)
201
showing in addition to the cellulose backbone between 60 and 110 ppm, the typical signal of
202
the (CH3)3N+ moiety appearing around 55 ppm. The resonances of the cellulose skeleton
203
appeared at 105 (C-1), 88 (crystalline C-4), 84 (amorphous C-4), 75, 72 (C-2, C-3, C-5), and
204
64 (C-6) ppm. Based on the peaks integration, the content of the trimethylammonium chloride
205
is determined with accuracy and found to be in good agreement with conductometric titration
206
of chloride ions using AgNO3. By changing the ratio of glycedyltrimethylammonium
207
chloride/cellulose fibers, three samples of cationized fibers with various trimethylammonium
208
contents
209
given in Table 1. It is worth noting that, when the reaction was performed in DMAC without
Ac ce p
te
d
M
193 194
were prepared. The content of TMAC according to the GMC/Cellulose ratio is
9 Page 9 of 29
swelling the fibers in water, the cationic content of the fibers was about five fold lower than
211
that obtained with the swelling treatment (C-NFC-210) (see Table 1). This difference might
212
be explained by the lower accessibility of the hydroxyl groups within the cellulose fibers.
213
Indeed, delignified cellulose fibers exhibit a multiscale hierarchical structure and are a porous
214
material which can absorb water and other molecular components. This internal porosity,
215
created during the lignin removal, opens the cell wall and allows the fibers to expanse in
216
water, making them accessible to a wide range of molecules with different size (Thompson,
217
Chen & Grethlein 1992). Since cationization is presently adopted to facilitate the fibrillation
218
process, it is crucial that the reaction occurred inside the fibers on the surface of the accessible
219
microfibrils.
M
an
us
cr
ip t
210
C ellu lose M icrof ib ril
Ac ce p
te
d
C2,3,5
O
CH2
CH
CH2
N(CH3)3+
OH
C1
C6
C4 N(CH3)3+
220 221
Figure 1 : CP-MAS 13C NMR spectra of quaternized fibers with TMAC content of 1140
222
µmol.g-1. (C1 to C6 stands for carbones of the glucosic unit of cellulose) 10 Page 10 of 29
223 Analysis of the crystalline structure of the fibers by X-ray diffraction, revealed no evolution
225
of the cellulose I polymorph, nor in the crystallinity degree after the cationization treatment
226
(see supplementary material), which indicates that the cationization reaction occurred mainly
227
on the surfaces of the cellulose microbrils. All the samples showed a typical diffraction
228
pattern of cellulose I, with the typical diffraction peaks at 2 16, 22.6°. The CrI remained
229
roughly constant around 75% irrespective of the TMAC content.
230 231
3.2 Nanofibrillation behaviour of the cationized fibers
232
To investigate how the cationic content affected the fibrillation behavior of the fibers, a high
233
pressure homogenization was progressively carried out, starting with several passes at 300 bar
234
until the suspension turned into a gel. Then, it was pursued at 600 bar to further promote the
235
breakdown of the cell walls. During the disintegration process, samples were withdrawn at
236
regular passes and the NFC suspension was analyzed in terms of its nanofibrillation yield,
237
transparency and rheological behavior.
238
A strong dependence of the fibrillation yield with the TMAC content of the fibers was noted
239
(Table 1, Figure 2 supplementary material), with a huge enhancement in the fibrillation
240
efficiency as the TMAC content increased. The fibrillation yield reached about 35, 70 and
241
85% as the cationicity grew from 350, to 750 and 1160 µmol.g-1, respectively. In the absence
242
of any pretreatment and adopting the same disintegration mode, the fibers failed to be
243
converted into NFC and multiple clogging occurred during high pressure homogenization.
244
This phenomenon was not observed in the presence of cationized treated fibers.
