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Natural Biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Nanocomposites with Multi-functional Cellulose Nanocrystals/ Graphene Oxide Hybrids for High-performance Food Packaging Fang Li, Hou-Yong Yu, Yan-Yan Wang, Ying Zhou, Heng Zhang, Juming Yao, Somia Yassin Hussain Abdalkarim, and Kam (Michael) Chiu Tam J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03110 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on August 3, 2019

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Journal of Agricultural and Food Chemistry

Natural Biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Nanocomposites with

Multi-functional

Cellulose

Nanocrystals/Graphene

Oxide

Hybrids

for

High-performance Food Packaging Fang Li,† Hou-Yong Yu, †*,‡ Yan-Yan Wang,† Ying Zhou†, Heng Zhang,† Ju-Ming Yao†, Somia Yassin Hussain Abdalkarim,†*Kam Chiu Tam‡

†

The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of

Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Xiasha Higher Education Park Avenue 2 No. 928, Hangzhou 310018, China. ‡

Department of Chemical Engineering, Waterloo Institute for Nanotechnology, University of

Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

____________________________

E–mail addresses: [email protected]; [email protected] *Corresponding authors. Tel: +86-571-86843618; Fax: +86-571-86843619

1 ACS Paragon Plus Environment


ABSTRACT: High-performance and useful graphene oxide (GO) and cellulose nanocrystals (CNC) are easily extracted from natural graphite and cellulose raw materials, and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is produced by bacterial fermentation from natural plant corn stalks, etc. In this study, novel ternary nanocomposites consisting of PHBV/cellulose nanocrystal-graphene oxide nanohybrids were prepared via a simple solution casting method. The synergistic effect of CNC/GO nanohybrids obtained by chemical grafting (covalent bonds) and physical blending (non-covalent bonds) on the physicochemical properties of PHBV nanocomposites were evaluated and their results compared with a single component nanofiller (CNC or GO) in binary nanocomposites. More interestingly, ternary nanocomposites displayed the highest thermal stability and mechanical properties. Compared to neat PHBV, the tensile strength and elongation to break increased by 170.2 and 52.1%, respectively, maximum degradation temperature (Tmax) increment by 26.3 oC were observed for the ternary nanocomposite with 1 wt % covalent bonded CNC-GO. Compared to neat PHBV, binary, and 1:0.5 wt % non-covalent CNC/GO based nanocomposites, the ternary nanocomposites with 1 wt % covalent bonded CNC-GO exhibited excellent barrier properties, good antibacterial activity (antibacterial ratio of 100.0%), reduced barrier properties and lower migration level for both food simulants. Such a synergistic effect yielded high-performance ternary nanocomposites with a great potential for bioactive food packaging materials.

KEYWORDS: Cellulose nanocrystals, Graphene oxide, Hybrids, Nanocomposites, Covalent bonds 2 ACS Paragon Plus Environment

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

INTRODUCTION As environmental pollution from traditional packaging materials is becoming a major

problem, the use of biodegradable nanocomposites with comparable properties in food packaging could replace petroleum-based plastic packaging materials.1 Biodegradable polymers, such as polyhydroxyalkanoates (PHAs) and polylactic acid (PLA), have received increasing attention as food packaging materials, tissue engineering, and biomedical fields due to good biocompatibility and enhanced properties.2,3 Poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as main members in PHAs family are used to replace petroleum-based traditional packaging plastic due to their biodegradability, non-toxicity,

friendly

environmental

effect

and

biocompatibility.4,5

Compared

to

high-crystallinity PHB with high melting temperature and brittleness, semi-crystalline PHBV produced by adding a few (3HV) units into PHB show larger toughness, lower melting temperature and crystallinity, thus PHBV is more suitable for the industrial packaging application.5,6,7 These advantages make PHBV bioplastics an ideal choice as nanocomposite packaging materials for food products, especially the global bioplastics market with economical values of US$43.8 billion in 2020 at a compound annual growth rate of 28.8%.1,7,8 However, PHBV polymer has some disadvantages, such as its brittleness, narrow processing window, poor thermal and mechanical properties, no antibacterial activity and low resistance to water vapor permeability. 6,9,10,11 These limitations can affect most of packaging and biomedical applications. Besides, PHBV has its high price depending on the HV amount,5,6 the used PHBV in this study contained a lower percentage of HV (2.57%) to reduce cost of the resulting nanocomposites. Also, some naturally renewable nanofillers are



good choices to be introduced into PHBV matrix for reducing their costs of the nanocomposite packaging materials. Recently, a wide range of enhancement strategies has been reported to improve physical and biological properties of PHBV matrix.6 One of this enhancement approaches consists of adding organic or inorganic nanofillers as reinforcing agents, especially cellulose nanocrystals (CNCs), graphene oxide (GO), zinc oxide (ZnO) and so on.6,10,11-17 Generally, CNC and GO are representative one-dimensional (1D) organic nanofillers and two-dimensional (2D) inorganic nanofillers, respectively, which could be used to enhance the property of PHBV matrix. Well-dispersed CNCs with high rigidity can improve the mechanical strength and thermal stability of PHBV polymers due to hydrogen bonds between CNC surface groups and PHBV,12 however the toughness and new functional properties (e.g. antibacterial activity) could not be introduced by the addition of pristine CNCs.11 Flexible GO with 2D nanostructure and excellent thermal conductivity18 could enhance the mechanical properties and crystallization temperature of PHBV nanocomposites.19,20,21 However, GO with few surface groups show poor compatibility with PHBV matrix, so surface modification of GO were used to improve dispersion and compatibility of nanofillers within PHBV matrix.19,21,22 Larsson et al. reported that alkylamine-modified GO improved the dispersion/compatibility of GO within PHBV matrix, and the crystallization temperature and elongation at break were obviously enhanced, due to more efficient nucleation effect of hydrophobically modified GO nanoparticles with alkyl chain length and increasing lengths of the alkyl chain modified on GO nanoparticles, respectively.21 However, compared to PHBV, the degradation temperature of the nanocomposite with 5 wt % GO content was reduced by 4 oC, due to the reduction in 4 ACS Paragon Plus Environment

