Accepted Manuscript Title: BIOSYNTHESIS OF BACTERIAL CELLULOSE IN THE PRESENCE OF DIFFERENT NANOPARTICLES TO CREATE NOVEL HYBRID MATERIALS Author: Esra Erbas Kiziltas Alper Kiziltas Melanie Blumentritt Douglas J. Gardner PII: DOI: Reference:
S0144-8617(15)00355-0 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.039 CARP 9870
To appear in: Received date: Accepted date:
7-4-2015 15-4-2015
Please cite this article as: Kiziltas, E. E., Kiziltas, A., Blumentritt, M., and Gardner, D. J.,BIOSYNTHESIS OF BACTERIAL CELLULOSE IN THE PRESENCE OF DIFFERENT NANOPARTICLES TO CREATE NOVEL HYBRID MATERIALS, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.039 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.
*Highlights (for review)
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BC-based novel hybrid nanocomposites were produced through an in situ approach with an aim to incorporate the multifunctional properties of BC and nanoclay, xGnP and CNFs for the development of nanostructed materials with designed functionalities. Nanocomposites exhibited good dispersion of the nanoparticles (NPs) within the BC matrix and the NPs were found embedded among the voids and microfibrils. The thermal stability and residual mass of BC-xGnP and BC-NC nanocomposites were significantly increased compared to the pristine BC. The ability to synthesize and manipulate bacterial cellulose in the presence of different nanoparticles allows tremendous versatility in creating new materials results in increased performance for a chosen application.
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BIOSYNTHESIS OF BACTERIAL CELLULOSE IN THE PRESENCE OF DIFFERENT
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NANOPARTICLES TO CREATE NOVEL HYBRID MATERIALS
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Esra Erbas Kiziltas1, 2†*, Alper Kiziltas1, 3, Melanie Blumentritt1 and
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Douglas J. Gardner 1
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Advanced Structures and Composites Center (AEWC), University of Maine, Orono,
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ME 04469, USA
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The Scientific and Technological Research Council of Turkey (TUBĐTAK), Tunus Cad, Kavaklıdere 06100, Ankara, TURKEY
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Department of Forest Industry Engineering, Faculty of Forestry, University of Bartin, 74100 Bartin, TURKEY
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†: Corresponding author.
E-mail: [email protected]
Tel: +1-207- 249-9346 Fax: +1-207-581-2074
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*: First author.
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E-mail: [email protected]
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Tel: +1-207- 249-9346 Fax: +1-207-581-2074
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Abstract
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The unique micro-nano porous three-dimensional network of bacterial cellulose (BC) can facilitate the incorporation of nanoparticles (NPs) into the BC matrix to create advanced BCbased functional nanomaterials for diverse applications. In this study, novel nanomaterials comprised of bacterial cellulose (BC) synthesized in the presence of different NPs (cellulose nanofibrils (CNF), exfoliated graphite nanoplatelets (xGnP) and nanoclay (NC)) were prepared using an in situ approach. NPs at 0.5 wt. % loading were added into the BC culture medium and their effect on the resulting nanocomposite structure was studied by field emission scanning electron microscopy (FE-SEM), X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). All produced BC-based nanomaterials exhibited good dispersion of the NPs within the BC matrix and the NPs were found embedded among the voids and microfibrils. The thermal stability and residual mass of BC-xGnP and BC-NC nanomaterials was significantly increased compared to the neat BC. CNF incorporation into the BC matrix did not change the thermal stability and residual mass of the BC matrix. This study also provides novel insights into the properties of the hybrid materials and shows the approach used to make these materials results in increased performance for chosen applications.
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Keywords: Nanoparticles; FT-IR; Thermal stability; Morphology; X-Ray diffraction
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1. Introduction Bacterial cellulose (BC) has received substantial interest because of its unique structural features and impressive physico-mechanical properties including high water holding capacity, tensile strength and modulus, crystallinity, porosity which consists of micro/nanoporous spaces, an ultrafine fiber network, biodegradability, excellent biological affinity and the ability to be molded into three-dimensional (3D) structures during synthesis (Ul-Islam et al. 2012a; Shah et al. 2013: Hu et al. 2014). Although BC has the same molecular formula as plant-based cellulose, it has a number of notable structural features and properties over the plant-based cellulose such as: high purity (absence of lignin and hemicellulose), high degree of polymerization (up to 8000), high crystallinity (of 70–80%), high water content to 99%, excellent biodegradability and biological affinity and high mechanical stability, which is quite different from the plant-based cellulose (Vitta and Thiruvengadam 2012; Hu et al. 2014). The unique micro/nanoporous three dimensional network of BC, which provides a natural methodology to control or restrict the size of the particles can facilitate the penetration of various particles made from different materials (polymers, metals and metal oxides and solid materials) into the interior, and thus, act as a template. The added particles can be anchored within the BC fiber network to create novel types of nanomaterials which represents a very specific and important modification method of BC (Vitta and Thiruvengadam 2012; Hu et al. 2014). These amazing and unique physico-mechanical properties of BC have inspired attempts to use it in a number of commercial products such as in tires, headphone membranes, high performance speaker diagrams, high-grade paper, make-up pads, diet-food, and textiles (Evans et al. 2003; Shah et al. 2013).
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Intensive research and exploration in the past few decades on BC nanomaterials mainly focused on their biosynthetic process to achieve the low-cost preparation and to enhance BC production with static and agitation cultures using a variety of BC production strains, carbon sources, alternative inexpensive sugar sources and supplementary materials (Hu et al. 2014; Shah et al. 2013). In the last few years, growing worldwide activity (increasing annual publication activity and increasing efforts for the practical use of BC materials) have led to the emergence of more diverse potential applications exploiting the functionality of BC nanomaterials (Hu et al. 2014). The functionalization of BC with organic, inorganic or polymeric materials enables the creation of materials with improved or new properties by mixing multiple constituents and exploiting synergistic effects (Hu et al. 2014). BC has been used as a matrix to incorporate nanoparticles for the preparation of novel functional nanomaterials that gather together excellent properties of BC with the ones displayed by typical inorganic or organic nanomaterials like antibacterial, optical, electrical and magnetic properties as well as catalytic and biomedical activity (Vitta and Thiruvengadam 2012; Hu et al. 2014). Several recent reviews giving a summary and discussion regarding the synthetic approaches and applications of functionalized BC have been published (Vitta and Thiruvengadam 2012; Shah et al. 2013; Hu et al. 2014).
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There are two basic synthesis approaches for creating BC matrix composites, in situ and ex situ. For the in situ method, secondary components such as NPs can be introduced into the BC culture media at the beginning of BC synthesis process. Using BC as a template for the in situ synthesis of BC-NPs has the following advantages: the shape, structure and properties can be easily adjusted during the biosynthesis, pretreatment and chemical modification process, does not require severe conditions, simple and easy to implement which can obtain the nano-particles with narrow size distribution (Shah et al. 2013; Hu et al. 2014). For the ex situ method, 3 Page 4 of 17
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secondary components such as NPs can be introduced into the BC matrix by the solution impregnation method. Ex situ synthesis of functionalized BC nanomaterials has the following limitations: only submicron to nano sized particles can penetrate into the BC, creating difficulties in achieving homogenous distribution of particles in the BC, and hydrophobic materials cannot be used for BC composites (Shah et al. 2013). Prior literature shows that most of the functionalized BC nanomaterials made with NPs including graphene and montmorillonite (MMT) are formed through either in situ addition using a reducing agent and chemical modification or the ex situ synthesis approach. The BC cultivation methods, which are either static or agitated processes, are important to produce good quality and good quantities of BC (Chen et al. 2010). Moreover, a change of cultivation methods can influence the BC microstructure such as crystalline structure and mechanical properties (Yan et al. 2008). The particles in a static BC culture cannot be entrapped in the surface of BC fibers after being precipitated in the media because the particles only remain suspended in the BC synthetic media for a short time and a BC sheet is formed on the surface of the media at the media air interface (Cheng et al. 2009). The agitation culture provides a much better environment for added materials to become entrapped in the BC fibrils thanks to constant movement in the media which prevents the particles from settling and provides homogenous BC formation (Shah et al. 2013).