245
How to explain the strong enhancement of the nanofibrillation efficiency with the TMAC
246
content? In fact, cellulose is a hierarchical material formed of elementary fibrils aggregated
247
into larger microfibrils 5–50 nm in diameter and several microns in length, sticking together
248
in a spiral manner within a multiple-layer-composite to form the cellulose fibers. Upon
Ac ce p
te
d
M
an
us
cr
ip t
224
11 Page 11 of 29
immersion in water, the fibers underwent expansion as a result of water diffusion inside the
250
fibers microspores. Then, after solvent exchange and addition of GMA, the accessible
251
microfibrils, namely those in the amorphous domain, were cationized with GMA, resulting in
252
the generation of quaternary trimethylammonium groups bounded on the surface of
253
microfibrils. The presence of these ionic groups within the cell wall resulted in an osmotic
254
swelling of the fibers that reduced the hydrogen bonding among the microfibrils by increasing
255
the gap between neighbouring fibrils as schematically illustrated in scheme 1. The
256
proportionality between the osmotic pressure and the content of ionic groups
257
Posm RT (
258
and V is the liquid volume of the fibre wall (m3/g) accounted for the enhancement in the
259
fibrillation yield as the content in TMC is going up.
us
cr
ip t
249
260
Ac ce p
OH OH
te
OH OH
d
M
an
N ion ) ( Posm = where Posm (N/m2), Nion is the moles of charged groups (moles/g), V
OH O CH2 CH CH2 N(CH3)3+ OH
Cl-
OH
Microfibril
Elementary fibril
261 262
P >P + osm+ ext +
+ +
+
+ +
Electrostatic repulsion
+
+
+
Osmotic pressure n Posm c RT V
263 264
Scheme 1: schematic illustration of how the fibers quaternization enhanced the
265
nanofibrillation process.
266 12 Page 12 of 29
The transparency degree of the C-NFC after the homogenization process was shown to be
268
greatly affected by the TMAC content of the fibers. As shown in Figure 2, at a TMAC content
269
of 350 µmol.g-1, the C-NFC gel is quite opaque and turned to a translucid gel as the TMAC
270
increased over 750 µmol.g-1. This change in the optical transparency is further highlighted by
271
the transmittance measurement of a diluted suspension of C-NFC. The transmittance level for
272
C-NFC-350 did not exceed 30% and grow to 65-70% and 72-80% for C-NFC-750 and C-
273
NFC-1160 respectively, which is indicative of the low fraction of micro-sized particles
274
responsible for light scattering. The evolution in the optical transparency is explained by the
275
change in the fibrillation yield and the dispersion of the cellulose nanofibrils in water. The
276
enhancement in the transmittance with increasing content of ionic groups is indicative of a
277
decrease in the amount of non-fibrillated and partially fibrillated fractions responsible for a
278
light-scattering phenomenon. In addition, the increase in the TMAC groups is expected to
279
enhance the colloidal stability of the cellulose nanofibrils through electrostatic stabilization
280
brought by the presence of cationic groups on the surface of the nanofibrils. This stabilization
281
prevents the likelihood tendency of nanosized fibrils to self-aggregate through hydrogen
282
interaction. The aggregation risk of nanosized particles is another phenomenon likely to affect
283
the transparency degree of the suspension.
284
The transmittance spectra of C-NFC also showed also a strong wavelength-dependence with
285
lower transmittance at shorter wavelength, namely with TMA content of 750 and1160
286
µmol.g-1. This phenomenon is explained by the dependence of scattering intensity to the
287
fourth power of wavelength ( I 1 / 4 ) for nanosized particles with square section lower than
288
50 nm.
Ac ce p
te
d
M
an
us
cr
ip t
267
289
13 Page 13 of 29
A 1140
300
760
ip t
760
300
1140
cr
290 90 80
1140 µmol.g-1
us
B
760 µmol.g-1
an
60 50
300 µmol.g-1
40
M
Transmittance (%)
70
30
291
500
600
700
800
Wavelength (nm)
Ac ce p
0 400
te
10
d
20
292
Figure 2. (A) Visual aspect of Q-NFC gel at a solid content of 1 wt% with different TMAC
293
amount, and (B) Visible transmittance spectra of the Q-NFC suspension for different TMAC
294
content after 5 passes at 300 bar and 5 additional passes at 600 bar
295 296
The AFM images of C-NFC samples deposited on wafer substrate and dried at room
297
temperature are shown in Figure 3. these observations were relative to C-NFC samples with a
298
TMAC content of 750 and 1160 %mol.g-1 and produced via high pressure homogenization
299
following the same pressure sequence (5 passes at 300 bar followed by 5 additional passes at
300
600 Bar). Both samples showed a network of entangled individual nanofibrils having typical
301
width of 20–40 nm and lengths in the micrometer range. The length evaluation of the fibril is 14 Page 14 of 29
qualitative as the long fibrils formed an entangled network. Besides, it is hard to evaluate the
303
entire length of the fibrils accurately.
