Polymer Degradation and Stability 107 (2014) 139e149 Contents lists available at ScienceDirect Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab PLA-PHB/cellulose based films: Mechanical, barrier and disintegration properties
n c, J. Lo
pez a, J.M. Kenny b, d M.P. Arrieta a, e, E. Fortunati b, *, F. Dominici b, E. Rayo a Instituto de Tecnología de Materiales, Universitat Polit ecnica de Valencia, 03801 Alcoy, Alicante, Spain Materials Engineering Centre, UdR INSTM, NIPLAB, University of Perugia, 05100 Terni, Italy c Instituto de Tecnología de Materiales, Universitat Polit ecnica de Valencia, E-46022 Valencia, Spain d Institute of Polymer Science and Technology, CSIC, Juan de la Cierva 3, Madrid 28006, Spain e Analytical Chemistry, Nutrition and Food Sciences Department, University of Alicante, P.O. Box 99, E-03080 Alicante, Spain b a r t i c l e i n f o a b s t r a c t Article history: Received 11 February 2014 Received in revised form 29 April 2014 Accepted 2 May 2014 Available online 29 May 2014 Nanocomposite films based on poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB) blends and synthesized cellulose nanocrystals (CNC) or surfactant modified cellulose nanocrystals (CNCs), as bio-based reinforcement, were prepared by melt extrusion followed by film forming. The obtained nanocomposites are intended for short-term food packaging. Thus, the mechanical, optical, barrier and wettability properties were studied. Functionalized CNCs contribute to enhance the interfacial adhesion between PLA and PHB, leading to improved mechanical stiffness and increased film stretchability. The synergic effects of the PHB and CNCs on the PLA barrier properties were confirmed by increases in oxygen barrier properties and reductions in surface wettability of the nanocomposites. In addition, the measurements of the viscosity molecular weight for ternary systems showed practically no degradation of PLA and smaller degradation of PHB during processing due to nanocrystal presence. The disintegration process in composting conditions of PLA was delayed by the addition of PHB, while CNC speeded it up. PLA-PHB-CNCs formulations showed enhanced mechanical performance, improved water resistance, reduced oxygen and UV-light transmission, as well as appropriate disintegration in compost suggesting possible applications as packaging materials. © 2014 Elsevier Ltd. All rights reserved. Keywords: Poly(lactic acid) Poly(hydroxybutyrate) Modified cellulose nanocrystals Nanocomposites Biodegradation Barrier properties 1. Introduction Many positive characteristics of PLA have situated it as the most used biopolyester for biodegradable food packaging industry. Among other properties, PLA compared to other biopolymers shows easy processability [1], superior transparency, availability in the market [2], excellent printability [3], high rate of disintegration in compost [4]. Conversely, the use of PLA films for food packaging has been strongly limited because of their poor mechanical and barrier properties [5]. Moreover, for large-scale industrial production PLA must guarantee adequate thermal stability or low thermal degradation during processing and use [6]. Initially, the materials for food applications were semi-rigid or flexible monolayer systems. Afterward, to improve the barrier properties they were replaced by more complex multilayer systems, which are still used in the market. However, its difficulty of * Corresponding author. Tel.: þ39 0744492921; fax: þ39 0744492950. E-mail address: elena.fortunati@unipg.it (E. Fortunati). http://dx.doi.org/10.1016/j.polymdegradstab.2014.05.010 0141-3910/© 2014 Elsevier Ltd. All rights reserved. recycling and the worldwide trend to reduce the polymer consumption per package unit, demand the development of simplest packaging formulations particularly focused on blending strategies [7]. Thus, melt blending PLA with another biopolymer can lead to significant improvement of the final properties through a cost effective, easy and readily available processing technology [8]. Food packaging, besides containment and information, should provide foodstuff protection against water, light or oxidative process [9]. It is known that the crystalline phase has an important impact on mechanical and permeation properties; as a result, considerable academic and industrial research efforts have been focused to increase PLA crystallinity. In this sense, the addition of poly(hydroxybutyrate) (PHB), a highly crystalline biopolymer, to the PLA matrix by melt blending has been considered as an easy way to increase PLA crystallinity and regulate its properties [10]. PHB, the most common representative of poly(hydroxyalkanoates) (PHA), with a high degree of crystallinity, has been also proposed for short-term food packaging applications [11]. PHB has a similar melting temperature to PLA, allowing blending both polymers in the melt state. In a previous work, PLA was melt blended with 25 wt 140 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 % of PHB showing an improvement in oxygen barrier and water resistant, whilst reducing the inherent high transparency of PLA [8,12]. Transparency is an essential issue to be considered in the development of materials intended for food packaging since seeing through the packaging is one of the most important requirements for the consumers [2]. Moreover, to preserve food products until they reach the consumer, packaging sometimes require protecting food products from ultraviolet light [13]. Thus, the design of transparent films with enhanced UV protection is particularly relevant in many packaging applications [14]. It has been reported that PHB acts as better light barrier in the visible and ultraviolet light regions [11] than PLA. On the other hand, novel and efficient polymer materials based on nanotechnology can provide innovative solutions to increase the polymers performance for food packaging. The incorporation of nanoparticles into polymers matrices has shown to improve mechanical properties and thermal stability, as well as it offers many additionally advantages such as reduction in raw materials and elimination of expensive lamination secondary processes, while the modification of the thermal properties of polymers leads to a reduction in machine cycle time and temperature [15]. The use of nanocomposites to improve the inherent shortcomings of PLA based packaging materials has proven to be a promising technology. The ideal nanoparticle should be biobased and biodegradable. In this sense, bioresources obtained from agriculturalrelated industries have received significant attention, particularly focused on cellulosic materials and especially to its specific form of cellulose nanocrystals (CNC), which have been revealed to be an interesting model filler [16] for various biopolymer matrices including PLA [3,17,18] and PHB [19], beside others biopolymers such as poly(vinyl alcohol) (PVA) [16] and poly(hydroxybutyrateco-hydroxyvalerate) (PHBV) [20]. Cellulose nanocrystals have shown better mechanical properties than a majority of the commonly used reinforcing materials and offer additional exceptional advantages such as biodegradability, high stiffness and low density [17] abundance in nature and low cost [21]. Although nanocelluloses have a great potential as mentioned above, the high amount of eOH on the surface of the crystals induces high attraction between them [22]. Thus, the high polarity of cellulose surface and the resultant low interfacial compatibility with hydrophobic polymer matrices make difficult the homogenous dispersion of nanocellulose in polymers [23]. For that reason, a surface modification of CNC by a surfactant (CNCs) has been proposed and successful dispersion of CNCs in the PLA matrix was achieved [3,17]. This specific type of modification enhances the interfacial adhesion polymer/nanofiller and thus improves some final properties of the final nanocomposites such as mechanical performance [17], oxygen barrier and water resistance [24], which are particularly interesting for materials intended for food packaging. In a previous work, the processing performance of PLA-PHB with CNC or CNCs was optimized and it was verified that the functionalization of CNCs favours the dispersion into PLA-PHB blend matrix enhancing the interfacial adhesion by means increasing the thermal stability [25]. In the current work, in order to address the positive effect of cellulose nanocrystals on the thermal stability of PLA and PHB, a more detailed study concerning the potential reduction of their molecular weight due thermal processing is reported. Moreover, since the main objective of this research is to propose this high performance nanocomposite films for biodegradable food packaging industry a complete characterization related with this field of application was conducted. For this purpose, the combination of ternary system based in PLA, PHB and cellulose nanocrystals blend was developed to enhanced PLA barrier and mechanical properties. Cellulose nanocrystals (CNC) were synthesized from microcrystalline cellulose (MCC) as well as further modified using a surfactant (CNCs) to improve the dispersion in the biopolymer matrix. Then nanocrystals were meltblended with a