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In the present investigation, BC-nanoclay (NC), BC-xGnP and BC-CNF nanomaterials were synthesized through in situ synthesis without using surface modification or reducing agents with an aim to obtain new promising BC nanomaterials with improved thermo-physical properties. The in situ synthesis method was selected for this research because of easy processing and the ability to promote homogenous distribution of particles in the BC matrix. This study will provide initial insight into the use BC-based nanomaterials for extremely large applications ranging from conducting paper to a flexible, electrically conducting display. BC-MMT, BCxGnP and BC-CNF composites were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) thermogravimetric analyses (TGA) and field emission scanning electron microscopy (FESEM). To the best of our knowledge, studies involving the preparation of materials containing BC and CNF have not been reported previously.
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2. Materials and Methods 2.1. Nanomaterials The xGnP fillers were supplied by XG Sciences Inc., USA. The xGnP fillers in powder form were used as the reinforcement having particle diameters of 5 µm. Average platelet thickness ranged from about 5 to 15 nanometers. This translates into an average particle surface area ranging from about 60 to 150 m2/g. The bulk density of xGnP fillers is reported to be 0.180.25 g/cm3. The NC, Nanomer I.44P, was supplied by Nanocore and it has a quartenary ammonium chemistry based surface modification and an average particle size 15-20 micrometers. CNF, trade name Celish- Rokameijin, was supplied by Daicel Chemical Industries, Ltd., Japan. This product consisted of a 35 wt. % fiber content suspension. SEM image in Figure 1. showed that the agglomeration phenomena happened in the nanoscale CNF, xGnP and NC materials when it was exposed to air. However, some of the nanomaterials would stay at the nanometer size.
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Figure 1. SEM micrograph of a) CNF and FESEM micrographs b) xGnP and c) NC. 2.2. Microorganism and Cultivation of Acetobacter xylinus Acetobacter xylinus 23769 strain was purchased from the American Type Culture Collection (ATTC), Manassas, VA, USA. Four types of culture media were used: 1) the Hestrin– Schramm culture medium (HS) (2 w/v% glucose, 0.5 w/v% bacto peptone, 0.5 w/v% yeast extract, 0.05 w/v% MgSO4.7H20, 0.115 w/v% citric acid and 0.27 w/v% disodium hydrogen phosphate), 2) nanoclay–HS medium (in the presence of final 0.5 w/v% nanoclay in HS medium), 3) xGnP–HS medium (in the presence of final 0.5 w/v% xGnP in HS medium) and 4) cellulose nanofibrils (CNF)–HS medium (in the presence of final 0.5 w/v% cellulose nanofibrils in HS medium). These culture media were sterilized at 121 °C in an autoclave for 20 min. For seed culture, 50 ml HS medium was inoculated with 1 ml of freeze dried stock culture and incubated in a 250 ml Erlenmeyer flask at 28°C for 48h at pH 5.5 under static conditions. These four cultures were inoculated with 10 v/v% of seed culture and incubated in static cultivation at 28 °C for 3 days until a cellulose pellicle formed. After static cultivation, cultures were transferred to a rotary shaker operating at a rotational speed of 120 rpm, for 6 days at 28 °C to prevent the NPs from settling and providing homogenous BC formation with the added NPs.
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2.3.2. FTIR Attenuated Total Reflectance Fourier-Transform InfraRed (ATR-FTIR) spectroscopy analysis was carried out on freeze-dried BC and BC composites samples on a Perkin Elmer Spectrum One FTIR spectrometer (Wellesley, MA, USA), equipped with a Universal ATR accessory, using 200 scans and a resolution of 4 cm−1, over the range 4000–400 cm−1. Before the analysis, samples were dried at 90 °C under vacuum for 24 h.
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2.3.3. XRD XRD was performed with a high resolution X-ray diffractometer (Model X’Pert PRO, Philips PANalytical, Netherlands) with a Ni-filtered Cu Kα (1.540562Å) radiation source operated at voltage 45 kV and 40 mA electric current. The samples were scanned from 4°-40° 2θ range with a step of 0.02 °, a step time of 2.5 s. Freeze-dried BC and BC composites samples were pressed manually using a Dake Press (Dake, Grand Falls, MI) into cylindrical wafers 2 cm in diameter and 0.05-0.1 cm thick. A silicon zero background plate was used to make sure that there were not any peaks resulting from the sample holder. The same sample holder position and sample holder (holder and silicon zero background plate) were used for both samples. Crystallinity index (C.I.) and percentage crystallinity (% Crystalline) of BC samples were calculated as follows (Rosli et al. 2013)
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2.3. Characterization of the Composites 2.3.1. Morphology-FESEM The freeze-dried BC and BC composites samples were coated with gold (E-1045, Hitachi, Tokyo, Japan). Analysis of the structure of the BC was performed by using a FieldEmission Scanning Electron Microscope (FESEM) (NVision 40, ZEIS) at 25 kV. Images were taken with 2000 X FESEM micrograph magnifications.
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where I200 is the maximum intensity of diffraction of the (200) lattice peak (2θ of 22º to 23º) and Iam is that of the amorphous material between 2θ of 18º to 19º where the intensity is minimum. 2.3.4. Thermal Stability-TGA TGA measurements were carried out using a Mettler Toledo analyzer on samples of about 8 mg. Each sample was scanned over a temperature range from ambient temperature to 600 °C at a heating rate of 10°C/min under nitrogen with a flow rate 20 ml/min to avoid sample oxidation. The samples used for the TGA measurement were randomly picked 5 individual samples from ground samples.
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3. Results and Discussion 3.1. Morphology The morphological properties of the BC composites are related to the materials’ microstructures, the state of nanoparticle dispersion, penetration and orientation in the BC matrix, as well as the interactions between nanoparticles and the BC matrix. Hence, the morphological properties should be characterized to confirm the synthesis of BC composites by assessing the attachment of nanoparticles onto the surface and their penetration into the matrix of the BC sheets and to optimize the synthesis conditions for achieving high performance BC composites (Ul-Islam et al. 2012b). The FESEM micrographs of a freeze-dried BC pellicle prepared from the reference medium and composites are shown in Figure 2.