304
The fibril size, estimated from the height of the fibrils directly attached to the wafer, was
305
found to be between 3 and 6 nm for the individual non-aggregated fibrils. The NFC produced
306
from fibers with a TMAC content of 1160 µmol.g-1 seems to exhibit lower square section than
307
that with lower TMAC content. The difference between the thickness of the fibrils and their
308
width is likely to be due to the dimensions and geometry effect of the AFM tip that led to
309
surface feature broadening. This latter effect became obvious when the surface features have
310
the same or smaller size than the AFM tip. Under this condition, the tip will not be able to
311
correctly draw the profile contours giving rise to the so called convolution effects with artifact
312
appearance results, and the height should give more reliable measurement of NFC width.
313
From the AFM analysis, it can be confirmed that cationized celluloses were efficiently
314
homogenized to individual nanofibrils having lateral dimensions lower than 8 nm. Further
315
confirmation of the nanoscale dimension of C-NFC was given by FE-SEM observation
316
(Figure 4) with a larger area view of C-NFC, and where the partially fibrillated material with
317
width within 100 nm was clearly shown.
319
cr
us
an
M
d
te
Ac ce p
318
ip t
302
15 Page 15 of 29
A
4.2 nm
3.5 nm
us
cr
ip t
2.5 nm
320
an
321
6.4 nm 4 nm
322 323
Ac ce p
te
d
M
B
324
Figure 3: AFM height images of Q-NFC with TMAC content of (A) 1140 and (B) 760 µmol.g-
325
1, and the corresponding height profile analysis (the arrow marks the points the points used
326
for the measurements of the height profile
327
16 Page 16 of 29
90 nm 10 nm
us
cr
ip t
Partially fibrillated material
Figure 4 : FE-SEM images of C-NFC produced from quaternized fibers with TMAC content of 350 µmol.g-1. (C-NFC-350)
333
3.3 Colloidal properties of C-NFC
334 335
The colloidal stability and the rheological behavior of NFC in water are highly dependant on
336
the zeta-potential of the cellulose nanofibrils. The electrokinetic properties and the rheological
337
behavior of C-NFC were studied according to the TMAC content. The evolution of the zeta-
338
potential versus pH for the C-NFC suspension is shown in Figure 5. For the different contents
339
in TMAC, the nanofibrils exhibited a positive ζ-potential over the whole range of pH.
340
However, the magnitude of the ζ-potential is shifted to higher value as the TMAC content
341
increased and showed a pH dependence between pH 5 and 11. The former effect is expected
342
since a higher content in TAMC led to an increase in the surface charge density of the fibrils.
343
The value of ζ-potential exceeding +30 mV for C-NFC with a TMA content of 760 and 1160
344
µmol.g-1 accounts for the good dispersion stability of the NFC suspension in water as attested
345
by the individualization of the nanofibrils in AFM observations and good transparency of the
346
C-NFC suspension, even at a solid content over 1wt.% .
Ac ce p
te
d
M
an
328 329 330 331 332
17 Page 17 of 29
40 1140 µmol.g
-1
30
760 µmol.g
-1
ip t
25 20 300 µmol.g
-1
cr
Zeta-potential (mV)
35
10 2
3
4
5
6
7 pH
348
8
9
10
11
an
347
us
15
Figure 5: Change in the zeta-potential versus pH of Q-NFC with different TMAC content.
M
349
d
1000
te
Ac ce p
Viscosity (Pa.s)
100
1140 µmol.g-1
10
760 µmol.g-1
1
300 µmol.g-1
0.1
0
2
4
6
8
10
12
Shear rate (s-1)
350 351
Figure 6. Viscosity as a function of shear rate of the Q-NFC gel at 1% solid content with
352
different TMAC content.
