Article

Cite This: Biomacromolecules XXXX, XXX, XXX−XXX pubs.acs.org/Biomac

Detailed Structural Analyses of Nanofibrillated Bacterial Cellulose

and Its Application as Binder Material for a Display Device
Kenji Tajima,*,† Koutaro Tahara,‡ Junya Ohba,‡ Ryo Kusumoto,‡ Ryota Kose,§ Hiroyuki Kono,∥
Tokuo Matsushima,⊥ Koji Fushimi,† Takuya Isono,† Takuya Yamamoto,† and Toshifumi Satoh†
†
Faculty of Engineering and ‡Graduate School of Chemical Sciences and Engineering, Hokkaido University, N13W8, Kita-ku,
Sapporo 060-8628, Japan
§
Institute of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu 183-8538, Japan
∥
National Institute of Technology, Tomakomai College, 443, Nishikioka, Tomakomai 059-1275, Japan
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⊥
Kusano Sakko Inc., 16, Nishi-machi, Kami-ebetsu, Ebetsu 067-0063, Japan
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*
S Supporting Information

ABSTRACT: Nanofibrillated bacterial cellulose (NFBC) is

produced by culturing a cellulose-producing bacterium under
agitated aerobic conditions in a carboxymethylcellulose (CMC)-
supplemented medium. Detailed structural analyses revealed that
NFBC fiber widths varied with the degree of substitution of the
CMC used, and zeta potential values decreased with the
increment of CMC concentration in the medium. Transmission
electron microscopy observation after immunostaining demon-
strated that CMC molecules were present on the NFBC
microfibril surfaces. We tested NFBC for utility as a binder for
a display device that uses electrochromic (EC) material.
Introduction of a quaternary ammonium group into the EC molecules enhanced their interactions with the negatively
charged NFBC microfibrils. A casting process homogeneously adsorbed the EC molecules onto the surface of a transparent
electrode with NFBC. A homogeneous color change was successfully observed upon applying an electric field, suggesting that
NFBC could be used as a binder material for uniform surface adsorption.

■ INTRODUCTION
One of the most abundant polymers in nature is cellulose, a
carbohydrate composed of β-1,4-linked glucose. Cellulose is
mainly produced by plants, and it can also be derived from
other sources, e.g., algae, some bacteria, and some animals.1−5
Compared to cellulose produced by plants and algae, bacterial
cellulose (BC), which is produced by bacteria including
Gluconacetobacter, has superior properties, such as high
mechanical strength, biocompatibility, and high biodegrad-
ability, although the chemical structure is identical.2,6,7 The
most representative characteristic of BC is its fiber width,
which is approximately 50−100 nm.8 In general, BC is
produced under static cultivation conditions, in which a
membranous gel sheet (pellicle) with a fine 3D network
structure is formed at the gas−liquid (medium) interface. BC
has been applied in several fields owing to the features of its
typical structure.9,10
Recently, nanofibrillated cellulose (NFC) has been receiving
attention as a new material.11 NFC is generally produced from
pulp via a top-down process, and, to date, various physical
and/or chemical production processes have been reported.12,13
NFC is usually obtained as a uniform water suspension. We
have developed a different NFC (NFBC) production system
via a bottom-up process using a cellulose-producing bacterium
[Gluconacetobacter intermedius (G. intermedius) NEDO-01] to
expand the applicability of BC.14 In this process, a cellulose-
producing bacterium was cultured with aerobic agitation in a
medium supplemented with carboxymethylcellulose (CMC) as
a dispersing agent of microfibrils. The NFBC prepared with
CMC (CM-NFBC) showed high dispersibility in water but
rapidly aggregated in organic solvents. To overcome this
problem and expand its applicability, we have also succeeded in
preparing an amphiphilic NFBC (HP-NFBC) via a one-step
process by using amphiphilic hydroxypropylcellulose (HPC)
instead of CMC, which improved the dispersibility of NFBC in
organic solvents.15
NFBC types produced via a bottom-up process by using a
cellulose-producing bacterium have superior features such as
high homogeneity and dispersibility and large surface area. At
present, various attempts are in progress to expand the
applications of NFBC, owing to these superior features.