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Figure 2. FESEM micrographs of a) BC, b) BC-CNF, c) BC-xGnP and d) BC-NC nanomaterials. The neat BC sample exhibited a highly fibrous three dimensional network-like structure consisting of ultrafine cellulose microfibrils and FESEM shows a random arrangement of ribbonshaped microfibrils without any preferential orientation which results in the formation of pores with different sizes on the surface and throughout the entire BC matrix. Small-sized nanoparticles can penetrate inside the BC matrix using these pores (Feng et al. 2012; Ul-Islam et al. 2012b). The typical lateral dimensions of the BC fibrils were in the range of 15–70 nm. Flakeshaped xGnP and NC and rod-shaped CNFs were observed to effectively be incorporated into the surface layer of the BC matrix. Figure 2b shows that the rod-shaped CNFs penetrated into the 7 Page 8 of 17
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BC matrix and some parts of the longer CNFs spreading on the BC surface or entangling with other CNFs or BC nanofibrils. On the other hand, the flake-shape xGnP and NC (Figure 2c and 2d), appeared to be woven within the surface layer of the weblike BC, and were indeed fixed or trapped by adjacent nanofibrils in the BC surface network (Zhou et al. 2013). The particles that penetrated into the BC matrix filled the empty space in entire BC matrix and the xGnP and NC showed larger surface coverage in the surface layer of the BC matrix because of their nearly fully extended 2-D morphology (Ul-Islam et al. 2012b; Zhou et al. 2013). The presence of hydroxyl groups in both CNF and BC can lead to strong hydrogen bonding interactions. On the other hand, NC may have weak hydrogen bonding interactions with BC because of inorganic-organic hydroxyl groups (NC hydroxyls cannot create hydrogen bond cooperatively with those of cellulose (they do not have the ca. 10 Å periodicity as for cellulose along the chain)) (Ul-Islam et al. 2012b). However the FT-IR spectra of pure xGnP incorporated BC matrix showed no significant shift in the peaks, suggesting that no interaction between xGnP and BC. This finding is in good agreement with Gopiraman’s et al. study (Gopiraman et al. 2013). Interactions of the nanoparticles with the BC matrix and nanoparticle attachment to the BC surface and penetration into the BC matrix can potentially enhance the physico-mechanical properties of the BC (UlIslam et al. 2012b, Shah et al. 2013; Hu et al. 2014).
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3.2. X-ray Diffraction The X-ray diffraction patterns of the BC and BC nanomaterials are shown in Figure 3. In the case of pure BC, four characteristic peaks centered at 14.5 º, 16.8º, 22.7º and 34.5º, indicating the typical cellulose I allomorph structure (Oh et al. 2005). BC-CNF composites showed a similar XRD pattern to the neat BC. The only difference between BC and BC-CNF nanomaterials was intensity changes in the peaks. BC-xGnP composites exhibited a large and sharp 2θ peak at 26.5º as well as BC characteristic peaks. In addition to the BC characteristic peaks, various diffraction peaks at 2θ angles of ∼7º, 20º, 26.6º and 27.7º were also observed in the BC-NC composites. As a result, it appeared that the xGnP and NC did not cause any significant change in the BC structure. The XRD patterns and FESEM images clearly indicate that the NPs were successfully incorporated into the nanofibrous BC matrix. In addition, the crystallinity index (CI) and percentage crystallinity calculated based on X-ray profiles are listed in Table 1. The proposed method (Segal Method or XRD peak height method) to calculate percentage crystallinity is convenient for empirical measurement to allow rapid comparison of cellulose samples (Park et al. 2010). Table 1 revealed a decrease in CI (0.75) and percentage crystallinity (80%) for BC-NC nanomaterials and an increased CI (0.86) and percentage crystallinity (88%) for BC-xGnP nanomaterials. On the other hand, BC and BC-CNF nanomaterials have the same CI (0.81) and percentage crystallinity (84). It is already known that the penetration and interaction of NC particles slightly interrupts the native hydrogen bonding interactions in BC resulting in lower crystallinity for BC-NC nanomaterials (Ul-Islam et al., 2013).
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3.3. FT-IR Figure 4. shows the FT-IR spectra of BC, CNF, xGnP, NC and BC nanomaterials. The FT-IR spectrum of BC in this research was similar to CNF. Table 2. summarizes FT-IR results for BC, NC and BC-NC nanomaterials. BC-CNF and BC-xGnP composites exhibited a similar spectra to BC and no differences could be resolved between the 2 cellulose samples. The xGnP, which is mostly composed of carbon atoms and a tiny amount of residual unreacted intercalant may also be present, but did not show any characteristic absorption peaks because it has no specific functional groups on its surface. As was mentioned above, the BC-xGnP matrix showed no significant shift in the peaks, suggesting that no interaction between xGnP and BC. In the BC FT-IR spectra, the bands at 3345 and 2922 cm-1 are attributed to the presence of OH-stretching and CH2 stretching, respectively. The bands at 1653 and 1372 cm-1 indicated H-O-H bending of adsorbed water and CH2 bending or O-H in plane bending and the band at 1035 to 1162 cm-1 9 Page 10 of 17
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could be associated with C-O symmetric stretching of primary alcohol and C-O-C antisymmetric bridge stretching, respectively (Ashori et al. 2012; Barsberg 2010; Marechal and Chanzy 2000).
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The most prominent bands in the IR spectrum of NC can be found at 3615 cm-1 which is attributed to the presence OH stretching. The absorption peak in the region of 1035 cm-1 is attributed Si-O stretching (in-plane) vibration for NC. The characteristic peaks at 618 and 918 cm-1 are attributed to Si-O bending and Al-Al-OH bending, respectively (Patel et al. 2006). The FT-IR spectrum of BC-NC nanomaterials exhibited the presence of characteristic peaks both from BC and NC but there are minor differences between the BC and BC-NC FT-IR results that may be related to the chemical interactions between the BC and NC. The important changes between the BC and BC-NC nanomaterials can be assigned to: 3300-3700 cm-1(OH-stretching); 2920 cm-1 (C-H stretching vibration), 920 cm-1 (Al-Al-OH bending) and 620 cm-1 (Si-O bending). This result is in good agreement with the work previously reported by Ul-islam et al. 2012b. In the FT-IR spectra of BC, the band at 3345 cm-1 was attributed to the intramolecular hydrogen bond which is important for elucidating hydrogen-bonding patterns because, in favorable cases, each distinct hydroxyl group gives a single stretching band at a frequency that decreases with increasing strength of hydrogen bonding (Poyrazoglu Coban and Biyik 2011; Sturcova et al. 2004). Although the OH peak of NC was present at 3615 cm−1, BC-NC showed a characteristic OH peak at 3347 cm−1 because of the merging of peaks for the hydrogen bonding of both BC and NC (Ul-islam et al. 2012b). The peak intensity of 2920 cm-1 (C-H stretching vibration), 920 cm-1 (Al-Al-OH bending) and 620 cm-1 (Si-O bending) deceased in BC-NC nanomaterials. It is believe that the sign of electrostatic interaction between BC and NC.