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hydrogen bonds between GO and PHBV, as the surface groups of GO were replaced by butyl-, octyl- and hexadecylamine molecules.19,21 Therefore, hybridization of GO with other functional nanofillers could be another effective way to their synergistic effect on the polymer properties. Recently, hydroxyapatite nanowhiskers and CNCs were combined with GO to prepare nanohybrids to add new functionals properties (e.g. antifouling, osteoblastic performances) for tissue regeneration and antifouling membrane application.22-25 However, the physical blending CNC with GO in the polymer matrix may not provide sufficient binding between nanohybrids and polymer, resulting in a weak synergistic reinforcement effect on polymer properties.13,25 To our best knowledge, no study on covalent bonding of CNC-GO or non-covalent bonding of CNC/GO reinforced PHBV nanocomposites has been reported. Thus, the main objective of the CNC-GO nanohybrids prepared via chemical grafting is to investigate their synergistic effect in PHBV matrix and compare them with non-covalent bonded CNC/GO, single GO or CNC nanofillers. Since the abundant oxygen-containing groups on CNCs can be used as support materials to modify the GO surfaces, forming more hydrogen bonds between nanohybrid and PHBV matrix to yield nanocomposites. Also, 2D layered GO may give rod-like CNC more interfacial barrier area in the nanohybrids to endow robust barrier and migration properties of biopolymer matrix. As a result, synergistic effect of CNC-GO with covalent bonds could enhance the crystallization and interfacial interaction of PHBV, producing robust nanocomposites with excellent performances for bioactive food packaging.



EXPERIMENTAL SECTION



Materials. Commercial PHBV (number-average molecular weight=5.90 x 104 Da, hydroxyvalerate (HV) = 2.57 mol %) was purchased from Tianan Biological Material Co., Ltd. (Ningbo, China). Graphene oxide (GO), chloroform, anhydrous dimethylacetamide, and hexamethylene-1,6 diisocyanate were purchased from Hangzhou Mick Chemical Instrument Co., Ltd. (Hangzhou, China). The carboxylated CNCs with rod-like shape and length of 100-200nm (Figure S1, Supporting Information) were prepared by the hydrolysis of MCC using citric acid (C6H8O7)/hydrochloric acid mixtures.26 All reagents and solvents were used as received or purified with standard procedures. Preparation of graphene oxide (GO) grafted CNC. The detailed grafting reaction (Scheme 1) is described as follows: GO (0.2 g) was dispersed in 200 mL of anhydrous dimethylacetamide, and hexamethylene-1,6 diisocyanate (2 g) was added to the GO dispersion at 110 oC and reacted for 3 h, which was followed by the addition of a CNC suspension. After reacting for another 3 h, the solution was poured onto glass plate, solidified with deionized water for 3 h, washed and vacuum dried at 60 oC, and designated as CNC-GO nanohybrids.


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Scheme1. Schematic diagram of graphene oxide (GO) grafted CNC (CNC-GO) Preparation of PHBV nanocomposites. The nanocomposites were prepared by solution casting. A known amount of PHBV was weighed and dissolved in chloroform (CHCl3) at room temperature, and it was mixed and kept in a water bath at 70 oC for 30 min. Different masses of CNC and GO were separately dispersed in chloroform, ultrasonicated at room temperature for 30 min, and details of composition are shown in (Table S1). The mass ratio of the CNC, GO, CNC/GO or CNC-GO and PHBV to chloroform was 1:9. A certain amount of CNC, GO, CNC/GO and CNC-GO suspensions were added to the PHBV solution, and the mixture was sonicated under ice water bath for 30 minutes, which was then onto a glass slide. 7 ACS Paragon Plus Environment


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Various PHBV nanocomposite systems (thickness of nanocomposites about 20-40 μm) designated as PHBV, 1 wt % CNC, 2 wt % CNC, 3 wt % CNC, 0.5 wt % GO, 0.6 wt % GO, 0.7 wt % GO, 1:0.5 wt % CNC/GO, 1 wt % CNC-GO were prepared. It should be noted that covalent CNC-GO nanohybrids showed intact morphological structure of layered GO coated nanorods, while the non-covalent 1:0.5 wt % CNC/GO exhibited relatively large size with some free CNCs (Figure S1, Supporting Information).



CHARACTERIZATION The cross-sectional morphology of PHBV nanocomposites was observed by field

emission scanning electron microscopy (FE-SEM, S4800 Hitachi), operating voltage at 3.0 kV. The system was frozen and chopped in liquid nitrogen to obtain a complete cross section. The optical properties of the PHBV nanocomposites were measured by a UV-vis spectrophotometer (U-3900 Hitachi) with a measurement wavelength range of 200-900 nm. Infrared spectra were recorded at room temperature on a FTIR spectrometer (Nlcolet iS50, Thermo Electron Corp, USA). A small amount of the dried PHBV nanocomposite system was taken and mixed with potassium bromide in a mass ratio of 1:100 to form a sheet. FT-IR spectroscopy test conditions: room temperature, 2 cm-1 resolution, 4000 to 400 cm-1 range of 32 scans. To investigate the changes in the hydrogen bonding interaction of nanocomposites, the FH−CO value is calculated by the following formula.17,23

𝐹

=

/ /

/

(1)

/

Where Aa and AH are the free peak area and the hydrogen bond component, respectively, and rH/a is the characteristic adsorption ratio of the above two bands. Depending on the strength of 8 ACS Paragon Plus Environment

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the hydrogen bond and for a semi-quantitative comparison, rH/a is in the range of 1.2 to 1.75. All samples were held at room temperature for 2 weeks prior to testing to achieve equilibrium crystallization. The X-ray diffraction test (XRD, ARL X’TRA, Thermo Electron Corp) was carried out by using Cu Ka (1.5418 Å) rays (40 kV, 40 mA) in a step size of 3 min-1 and room temperature. The samples (5-10 mg) were weighed and raised from room temperature to 600 oC at a heating rate of 20 oC min-1 in a N2 (30 mL min-1) atmosphere. The initial degradation temperature (T0) and the maximum degradation temperature (Tmax) can be obtained from the TGA curves. The thermogravimetric analyzer coupled with Fourier transform infrared spectrometer (TG/FT-IR) was first heated to 200 oC and stabilized for 4 hours before TG operation. Under a nitrogen atmosphere, the flow rate was 30 mLmin-1, and thermal degradation was performed at a scan rate of 20 oC min-1 in the range of 30-600 oC, and the obtained series of FT-IR spectra were reordered according to the corresponding time/temperature. The non-isothermal crystallization and melting behavior were investigated (DSC Q20 differential scanning calorimeter). We weigh the sample (5-10 mg) into the sample cell. The test conditions were as follows: the sample was heated to 200 oC at a heating rate of 20 oC min-1 and kept at this temperature for 5 min, then cooled to -20 oC at a rate of 10 oC min-1. Finally, the temperature is raised to 200 oC at a rate of 10 oC min-1. The melt crystallization temperature (Tm) and the cold crystallization temperature (Tcc) can be obtained from DSC curves. To further investigate the change in relative crystallinity (Xc) of nanocomposites, the Xc value was calculated by the following formula.12,13 9 ACS Paragon Plus Environment