353
NFC suspension is known to exhibit a gel-like aspect as the solid content exceeds a critical
354
threshold in the range of 0.1-5 wt%, depending on the origin and the morphological features 18 Page 18 of 29
of the cellulose nanofibrils. The evolution of the viscosity vs. shear rate of C-NFC at different
356
TMAC contents is shown in Figure 6. All of the C-NFC suspensions exhibited a strong shear
357
thinning behavior, namely when the TMAC content exceeds 750 µmol.g-1, as attested by the
358
intense decrease in the viscosity as the shear rate increases. For instance, the viscosity of the
359
C-NFC gel with a cationicity of 1160 µmol.g-1, falls down from 725 to about 35 Pa.s, as the
360
shear rate goes from 0.4 to 5 s-1. This behaviour, typical of most NFC suspensions in water, is
361
explained by the hydrodynamic properties of charged cellulose nanofibrils with a high aspect
362
ratio. Indeed, , the NFCs flexibility along with their strong tendency to self interact through
363
hydrogen bonding led to the formation of coiled network formed by entangled cellulose
364
fibrils. Upon the application of a shear rate the network is progressively broken down, the
365
elementary nanofibrils start to be oriented in the shear direction leading to their
366
disentanglement. Consequently, the flow resistance is lowered and the viscosity is decreased.
367
Once in rest, the nanofibrils network is reformed and the viscosity rebuilt again. The shift in
368
the viscosity toward higher value as the cationic content increases is mainly due to the
369
enhancement in the fibrillation extent- as the TMAC content exceeded 700 µmol.g-1.
370 371
3.4. Antibacterial properties.
372 373
To evaluate the antibacterial activity of the C-NFC when incorporated in a polymer matrix,
374
the diffusion method using Gram+ and Gram- bacteria was adopted. Pure PVA matrix and
375
nanocomposite films prepared from negatively charged A-NFC and PVA matrix was used as
376
a reference for the sake of comparison.
377
Figure 7 presents the picture of the diffusion assay resulting from pure PVA films, C-NFC-
378
PVA and A-NFC-PVA nancomposite films with 3 and 7% nanofiller loading. Pure PVA film
379
did not show any inhibitory effects on .all the bacterial tested. The same tendency was
380
observed for nanocomposite films prepared with A-NFC, with colonies growing of BC and E-
Ac ce p
te
d
M
an
us
cr
ip t
355
19 Page 19 of 29
colis growing on the agar plates as well as on the surface of the A-NFC-PVA films. On the
382
other hand, for all nanocomposite films prepared with C-NFC, bacteria increased on the agar
383
plates around the films, but not on top or under the films, and no trace of colonisation of the
384
film was observed. In the presence of Staphylococcus aureus and Salmonella enterica, better
385
antibacterial activity was observed as attested by the appearance of inhibition zone around the
386
film. The absence of a clear and large inhibition zone is probably due to the immobilization
387
of antibacterial agent (C-NFC) on the polymer matrix restricting the diffusion of the
388
antibacterial agent to areas close to the specimen. The antibacterial activity of C-NFC-PVA
389
seems to be independent on the NFC loading over the range between 3 to 10wt. %. These
390
results clearly demonstrate the trivial role of C-NFC in preventing bacteria proliferation when
391
incorporation into a polymer matrix at a content as low as 4wt%. The efficient antibacterial
392
activity of C-NFC against a board range of bacteria is likely due to the presence of quaternary
393
ammonium groups on the surface of the C-NFC. These groups are known to be one of the
394
most useful antiseptics and disinfectants used in wide range of applications in cosmetic
395
products, clinical purposes, textiles and active packaging. The mechanism proposed to
396
account for these strong antibacterial properties is the interaction with the lipids and proteins
397
that compose the cytoplasmic membrane damaging the outer layer of bacterial disturbing their
398
metabolism (Denyer 1995).
cr
us
an
M
d
te
Ac ce p
399
ip t
381
20 Page 20 of 29
399 a
C-NFC
b
c
1 401 4 3
4
403
405 406
Bacillus cereus
C-NFC Staphylococcus aureus
C-NFC
A-NFC
d
an
408 409 410 Escherichia coli
M
Bacillus cereus
412 413
418
A-NFC
te
d
Figure 7. The antibacterial activity of control NFC-PVA nanocomposite film prepared from quationized NFC C-NFC (a, b and c) and from anionic A-NFC (d, e) tested against Gram- and Gram+ bacteria: (1) neat PVA matrix, (2) 3% NFC, (3) 5% NFC and (4) 7% NFC.