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Table 2. FT-IR results for BC, NC and BC-NC nanomaterials. Assignment Wavenumber (cm-1) BC and CNF NC O-H Bending 400-700 b - glucosidic linkages between the glucose units ∼896 C-O symmetric stretching of primary alcohol 1035 C-O-C antisymmetric bridge stretching 1162 C-H deformation 1316 CH2 bending or O-H in plane bending 1372 H-O-H bending of adsorbed water 1653 CH2 2852 CH2 stretching 2922 OH-stretching 3345 Si-O bending 618 Al-Al-OH bending 918 Si-O stretching (in-plane) 1035 CH2 bending 1467 C-H stretching 2923 OH stretching or Al-Al-OH stretching ∼ 3615 Si-O bending Al-Al-OH bending Si-O stretching (in-plane) C-O-C antisymmetric bridge stretching CH2 stretching OH-stretching
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3.4. Thermal Stability Figure 5 shows the TGA and derivative TGA of the BC and CNF in an inert TGA atmosphere. Three stages of mass loss are associated with the TGA curve. A first stage, up to 200°C, corresponds to less than 10% of loss for BC and CNF samples and is followed by second stage with more than 80 and 85 wt. % of loss for BC and CNF respectively, up to about 500°C. The final third stage extends to the usual ending test temperature at 600 °C in association with more than 81 and 86 wt. % of loss for BC and CNF, respectively. It is very well know that the first initial loss, below 200°C is usually associated with a relatively small DTG peak assigned to the release of water (moisture, humidity) or other volatiles from the BC. The peaks in the DTG graphs in the range of 200–500°C correspond to volatilization stages (El-Saied et al., 2008; Peng et al., 2013). BC has slightly higher thermal stability compared to CNF because of its high purity (absence of lignin and hemicellulose). In general, the DTG scan of pristine clay displays four distinct regions. These are the elimination of the free and physisorbed water in the internal layer of the clay and the dis-aggregation or de-crystallization melting of the long chain tails of the surfactant molecules occurring at temperatures up to 200°C, surfactant decomposition occurred between 200 and 500°C, the dehydroxylation of the clay occurred between 500 and 700°C and decomposition of organic carbonaceous residue occurred after 700 to 1000°C (Santos et al. 2009; Xie et al., 2001; Xie et al,. 2002). The most important regions influencing the thermal behavior of the clay-filled nanomaterials are region 1 and region 2 where the release of small molecules associated with fabrication and storage of the clay-filled nanomaterials (Xie et al., 2002). A first stage, up to 200°C, corresponds to less than 1% of loss for NC sample and is followed by second stage with more than 35 wt. % of loss for NC, up to about 500°C. The xGnP has higher thermal stability compared to other nanofillers and did not show any significant loss (less than 3.5 wt. %) at 600°C. All the BC composites exhibit a first mass loss step associated with water release (dehydration process occurring up to about 200°C). The second weight loss step shows a small shift towards higher temperatures when xGnP and NC are introduced into the BC matrix, beginning at 225 °C for BC and CNF and at 265 °C for the BC-xGnP and BC-NC composites, according to DTG data. Figure 5. shows that CNF incorporation into the BC matrix did not significantly change the thermal stability and residual mass of the BC matrix. On the other hand, the thermal stability and residual mass of BC-xGnP nanomaterials is significantly increased, which is because of the high thermal stability of xGnP and the shielding effect of xGnP on the diffusion of combustion gases into and out of the BC matrix during its thermal decomposition (Yang et al., 2007). We also estimated the amount of xGnPs incorporated in the bacterial cellulose membrane using thermogravimetric analysis (TGA) based on ash content determination at 600°C. The integration of xGnP content for 0.5 % wt was found 38% in BC-xGnP nanomaterials. This result may also explain the reason of higher thermal stability. As expected, the addition of NC in the BC matrix increased of thermal stability and residual mass of BC-NC nanomaterials because of the homogeneous incorporation of clay sheets, to a barrier of these high-aspect ratio fillers, and char formation (Ray and Bousmina 2005). The establishment of hydrogen bonds between the organic BC matrix and inorganic NC particles can also cause difficulties in the release of water molecules produced by the condensation of hydroxyl groups of cellulose and may also lead to an improvement in the thermal stability of the BC matrix (Perotti et al., 2011; Ul-Islam et al., 2012b).
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4. Conclusions The added NPs appeared to be dispersed uniformly in the BC matrix. The BC ribbons interwound with the CNF and formed a three-dimensional network. The flake-shaped xGnP and NC were indeed fixed or trapped by adjacent nanofibrils in the BC surface network. FT-IR and XRD spectra showed some differences between the neat BC and BC nanomaterials which are related to the physical and chemical interactions between the BC and NPs. TGA data showed a shift to higher temperature of the beginning of decomposition process of the BC-NC and BCxGnP nanomaterials in comparison to the pristine BC. The thermal stability of BC-CNF did not change compared to pure BC. The incorporation of NPs into BC provides new opportunities for the development of novel nanostructured materials with improved properties.
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5. Acknowledgements The republic of Turkey, The Scientific and Technological Research Council of Turkey (TUBITAK) is greatly acknowledged for support of the scholarship of the researcher Esra Erbas Kiziltas to do this study at the University of Maine. The authors would like to acknowledge the contributions of Justin Crouse and Chris West whose hard work made this paper possible. The authors would also like to thank U.S. Army Corps of Engineers, Engineer Research and Development Center project 912HZ-07-2-0013 and Maine Agricultural and Forest Experiment Station (MAFES) project ME09615-08MS and the Wood Utilization Research Hatch 2007-2008 project.
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6. References Ashori, A., Sheykhnazari, S., Tabarsa, T., Shakeri, A., & Golalipour, M. (2012). Bacterial cellulose/silica nanocomposites: Preparation and characterization. Carbohydrate Polymers, 90(1), 413–418. Barsberg S. (2010). Prediction of vibrational spectra of polysaccharides-simulated IR spectrum of cellulose based on density functional theory (DFT). J. Phys. Chem. B., 114(36):11703-8. Chen P., Cho S.Y. & Jin H.-J. (2010). Modification and applications of bacterial cellulose in polymer science. Macromol Res 18 (4): 309-320. Cheng, K. C., Catchmark, J. M., & Demirci, A. (2009). Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis. Journal of Biological Engineering, 3, 12. El-Saied H., El-Diwany A.I., Basta A.H., Atwa N.A. & El-Ghwas DE (2008). Production and characterization of economical bacterial cellulose. BioResources, 3:1196-1217. Evans, B. R., O‘Neill, H. M., Malyvanh, V. P., Lee, I., & Woodward, J. (2003). Palladiumbacterial cellulose membranes for fuel cells. Biosensors and Bioelectronics, 18,917–923. Feng, Y., Zhang, X., Shen, Y., Yoshino, K., & Feng, W. (2012). A mechanically strong,flexible and conductive film based on bacterial cellulose/graphene nanocom-posite. Carbohydrate Polymers, 87, 644–649. Gopiraman M., Fujimori K., Zeeshan K., Kim B.S. and Kim I.S. (2013) Structural and mechanical properties of cellulose acetate/graphene hybrid nanofibers: Spectroscopic investigation. eXPRESS Polymer Letters, 7(6):554-563. Hu W., Chen S., Yang J., Li Z. & Wang H. (2014). Functionalized bacterial cellulose derivatives and nanocomposites. Carbohydrate Polymers 101 (2014) 1043-1060. Marechal Y. & Chanzy H. (2000). The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry. Journal of Molecular Structure, 523 (1–3):183–196.