However, although NFBC was analyzed using transmission
electron microscopy (TEM), Fourier-transform infrared (FT-
Received: September 26, 2019
Revised: November 15, 2019
Published: November 21, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.biomac.9b01328

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Scheme 1. Synthesis of Ethynyl-CMC via a Condensation Reaction
Scheme 2. Introduction of Fluorescein Group via a Click Reaction
IR) spectroscopy, and wide-angle X-ray diffraction analysis,
detailed structural analyses have not yet been performed.
In this study, detailed structural analyses of NFBC were
performed to elucidate its characteristics and develop a new
application of NFBC. Because of its characteristics, NFBC was
used as a binder material for a display device by using an
electrochromic (EC) material that shows reversible color
change under electric field.16,17 Because homogeneous color
development is remarkably important for a display device,
adsorbing EC molecules on the surface of a transparent
electrode is necessary. A quaternary ammonium (QA) group
was introduced into an EC molecule to enhance its interaction
with the negatively charged NFBC microfibrils.18 EC
combined with QA and NFBC mixture was cast on the
surface of a transparent electrode to form a color-developable
layer. A filter paper embedded with an electrolyte was held by
the transparent electrode and an opposite electrode. Finally, a
coloring test was performed to evaluate the potential of NFBC
as a binder material. To our knowledge, this is the first study to
consider NFBC as a binder material for display devices
containing EC materials.
■
MATERIALS AND METHODS
Chemicals. Bacto-peptone, yeast extract, glucose, disodium
hydrogen phosphate (Na2HPO4), citric acid, and diethyl ether were
purchased from Kanto Chemical (Tokyo, Japan). CMC (sodium salt,
degrees of substitution (DSs) = 1.24 and 1.44), and CMC (sodium
salt, DS = 0.72) were kindly provided by DKS (Kyoto, Japan) and
Sansyo (Osaka, Japan), respectively. Degrees of substitution (DSs)
B DOI: 10.1021/acs.biomac.9b01328
Article
were determined by quantitative 13C NMR analyses (Figure S1).
Propargylamine, 3-azidopropylamine, 1,8-dibromooctane, and trime-
thylamine were the products of Tokyo Chemical Industry (TCI).
Methanol, acetone, copper(II) sulfate pentahydrate, L-ascorbic acid,
and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide
were purchased from Kanto Chemical. N-(3-Azidopropyl)-3′,6′-
dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxa-
mide (5-FAM-azide) was purchased from Funakoshi (Tokyo, Japan).
Electrochromic (EC) N-phenyl-1,4-dipyridyl-pyrrole was kindly
provided by Ricoh (Tokyo, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium chloride (DMT-MM) was prepared
according to the method previously reported.19
Bacteria and Media. G. intermedius NEDO-01 (NITE P-1495)
was used for NFBC production.14 Hestrin and Schramm’s (HS)
medium20 was used as the standard medium, and CMC was added to
prepare NFBC types.
Preparation of NFBC. NFBCs were prepared according the
method reported previously.14,15
Preparation of Ethynyl-CMC. CMC (DS = 1.44, 2.5 g) was
dissolved in 200 mL of deionized water in a round-bottom flask. The
solution was heated to 50 °C, and then propargylamine (1.25 mL,
20.9 mmol) was added to the flask. Under stirring, DMT-MM (4.61 g,
16.7 mmol) was added to the flask and then incubated for 48 h
(Scheme 1). The reaction mixture was poured into a large amount of
acetone to collect the product as a precipitate. After washing with
methanol, the precipitate obtained was dissolved in water. The
product-containing solution was dialyzed against distilled water for
purification. A white spongy product was obtained after lyophilization.