X =(

∆ ∅

)∆

× 100%

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(2)

Where XC is the degree of crystallinity and ØCNC+GO is the weight fraction of the nanofillers (CNC+GO). ΔHm is the measured melting enthalpy, and ΔH100 is 146.6 J g-1 refers to the melting enthalpy of PHBV at 100% crystallization.23 The sample was dried overnight in a vacuum oven before testing for Polarized light microscope (POM, WT4000). A complete sample was placed on the sample stage, raised from room temperature to 200 oC at a rate of 30 oC min-1 for 3 min, then cooled to room temperature at a rate of 40 oC min-1. The spherulite growths of the PHBV nanocomposites were observed at room temperature. The tensile properties of the PHBV nanocomposites were tested using an Instron 5943 universal tester. The sample with dumbbell shapes (length of 50 mm, width of 10 mm, thickness about 20-40 μm), was tested at ambient temperature (25 oC) and tensile rate of 1 mm min-1, and then repeated test for each sample eight times to get average value. The water absorption was determined by previously reported methods.24 The sample was a rectangular strip with an area of 10 cm2 and was dried in a desiccator at 0% relative humidity for one week until constant weight. The system was then placed in a beaker at 100% relative humidity and allowed to absorb water until constant weight. The water absorption rate is calculated as follows:

Water Uptake =

× 100%

(3)

Where w0 and wt are the weights of the sample before and after water absorption, respectively, take the average of 5 replicates. The water vapor transmission rate (WVP, kg m m- 2 s-1 Pa-1) was determined according to 10 ACS Paragon Plus Environment

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the previously reported method.24 Briefly, a 10 cm2 sample was packaged in a 10 mL vial and placed in an autoclave at 120 oC for 10 min at 110 kPa. When the temperature was cooled to room temperature, the penetration of water vapor through the systems was almost completed, and the condensed water in the bottle was weighed. Finally, WVP can be calculated as follows:

WVP =

∆ × ×

×𝑒

(4)

Where Δm is the mass change (kg) of the sample at time t(s), S is the test area (m2), e is the systems thickness (m), and P is the saturation pressure (Pa). According to the Regulation EU no.10/20111,6,24, overall migration tests were performed in two liquid food simulants (isooctane and 10% (v/v) ethanol). About 10 cm2 sample was impregnated into a glass tube containing 10 mL of food simulant. The glass tube was then completely sealed to avoid loss of simulated fluid. The sample in 10% ethanol was kept in an oven at 40 oC for 10 days (Commission Regulation EU 10/2011),1,6,24 and while the sample in isooctane was kept at 20 oC for 2 days (European Standard EN 1186-1:2002).1,6,24 At the end of the test period, the sample was removed, the simulated solution was evaporated and the residue was weighed by using an analytical balance. Each sample was tested in triplicate and averaged. 100 μL of E. coli and S. aureus solution were applied to a solid medium prepared by agar or the like, and the sample was placed on a medium coated with a bacterial liquid. Then, it was placed in a 37 oC incubator for 12 h. The antibacterial properties of the samples were evaluated by measuring the size of the inhibition zone, and 6 parallel samples were tested to obtain accurate results. 11 ACS Paragon Plus Environment


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The antibacterial rate was the average unit colony (CFU). After the sample was placed in a liquid medium and incubated in a constant temperature incubator at 37 oC, the obtained columnar gels on the culture dishes coated with approximately 1×106 CFU/mL for bacteria and 5×104 CFU/mL for yeast at 37 oC for 24 h to count the amount of viable microorganism colonies. The calculation formula of antibacterial rate (AR) is as follows:17

AR (%) =

(5)

Among them, N0 is the average number of colonies on neat PHBV, N is the average number of colonies on the nanocomposites, and the experiment is repeated 6 times.



RESULTS AND DISCUSSION Figure 1a-d shows the morphologies of PHBV nanocomposites containing various CNC

contents (1-3 wt %), and a smooth fractured morphology of PHBV matrix was observed. More and more white dots with good dispersion (Red circle show cellulose nanocrystals) were found for the PHBV/CNC nanocomposites, this indicates that the CNC had good dispersibility and compatibility in the PHBV matrix (Figure1b-d). However, the binary nanocomposites with low GO content (0.5-0.7 wt %) revealed a homogeneously dispersed GO within the PHBV matrix as shown in Figure 1e-i. By using less than 1 wt % GO the surface topography of GO was significantly modified, leading to the creation of a wavy surface within the PHBV/GO binary nanocomposite (Figure 1e-g). Complete exfoliation of GO nanosheets occurred due to the strong interfacial bonding,27 and good interface adhesion between GO and PHBV (hydrogen bond interaction),20 exceeding that of only PHBV/CNC nanocomposites. The fractured morphologies of PHBV ternary nanocomposites are shown in


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Figure 1h-i, the PHBV nanocomposite with CNC/GO (non-covalent bond) showed similarly rough and pore structure as the nanocomposites with 0.7 wt % GO, while the PHBV nanocomposite with CNC-GO (covalent bond) gave the relatively smooth cross-sectional morphology where PHBV seem likely embedded into the network structure of CNC-GO nanofillers. Thus, in this work, we elucidated that CNC-GO nanofillers with covalent amide linkages between GO layers and CNC were easily to promote the better dispersion of only GO nanofiller into PHBV polymer matrices 4,8 to form homogenously interconnected network between various components, compared to CNC/GO nanofillers.