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Oh, S. Y., Yoo, D. I., Shin, Y., & Kim, H. C. (2005). Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydrate Research, 340(15), 2376–2391. Park S., Baker J.O., Himmel M.E., Parilla P.A. and Johnson D.K. (2010) Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance, Biotechnology for Biofuels, 3:10. Patel H.A., Somani R.S., Bajaj H.C. & Jasra R.V. (2006). Nanoclays for polymer nanocomposites, paints, inks, greases and cosmetics formulations, drug delivery vehicle and waste water treatment. Bull. Mater. Sci., 29(2):133-145. Peng Y., Gardner D.J., Han Y., Kiziltas A., Cai Z., Tshabalala M.A. (2013) Influence of drying method on the material properties of nanocellulose I: Thermostability and crystallinity, Cellulose, 20(5):2379-2392. Perotti G.F., Barud H.S., Messaddeq Y., Ribeiro S.J.L. & Constantino V.R.L. (2011). Bacterial cellulose–laponite clay nanocomposites. Polymer, 52 (1):157–163. Poyrazoglu Coban E. & Biyik H. (2011). Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter lovaniensis HBB5. African Journal of Biotechnology, 10(27):1037-1045. Ray S.S. & Bousmina M. (2005). Biodegradable polymers and their layered silicate nanocomposites: In greening the 21st century materials world. Progress in Materials Science, 50(8):962-1079. Rosli NA, Ahmad I and Abdullah I (2013). Isolation and characterization of cellulose nanocrystals from Agave Angustifolia fibre. BioResources, 8:1893-1908. Santos K.S., Liberman S.A., Oviedo M.A.S. & Mauler R.S. (2009). Optimization of the mechanical properties of polypropylene-based nanocomposite via the addition of a combination of organoclays." Composites: Part A, 40 :(8) 1199-1209. Shah N., Ul-Islam M., Khattak W.A. & Park J.K. (2013). Overview of bacterial cellulose composites: A multipurpose advanced material. Carbohydrate Polymers 98 (2013):1585-1598. Sturcova A., His I., Apperley D.C., Sugiyama J. &Jarvis M.C. (2004). Structural details of crystalline cellulose from higher plants. Biomacromolecules, 5: 1333-1339. Ul-Islam, M., Khan, T., & Park, J. K. (2012a). Water holding and release properties of bacterial cellulose obtained by in situ and ex situ modification. Carbohydrate Polymers, 88(2), 596–603. Ul-Islam, M., Khan, T., & Park, J. K. (2012b). Nanoreinforced bacterial cellulose– montmorillonite composites for biomedical applications. Carbohydrate Polymers, 89(4), 1189– 1197. Ul-Islam, M., Khan, T., Khattak, W. A., & Park, J. K. (2013). Bacterial cellulose-MMTs nanoreinforced composite films: Novel wound dressing material with antibacterial properties. Cellulose, 20, 589–596. Vitta S. & Thiruvengadam V. (2012). Multifunctional bacterial cellulose and nanoparticleembedded composites. Current Science, 102(10):1398-1405. Wang, W., Li, H. Y., Zhang, D. W., Jiang, J., Cui, Y. R., Qiu, S., et al. (2010). Fabrication of bienzymatic glucose biosensor based on novel gold nanoparticles-bacterial cellulose nanofibers nanocomposite. Electroanalysis, 22(21), 2543–2550. Wang, W., Zhang, T. J., Zhang, D. W., Li, H. Y., Ma, Y. R., Qi, L. M., et al. (2011). Amperometric hydrogen peroxide biosensor based on the immobilization of hemeproteins on gold nanoparticles-bacteria cellulose nanofibers nanocomposite.Talanta, 84(1), 71–77.
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Xie W., Gao Z., Pan W.-P., Hunter D., Singh A. & Vaia R. (2001). Thermal degradation chemistry of alkyl quaternary ammonium montmorillonite." Chem. Mater., 13: (9) 2979-2990. Xie W., Xie R., Pan W.P., Hunter D., Koene B., Tan L.-S. & Vaia R. (2002). Thermal stability of quaternary phosphonium modified montmorillonites. Chem. Mater,: 14: (11) 4837-4845. Yan Z., Chen S., Wang H.W., Wang B., Wang C. &Jiang J. (2008b). Cellulose synthesized by Acetobacter xylinum in the presence of multi-walled carbon nanotubes. Carbohydrate Research 343 (2008):73–80. Yan, Z. Y., Chen, S. Y., Wang, H. P., Wang, B. A., & Jiang, J. M. (2008a). Biosynthesis of bacterial cellulose/multi-walled carbon nanotubes in agitated culture. Carbohydrate Polymers, 74(3), 659-665. Yang, S., Taha-Tijerina, J., Serrato-Diaz, V., Hernandez, K., & Lozano, K. (2007). Dynamic mechanical and thermal analysis of aligned vapor grown carbon nanofiber reinforced polyethylene. Composites Part B: Engineering, 38(228-235):1359-8368. Zhou T., Chen D., Jiu J., Nge T.T., Sugahara T., Nagao S., Koga H., Nogi M., Suganuma K., Wang X., Liu X., Cheng P., Wang T. & Xiong D. (2013). Electrically conductive bacterial cellulose composite membranes produced by the incorporation of graphite nanoplatelets in pristine bacterial cellulose membranes. eXPRESS Polymer Letters, 7 (9):756–766.
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S0144-8617(15)00355-0 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.039 CARP 9870
To appear in: Received date: Accepted date:
7-4-2015 15-4-2015
Please cite this article as: Kiziltas, E. E., Kiziltas, A., Blumentritt, M., and Gardner, D. J.,BIOSYNTHESIS OF BACTERIAL CELLULOSE IN THE PRESENCE OF DIFFERENT NANOPARTICLES TO CREATE NOVEL HYBRID MATERIALS, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.039 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.
*Highlights (for review)
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BC-based novel hybrid nanocomposites were produced through an in situ approach with an aim to incorporate the multifunctional properties of BC and nanoclay, xGnP and CNFs for the development of nanostructed materials with designed functionalities. Nanocomposites exhibited good dispersion of the nanoparticles (NPs) within the BC matrix and the NPs were found embedded among the voids and microfibrils. The thermal stability and residual mass of BC-xGnP and BC-NC nanocomposites were significantly increased compared to the pristine BC. The ability to synthesize and manipulate bacterial cellulose in the presence of different nanoparticles allows tremendous versatility in creating new materials results in increased performance for a chosen application.
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BIOSYNTHESIS OF BACTERIAL CELLULOSE IN THE PRESENCE OF DIFFERENT
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NANOPARTICLES TO CREATE NOVEL HYBRID MATERIALS
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Esra Erbas Kiziltas1, 2†*, Alper Kiziltas1, 3, Melanie Blumentritt1 and
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Douglas J. Gardner 1
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Advanced Structures and Composites Center (AEWC), University of Maine, Orono,
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ME 04469, USA
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The Scientific and Technological Research Council of Turkey (TUBĐTAK), Tunus Cad, Kavaklıdere 06100, Ankara, TURKEY
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Department of Forest Industry Engineering, Faculty of Forestry, University of Bartin, 74100 Bartin, TURKEY
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†: Corresponding author.
E-mail: [email protected]
Tel: +1-207- 249-9346 Fax: +1-207-581-2074
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*: First author.
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E-mail: [email protected]
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Tel: +1-207- 249-9346 Fax: +1-207-581-2074
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Abstract
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The unique micro-nano porous three-dimensional network of bacterial cellulose (BC) can facilitate the incorporation of nanoparticles (NPs) into the BC matrix to create advanced BCbased functional nanomaterials for diverse applications. In this study, novel nanomaterials comprised of bacterial cellulose (BC) synthesized in the presence of different NPs (cellulose nanofibrils (CNF), exfoliated graphite nanoplatelets (xGnP) and nanoclay (NC)) were prepared using an in situ approach. NPs at 0.5 wt. % loading were added into the BC culture medium and their effect on the resulting nanocomposite structure was studied by field emission scanning electron microscopy (FE-SEM), X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). All produced BC-based nanomaterials exhibited good dispersion of the NPs within the BC matrix and the NPs were found embedded among the voids and microfibrils. The thermal stability and residual mass of BC-xGnP and BC-NC nanomaterials was significantly increased compared to the neat BC. CNF incorporation into the BC matrix did not change the thermal stability and residual mass of the BC matrix. This study also provides novel insights into the properties of the hybrid materials and shows the approach used to make these materials results in increased performance for chosen applications.