Ac ce p
414 415 416 417
C-NFC Salmonella enterica
e
407
411
3
3
us
404
4
ip t
402
1
1
cr
400
C-NFC
419
3.5 Reinforcing potential
420
In addition to the antibacterial effect imparted by the inclusion of C-NFC into a polymer
421
matrix, a reinforcing effect is also expected by the addition of NFC. To investigate the
422
reinforcing potential of the C-NFC, nanocomposite films based on PVA matrix and different
423
nanofiller loadings were prepared by solvent-casting and were analysed by DMA. For the
424
sake of comparison, similar films were also prepared using nanofibrillar cellulose bearing
425
carboxylic groups (A-NFC). The temperature dependence of the storage tensile modulus, E΄ ,
426
as a function of temperature at 1 Hz for PVA-NFC (C-NFC and A-NFC) nanocomposite with
427
different NFC are reported in the supplementary material. 21 Page 21 of 29
30
(A)
At 15% A-NFC 27-fold matrix
25
E'c/E'm.100
20
ip t
15 10
cr
At 15% Q-NFC 11 fold matrix
5
0
1
2
3
4
5
6
7
us
0 8
9 10 11 12 13 14 15 16
429 (B)
an
NFC content (wt%)
428
Cationized Cellulose nanofibril
OH
OH
OH CH2
CH2 CH O
OH
N+
H3C OH CH3
OH
OH
CH3 N+ H3C OH CH3
OH
d
OH
OH OH CH CH3 CH2 CH2 O
M
OH
OH
te
Cationized Cellulose nanofibril
430 431
OH
Ac ce p
OH
OH
OH
OH
OH
OH
OH
OH
PVA matrix
(C)
Carboxylated Cellulose nanofibril
OH
OH
O
OH
HO
C
O
OH
OH
OH
C
OH
OH O
OH
OH
HO
C
O
OH
OH C
OH
OH
Carboxylated Cellulose nanofibril
OH
OH
COOH
OH
OH
OH OH
432 433
OH OH
PVA matrix
22 Page 22 of 29
Figure 8. (A) The relative storage modulus versus NFC content at 70 °C for nanocomposite films prepared from C-NFC and A-NFC and schematic illustration of the hydrogen potential interaction among (B) C-NFC, and(C) A-NFC, when embedded into a polymer matrix.
438
At a temperature lower than the glass transition, the storage modulus undergoes only a slight
439
enhancement with respect to the unfilled matrix. For instance, at 15% NFC E΄ is about two
440
fold higher. Besides, at 20°C the nanocomposites containing 15 wt% C-NFC and A-NFC
441
exhibit a storage modulus around 1.2 and 1.4 GPa, which represents a 2.8 and 3.3-fold
442
enhancement, respectively, over that of the neat matrix (0.42 GPa). This behavior, common to
443
nanocomposite materials based on nanosized cellulose particles, is explained by the low
444
difference between the modulus of the glassy matrix (close to 1 GPa) and that of the
445
nanofiller network (being in the range of 10–15 GPa).
446
Above the glass transition, however, the storage modulus is more sensitive to the presence of
447
the nanofiller and increased significantly with NFC addition, which is in line with the well-
448
known reinforcing effect of nanocellulose based nanofiller.(Boufi 2014)
449
The stiffening effect imparted by the addition of NFC can be expressed by the evolution of
450
the relative modulus Er
451
and unfilled matrix respectively measured in the rubbery region taken here at 70°C), versus
452
the NFC content (Figure 8A). The continuous rise in the relative modulus clearly confirms the
453
reinforcing potential of the NFC. However, the extent of the reinforcing potential is different
454
from cationic to anionic NFC. At the same nanofiller loading, the modulus enhancement is
455
lower for C-NFC than that observed for A-NFC. This is namely observed when the NFC
456
content exceeded 2%. For instance, at 15 wt %, the modulus is about 27 and 11 times higher
457
for samples prepared with A-NFC and C-NFC respectively.