Introduction of a Fluorescent Group into NFBC via a Click
Reaction. 5-FAM-azide (2.2 mM in methanol, 20 μL), copper sulfate
solution (4.0 mM, 17.2 μL), and ascorbic acid solution (2.3 mM, 60
μL) were added to a 0.1% (w/v) ethynyl-CM-NFBC aqueous
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Biomacromolecules
Figure 1. TEM images and histograms of NFBC fiber widths prepared using CMC with DS = 0.72, 1.24, and 1.44.
solution under stirring. The mixture was incubated at 25 °C for 24 h
to complete a click reaction (Scheme 2). The product was washed
with distilled water several times by repeating centrifugation and
resuspension.
NMR Spectroscopy. Each type of CMC (∼35 mg) was dissolved
in 700 μL of deuterium oxide (99.8 atom % D) containing 0.03% 4,4-
dimethyl-4-silapentane-1-sulfonic acid (DSS) and then transferred to
an NMR tube (diameter = 5 mm, Wilmad-LabGlass, Vineland, NJ).
The NMR data were acquired on a two-channel 500 MHz Bruker
AVIII spectrometer equipped with a Bruker z-gradient dual-resonance
BBFO probe (Bruker BioSpin, GmbH, Germany). Data were
accumulated at 363 K. Quantitative 13C NMR spectra were obtained
using the 1H inverse-gated decoupling method with a 13C flip angle,
repetition time, and scans of 30°, 30 s, and 6144, respectively.21
The 1H nuclear magnetic resonance (NMR) spectra of the
polymers were obtained using a JEOL JNM-ESC400 instrument
(400 MHz; JEOL, Tokyo, Japan). Chemical shifts were reported in
parts per million using tetramethylsilane (TMS) as an internal
reference.
FT-IR Spectroscopy. Fourier-transform infrared (FT-IR) spectra
for the film samples were obtained according the method reported
previously.14,15
Raman Spectroscopy. Raman spectra for samples were obtained
using a Horiba XploRA spectrophotometer (Horiba, Kyoto). The
conditions for Raman measurements were as follows: 532 nm, 64
scans, 1200 g·mm−1, and a 4000−500 cm−1 scan range.
Measurement of Zeta Potential. The zeta potential of NFBC
was measured with a particle size and zeta potential analyzer Delsa
Nano HC (Beckman Coulter, Atlanta, GA). Concentrations of all
NFBC types were adjusted to 0.10 wt %. Zeta potential was
determined as a mean value by repeating the measurement 10 times.
Microscopic Evaluation. Polarizing microscope observation
(POM): Drops of diluted sample were added to glass plates and
covered with glass coverslips. The specimens were observed with a BX
40 light microscope (Olympus, Tokyo, Japan) equipped with a
polarizer and an analyzer.
Fluorescent microscope observation (FM): A small portion of
NFBC dispersion was dropped onto a glass slide. Slow Fade (Life
Technologies Japan) was dropped onto the slide to preserve
fluorescence. A coverslip was placed on the sample and sealed with
nail polish to keep the sample from drying out. The NFBC-containing
slides were observed under a BZ-9000 fluorescence microscope
(Keyence Corporation, Osaka, Japan). The microscope settings were
as follows: excitation at 488 nm, emission at 530/30 BP, and 100× 1.4
NA oil-immersion lens.
C DOI: 10.1021/acs.biomac.9b01328
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Transmission electron microscope (TEM) observation: Five
microliters of NFBC aqueous dispersion (0.0002 wt %) was mounted
onto copper grids and dried at 30 °C. The specimen was negative-
stained with 5% (w/v) aqueous gadolinium acetate and washed with
distilled water. The specimen was observed with a JEM-2000FX
microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.
Immunoelectron microscope observation: Immunoelectron micro-
scope observation was carried out according the method reported
previously.22
Scanning electron microscope (SEM) observation: After freeze-
drying, the samples were mounted on SEM grids, and gold particles
were evaporated to the sample surfaces. The specimens were observed
with a JSM-6500F scanning electron microscope (JEOL, Tokyo,
Japan) at an accelerating voltage of 5 kV.