previous prepared PLA-PHB masterbatch and finally processed into films. The processing of these systems and their crystalline and thermal stability properties were reported previously [25]. The mechanical, optical and barrier properties were tested with the aim to evaluate their suitability for the food packaging sector and are reported here. Additionally, the disintegrability under composting of the multifunctional materials was evaluated to get information about their post-use. 2. Experimental 2.1. Materials Poly(lactic acid) (PLA 2002D, Mn ¼ 98,000 g mol 1, 4 wt% D-isomer) was supplied by NatureWorks (USA). Poly(hydroxybutyrate) (PHB, under the trade name PHI002) was acquired from NaturePlast (France) and microcrystalline cellulose (MCC, dimensions of 10e15 mm) was purchased from SigmaeAldrich. 2.2. Nanocrystal synthesis and modification Acid hydrolysis of microcrystalline cellulose (MCC) was carried out by using sulphuric acid 64% (wt/wt) at 45  C for 30 min with continuous stirring [17]. The obtained cellulose nanocrystals (CNC) in an acid solution were washed with ultrapure water (1:200), centrifugated and dialyzed until neutral pH. An ion exchange resin was added to the cellulose suspension for 24 h and then was removed by filtration in order to ensure that all ionic materials were removed except the Hþ counter ions associated with the sulphate groups on the CNC surfaces. After that, nanocrystal suspensions were ultrasonicated (Vibracell 75043, 750 W, Bioblock Scien-tific) for 2 min in an ice bath. Surface modified cellulose nanocrystals (CNCs) were also prepared by adding a surfactant (STEFAC TM 8170, Stepan Company Northfield) in 1/1 (wt/wt). Finally, cellulose nanocrystals in powder were obtained by a freezedrying process of previously neutralized solutions (1.0% (wt/wt) of 0.25 mol l 1 NaOH). 2.3. PLA-PHB-nanocomposite preparation PLA (75 wt%) was blended with 25 wt% of PHB and then reinforced with 5wt% of pristine (CNC) or surfactant modified (CNCs) cellulose nanocrystals. Masterbatches were prepared by using a twin-screw microextruder (DSM explorer 5&15 CC Micro Compounder) by following the same processing conditions as described in a previous work [25]. Briefly, using a temperature profile of mixing process with a maximum temperature of 200  C with three-step temperature procedure of 180e190e200  C and a screw speed of 150 rpm for 2 min. Masterbatches were pelletized and mixed for 1 min and directly processed in films with a head force of 3000N. Then, a film procedure was conducted to obtain six formulations, including the neat PLA and PLA-PHB blend with a thickness ranged from 10 to 30 mm. The obtained formulations and the proportion of each component are summarized in Table 1. 2.4. Characterization techniques The capillary viscosity was measured at room temperature using a Ubbelohde viscometer (type 1C) according to ISO 1628 [26] for all film sample diluted in chloroform (SigmaeAldrich 99% purity) and at least three concentrations were used. PLA and PHB pellet were also measured as control. The concentration of PLA and/or PHB in 141 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 Table 1 PLA and PLA-PHB nanocomposite film formulations and their viscosity molecular weight (Mv). Film formulations PLA PLA-CNC PLA-CNCs PLA-PHB PLA-PHB-CNC PLA-PHB-CNCs PLA Mv (g mol Materials (wt %) PLA PHB CNC CNCs 100 95 95 75 71.25 71.25 e e e 25 23.75 23.75 e 5 e e 5 e e e 5 e e 5 the final film formulations were expressed taken into account the proportion reported in Table 1. The intrinsic viscosity [h] of samples was determined to estimate the viscosity molecular weight by means the MarkeHouwink relation: ½hŠ ¼ K  MVa (1) were K and a are 1.53  10 2 and 0.759 for PLA, respectively [27] as well as K and a for PHB are 1.18  10 2 and 0.780 in that order [28]. The mechanical behaviour was investigated by tensile test in a digital Lloyd instrument LR 30K, performed on rectangular probes (100 mm  10 mm) at room temperature by following the UNE-EN ISO 527-3 standard [29] with a crosshead speed of 5 mm/min, a load cell of 500N and an initial gauge length of 50 mm. Average tensile strength (TS), percentage elongation at break (εb %) and Young's modulus (E) were calculated from the resulting stressestrain curves as the average of five measurements of each composition. The absorption spectra of nanocomposites, obtained in the 700e250 nm region, were investigated by a PerkineElmer (Lambda 35, USA) UVeVIS spectrophotometer. Nanocomposite film colour properties were evaluated in the CIELAB colour space by using a KONICA CM-3600d COLORFLEX-DIFF2, HunterLab, Hunter