Figure 1. FE-SEM images of (a) PHBV, (b) 1 wt % CNC, (c) 2 wt % CNC, (d) 3 wt % CNC, (e) 0.5 wt % GO, (f) 0.6 wt % GO, (g) 0.7 wt % GO, (h) 1:0.5 wt % CNC/GO, (i) 1 wt % CNC-GO, (j) UV-visible spectra of PHBV nanocomposites

Figure 1j shows the optical properties of PHBV binary and ternary nanocomposites, and neat PHBV possessed a transmittance of 80% at 800 nm. The transmittance decreased to 58% 14 ACS Paragon Plus Environment

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for PHBV binary nanocomposites containing 3 wt % CNC. For the PHBV binary nanocomposites with 0.7 wt % GO, the transmittance reduced to 37% due to the electronic structure of graphene oxide, that absorb more ultraviolet rays.4 In addition, the transmittance of ternary nanocomposite containing 1:0.5 wt % CNC/GO (non-covalent bond) was reduced to 34 %. In contrast, the transparency of ternary nanocomposite containing 1 wt % CNC-GO (covalent bond) decreased to 22%, confirming the good UV-shielding properties. This makes them attractive for use as flexible UV-barrier packaging materials.1,10,17,28 By comparing the binary and ternary PHBV nanocomposites, the light transmission of the ternary nanocomposites containing CNC and GO was lower than the binary nanocomposites with the only CNC or GO. Since CNCs and GO possessed absorption properties for ultraviolet light, particularly GO incorporation into PHBV nanocomposites enhanced their ultraviolet shielding characteristics.27 The FT-IR spectra of neat PHBV and its binary or ternary nanocomposites are shown in Figure 2a. The two absorption peaks at 2900 and 1636 cm-1 correspond to the C-H stretching and O-H bending vibration peaks of CNC. Additionally, the peak at 3436 cm-1 is associated to the free and hydrogen bonded O-H stretching vibration.29 When the concentration of CNC was increased, the peak height of the C-O-C and C-C stretching vibration peaks at 1054 and 975 cm-1 increased gradually due to the increase in the crystalline component of the nanocomposites.17 The appearance of carbonyl band in the obtained composite system was further studied in the range from 1800 and 1680 cm−1 as shown in Figure 2b and curve-fitted by Gauss/Lorentz spectral function to estimate the hydrogen bond fraction (FH−CO) using the similar reported equation of the composites elsewhere.17,23,24 As the content of CNC or GO 15 ACS Paragon Plus Environment


increased, the position of the hydrogen bonded components of the PHBV binary or ternary nanocomposites shifted from 1723 cm-1 to 1726 cm-1. This was caused by the hydrogen bond interaction between the nanofillers and PHBV that altered the polarity of the carbonyl group, resulting in an increase in the C=O band position.10 When the concentration of CNC was increased (1-3 wt %), the intensity of the hydrogen bonded O-H band increased, and the FH-CO of the PHBV binary nanocomposites increased from 0.12 to 0.15 (Table 1). This result indicates that more hydrogen bonds between residual hydroxyl groups of the CNC as proton donors and ester carbonyl groups of PHBV as proton acceptors were formed.24 Moreover, a FH–CO value of 0.14 was observed for the binary nanocomposite containing 0.5 wt % GO, which increased to 0.16 in the presence of 0.7 wt % GO. This trend was also evident when GO was added to the binary nanocomposites. For more details, peak deconvolution for the nanocomposites with 1 wt % CNC-GO in Figure 2c shows good fitness, suggesting good data credibility. In Figure 2b-c, the amorphous and free C=O segments were dominant in neat PHBV, and the addition of CNC-GO induced a reduction in the ratio of amorphous and free C=O segments in the nanocomposites. The nanofiller controls the amounts of C=O functionalities in the crystalline regions, where the FH-CO were increased to 0.21 for the nanocomposites with CNC/GO (non-covalent bond) and maximum value of 0.27 for the ternary nanocomposites with CNC-GO (covalent bond). XRD test was used to investigate the crystal structures of binary or ternary PHBV nanocomposites in Figure 2d. The XRD patterns of neat PHBV possessed multiple characteristics peaks at 2θ = 13.5°, 16.9°, 19.8°, and 25.5° assigning to (020), (110), (021), and (121) planes, respectively.30 In addition, GO displays a characteristic peak at 2θ = 10.0° 16 ACS Paragon Plus Environment

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due to the interlayer spacing between the GO sheets.22 Compared to neat PHBV, the intensities of (020) and (110) planes of the binary or ternary nanocomposites were significantly reduced with the addition of CNC, GO, CNC/GO, and CNC-GO. This indicated that the PHBV crystal growth of the binary or ternary PHBV nanocomposites along the (020) and (110) planes were inhibited due to interfacial interaction between the various components.24 As we known, it has been reported that CNCs are the most efficient nucleation agent to promote the crystallization rate, but not the crystallinity and crystal growth direction. Thus, we think that the CNC-GO like CNCs can influence the crystallization of PHBV in two opposite ways.23 On the one hand; the nanofillers can act as a nucleating agent to increase the nucleation and overall crystallization rate. On the other hand; owing to the strong interactions between the nanofillers and PHBV chains, the chain motion of the PHBV along some planes/directions is restricted.10,15,23 However, the crystals preferentially grew on other crystal planes, such as (021) crystal plane. In addition, the full width at half maximum of the diffraction peak of the (101) plane in the nanocomposites with the CNC was wider than the neat PHBV. It hints that smaller spherulites were formed, and this was also observed for the binary or ternary nanocomposites containing GO or CNC/GO and CNC-GO, thus the addition of CNC and GO or their hybrids would promote the crystallization of PHBV.



Figure 2. (a) FT-IR spectra; (b) carbonyl stretching region (VC=O) in the infrared spectra for the nanocomposites; (c) peak deconvolution for the nanocomposites with 1 wt % CNC-GO (covalent bond); (d) XRD spectra of PHBV nanocomposites.