Scanning probe microscope (SPM) observation: The overall
structure of microfibrils was observed by a scanning probe microscope
SPM-9700HT (Shimadzu Corporation, Kyoto, Japan). NFC
suspension was diluted to a concentration of 0.001 wt % and then
cast onto a new cleavage surface of mica. NFC (BiNFi-s: Sugino
Machine Limited, Uozu, Japan) obtained from pulp via a top-down
process was used as a control to compare fiber lengths. The SPM
image of BiNFi-s has been published in Application News No. S30 of
Shimadzu Corporation.
Introduction of QA Structure into an EC Molecule. A
quaternary ammonium (QA) structure was introduced into an EC
molecule to enhance the interaction between the surface of NFBC
microfibrils and electrochromic molecules.
Preparation of N-(8-bromooctyl)trimethylammonium bromide
(BOTMAB): 1,8-Dibromooctane (1.85 mL, 10 mM) was dissolved
with 20 mL of toluene in a three-necked round-bottom flask.
Trimethylamine (2.27 mL of 25% in MeOH) was added to the flask
and then stirred for 48 h under nitrogen atmosphere (Scheme S1). An
excess amount of diethyl ether was added to the reaction mixture to
form a white precipitate. A white powdery solid was obtained by
filtration and vacuum drying (0.85 g, yield 30%).
Introduction of BOTMAB into EC (N-phenyl-1,4-dipyridyl-
pyrrole): EC and BOTMAB were dissolved with DMF in a three-
necked round-bottom flask and then incubated at 90 °C for 5 h. After
condensation by evaporation, the product was purified by silica
column chromatography. By using 1-bromooctane, EC with an octyl
group was synthesized as a control sample which has no QA group
(Scheme S1).
Formation of a Color-Developable Layer and Coloring Test.
The mixture of QA-containing EC and NFBC was cast on a
transparent electrode to make a color-developable layer, the thickness
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of which was determined by scanning electron microscope
observation after focused ion beam (FIB) treatment. A filter paper
embedded with electrolyte was held by the transparent electrode and
an opposite electrode (Figure 8a). Finally, a coloring test was
performed by alternately applying a constant voltage (3 V). The
homogeneity of coloring was visually observed to evaluate the
potential of NFBC as a binder material.
■
RESULTS AND DISCUSSION
Preparation of NFBC Types Using CMC Molecules
with DS = 0.72, 1.24, and 1.44. Structural Analyses by
FT-IR and TEM. CM-NFBC is prepared by rotating
cultivation of strain NEDO-01 in HS medium supplemented
with various concentrations of CMC [0.2, 0.4, 0.6, 0.8, 1.0, and
2.0% (w/v)] to analyze the dispersing mechanism for NFBC
production. The degree of substitution (DS) of each CMC was
determined by 13C NMR (Figure S1). No drastic changes in
medium viscosity were observed for differences in DSs.
Homogeneous NFC dispersion was obtained at a CMC
concentration of 0.6% (w/v) or higher (Figure S2).
The yields of NFBCs prepared using CMCs with different
DSs were almost the same (approximately 2 g·L−1), and no
effect of DS on the yields of NFBCs was observed. TEM
observation was carried out to measure the width of each
NFBC fiber prepared with 2.0% (w/v) of CMC (Figure 1).
Highly homogeneous nanofibers were observed for all NFBC
types. The fiber widths varied with the degree of substitution
(DS) of CMC used for NFBC preparation. The values were 27
± 5 nm (DS = 0.72), 29 ± 5 nm (DS = 1.24), and 39 ± 6 nm
(DS = 1.44), respectively; the NFBC fibers prepared using
CMC with DS = 1.44 were slightly larger than the ones
prepared using CMC with DSs = 0.72 and 1.24.