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Keywords: Nanoparticles; FT-IR; Thermal stability; Morphology; X-Ray diffraction
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1. Introduction Bacterial cellulose (BC) has received substantial interest because of its unique structural features and impressive physico-mechanical properties including high water holding capacity, tensile strength and modulus, crystallinity, porosity which consists of micro/nanoporous spaces, an ultrafine fiber network, biodegradability, excellent biological affinity and the ability to be molded into three-dimensional (3D) structures during synthesis (Ul-Islam et al. 2012a; Shah et al. 2013: Hu et al. 2014). Although BC has the same molecular formula as plant-based cellulose, it has a number of notable structural features and properties over the plant-based cellulose such as: high purity (absence of lignin and hemicellulose), high degree of polymerization (up to 8000), high crystallinity (of 70–80%), high water content to 99%, excellent biodegradability and biological affinity and high mechanical stability, which is quite different from the plant-based cellulose (Vitta and Thiruvengadam 2012; Hu et al. 2014). The unique micro/nanoporous three dimensional network of BC, which provides a natural methodology to control or restrict the size of the particles can facilitate the penetration of various particles made from different materials (polymers, metals and metal oxides and solid materials) into the interior, and thus, act as a template. The added particles can be anchored within the BC fiber network to create novel types of nanomaterials which represents a very specific and important modification method of BC (Vitta and Thiruvengadam 2012; Hu et al. 2014). These amazing and unique physico-mechanical properties of BC have inspired attempts to use it in a number of commercial products such as in tires, headphone membranes, high performance speaker diagrams, high-grade paper, make-up pads, diet-food, and textiles (Evans et al. 2003; Shah et al. 2013).
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Intensive research and exploration in the past few decades on BC nanomaterials mainly focused on their biosynthetic process to achieve the low-cost preparation and to enhance BC production with static and agitation cultures using a variety of BC production strains, carbon sources, alternative inexpensive sugar sources and supplementary materials (Hu et al. 2014; Shah et al. 2013). In the last few years, growing worldwide activity (increasing annual publication activity and increasing efforts for the practical use of BC materials) have led to the emergence of more diverse potential applications exploiting the functionality of BC nanomaterials (Hu et al. 2014). The functionalization of BC with organic, inorganic or polymeric materials enables the creation of materials with improved or new properties by mixing multiple constituents and exploiting synergistic effects (Hu et al. 2014). BC has been used as a matrix to incorporate nanoparticles for the preparation of novel functional nanomaterials that gather together excellent properties of BC with the ones displayed by typical inorganic or organic nanomaterials like antibacterial, optical, electrical and magnetic properties as well as catalytic and biomedical activity (Vitta and Thiruvengadam 2012; Hu et al. 2014). Several recent reviews giving a summary and discussion regarding the synthetic approaches and applications of functionalized BC have been published (Vitta and Thiruvengadam 2012; Shah et al. 2013; Hu et al. 2014).
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There are two basic synthesis approaches for creating BC matrix composites, in situ and ex situ. For the in situ method, secondary components such as NPs can be introduced into the BC culture media at the beginning of BC synthesis process. Using BC as a template for the in situ synthesis of BC-NPs has the following advantages: the shape, structure and properties can be easily adjusted during the biosynthesis, pretreatment and chemical modification process, does not require severe conditions, simple and easy to implement which can obtain the nano-particles with narrow size distribution (Shah et al. 2013; Hu et al. 2014). For the ex situ method, 3 Page 4 of 17
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secondary components such as NPs can be introduced into the BC matrix by the solution impregnation method. Ex situ synthesis of functionalized BC nanomaterials has the following limitations: only submicron to nano sized particles can penetrate into the BC, creating difficulties in achieving homogenous distribution of particles in the BC, and hydrophobic materials cannot be used for BC composites (Shah et al. 2013). Prior literature shows that most of the functionalized BC nanomaterials made with NPs including graphene and montmorillonite (MMT) are formed through either in situ addition using a reducing agent and chemical modification or the ex situ synthesis approach. The BC cultivation methods, which are either static or agitated processes, are important to produce good quality and good quantities of BC (Chen et al. 2010). Moreover, a change of cultivation methods can influence the BC microstructure such as crystalline structure and mechanical properties (Yan et al. 2008). The particles in a static BC culture cannot be entrapped in the surface of BC fibers after being precipitated in the media because the particles only remain suspended in the BC synthetic media for a short time and a BC sheet is formed on the surface of the media at the media air interface (Cheng et al. 2009). The agitation culture provides a much better environment for added materials to become entrapped in the BC fibrils thanks to constant movement in the media which prevents the particles from settling and provides homogenous BC formation (Shah et al. 2013).
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In the present investigation, BC-nanoclay (NC), BC-xGnP and BC-CNF nanomaterials were synthesized through in situ synthesis without using surface modification or reducing agents with an aim to obtain new promising BC nanomaterials with improved thermo-physical properties. The in situ synthesis method was selected for this research because of easy processing and the ability to promote homogenous distribution of particles in the BC matrix. This study will provide initial insight into the use BC-based nanomaterials for extremely large applications ranging from conducting paper to a flexible, electrically conducting display. BC-MMT, BCxGnP and BC-CNF composites were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) thermogravimetric analyses (TGA) and field emission scanning electron microscopy (FESEM). To the best of our knowledge, studies involving the preparation of materials containing BC and CNF have not been reported previously.
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2. Materials and Methods 2.1. Nanomaterials The xGnP fillers were supplied by XG Sciences Inc., USA. The xGnP fillers in powder form were used as the reinforcement having particle diameters of 5 µm. Average platelet thickness ranged from about 5 to 15 nanometers. This translates into an average particle surface area ranging from about 60 to 150 m2/g. The bulk density of xGnP fillers is reported to be 0.180.25 g/cm3. The NC, Nanomer I.44P, was supplied by Nanocore and it has a quartenary ammonium chemistry based surface modification and an average particle size 15-20 micrometers. CNF, trade name Celish- Rokameijin, was supplied by Daicel Chemical Industries, Ltd., Japan. This product consisted of a 35 wt. % fiber content suspension. SEM image in Figure 1. showed that the agglomeration phenomena happened in the nanoscale CNF, xGnP and NC materials when it was exposed to air. However, some of the nanomaterials would stay at the nanometer size.
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Figure 1. SEM micrograph of a) CNF and FESEM micrographs b) xGnP and c) NC. 2.2. Microorganism and Cultivation of Acetobacter xylinus Acetobacter xylinus 23769 strain was purchased from the American Type Culture Collection (ATTC), Manassas, VA, USA. Four types of culture media were used: 1) the Hestrin– Schramm culture medium (HS) (2 w/v% glucose, 0.5 w/v% bacto peptone, 0.5 w/v% yeast extract, 0.05 w/v% MgSO4.7H20, 0.115 w/v% citric acid and 0.27 w/v% disodium hydrogen phosphate), 2) nanoclay–HS medium (in the presence of final 0.5 w/v% nanoclay in HS medium), 3) xGnP–HS medium (in the presence of final 0.5 w/v% xGnP in HS medium) and 4) cellulose nanofibrils (CNF)–HS medium (in the presence of final 0.5 w/v% cellulose nanofibrils in HS medium). These culture media were sterilized at 121 °C in an autoclave for 20 min. For seed culture, 50 ml HS medium was inoculated with 1 ml of freeze dried stock culture and incubated in a 250 ml Erlenmeyer flask at 28°C for 48h at pH 5.5 under static conditions. These four cultures were inoculated with 10 v/v% of seed culture and incubated in static cultivation at 28 °C for 3 days until a cellulose pellicle formed. After static cultivation, cultures were transferred to a rotary shaker operating at a rotational speed of 120 rpm, for 6 days at 28 °C to prevent the NPs from settling and providing homogenous BC formation with the added NPs.
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2.3.2. FTIR Attenuated Total Reflectance Fourier-Transform InfraRed (ATR-FTIR) spectroscopy analysis was carried out on freeze-dried BC and BC composites samples on a Perkin Elmer Spectrum One FTIR spectrometer (Wellesley, MA, USA), equipped with a Universal ATR accessory, using 200 scans and a resolution of 4 cm−1, over the range 4000–400 cm−1. Before the analysis, samples were dried at 90 °C under vacuum for 24 h.