458
To explain the lower reinforcing potential of C-NFC compared to that of A-NFC, it is worth
459
noting that the unusual enhancement of the stiffness and strength in the nanocellulose
te
d
M
an
us
cr
ip t
434 435 436 437
Ac ce p
E 'c (with Ec and Em are the storage modulus of the nanocomposite E 'm
23 Page 23 of 29
reinforced nanocomposites arouse mainly from the setup of an entangled interconnected
461
network held-up by strong hydrogen bonding. The strength of the NFC network is dependant
462
on the aspect ratio of the cellulose nanofibrils and their capacity to interact through hydrogen
463
bonding. Presently, we infer that the presence of a relatively high surface density of appended
464
2-hydroxypropyltrimethylammonium chloride moiety reduced the potential of hydrogen
465
bonding among neighbouring nanofibrils and weakened the network cohesion. This can be
466
deduced from the following analysis. Indeed, assuming that the square section of the
467
nanofibrils is 5 nm and that the average values between cellulose chains within a transversal
468
cross section ranges from 0.54 to 0.61 nm, then for a content of 1100 µmol.g-1, the surface
469
substitution degree should be DSS=DS/ratio of surface chains=0.19/0.4=0.47 . This means
470
that
471
hydroxypropyltrimethylammonium
472
trimethylammonium group to share hydrogen bonding with hydroxyl groups, the interaction
473
among entangled cellulose nanofibrils network is expected to weaken due to the shielding
474
effect imparted by the trimethylammonium group. For A-NFC, the presence of carboxylic
475
groups on the surface of the nanofibrils will, on the contrary, favour hydrogen interaction
476
among NFC. A schematic illustration of this phenomenon is given in Figure 8Aand B.
477
Though, the production of C-NFC via the quaternization of cellulose fibers as a pretreatment
478
was already reported in several paper as noted in the introduction (Olszewska et al 2011, Ho
479
et al 2011 and Pei et al 2013), the main finding of the present work is twofold; correlate the
480
fibrillation efficiency with the content of cationic groups, and highlight the great potential of
481
C-NFC to enhance the antibacterial resistance of the material when included as additive into a
482
polymer matrix. The potential usefulness of C-NFC gel as wound healing might be also
483
considered. It follows that the quaterization of NFC as both a pretreatment and method for
each
two
anhydroglucosic
M
of
chloride
units
moiety.
is
Given
linked the
to lack
the
2-
of
the
Ac ce p
te
d
one
an
us
cr
ip t
460
24 Page 24 of 29
surface modification open the way for usefulness of C-NFC in biomedical application. This
485
work is under investigation.
486
4. Conclusion
487
Cationized NFC from eucalyptus pulps was prepared by cationization of the cellulose fibers
488
with GTMAC in DMF and mechanical disintegration using a high pressure homogenization.
489
What can be concluded from the present study is that:
cr
490
ip t
484
1- The cationization of the bleached cellulose fibers is an efficient approach to facilitate the breakup of the cell wall and the release of cellulose nanofibrils.
us
491
2- The fibrillation efficiency is meaningfully enhanced as the TMAC content exceeded
493
700 µmol.g-1. Over this content, thick transparent gel was obtained with good
494
colloidal stability for long-term storage.
M
495
an
492
3- AFM observation confirmed the nanosized scale of C-NFC with a square section in the range of 5 nm.
d
496
4- The inclusion of C-NFC within a polymer matrix strongly enhanced the antibacterial
498
activity of the nanocomposite films by imparting a reinforcing effect. These
499
nanocomposites films could be useful in active packaging material for food
501 502
Ac ce p
500
te
497
preservation. These results clearly suggested the great potential of cationic NFC as reinforcement additive for polymer with effective antibacterial activity.
503
References
504
Besbes I., Rei Vilar M., & Boufi S.(2011) Nanofibrillated cellulose from alfa, eucalyptus and
505
pine fibres: preparation, characteristics and reinforcing potential. Carbohyd. Polym. 86, 1198-
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Boufi S. Chapter 14 in . Biomass and Bioenergy. Khalid Rehman Hakeem,Mohammad
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Jawaid,Umer Rashid. Springer 2014, pp 267-305.