The effects of CNCs and GOs or their hybrids on the thermal stability of binary or ternary PHBV nanocomposites are shown in Figure 3. The T0 and Tmax values were summarized in Table 1. All samples exhibited a single thermal degradation process, and the Tmax value of neat PHBV was about 245.8 oC. The thermal degradation temperature of binary PHBV nanocomposites was increased by the addition of CNC contents (1-3wt %). For example, the Tmax value was increased to a maximum of 271.8 oC for the nanocomposites with 3 wt % CNC. A similar effect was observed for the T0 values. This result could be due to 18 ACS Paragon Plus Environment

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that more hydrogen bonds between PHBV and CNC with increasing amount of CNCs, that suppressed the elimination mechanism of the six-membered ring in PHBV degradation process, and thus a higher thermal stability was achieved.31 In addition, the T0 and Tmax of binary PHBV nanocomposites with 0.6 wt % GO were 233.6 and 257.9 oC, respectively, which were significantly higher than neat PHBV, but lower than the binary PHBV nanocomposite with various amounts of CNCs. It indicated that the high heat-resistance of GO or other inorganic nanofillers can contribute to improve thermal stability of the nanocomposites.32 In contrast, the ternary PHBV nanocomposite with 1:0.5 wt % CNC/GO (non-covalent bond) yielded a lower T0 (234.4 oC) and Tmax (258.3 oC) than the binary PHBV/CNC nanocomposite, since the CNC surface groups enhanced the interactions with GO. The ternary PHBV nanocomposite containing 1 wt % CNC-GO (covalent bond) displayed the highest thermal degradation temperature (T0=243.7 oC and Tmax=272.1 oC), where more surface groups (hydroxyl and carboxyl groups) on the CNC-GO hybrids (see structure in Scheme 1) formed hydrogen bonds with PHBV matrix to improve the thermal stability of the nanocomposites.

Figure 3. (a) TGA and (b) DTG curves of PHBV nanocomposites



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Table 1. Hydrogen bond fractions (FH-CO), thermal degradation parameters, and antibacterial ratio of PHBV nanocomposites. Sample

aF H-CO

T0 (oC)b

Tmax (oC)b

Antibacterial ratio S. aureus (%)c E. coil (%)c

PHBV

-

229.4 ± 1.5

245.8 ± 1.9

0.0±0.1

0.0±0.1

1 wt % CNC

0.12

238.4 ± 1.7

267.5 ± 1.8

0.0±0.2

0.0±0.1

2 wt % CNC

0.13

241.7 ± 1.3

268.1 ± 2.1

-

-

3 wt % CNC

0.15

243.1 ± 1.9

271.8 ± 1.6

-

-

0.5 wt % GO

0.14

232.3 ± 1.8

257.5 ± 2.0

99.8±0.4

99.7±0.3

0.6 wt % GO

0.16

233.6 ± 1.9

257.9 ± 1.7

-

-

0.7 wt % GO

0.16

231.5 ± 2.1

254.6 ± 2.1

-

-

1:0.5 wt % CNC/GO

0.21

234.4 ± 1.9

258.3 ± 2.0

98.7±0.3

98.9±0.2

0.27

243.7 ± 1.8

272.1 ± 2.2

99.9±0.6

100.0±0.5

(non-covalent bond) 1 wt % CNC-GO (covalent bond) a

FH-CO was obtained from de-convoluted FT-IR spectra.

b

T0, and Tmax were obtained from TGA curves at the heating rate of 20 oC /min

c

Antibacterial ratios of S. aureus and E. coli were obtained from antibacterial activity test.

TG/FT-IR was used to investigate the thermal degradation mechanism of the binary and ternary PHBV nanocomposites, and the TG/FT-IR stack plots are shown in Figure 4. From Figure 4a-b, similar infrared bands were observed for neat PHBV, binary and ternary PHBV nanocomposites, indicating that the approximately same by-products were produced during the thermal decomposition of PHBV. For neat PHBV, the temperature was raised to 220 oC and a strong infrared absorption peak appeared, such as O-H vibrational peaks (969 and 3580 20 ACS Paragon Plus Environment

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cm-1), C=C stretching vibration peak (1660 cm-1), and C=O stretching vibration peaks (1760 and 1768 cm-1) for unsaturated ester and unsaturated carboxyl acid resulting from the PHBV degradation.29,30 When 1 wt % CNC was added to the binary PHBV nanocomposite, only a weak C=O stretching vibration peak appeared when the temperature was raised to 250 oC, and the total degradation temperature range were between 260.0-490.0 oC (Figure 4b). When 0.5 wt % GO was added to PHBV, the degradation temperature ranged from 240.0-490.0 oC (Figure 4c). However, in the presence of 1:0.5 wt % CNC/GO, the total degradation temperature range were about 240.0-500.0 oC (Figure 4d), whereas the nanocomposite with 1 wt % CNC-GO exhibited degradation temperature range of 280.0-450.0 oC (Figure 4e). These results suggested that the decomposition pathway of PHBV was not affected by the addition of CNC or GO, whereas the volatile product evolution temperature was increased from 246.3 o

C for neat PHBV to 274.1 oC for ternary PHBV nanocomposites with 1 wt % CNC-GO. In

addition, the shorter degradation time and narrower degradation temperature range were observed for the PHBV nanocomposites compared to neat PHBV. The presence of CNC or GO and their hybrids promoted the intermolecular hydrogen bonding interactions with the PHBV matrix that inhibited the chain scission of the six-membered intermediates during the degradation of PHBV.17,31 Moreover, a lower degradation temperature was observed for the binary PHBV nanocomposites with 0.5 wt % GO, compared to the nanocomposites with 1 wt % CNC. These results revealed that compared to CNC, the GO surface with limited groups formed less hydrogen bonds with PHBV to induce slight reduction in degradation temperature.20,29,32 However, ternary PHBV nanocomposites with 1:0.5 wt % CNC/GO showed almost similar results with the binary PHBV nanocomposites containing 0.5 wt % GO, 21 ACS Paragon Plus Environment


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where the GO would produce free radicals causing the thermal degradation of cellulose.4 Compared to binary and ternary PHBV nanocomposites, ternary PHBV nanocomposites with 1 wt % CNC-GO showed better thermal stability, where the Tmax increased 27.8 oC, which was related to the synergetic effect induced by the formation of hydrogen bonds.