Associates Laboratory, Inc, (Reston, Virginia, USA). The instrument was calibrated with a white standard tile. Yellowness index (YI) and colour coordinates, L (lightness), a* (red-green) and b* (yellowblue) were measured at random positions over the film surface. Average values of five measurements were calculated. Total colour difference (DE) was calculated with respect to the control pure PLA film or PLA-PHB film as: pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DE ¼ Da*2 þ Db*2 þ DL*2 (2) The oxygen transmission rate (OTR) was measured to study the oxygen permeability of the nanocomposites by using a Systech Instruments 8500 oxygen permeation analyzer (Metrotec S.A, Spain) at room temperature and 2.5 atm. 14 cm diameter circle films were compressed between the upper and lower diffusion chamber. Pure oxygen (99.9% purity) was introduced into the upper half of the sample chamber while nitrogen was injected into the lower half. To prepare the appropriate samples for OTR measurements, masterbatch pellets were set in discs by using a DSM Xplore 10-ml injection moulding machine at 175, 180 and 190  C with a pressure profile in three steps: 6 bar for 4 min, 8 bar for 5 min and 8 bar for 3 min. Discs were then processed into films by compression moulding process at 180  C in a hot press (Mini C 3850, Caver, Inc., Wabash, IN, USA) with a pressure cycle of 3 MPa for 1 min, 5 MPa for 1 min, and 10 MPa for 2 min. Nanocomposite films were then quenched to room temperature at atmospheric pressure. Their average thickness was between 180 and 250 mm. Surface wettability of films was studied through static water contact angle measurements with a standard goniometer 90,600 86,200 81,100 91,900 95,000 95,600 ± ± ± ± ± ± 1 ) 10,700 18,800 14,400 19,000 19,500 18,800 PHB Mv (g mol 1 ) e e e 224,300 ± 45,150 231,700 ± 46,400 233,000 ± 44,700 (EasyDrop-FM140, KRÜSS GmbH, Hamburg, Germany) equipped with a camera and Drop Shape Analysis SW21; DSA1 software was used to test the water contact angle (q ) at room temperature. The contact angle was determined by randomly putting 5 drops of distilled water (z2 mL) with a syringe onto the film surfaces and, after 30 s, the average values of ten measurements for each drop were used. The maximum standard deviation in the water contact angle measurements did not exceed ±3% [30]. 2.5. Disintegrability under composting conditions The disintegration under composting conditions of PLA and PLA-PHB nanocomposites was investigated on the basis of the ISO 20200 standard [31]. A solid synthetic waste was prepared by mixing 10% of compost supplied by Gesenu S.p.a. (Perugia, Italy), with 30% rabbit food, 10% starch, 5% sugar, 1% urea, 4% corn oil and 40% sawdust. The water content of the substrate was around 50 wt% and the aerobic conditions were guaranteed by mixing it softly [4]. Nanocomposite films (cut in 15  15 mm2) were weighed and buried at 4e6 cm depth in perforated plastic boxes, containing the prepared mix, and incubated at 58  C. Each nanocomposite film was recovered at 1, 2, 3, 7, 10, 14 and 21 days of disintegration, cleaned with distilled water, dried in an oven at 37  C during 24 h and reweighed. The disintegration degree was calculated by normalizing the sample weight, at different days of incubation, to the initial weight. In order to determine the time at which 50% of each film was degraded, disintegrability degree values were then fitted using the Boltzmann equation (OriginPro 8.1.software) as follows: m¼ ðmi m∞ Þ 1 þ eð1 ðt50 =dt ÞÞ (3) where mi and m∞ are the initial and final mass values measured respectively at the beginning of the exposition to compost and after the final asymptotes of the disintegrability test, and t50 is the time at which materials disintegrability reaches the average value between mi and m∞, known as the half-maximal degradation, dt is a parameter that describes the shape of the curve between the upper and lower asymptotes [32]. Photographs of recovered samples were taken for visual comparison. Surface microstructure of PLA and PLA-PHB nanocomposites before and after 3 days of incubation in composting were studied by optical microscopy using a LV-100 Nikon Eclipse equipped with a Nikon sight camera at 20 magnifications using the extended depth of field (EDF-z) imaging technique to obtain a tridimensional vision of films surfaces. This technique uses a motorized z-axis (height of focus) to take images at different height planes. Subsequently, by means of a dedicated algorithm installed in the NIS-Elements software a 3D image is reconstructed following the original texture of sample. The