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nanocellulose reinforced polymer nanocomposites. Macromol. Mater. Eng. 299, 560–568
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Boufi S.,& Gandini A.. Triticale crop residue: a cheap material for high performance
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González I., Vilaseca F., Alcalá M., Pèlach M., Boufi S.,& Mutjé P. (2013) Effect of the
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Ho T. T. T., Zimmermann T., Hauert R.,& Caseri W.,(2011) Preparation and characterization
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Kalia S., Boufi S., Celli A., & Kango S. (2014) Nanofibrillated cellulose: surface
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Liimatainen H., Suopajärvia T., Sirviöa J., Hormib O.,& Niinimäki J. (2014) Fabrication of
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colloid aggregation, Carbohydrate Polymers 103, 187– 192
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Liimatainen H., Visanko M., J Sirvio¨ J., Hormi O.,& Niinima¨ki J., (2013) Sulfonated
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cellulose nanofibrils obtained from wood pulp through regioselective oxidative bisulfite pre-
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treatment, Cellulose 20,741–749
549 Liimatainen H., Visanko M., Sirvio¨ J., Hormi O., Niinima¨ki J (2012) Enhancement of the
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nanofibrillation of wood cellulose through sequential periodate–chlorite oxidation.
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Biomacromolecules 13, 1592–1597
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Maatar W., Alila S.,& Boufi S. (2013) Cellulose based organogel as an adsorbent for
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dissolved organic compounds. Ind Crop Prod 49, 33–42
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Mathew A.P, Oksman K., Pierron D.,& Harmand M.F. (2012) Fibrous cellulose
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nanocomposite scaffolds prepared by partial dissolution for potential use as ligament or
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tendon substitutes. Carbohydr Polym 87:2291–2298
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Olszewska A., Eronen P., Johansson L.S., Malho J.M., Ankerfors M., Lindstrom T.,
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Ruokolainen J., Laine J.,& Osterberg M. (2011) The behaviour of cationic NanoFibrillar
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Cellulose in aqueous media, Cellulose 18, 1213–1226
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Pei A., Butchosa N., Berglund L.A., Zhou Q.(2013) Surface quaternized cellulose nanofibrils
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with high water absorbency and adsorption capacity for anionic dyes, Soft Matter, 9, 2047–
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Saito, T., Nishiyama, Y., Putaux, J. L., Vignon, M., & Isogai, A. (2006). Homogeneous
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Valo H., Kovalainen M., Laaksonen P, Hakkinen M, Auriola S, Peltonen L, Linder M,
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Jarvinen K, Hirvonen J,& Laaksonen T (2011) Immobilization of protein-coated drug
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578
Wågberg L, Decher G, Norgren M, Lindstro¨m T, Ankerfors M,& Axna¨s K (2008) The
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583
Figure captions
584 Figure 1 : CP-MAS 13C NMR spectra of quaternized fibers with TMAC content of 1140
586
µmol.g-1. (C1 to C6 stands for carbones of the glucosic unit of cellulose)
587
Scheme 1: schematic illustration of how the fibers quaternization enhanced the
588
nanofibrillation process.
589
Figure 2. (A) Visual aspect of Q-NFC gel at a solid content of 1 wt% with different TMAC
590
amount, and (B) Visible transmittance spectra of the Q-NFC suspension for different TMAC
591
content after 5 passes at 300 bar and 5 additional passes at 600 bar
592
Figure 3: AFM height images of Q-NFC with TMAC content of (A) 1140 and (B) 760
593
µmol.g-1, and the corresponding height profile analysis (the arrow marks the points the points
594
used for the measurements of the height profile
595
Figure 4 : Change in the zeta-potential versus pH of Q-NFC with different TMAC content.
596
Figure 5. Viscosity as a function of shear rate of the Q-NFC gel at 1% solid content with
597
different TMAC content.
598 599 600 601 602 603 604 605 606
Figure 6. The antibacterial activity of control NFC-PVA nanocomposite film prepared from C-NFC (a, b and c) and from A-NFCtested against Gram- and Gram+ bacteria: (1) neat PVA matrix, (2) 3% NFC, (3) 5% NFC and (4) 7% NFC.
te
d
M
an
us
cr
ip t
585
Ac ce p
Figure 7. the relative storage modulus versus NFC content at 70 °C for nanocomposite films prepared from Q-NFC and A-NFC, and schematic illustration of the hydrogen potential interaction among (A) C-NFC, and A-NFC, when embedded into a polymer matrix
29 Page 29 of 29