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2.3.3. XRD XRD was performed with a high resolution X-ray diffractometer (Model X’Pert PRO, Philips PANalytical, Netherlands) with a Ni-filtered Cu Kα (1.540562Å) radiation source operated at voltage 45 kV and 40 mA electric current. The samples were scanned from 4°-40° 2θ range with a step of 0.02 °, a step time of 2.5 s. Freeze-dried BC and BC composites samples were pressed manually using a Dake Press (Dake, Grand Falls, MI) into cylindrical wafers 2 cm in diameter and 0.05-0.1 cm thick. A silicon zero background plate was used to make sure that there were not any peaks resulting from the sample holder. The same sample holder position and sample holder (holder and silicon zero background plate) were used for both samples. Crystallinity index (C.I.) and percentage crystallinity (% Crystalline) of BC samples were calculated as follows (Rosli et al. 2013)
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2.3. Characterization of the Composites 2.3.1. Morphology-FESEM The freeze-dried BC and BC composites samples were coated with gold (E-1045, Hitachi, Tokyo, Japan). Analysis of the structure of the BC was performed by using a FieldEmission Scanning Electron Microscope (FESEM) (NVision 40, ZEIS) at 25 kV. Images were taken with 2000 X FESEM micrograph magnifications.
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where I200 is the maximum intensity of diffraction of the (200) lattice peak (2θ of 22º to 23º) and Iam is that of the amorphous material between 2θ of 18º to 19º where the intensity is minimum. 2.3.4. Thermal Stability-TGA TGA measurements were carried out using a Mettler Toledo analyzer on samples of about 8 mg. Each sample was scanned over a temperature range from ambient temperature to 600 °C at a heating rate of 10°C/min under nitrogen with a flow rate 20 ml/min to avoid sample oxidation. The samples used for the TGA measurement were randomly picked 5 individual samples from ground samples.
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3. Results and Discussion 3.1. Morphology The morphological properties of the BC composites are related to the materials’ microstructures, the state of nanoparticle dispersion, penetration and orientation in the BC matrix, as well as the interactions between nanoparticles and the BC matrix. Hence, the morphological properties should be characterized to confirm the synthesis of BC composites by assessing the attachment of nanoparticles onto the surface and their penetration into the matrix of the BC sheets and to optimize the synthesis conditions for achieving high performance BC composites (Ul-Islam et al. 2012b). The FESEM micrographs of a freeze-dried BC pellicle prepared from the reference medium and composites are shown in Figure 2.
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Figure 2. FESEM micrographs of a) BC, b) BC-CNF, c) BC-xGnP and d) BC-NC nanomaterials. The neat BC sample exhibited a highly fibrous three dimensional network-like structure consisting of ultrafine cellulose microfibrils and FESEM shows a random arrangement of ribbonshaped microfibrils without any preferential orientation which results in the formation of pores with different sizes on the surface and throughout the entire BC matrix. Small-sized nanoparticles can penetrate inside the BC matrix using these pores (Feng et al. 2012; Ul-Islam et al. 2012b). The typical lateral dimensions of the BC fibrils were in the range of 15–70 nm. Flakeshaped xGnP and NC and rod-shaped CNFs were observed to effectively be incorporated into the surface layer of the BC matrix. Figure 2b shows that the rod-shaped CNFs penetrated into the 7 Page 8 of 17
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BC matrix and some parts of the longer CNFs spreading on the BC surface or entangling with other CNFs or BC nanofibrils. On the other hand, the flake-shape xGnP and NC (Figure 2c and 2d), appeared to be woven within the surface layer of the weblike BC, and were indeed fixed or trapped by adjacent nanofibrils in the BC surface network (Zhou et al. 2013). The particles that penetrated into the BC matrix filled the empty space in entire BC matrix and the xGnP and NC showed larger surface coverage in the surface layer of the BC matrix because of their nearly fully extended 2-D morphology (Ul-Islam et al. 2012b; Zhou et al. 2013). The presence of hydroxyl groups in both CNF and BC can lead to strong hydrogen bonding interactions. On the other hand, NC may have weak hydrogen bonding interactions with BC because of inorganic-organic hydroxyl groups (NC hydroxyls cannot create hydrogen bond cooperatively with those of cellulose (they do not have the ca. 10 Å periodicity as for cellulose along the chain)) (Ul-Islam et al. 2012b). However the FT-IR spectra of pure xGnP incorporated BC matrix showed no significant shift in the peaks, suggesting that no interaction between xGnP and BC. This finding is in good agreement with Gopiraman’s et al. study (Gopiraman et al. 2013). Interactions of the nanoparticles with the BC matrix and nanoparticle attachment to the BC surface and penetration into the BC matrix can potentially enhance the physico-mechanical properties of the BC (UlIslam et al. 2012b, Shah et al. 2013; Hu et al. 2014).
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3.2. X-ray Diffraction The X-ray diffraction patterns of the BC and BC nanomaterials are shown in Figure 3. In the case of pure BC, four characteristic peaks centered at 14.5 º, 16.8º, 22.7º and 34.5º, indicating the typical cellulose I allomorph structure (Oh et al. 2005). BC-CNF composites showed a similar XRD pattern to the neat BC. The only difference between BC and BC-CNF nanomaterials was intensity changes in the peaks. BC-xGnP composites exhibited a large and sharp 2θ peak at 26.5º as well as BC characteristic peaks. In addition to the BC characteristic peaks, various diffraction peaks at 2θ angles of ∼7º, 20º, 26.6º and 27.7º were also observed in the BC-NC composites. As a result, it appeared that the xGnP and NC did not cause any significant change in the BC structure. The XRD patterns and FESEM images clearly indicate that the NPs were successfully incorporated into the nanofibrous BC matrix. In addition, the crystallinity index (CI) and percentage crystallinity calculated based on X-ray profiles are listed in Table 1. The proposed method (Segal Method or XRD peak height method) to calculate percentage crystallinity is convenient for empirical measurement to allow rapid comparison of cellulose samples (Park et al. 2010). Table 1 revealed a decrease in CI (0.75) and percentage crystallinity (80%) for BC-NC nanomaterials and an increased CI (0.86) and percentage crystallinity (88%) for BC-xGnP nanomaterials. On the other hand, BC and BC-CNF nanomaterials have the same CI (0.81) and percentage crystallinity (84). It is already known that the penetration and interaction of NC particles slightly interrupts the native hydrogen bonding interactions in BC resulting in lower crystallinity for BC-NC nanomaterials (Ul-Islam et al., 2013).
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3.3. FT-IR Figure 4. shows the FT-IR spectra of BC, CNF, xGnP, NC and BC nanomaterials. The FT-IR spectrum of BC in this research was similar to CNF. Table 2. summarizes FT-IR results for BC, NC and BC-NC nanomaterials. BC-CNF and BC-xGnP composites exhibited a similar spectra to BC and no differences could be resolved between the 2 cellulose samples. The xGnP, which is mostly composed of carbon atoms and a tiny amount of residual unreacted intercalant may also be present, but did not show any characteristic absorption peaks because it has no specific functional groups on its surface. As was mentioned above, the BC-xGnP matrix showed no significant shift in the peaks, suggesting that no interaction between xGnP and BC. In the BC FT-IR spectra, the bands at 3345 and 2922 cm-1 are attributed to the presence of OH-stretching and CH2 stretching, respectively. The bands at 1653 and 1372 cm-1 indicated H-O-H bending of adsorbed water and CH2 bending or O-H in plane bending and the band at 1035 to 1162 cm-1 9 Page 10 of 17
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could be associated with C-O symmetric stretching of primary alcohol and C-O-C antisymmetric bridge stretching, respectively (Ashori et al. 2012; Barsberg 2010; Marechal and Chanzy 2000).