We have previously reported the preparation of NFBC using
hydroxyethylcellulose (HEC) and hydroxypropylcellulose
(HPC) as well as CMC,15 which has been reported to prevent
the formation of higher-order structure (ribbon) by absorbing
onto the microfibril surface.22 These cellulose derivatives
adsorb on the surface of microfibrils to alter the ribbon
structure. The NFBC microfibril widths varied with the
cellulose derivatives used for the preparation,15 and the value
increased with the increment of the derivatives’ substituent
bulkiness (42 ± 8 nm for HPC > 33 ± 7 nm for HEC > 27 ± 7
nm for CMC). These results suggest that HEC and HPC have
distinct effects on fiber formation, which could result from the
differences in their accessibility to the microfibrils derived from
substituent structures.15
Similarly, changes in the microfibril widths were observed
for NFBC types prepared using CMCs with various DSs
(Figure 1). The fiber widths of microfibrils increased with the
increment of their DS values.15 BC is thought to be
synthesized by the terminal (cellulose synthase) complex
(TC) present in the cell membrane of Gluconacetobacter
species.23 Although the complete TC structure in Gluconace-
tobacter has not yet been determined, it is known that 1.5-nm-
wide subelementary fibrils (SEFs) are extruded by each TC
and aggregate into microfibrils that self-assemble into fibers
(ribbons).23 The microfibrils and the cellulose derivatives are
thought to interact through hydrogen bonds.8 Indeed, the
contents of CMC in NFBC types decreased as the CMC DS
values increased (CMC with DS = 0.72 > CMC with DS =
1.24 > CMC with DS = 1.44).15 This could relate to the
interaction between the microfibril surface and CMC, with
stronger interactions forming smaller width fibers and weaker
D DOI: 10.1021/acs.biomac.9b01328
Article
interactions forming larger width fibers. Because the
interaction between the microfibrils and the cellulose
derivatives is an essential factor for controlling the microfibril
widths in NFBC, it would be possible to create multiple NFBC
types by selecting different cellulose derivatives.
Measurement of Zeta Potential. The presence of CMC
on the surface of microfibrils has been suggested, but any
direct evidence has not been obtained so far. Because the
carboxyl group in CMC has a negative charge, the zeta
potential of NFBC was measured to obtain evidence to
support the presence of CMC on the surface of microfibrils.
The zeta potential decreased with the increment of CMC
concentration in a medium (Figure 2). The zeta values for
Figure 2. Concentration dependency of zeta potential values of
NFBC types containing CMC with DS = 0.72, 1.24, and 1.44.
NFBC types with CMC DSs of 0.72 or 1.24 were larger than
those with a CMC DS of 1.44. The order of CMC contents in
NFBC types was CMC with DS = 0.72 > CMC with DS = 1.24
> CMC with DS = 1.44;15 both the concentrations and the DS
values relate to the zeta potential values. The interaction
between the microfibril surfaces and the CMC molecules is
thought to be hydrogen bonds,8 and the decrease of hydroxyl
groups could cause a reduction of the interaction. All NFBC
types prepared using CMC had a negative charge, suggesting
that CMC existed on the surface of microfibrils.
Preparation of NFBC with Fluorescent Groups
Attached. CMC is thought to prevent the assembly of a
cellulose ribbon by absorbing on the surface of microfibrils.8,23
In addition, the results of the zeta potential observations
strongly suggested that CMC existed on the surface of
microfibrils. A fluorescein (FL) group was introduced into
CMC, and the NFBC was prepared using the modified CMC
and observed by fluorescent and transmission electron
microscopes to verify that CMC existed on the surface of
microfibrils.
First, CMC containing ethynyl groups (ethynyl-CMC) was
synthesized to prepare NFBC with ethynyl groups (Scheme 1).
After the condensation reaction between a carboxyl group in
CMC and propargylamine using DMT-MM as a condensation
reagent, the introduction of ethynyl groups was confirmed by
IR and Raman spectroscopies (Figure S3). Although only one
carbonyl peak was observed in the IR spectrum of CMC
(Figure S3a, upper), two carbonyl peaks were observed after
the condensation reaction (Figure S3a, lower). One peak
corresponded to the unreacted carboxyl group (1600 cm−1)
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and the other corresponded to the carbonyl group (1680
cm−1) in the newly formed amide linkages (Figure S3a, lower).