relationship between meso-lactide and L,D-lactide form in the polymer after compost incubation was also studied by Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) by 142 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 Significance in the mechanical, wettability and colour parameters differences were statistically by one-way analysis of variance (ANOVA) using OriginPro 8 software. To identify which groups were significantly different from other groups, means comparison were done employing a Tukey's test with a 95% confidence level. The addition of PHB also produced a reduction on the Mv of PLA of about 4% and a higher reduction (11%) was showed, as expected, in the case of the Mv value of PHB. This higher reduction in the Mv value of PHB than in the Mv value of PLA can be explained by somewhat PHB thermal degradation that might take place at the processing temperature used here, in the range of 180e200  C [33]. The viscosity molecular weight of PLA in ternary PLA-PHB based nanocomposites was almost constant. Meanwhile, the viscosity molecular weight of PHB was reduced by approximately 9% in these formulations (PLA-PHB-CNC and PLA-PHB-CNCs). The addition of cellulose nanocrystals into PLA-PHB blends lead to a enhancement the interface interaction between PLA and PHB leading to an improvement of the thermal stability of both polymers, particularly for PHB that usually shows a small processing window [25]. Thus smaller reductions on PHB Mv values were observed after processing due the positive effect of CNC and CNCs presence. 3. Results and discussion 3.2. Mechanical properties 3.1. Viscosity molecular weight As it can be seen from tensile curves (Fig. 1-a), the neat PLA film showed a characteristic plastic deformation that it was reduced with both, PHB and CNC incorporation. CNC and PHB proved to be effective to increase PLA modulus (Fig. 1-b), but no significant differences were observed between the Young's modulus of PLA-CNCs and PLA films. While CNCs or PHB produced a decrease on the tensile strength (TS) of PLA, the combination of PHB and CNCs produce a nanocomposite (PLA-PHB-CNCs) with comparable TS with respect to PLA. This behaviour can be related with the more efficient dispersion of functionalized cellulose nanocrystals (CNCs) [3] resulting in an enhancement in the interfacial adhesion and therefore in a better interaction between PLA and PHB [25]. Moreover, the PLA-PHB-CNCs film revealed the highest deformation at break, showing an increase of 175% with means of a Pyroprobe 1000 pyrolyzer (CDS Analytical, Oxford, Pennsylvania, USA) at 1000  C for 0.5 s, coupled with a gas chromatograph (6890N, Agilent Technologies) and a mass selective detector (Agilent 5973N) on the basis of a previous developed method [32]. Fourier infrared spectra of the samples in the 400e4000 cm 1 range were recorded by a Jasco FTIR 615 spectrometer, in transmission mode. 2.6. Statistical analysis The estimated viscosity molecular weight (Mv) of PLA and PHB pellets were 95,800 ± 4400 g mol 1and 255,300 ± 39,000 g mol 1, respectively. It is know that polymers can undergo thermal degradation during processing and diminution of Mv values for all film formulations with respect of PLA pellet and/or PHB pellet were detected (Table 1). PLA processed into film resulted in a reduction of the Mv of PLA around 5%. Further decrease occurred in binary PLA based nanocomposites. While, a reduction of PLA Mv value of 10% was detected for PLA-CNC, PLA-CNCs showed a higher reduction of 15%. This results is in accordance with previous work where we showed that the thermal stability of PLA was reduced with the addition of cellulose nanocrystals, particularly for PLA-CNCs [25]. Fig. 1. Tensile test results of PLA, PLA-PHB and nanocomposite films: a) Stress-strain curves, b) Young's Modulus (E), c) Tensile strength (TS) and d) Elongation and break (εB). aed Different letters on the bars within the same image indicate significant differences between formulations (p < 0.05). 143 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 Fig. 2. PLA, PLA-PHB and nanocomposite films: a) UVeVis spectra, b) Visual appearance, c) contact angle measurements. aedDifferent letters on the bars within the same image indicate significant differences between formulations (p < 0.05). respect to the neat PLA film, while significant lower elongations at break respect to the PLA film were measured for the other nanocomposite formulations. Films for food packaging are required to maintain their integrity in order to withstand the stress that occurs during shipping, handling and storage [34] and PLA-PHBCNCs is therefore far stretchable and stiff with comparable strength than PLA and could be defined as the best formulation for food packaging applications. 