Figure 4. (a) TG/FT-IR stack plots for neat PHBV, and PHBV nanocomposites with (b) 1 wt % CNC, (c) 0.5 wt % GO, (d) 1:0.5 wt % CNC/GO and (e) 1 wt % CNC-GO 22 ACS Paragon Plus Environment

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The effect of various nanofillers on the melt crystallization temperature (Tmc), cold crystallization temperature (Tcc) and melting temperature (Tm1, Tm2, Tm3) of the binary and ternary PHBV nanocomposites were determined from DSC analysis in Figure 5. Typical cooling–heating scans of all PHBV nanocomposites are shown in Figure 5a-b and the measured thermal parameters are listed in Table 2. In Figure 5a, no significant melting crystallization peak in the neat PHBV was observed during the first cooling scan, rather only the cold crystallization peak was evident during the second heating scan in Figure 5b. However, the melting crystallization temperature (Tmc) increased from 36.2 to 42.8 oC for the binary PHBV nanocomposites when the CNC content was increased from 1 to 3 wt %). Also, the Tmc also increased from 52.0 oC for 0.5 wt % GO to 69.6 oC for 0.7 wt % GO. These results suggest that the crystallization of PHBV was promoted with the addition of CNC or GO. Additionally, for the non-covalent bonded 1:0.5 wt % CNC/GO within PHBV, the Tmc was increased to 78.9 oC, while the ternary PHBV nanocomposites with covalent bonded 1 wt % CNC-GO possessed the highest Tmc of 81.1 oC among all the nanocomposites (Figure 5a). The GO with the same loading levels exhibited stronger heterogeneous nucleation on the PHBV crystallization than CNC, due to the higher thermal conductivity of GO structure to induce conformation changes and folding of PHBV crystalline chains.20,21,29 Besides, both non-covalent bonded CNC/GO and covalent bonded CNC-GO displayed synergistic nucleation on the PHBV crystallization, resulting in higher Tmc values due to the combined effect of CNC and GO. Similar results were reported for in PVA nanocomposites containing CNC-GO as nanofillers.33



Figure 5. DSC curves (a) first cooling and (b) second heating, of binary and ternary PHBV nanocomposites with various CNC or GO or CNC/GO or CNC-GO contents, (c) relative crystallinity crystallization time and (d) Avrami diagram.

In Figure 5b, neat PHBV and its nanocomposites exhibited two or three melting peaks (Tm1, Tm2, and Tm3), caused by the melting of incomplete PHBV crystals and the melting-recrystallization-melting process of PHBV crystals.30 When the amounts of CNC were increased, the Tm3 increased from 130.6 oC for neat PHBV to 138.2 oC for binary nanocomposites containing 3 wt % CNC, while the crystallinity of neat PHBV (58.2%) was maintained for the binary nanocomposites with CNC. In the presence of various GO contents, Tm3 increased from 131.4 oC for 0.5 wt % GO to 135.6 oC for 0.7 wt % GO, confirming that 24 ACS Paragon Plus Environment

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the addition of conductive GO promoted the formation of PHBV crystals. We also observed that the Tm3 and crystallinity of the ternary nanocomposites were 136.1 oC and 61.3% for 1:0.5 wt % CNC/GO, and 137.7 oC and 62.7 % for 1 wt % CNC-GO. The ternary nanocomposites containing 1 wt % CNC-GO possessed the highest crystallinity since the well-dispersed grafted CNC-GO acted as an efficient nucleating agent that improved and enhanced the nucleation and crystallization rate, resulting in more perfect PHBV crystals. The observed higher melting temperatures correlated with the high crystallinity of polymer crystals.12, 21,24,29,34 In Figure 5c-d, the isothermal crystallization behavior of the binary and ternary PHBV nanocomposites was examined using the Avrami relationship:35

log[− ln(1 − 𝑋 )] = logk + nlogt , 𝑋 𝑡

/

=(

)

∫(

/

)

∫ (

/

)

/

(6) (7)

And n provides information on the nucleation mechanism and crystal growth size24,35. Figure 5c shows the evolution of relative crystallinity (Xt) with time (t) for the binary and ternary PHBV nanocomposites. Table 2 summarizes the isothermal crystallization kinetic parameters. Clearly, the crystallization of neat PHBV was completed within 5.6 min, while the crystallization of binary nanocomposites with CNC contents (1 and 3 wt %) was completed within 5.24 and 4.66 min, respectively. In the binary nanocomposites containing various amounts of GO, the crystallization was completed within 4.53 and 4.02 min for GO contents with 0.5 and 0.7 wt %, respectively. Additionally, the crystallization for 1:0.5 wt % CNC/GO and 1 wt % CNC-GO was completed within 3.81 and 3.55 min, respectively. The ternary nanocomposites with 1:0.5 wt % CNC/GO and 1 wt % CNC-GO yielded a shorter crystallization time compared to neat PHBV and binary nanocomposites. These results 25 ACS Paragon Plus Environment


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indicated both well-dispersed GO and CNC enhanced the PHBV crystallization rate. The highest crystallization rate of ternary PHBV/CNC-GO nanocomposites was achieved due to that CNC-GO (grafted) induced a higher mobility in the PHBV chain segments.20,24,32 The shortest half-crystallization time (t1/2) of the ternary PHBV/CNC-GO nanocomposites supported the above observations (Table 2). Besides, the n value of the neat PHBV was 2.50, which was assigned to the three-dimensional homogeneous nucleation and crystallization of the PHBV crystals,5,11 while the binary PHBV/CNC nanocomposites exhibited higher n values of 2.52-2.62, and the binary PHBV/GO nanocomposites and the ternary nanocomposites exhibited lower n values of 2.21-2.47, indicating the heterogeneous nucleation of the PHBV crystals in these nanocomposites.24,29 Table 2. Crystallization and melting temperatures, crystallinity (Xc) and isothermal crystallization parameters of PHBV and PHBV nanocomposites. Sample

Tmc

Tcc

Tm1

Tm2

Tm3

（oC）（℃）（oC）（oC）（oC）

Xc (%)

n

k×10-2

（oC）

t1/2 （min）

PHBV

-

41.5

98.1

111.2

130.6

58.2

2.50±0.12

2.77

3.62±0.20

1 wt %

36.2

-

122.3

135.2

-

58.3

2.62±0.07

3.42

3.15±0.08

39.9

-

123.2

136.8

-

58.5

2.52±0.05

4.77

2.89±0.06

42.8

-

125.4

138.2

-

58.8

2.58±0.09

5.44

2.68±0.09

52.0

-

101.8

112.1

131.4

58.6

2.47±0.11

6.84

2.55±0.10

69.7

-

105.8

122.5

134.9

58.9

2.42±0.03

7.48

2.51±0.03

CNC 2 wt % CNC 3 wt % CNC 0.5 wt % GO 0.6 wt % GO 26 ACS Paragon Plus Environment