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The most prominent bands in the IR spectrum of NC can be found at 3615 cm-1 which is attributed to the presence OH stretching. The absorption peak in the region of 1035 cm-1 is attributed Si-O stretching (in-plane) vibration for NC. The characteristic peaks at 618 and 918 cm-1 are attributed to Si-O bending and Al-Al-OH bending, respectively (Patel et al. 2006). The FT-IR spectrum of BC-NC nanomaterials exhibited the presence of characteristic peaks both from BC and NC but there are minor differences between the BC and BC-NC FT-IR results that may be related to the chemical interactions between the BC and NC. The important changes between the BC and BC-NC nanomaterials can be assigned to: 3300-3700 cm-1(OH-stretching); 2920 cm-1 (C-H stretching vibration), 920 cm-1 (Al-Al-OH bending) and 620 cm-1 (Si-O bending). This result is in good agreement with the work previously reported by Ul-islam et al. 2012b. In the FT-IR spectra of BC, the band at 3345 cm-1 was attributed to the intramolecular hydrogen bond which is important for elucidating hydrogen-bonding patterns because, in favorable cases, each distinct hydroxyl group gives a single stretching band at a frequency that decreases with increasing strength of hydrogen bonding (Poyrazoglu Coban and Biyik 2011; Sturcova et al. 2004). Although the OH peak of NC was present at 3615 cm−1, BC-NC showed a characteristic OH peak at 3347 cm−1 because of the merging of peaks for the hydrogen bonding of both BC and NC (Ul-islam et al. 2012b). The peak intensity of 2920 cm-1 (C-H stretching vibration), 920 cm-1 (Al-Al-OH bending) and 620 cm-1 (Si-O bending) deceased in BC-NC nanomaterials. It is believe that the sign of electrostatic interaction between BC and NC.
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Table 2. FT-IR results for BC, NC and BC-NC nanomaterials. Assignment Wavenumber (cm-1) BC and CNF NC O-H Bending 400-700 b - glucosidic linkages between the glucose units ∼896 C-O symmetric stretching of primary alcohol 1035 C-O-C antisymmetric bridge stretching 1162 C-H deformation 1316 CH2 bending or O-H in plane bending 1372 H-O-H bending of adsorbed water 1653 CH2 2852 CH2 stretching 2922 OH-stretching 3345 Si-O bending 618 Al-Al-OH bending 918 Si-O stretching (in-plane) 1035 CH2 bending 1467 C-H stretching 2923 OH stretching or Al-Al-OH stretching ∼ 3615 Si-O bending Al-Al-OH bending Si-O stretching (in-plane) C-O-C antisymmetric bridge stretching CH2 stretching OH-stretching
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619 918 1035 1162 2918 3347
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3.4. Thermal Stability Figure 5 shows the TGA and derivative TGA of the BC and CNF in an inert TGA atmosphere. Three stages of mass loss are associated with the TGA curve. A first stage, up to 200°C, corresponds to less than 10% of loss for BC and CNF samples and is followed by second stage with more than 80 and 85 wt. % of loss for BC and CNF respectively, up to about 500°C. The final third stage extends to the usual ending test temperature at 600 °C in association with more than 81 and 86 wt. % of loss for BC and CNF, respectively. It is very well know that the first initial loss, below 200°C is usually associated with a relatively small DTG peak assigned to the release of water (moisture, humidity) or other volatiles from the BC. The peaks in the DTG graphs in the range of 200–500°C correspond to volatilization stages (El-Saied et al., 2008; Peng et al., 2013). BC has slightly higher thermal stability compared to CNF because of its high purity (absence of lignin and hemicellulose). In general, the DTG scan of pristine clay displays four distinct regions. These are the elimination of the free and physisorbed water in the internal layer of the clay and the dis-aggregation or de-crystallization melting of the long chain tails of the surfactant molecules occurring at temperatures up to 200°C, surfactant decomposition occurred between 200 and 500°C, the dehydroxylation of the clay occurred between 500 and 700°C and decomposition of organic carbonaceous residue occurred after 700 to 1000°C (Santos et al. 2009; Xie et al., 2001; Xie et al,. 2002). The most important regions influencing the thermal behavior of the clay-filled nanomaterials are region 1 and region 2 where the release of small molecules associated with fabrication and storage of the clay-filled nanomaterials (Xie et al., 2002). A first stage, up to 200°C, corresponds to less than 1% of loss for NC sample and is followed by second stage with more than 35 wt. % of loss for NC, up to about 500°C. The xGnP has higher thermal stability compared to other nanofillers and did not show any significant loss (less than 3.5 wt. %) at 600°C. All the BC composites exhibit a first mass loss step associated with water release (dehydration process occurring up to about 200°C). The second weight loss step shows a small shift towards higher temperatures when xGnP and NC are introduced into the BC matrix, beginning at 225 °C for BC and CNF and at 265 °C for the BC-xGnP and BC-NC composites, according to DTG data. Figure 5. shows that CNF incorporation into the BC matrix did not significantly change the thermal stability and residual mass of the BC matrix. On the other hand, the thermal stability and residual mass of BC-xGnP nanomaterials is significantly increased, which is because of the high thermal stability of xGnP and the shielding effect of xGnP on the diffusion of combustion gases into and out of the BC matrix during its thermal decomposition (Yang et al., 2007). We also estimated the amount of xGnPs incorporated in the bacterial cellulose membrane using thermogravimetric analysis (TGA) based on ash content determination at 600°C. The integration of xGnP content for 0.5 % wt was found 38% in BC-xGnP nanomaterials. This result may also explain the reason of higher thermal stability. As expected, the addition of NC in the BC matrix increased of thermal stability and residual mass of BC-NC nanomaterials because of the homogeneous incorporation of clay sheets, to a barrier of these high-aspect ratio fillers, and char formation (Ray and Bousmina 2005). The establishment of hydrogen bonds between the organic BC matrix and inorganic NC particles can also cause difficulties in the release of water molecules produced by the condensation of hydroxyl groups of cellulose and may also lead to an improvement in the thermal stability of the BC matrix (Perotti et al., 2011; Ul-Islam et al., 2012b).
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4. Conclusions The added NPs appeared to be dispersed uniformly in the BC matrix. The BC ribbons interwound with the CNF and formed a three-dimensional network. The flake-shaped xGnP and NC were indeed fixed or trapped by adjacent nanofibrils in the BC surface network. FT-IR and XRD spectra showed some differences between the neat BC and BC nanomaterials which are related to the physical and chemical interactions between the BC and NPs. TGA data showed a shift to higher temperature of the beginning of decomposition process of the BC-NC and BCxGnP nanomaterials in comparison to the pristine BC. The thermal stability of BC-CNF did not change compared to pure BC. The incorporation of NPs into BC provides new opportunities for the development of novel nanostructured materials with improved properties.
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5. Acknowledgements The republic of Turkey, The Scientific and Technological Research Council of Turkey (TUBITAK) is greatly acknowledged for support of the scholarship of the researcher Esra Erbas Kiziltas to do this study at the University of Maine. The authors would like to acknowledge the contributions of Justin Crouse and Chris West whose hard work made this paper possible. The authors would also like to thank U.S. Army Corps of Engineers, Engineer Research and Development Center project 912HZ-07-2-0013 and Maine Agricultural and Forest Experiment Station (MAFES) project ME09615-08MS and the Wood Utilization Research Hatch 2007-2008 project.
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