3.3. Optical and colourimetric properties The absorption spectra of PLA, PLA-PHB and nanocomposites are shown in Fig. 2(a) while the visual appearances of the films are displayed in Fig. 2(b). Neat PLA film proved to be the most transparent showing the highest transmission in the visible region of the spectra (400e700 nm). No significant changes were observed due to the presence of CNC or CNCs in the visible region of the spectra, thus PLA-CNC and PLA-CNCs resulted in highly transparent films, referred to the light transmission in the range of 540e560 nm [13,35], as it can be observed in Fig. 2(b). The good transparency of PLA-CNC films has been related with the good dispersion of cellulose nanocrystals into PLA matrix [17,35]. On the other hand, a 25 wt% of PHB, provokes a reduction of the light transmission of the films. Both, PHB and cellulose nanocrystals show a blocking effect on the virtually transparent PLA matrix at the UV spectra region (250e400 nm). Cellulose nanocrystals reduced the UV light transmission with a maximum centered at 275 nm which corresponds to the UV-C region (280e100 nm), generally created from artificial light sources [13]. This behaviour was more evident with surface modified nanocrystals (CNCs). The PLA-PHB-CNCs film showed a blocking effect in the UV light spectra region with the lowest UV-C light transmission, while maintaining the high transparency in the visible spectra region. Table 2 summarizes the colour parameters obtained for PLA, PLA-PHB and cellulose nanocrystal based nanocomposites. PLA showed the highest L value confirming it characteristic high brightness. L is significantly affected by CNC, CNCs and PHB presence, although all film samples present still higher L values than commercial low density polyethylene (LDPE) and poly(ethylene terephthalate) (PET) films [13]. Negative values of the a* coordinate reveal a deviation towards green, while positive values for b* are indicative of a deviation towards yellow. As a consequence of the PHB presence, the highest deviations towards green and yellow colours were observed in the PLA-PHB blend, in accordance to previous studies [36]. As a result, the yellowness index (YI) showed the maximum value for the PLA-PHB film, followed by PLA-PHBCNCs and PLA-PHB-CNC. The YI is used to describe the change in colour of a sample from clear toward yellow. It must be noticed, that the obtained YI values are significant lower than those previously reported for PLA [12,13] and PLA-PHB (75:25) blends [12,36]. The main reason for this important reduction in YI is due to the lower films thickness obtained by the processing film methodology used in the present work. Despite the total colour differences obtained with respect to the neat PLA film were significant different in all cases, the total colour differences were in general smaller than 2.0, being this value the threshold of perceptible colour difference for the human eye [37], with the exception of the PLA-PHB film as can be confirmed in Fig. 2b. 3.4. Oxygen transmission rate and wettability In a previous reported work the incorporation of CNC and functionalized CNCs had shown reductions in OTR values of neat PLA film (30.5 cm3 * mm * m 2 * day 1) of about 43% and 48%, respectively [24]. In this case, the addition of PHB reduced the oxygen permeation of PLA to 13.3 cm3 * mm * m 2 * day 1 (reduction of 56%) due to the increased crystallinity in the system Table 2 Colour parameters from CIELab space and YI of PLA, PLA-PHB and nanocomposite films. Samples L PLA PLA-CNC PLA-CNCs PLA-PHB PLA-PHB-CNC PLA-PHB-CNCs 94.64 93.57 94.28 93.79 93.55 93.69 a* ± ± ± ± ± ± 0.01a 0.01b 0.01c 0.01d 0.01e 0.01f 0.98 1.08 1.01 1.07 0.91 1.04 b* ± ± ± ± ± ± 0.01a 0.02b 0.01c 0.01b 0.01d 0.01c 0.76 0.62 0.83 1.54 1.03 1.48 ± ± ± ± ± ± 0.02a 0.02b 0.01c 0.01d 0.01e 0.01f DE Calculated by using PLA film colour coordinates as reference. aef Different superscripts within the same column indicate significant differences between formulations (p < 0.05). DE YI e 1.08 0.37 1.15 1.12 1.19 0.70 0.52 0.82 2.14 1.29 2.07 ± ± ± ± ± 0.01 0.01 0.01 0.01 0.01 ± ± ± ± ± ± 0.03a 0.02b 0.02c 0.02d 0.01e 0.03f 144 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 [12,25]. Nevertheless, from the OTR values obtained in ternary systems (15.3 cm3 * mm * m 2 * day 1 for PLA-PHB-CNC and 13 cm3 * mm * m 2 * day 1 for PLA-PHB-CNCs), it can be noticed that the incorporation of CNC or CNCs to PLA-PHB blend did not provoke major changes