In addition, a peak corresponding to ethynyl groups was
observed at around 3250 cm−1 in IR (Figure S3a, lower) and
2120 cm−1 in Raman spectra after the condensation reaction
(Figure S3b). These results suggested that ethynyl groups were
successfully introduced into CMC. Then NFBC was prepared
by using ethynyl-CMC instead of CMC.
IR spectra of NFBC prepared with ethynyl-CMC and
bacterial cellulose are shown in Figure S4a. A peak
corresponding to CO from ethynyl-CMC was observed at
around 1600 cm−1 in IR spectrum (Figure S4a, upper).
Furthermore, a peak corresponding to ethynyl groups from
ethynyl-CMC was observed at around 2123 cm−1 in the
Raman spectrum (Figure 4b). These results suggested that the
prepared NFBC contained ethynyl-CMC.
After NFBC with ethynyl-CMC (ethynyl-NFBC) was
successfully created, it was reacted with 5-FAM-azide via a
Cu-catalyzed azido alkyne cycloaddition (CuAAC) (Scheme
2). After the reaction, obvious fluorescence was observed for
ethynyl-NFBC reacted with 5-FAM-azide (Figure 3, right),
suggesting that fluorescent groups were successfully introduced
into the NFBC.
Figure 3. Fluorescence microscopy images of (a) ethynyl-NFBC and
(b) FL-NFBC.
TEM Observation Confirming the Presence of CMC
on the Microfibril Surface. By using the click reaction, a
fluorescein (FL) group could be successfully introduced into
NFBC. Although the result of fluorescent microscope
observation confirmed the introduction of FL groups into
NFBC, the presence of FL-CMC on the surface of microfibrils
has not been confirmed. Immunostaining using antifluorescein
antibody and gold-nanoparticle-labeled anti IgG antibody and
TEM observation were carried out to confirm the presence of
FL-CMC on the surface of microfibrils. Gold nanoparticles
were observed on the surface of microfibrils of ethynyl-NFBC
reacted with 5-FAM-azide (FL-NFBC) (Figure 4b). In
Figure 4. TEM images of (a) ethynyl-NFBC and (b) FL-NFBC after click (CuAAC) reaction and immuno-staining using appropriate antibodies.
E DOI: 10.1021/acs.biomac.9b01328
Article
contrast, gold nanoparticles were not observed on the surface
of microfibrils of ethynyl-NFBC without fluorescein (Figure
4a). These results clearly demonstrated that CMC with FL
groups existed on the surface of microfibrils.
Click reactions are generally high-yielding, wide in scope,
stereospecific, and simple and require reagents that can be
easily removed without using chromatographic methods.24
One representative example of a click reaction is the Huisgen
copper(I)-catalyzed azide−alkyne 1,3-dipolar cycloaddition
(CuAAC), which yields a 1,4-disubstituted five-membered
1,2,3-triazole ring.24 Because ethynyl-NFBC has ethynyl
groups on the microfibril surface which can be easily modified
via a click reaction (CuAAC), it can provide a new platform to
create fascinating NFBC types with useful functions.
SPM Observations of NFC and NFBC. The fiber lengths
of NFC prepared via a top-down process were reported to
range from several hundred nanometers to several micro-
meters,25 although the values depend on the preparation
procedures. Figure 5a is an SPM image of NFC, and the fiber
Figure 5. Scanning probe microscope height images of (a) NFC
(BinFi-s) prepared from pulp via a top-down process and (b) NFBC.
length is up to several micrometers. In contrast, the fibers of
NFBC were longer than the observation field (Figure 5b),
suggesting that the NFBC fibers are clearly longer than that of
NFC prepared via a top-down process. Additionally, NFBC has
a very high aspect ratio, and this could be a big advantage for
various applications, including utilization as a binder material.
To date, NFC has been prepared by various methods such as
TEMPO oxidation,26,27 aqueous counter collision,28,29 grind-
ing,30 and homogenizing.31 However, these methods decrease
the fiber length as well as fiber width. In contrast, NFBC can
be obtained with very long fibers based on the bottom-up
preparation using cellulose-producing bacterium. The produc-
tion of microfibrils continues during cultivation, which could
be a reason why such significantly long fibers are produced.