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0.7 wt %

69.6

-

107.0

125.4

135.6

59.0

2.21±0.06

9.28

2.48±0.06

78.9

-

111.7

126.9

136.1

61.3

2.21±0.08

11.24

2.27±0.06

81.1

-

112.8

127.4

137.7

62.7

2.28±0.10

17.12

1.85±0.05

GO 1:0.5wt % CNC/GO 1 wt % CNC-GO

The spherulite morphologies of PHBV in various nanocomposites were investigated using a polarizing optical microscope (POM), as shown in Figure 6. Neat PHBV possessed a large spherulite size of 59.1 μm due to the low nucleation density caused by larger separation of PHBV spherulites during the crystallization process.17 However, with the addition of 1 wt % CNC, the number of spherulites in the binary nanocomposites increased, while the spherulite size decreased to 37.9 μm, while the binary nanocomposites containing 0.5 wt % GO possessed a smaller spherulite size of around 19.6μm. When grafted CNC-GO or blended CNC/GO was added to the PHBV, the spherulite size of the ternary nanocomposites with non-covalent bond 1:0.5 wt % CNC/GO was about 7.8 μm, while the ternary PHBV nanocomposites with covalent bonded 1 wt % CNC-GO displayed the smallest spherulite size of about 3.2 μm among all PHBV nanocomposites. Besides, the growth rates (G) of the spherulite were determined from the slope of the spherulite radius versus time profile. It was obvious that the G value of neat PHBV was 3.743 μm/s, while that of nanocomposites decreased gradually to 2.712 μm/s and 2.413 μm/s in the presence of 1 wt % CNC and 0.5 wt % GO. Finally, the decreased G values reduced to 1.879 μm/s and 1.426 μm/s for non-covalent 1:0.5 wt % CNC/GO and covalent 1 wt % CNC-GO, respectively. These observations suggested that the addition of CNC, GO, CNC/GO and CNC-GO nanofillers could improve 27 ACS Paragon Plus Environment


the nucleation capabilities of the nucleus of PHBV crystals that restricted the diffusion and growth of the spherulites. Especially, compared to CNC or GO and CNC/GO nanofillers, CNC-GO with denser nanostructure and the strongest heterogeneous nucleation effect can give more nucleation sites to induce chain folding of PHBV chains to form smaller PHBV spherulites. The formation of hydrogen bonding interaction in the nanocomposite could inhibit the orientation or extension of the PHBV crystalline chain, and thus result in the reduction in the spherulite size.6,8,17,23,28 Overall, the nanofillers induced nucleation and crystallization rates were larger than the limitation rate of PHBV spherulite growth, resulting in an overall increase in the crystallization rate of PHBV nanocomposites, supported by the increases in crystallization temperature from DSC results (Figure 5).


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Figure 6. POM image and spherulites size for (a) neat PHBV, (b) 1 wt % CNC, (c) 0.5 wt % GO, (d) 1:0.5 wt % CNC/GO, (e) 1 wt % CNC-GO, (f) spherulites size diameter for PHBV nanocomposites.

In Figure 7, the tensile properties of PHBV nanocomposites were affected by the contents of CNC, GO, CNC/GO (blended) and CNC-GO (grafted) nanofillers. By adding 3 wt % CNC, the tensile strength and Young's modulus were enhanced by 34.2 and 25%, respectively; while the elongation to break deceased by 44% in Figure 7a-b. Nevertheless, the tensile strength, Young's modulus and elongation to break of the binary nanocomposites containing 0.7 wt % GO increased by 119.2, 100.0 and 23.8%, respectively. Clearly, the addition of a low content of GO with two-dimensional (2D) flexible sheet structure20,36,37 enhanced the tensile strength and toughness of the nanocomposites better than CNC with 1D rigid rod structure. Furthermore, with increasing GO content, the enhancement effect became more noticeable in the binary nanocomposites. The synergistic reinforcing effect of 1:0.5 wt % CNC/GO (blended) and 1 wt % CNC-GO (grafted) on the tensile strength and toughness of the PHBV matrix were greater than the effect of CNCs or GOs alone. Because the ternary nanocomposites with 1 wt % CNC-GO (grafted) demonstrated larger enhancements in the 29 ACS Paragon Plus Environment


tensile properties, the tensile strength, Young's modulus and elongation to break were increased by 170.2, 137.5, and 52.1%, respectively. However, adding 1:0.5 wt % CNC/GO (blended) resulted in a slight improvement, where the tensile strength Young's modulus and elongation to break increased by 143.1, 134.7, and 13.6%, respectively (Figure 7a-b). A stronger synergetic effect was more noticeable by addition 1 wt % CNC-GO into PHBV matrix due to the efficient dispersion of grafted CNC-GO resulting in the interphase content through hydrogen bonding interactions between nanofiller-PHBV matrix interfaces33. This effect in the ternary nanocomposites containing 1:0.5 wt % CNC/GO (non-covalent) was smaller than 1 wt % CNC-GO (covalent), as physical blending only allowed CNC to coat the surface of GO, yielding a reduced interphase area between two phases. More significantly, synergistic strengthening and toughening of PHBV nanocomposites could be achieved by adding low weight fraction of grafted CNC-GO.


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Figure 7. (a) Tensile strength, (b) Elongation to break, (c) Possible penetration paths of water molecules, (d) Barrier properties against water/water vapor, and (e) Migration properties of PHBV nanocomposites with different types of nanofillers.

The water absorption and water vapor permeability (WVP) are important properties for food packaging systems, where the nanocomposites should avoid or at minimizing the water absorption and water vapor permeability (shown in Figure 7c) needed for food protection. Consequently, the water absorption and WVP of PHBV nanocomposites were investigated and shown in Figure 7d, where the neat PHBV had the highest water absorption and WVP values. With different nanofillers, the water absorption and WVP of the binary nanocomposites decreased gradually with increasing CNC contents. By adding 3 wt % CNC, the water absorption and VWP values decreased by 23.1 and 43.4%, respectively. However, for the binary nanocomposite containing 0.7 wt % GO, we found the reduction in water absorption and WVP values by 20.3 and 46.8%, respectively. The effect of GO on the barrier properties was identical to CNC. This improvement could be attributed to the increased crystallinity and improved interfacial interactions between the PHBV and nanofillers that strongly restricted the motions of the polymeric chains in water and improved tortuosity 31 ACS Paragon Plus Environment