in OTR values. The low OTR values obtained for the PLA-PHB blend and ternary nanocomposites highlight the advantage of blending PLA with crystalline PHB. These films are therefore attractive for food packaging applications were barrier to oxygen is critical to avoid or reduce oxidative processes. Additionally, films for food packaging are required to protect foodstuff from humidity during transport, handling and storage. Thus, water contact angle measurements were carried out to evaluate the hydrophilic/hydrophobic character of films and the results are shown in Fig. 2(c) [37]. It should be noticed that all formulations showed values higher than 65 , being materials acceptable for the intended end-use applications. PHB has a hydrophobic character due to the poor affinity of the water to the nonpolar polymer surface [38]. In this way, the PLA-PHB blend showed significant increased water resistance in comparison with neat PLA, in good accordance with a previous reported work [12]. The presence of CNC in PLA and PLA-PHB caused an increase in wettability, while functionalized CNCs did not significantly change PLA or PLAPHB wettability. The positive effect of cellulose nonocrystal chemical modification in the wettability of PLA and PLA-PHB films is mainly due to the presence of sulphate groups with low polarity on the surface that increase the surface hydrophobicity of the final material. 3.5. Disintegration under composting Fig. 3 (a) shows the visual appearance of PLA, PLA-PHB and cellulose nanocrystal based nanocomposites after different time of disintegration in composting conditions where it is possible to confirm the biodegradable character of all the formulations studied. After only 1 day of incubation, films become smaller, with the exception of PLA-PHB blend, which started the film size reduction on the second day of incubation. After 7 days of incubation binary and ternary formulation films became breakable and small pieces of films were recovered. It also could be noticed that they changed their colour and became more opaque after 7 days. When the degradation process of the polymer matrices started, a change in the refraction index of the materials was observed as a result of water absorption and/or presence of products formed by the hydrolytic process [39]. Additionally, the films disintegrability was evaluated in terms of mass loss as a function of incubation time (Fig. 3 (b)), in which the line at 90% of disintegration represents the goal of disintegrability test [4]. Unmodified cellulose nanocrystals (CNC) speed up the disintegration of PLA and PLA-PHB blend from 14 days to 21 days, respectively, to 10 days. Comparable findings were previously reported for PLA nano-biocomposite films with functionalized cellulose nanocrystals and silver nanoparticles [40]. Accordingly, after 10 days CNC incorporated films were visibly disintegrated (Fig. 3 (a)), while CNCs incorporated counterparts reached between 50 and 60% of disintegrability and need 14 days to reach the goal of the disintegrability test (Fig. 3 (b)). It is known that Fig. 3. a) Visual appearance of film samples before and after different incubation days under composting conditions. b) Degree of disintegration of films under composting conditions as a function of time. M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 the PLA disintegration in compost starts by a hydrolysis process [41], thus this different behaviour observed between the pristine and modified cellulose nanocrystals could be ascribed to the more hydrophobic character of functionalized cellulose nanocrystals that protect the polymer matrix from the water attack. In brief, PLA-CNC and PLA-PHB-CNC lost more than 90% of the initial matter in 10 days; PLA, PLA-PHB-CNC and PLA-PHB-CNC in 14 days and PLA-PHB in 21 days. These short degradation times have been related to the low thickness of tested samples [4]. Some changes in compost colour were observed (Fig. 3 (a)) due to the aerobic fermentation that results in dark humus soil. However, all formulations showed different rate of disintegration and thus the Boltzmann function was used to correlate the sigmoidal behaviour of the mass loss during the disintegrability in the composting process (Fig. 4). The estimated regression parameters of the fitted results of the non-linear model and t50 were calculated, while 145 mi and m∞ values were assigned as 0% and 100% of disintegrability, respectively. The correlation coefficients between theoretical and experimental data (R2) were higher than 0.990 in all cases, indicating that only minor differences were observed in the fitting of