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Figure 6. Chemical structure of (a) EC combined with N-(8-bromooctyl)trimethylammonium bromide (EC-BOTMAB) and (b) EC combined
with 1-bromooctane (EC-BO).

Figure 7. IR spectra of (a) EC-BOTMAB/NFBC and (b) EC-BO/NFBC layers before washing and after washing with acetone three times.

Application of NFBC as a Binder for a Display Device
Using Electrochromic Material. The result of zeta-potential
measurement confirmed that the surface of microfibrils has
high negative potential based on the adsorption of CMC.
Furthermore, NFBC has an extremely long fiber length and a
very high aspect ratio. To take advantage of these character-
istics, we applied NFBC as a binder material for a display
device that uses EC material to undergo reversible color
changes when exposed to an electric field.16,17
In this study, N-phenyl-1,4-dipyridyl-pyrrole and 1-ethyl-3-
methylimidazolium bis(trifluoromethylsulfonyl) imide were
used as an EC and electrolyte, respectively. Indium tin oxide
(ITO) is an electron conductive and transparent material that
is widely used as a transparent electrode. EC molecules should
be homogeneously dispersed on the surface of an ITO
transparent electrode to obtain uniform color development.
However, interactions between the electrode and EC
molecules are very weak; therefore, it is impossible to spread
the EC molecules homogeneously without a binder material.
Because NFBC has superior features such as high dispersibility,
homogeneous fiber width [27 ± 5 nm (DS = 0.72)], very high
aspect ratio (large surface area), and very fine network
structure, NFBC appeared to be a good candidate as binder
material for a display device using EC to estimate this
possibility.
Shimizu et al. reported surface modification of microfibrils
for TMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-oxidized cel-
lulose nanofibrils (TOCNs) by using QAs.18 The microfibril
surface of NFBC with CMC has carboxylate groups as well as
TOCNs. Therefore, QA was introduced into an electro-
chromic (EC) molecule to enhance the interaction between
F DOI: 10.1021/acs.biomac.9b01328
the microfibril surfaces and the EC molecules (Scheme S1).
Peaks from the EC moiety were observed at the 7 to 9 ppm
range in the 1H NMR spectrum (Figure S5a). Peaks from
BOTMAB were observed at the 1 to 5 ppm range (Figure
S5a). All peaks were assigned without contradiction,
confirming that EC-BOTMAB was achieved (Figure 6a).
Preparation of EC-OB was also confirmed by 1H NMR
analysis (Figure S5b). EC combined with QA (EC-
BOTMAB), and the NFBC mixture was cast onto a
transparent electrode to make a color-developable layer. EC-
BO without a QA group (Figure 6b) was used as a control
Figure 8. SEM images of (a) the surface of an EC-BOTMAB/NFBC
layer and (b) the cross-section.
sample, and an EC-BO/NFBC layer was formed by the same
procedure. After drying, the layers were washed with acetone
three times to remove excessive EC molecules and then
analyzed by FT-IR (Figure 7). The peaks from EC-BOTMAB
still remained after washing with acetone (Figure 7a). In
contrast, the peaks from EC-BO disappeared after washing
(Figure 7b). These results clearly demonstrated that the
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Figure 9. (a) Experimental equipment for the coloring test and (b) images of the EC-BOTMAB/NFBC and EC-BO/NFBC layers after applying
voltage.
introduction of a QA group was a significantly effective surface
modification for NFBC with CMC.
The surface structure of the EC-BOTMAB/NFBC layer was
analyzed by SEM. The result of SEM observation showed the
homogeneous fine network structure and the presence of small
pores (Figure 8a). Excavation by FIB and SEM observation
was carried out to determine the thickness of the EC-
BOTMAB/NFBC layer, which was 13.6 μm (Figure 8b).