(shown in Figure 7c) in the binary nanocomposites.7,20,24 When 1:0.5 wt % CNC/GO was added, the water absorption and WVP values of the ternary nanocomposites decreased by 68.4 and 72.3%, respectively. In contrast, with 1 wt % CNC-GO (covalent bond), the water absorption and WVP values decreased by 70.2, and 72.6%, where the reduction was slightly higher than the other binary nanocomposites and neat PHBV. We concluded that the barrier properties of PHBV were enhanced with the addition of blended CNC/GO or grafted CNC-GO, particularly for grafted CNC-GO. This enhancement was related to the strong hydrogen bond interactions between the PHBV matrix and CNC-GO nanohybrids, which increased the crystallinity that hindered the passage of water molecules through the nanocomposites.24,27,29 The overall migration levels in two food simulants (isooctane and ethanol 10% (v/v)) are show in Figure 7e. The migration levels of neat PHBV in ethanol and isooctane were 162.1 and 118.5 μg kg -1, respectively. However, with 3 wt % CNC, the migration in both ethanol and isooctane decreased by 35.8 and 22.9%, respectively. For the binary nanocomposites with 0.7 wt % GO the migration for both food simulants decreased by 35.2 and 19.5%, respectively. The migration levels of the nanocomposite with 1 wt % CNC-GO yielded the largest reduction (61.7 and 49.2%, respectively), and all the maximum migration levels of various components (Table S2 and Table S3) in the nanocomposites were below the international migration limits for food contact packaging materials (60 mg/kg for Commission Regulation EU 10/2011 for requirement in the legislation of European commission).24,30 It concludes that the addition of grafted CNC-GO yielded better barrier effect on the migration properties of PHBV, compared to the addition of only CNC or GO or blended CNC/GO as nanofillers. 32 ACS Paragon Plus Environment

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The antibacterial activity of PHBV nanocomposites with different nanofillers against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was detected using the agar diffusion method.38 Figure 8 shows that the antibacterial performance of the sample after 12 h of culture, and there was no inhibitory zone in the neat PHBV and binary nanocomposites (1 wt % CNC). After the additions of 0.5 wt % GO, a significant inhibition zone appeared around the binary nanocomposite. The inhibition zones for Escherichia coli and Staphylococcus aureus were about 2.83 and 2.79 mm, respectively, which demonstrated that the addition of GO yielded good antibacterial properties for PHBV. Indeed, GO displays antimicrobial properties since GO is capable of disrupting cell wall and membrane of bacteria.38,39,40 The inhibition zone was evident in the ternary nanocomposites when non-covalent bonded 1:0.5 wt % CNC/GO was added, while the inhibition zone was slightly smaller than with GO alone. The PHBV nanocomposites with 1 wt % CNC-GO yielded relatively larger inhibition zones of 3.49 and 3.32 mm, respectively. The antibacterial rate of the PHBV nanocomposites was quantitatively determined as shown in Table 1. By adding GO, the antibacterial ratio of binary nanocomposites was as high as 99.7%, while the ternary nanocomposites with grafted (1 wt % CNC-GO) showed antibacterial ratio of almost 100%. This result was attributed to two factors: firstly, the GO in the matrix damaged the bacterial cell membranes through oxidative stress or free radicals.39,40 Secondly, bacteria isolated from the normal environment reduced its ability to extract nutrients needed for growth, and the combined effects would lead to the death of bacteria.6,37,41 In summary, the best antibacterial activity was observed for ternary nanocomposites with 1 wt % CNC-GO, owing to their compact structure and favorable interfacial adhesion between GO and CNC. 33 ACS Paragon Plus Environment


Figure 8. Antibacterial activity of PHBV nanocomposites with different nanofillers against (a, b, c) Escherichia coli and (d, e, f) Staphylococcus aureus



CONCLUSIONS In summary, a series of PHBV nanocomposites with multi-functional cellulose

nanocrystals/graphene oxide hybrids were evaluated as potential food packaging materials. Covalent grafted CNC-GO was well-dispersed and it functioned as heterogeneous nucleating agents in the PHBV matrix, accelerating the crystallization of PHBV. The synergistic effect between CNC and GO through covalent CNC-GO or noncovalent CNC/GO bonds formed strong interfacial interactions, as a result, synchronously significant enhancements in the tensile strength, Young’s modulus, and toughness of ternary nanocomposites. Compared to neat PHBV, a 170.2% increase in the tensile strength, 137.5% enhancement in the Young's


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modulus, and 52.1% improvement in the elongation to break were reported for covalent bonded 1 wt % CNC-GO. Besides, Tmax for the ternary nanocomposite with covalent CNC-GO (grafted) increased by 26.3 oC, and this ternary nanocomposite displayed good excellent crystallization ability, robust barrier properties, good antibacterial activity (antibacterial ratio of 100.0%) and lower migration levels (far below 60 mg/kg for the international migration limits for food contact packaging materials required by the legislation of European commission), which was attributed to the covalent bonded CNC-GO with more compact hybrid structure, and their synergistically combined effect of GO and CNC. Such novel nanocomposites possess great potentials as green high-performance food packaging materials.



ASSOCIATED CONTENT Supporting Information The characterizations properties of PHBV nanocomposites including the SEM and

TEM morphologies (Figure S1) and microstructure, the designated percentages of PHBV nanocomposites (Table S1), the migration values of various components in PHBV nanocomposites in 10% ethanol (Table S2) and in isooctane (Table S3) are provided in Supporting Information.



AUTHOR INFORMATION

Corresponding Author *Hou-Yong Yu (H.Y. Yu); Tel.: 86 571 86843618; E–mail addresses: [email protected]



ORCID Hou-Yong Yu: 0000-0002-6543-5924 Kam Chiu Tam: 0000-0002-7603-5635 Notes The authors declare no competing financial interest.



ACKNOWLEDGMENTS The work is supported by international cooperation of Prof. Jaromir Marek and Key

Program for International S & T Innovation Cooperation Projects of China [2016YFE 0131400], the Scientific Research Foundation of Zhejiang Sci-Tech University (ZSTU) under Grant. no. 19012099-Y, Candidates of Young and Middle Aged Academic Leader of Zhejiang Province, “521” Talent Project of Zhejiang Sci-Tech University, and the Young Elite Scientists Sponsorship Program by CAST(2018QNRC001).



REFERENCES

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