Finally, a coloring test was performed to evaluate the
potential of NFBC as a binder material. A filter paper
embedded with electrolyte was held by the transparent
electrode and an counter electrode (Figure 9a). Homogeneous
color change was successfully observed by applying a voltage of
3 V to the EC-BOTMAB/NFBC (Figure 9b, left), though
obscure color development based on the surface roughness of
filter paper was observed at a portion of the outer edge. In
contrast, only partial color development was observed for EC-
BO/NFBC (Figure 9b, right) as speculated from the result of
IR analysis shown in Figure 7b. NFBC with EC-BOTMAB
formed a fine network structure and homogeneously adsorbed
onto the surface of the transparent electrode (Figure 8a). Small
pores were observed on the surface of the EC-BOTMAB/
NFBC layer, which may have provided a path for the ionic
liquid to permeate and contact the EC molecules. These results
suggest that NFBC could be used as a binder material for
uniform surface adsorption. For display devices developed
using EC material, a screen is required to reflect the irradiated
light. The NFBC cast film is white; therefore, NFBC can
function as a screen as well as a binder material. Furthermore,
the mechanical strength of NFBC is extremely high; therefore,
the strength of the display device would also be increased by
adsorption of NFBC.
NFBC can be easily modified by changing the dispersing
agent. For example, amphiphilic-type NFBC (HP-NFBC) can
be obtained using HPC as a dispersing agent. Because the
surface of HP-NFBC microfibrils is amphiphilic, interactions
with both hydrophilic and hydrophobic materials are possible.
For CM-NFBC, EC was combined with QA absorbed on
NFBC microfibrils via electrostatic interactions. HP-NFBC can
be used as a binder material to allow the adsorption of various
hydrophobic materials on the surface of a matrix.
■
CONCLUSIONS
In this study, we prepared three NFBC types by using CMC
with different degrees of substitution (DSs) to understand the
detailed structure and characteristics of NFBC. The results of
our analyses suggested that CMC existed on the microfibril
surface, and that the DS values affected the NFBC structure,
especially the fiber width. SPM observation confirmed that
NFBC has very long fibers, which is a unique property to NFC
G DOI: 10.1021/acs.biomac.9b01328
Article
prepared via a bottom-up process. Furthermore, NFBC has
other superior features such as homogeneity, dispersibility,
safety, and a fine network structure. We took advantage of
these characteristics by applying NFBC as a binder material for
a display device by using EC. To our knowledge, this is the first
study to consider NFBC as a binder material for display
devices containing EC materials. Homogeneous color change
was successfully observed by applying an electric field,
suggesting that NFBC could be used as a binder material for
uniform surface adsorption.
NFBC is a sustainable material and has superior and unique
characteristics. A simpler and more versatile development of
novel functional NFBC is in progress by our research group
and will be reported in the near future.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.9b01328.
Additional figures (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: ktajima@eng.hokudai.ac.jp.
ORCID
Kenji Tajima: 0000-0002-3238-813X
Takuya Isono: 0000-0003-3746-2084
Takuya Yamamoto: 0000-0001-9716-8237
Toshifumi Satoh: 0000-0001-5449-9642
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Dr. Shigenobu Hirano of Ricoh Co., Ltd., for his
valuable comments on the experiments. We also thank Mr.
Kenji Ohkubo and Ms. Emiko Obari of Hokkaido University
for his technical support for TEM observations. Scanning
probe microscope images were provided by Ms. Risa Fuji of
Shimadzu Corporation. This work was supported by the
Strategic Foundational Technology Improvement Support
Operation program of the Ministry of Economy, Trade and
Industry (no. Hokkaido 1607005) and by the Japan Society for
the Promotion of Science (JSPS; grant number JP16K05802,
H.K., K.T.; JP19H02549, K.T.).
■
ABBREVIATIONS
BC, bacterial cellulose; NFC, nanofibrillated cellulose; NFBC,
nanofibrillated bacterial cellulose; CMC, carboxymethylcellu-
lose; HEC, hydroxyethylcellulose;; HPC, hydroxypropylcellu-
Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules Article

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H DOI: 10.1021/acs.biomac.9b01328
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