the model to experimental values. The rate of disintegration under composting conditions was longer for PLA-PHB with a half-maximal degradation (t50) at about 14 days, with respect to neat PLA that showed t50 at about 10 days, due to the fact that the polymer disintegrability in composting starts in the amorphous phase of the polymers [42] and the increasing crystallinity in PLA-PHB blend due to the PHB presence delays the PLA degradation rate [12,36]. Both cellulose nanocrystals speed up the rate of disintegration of PLA and PLA-PHB shifting half-maximal degradation to lower values. The t50 of PLA was shifted from 10 days to 7 days in PLA-CNC and remains practically constant in PLA-CNCs, while the t50 of PLAPHB was shifted from 14 days to 7 days and to 10 days in PLA-PHB- Fig. 4. Disintegrability of PLA, PLA-PHB and nanocomposite films as a function of time. 146 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 CNC and PLA-PHB-CNCs, respectively. As a result, PLA-PHB-CNC showed higher rate of disintegration than PLA-CNCs, even when PLA-PHB-CNC (cc ¼ 18.5%) is more crystalline than PLA-CNCs (cc ¼ 11.0%) [25]. This unexpected result could be explained with the fact that during disintegration in compost, PLA surface is firstly attacked by water where polymer chain are hydrolysed [4] and as a result the smaller molecules become susceptible for enzymatic degradation mediated by microorganisms [41]. Meanwhile, PHB disintegration is firstly caused by polymer surface erosion mediated by microorganisms which then are able to spread gradually inside the polymer matrix [43]. PLA-PHB-CNC showed higher surface polarity than PLA-CNCs. Thus, the water attack starts on the more susceptible component, CNC, with hydroxyl groups available on the surface, allowing the hydrolysis in PLA-PHB-CNC, which is followed by the microorganisms attack, while CNCs is protecting PLA in PLACNCs. In the meantime, available eOH are now able to attack he carbon of the ester group and produce intramolecular degradation, followed by the hydrolysis of the ester link [44]. As a consequence, the higher surface polarity of PLA-PHB-CNC and the eOH presence that catalyse the hydrolysis process, leading to a higher disintegration rate for PLA-PHB-CNC than PLA-CNCs. Micrograph observations of film surfaces before and after 3 days in composting, and their profiles measured by the EDF-z technique, are shown in Fig. 5. PLA and PLA nanocomposite films before composting showed smoother profile than PLA-PHB counterparts. Similar behaviour was observed in a previous work where neat PLA, neat PHB films roughness were investigated by means of confocal microscopy. The PLA film showed a smoother roughness profile than PHB [12]. In general, after 3 days in composting all formulations showed a more irregular EDF-z profile with respect to the same formulation before composting. The chemical changes of nanocomposite films before and after 1, 2 and 3 days in composting were followed by FTIR analysis. At higher time of disintegration, film samples could not be studied in the FTIR spectrometer due the small portions of films recovered. The main differences found were in the 2000e1200 cm 1 region of the FTIR spectra as shown in Fig. 6. The typical asymmetric stretching of the carbonyl group (eC]O) of PLA centered at 1760 cm 1 become broader during composting due to an increase in the number of carboxylic end groups in the polymer chain during the hydrolytic degradation [39]. Moreover, a band at 1722 cm 1 was also apparent (shown by grey arrows) that has been associated with crystalline C]O stretching vibration of PHB [45]. In some samples, this band appears as a shoulder due to its low intensity and the strong stretching vibration of carbonyl group. A small band at 1687 cm 1 was apparent (shown by black arrows) in PLA-PHB and PLA-PHB-CNCs after three days of composting. This band has been reported to be a crystalline band, although its spectral origin is not yet assigned [45,46]. The clear appearance of this band in PLA-PHB and PLA-PHB-CNCs is supported by the early degradation of the amorphous phase of the polymer blend while the crystalline PHB remains in the polymer matrix. Similar results indicating that PHB slow down the disintegration rate of PLA in PLA-PHB blends have been previously reported [36]. In the region between 1550 and 1650 cm 1 the appearance of a broad band was observed for all formulations. The appearance of this band has been previously observed during the degradation of PLA-MCC based composites and was related to the presence of carboxylate ions in degraded PLA composites [4]. Fig. 7(a) shows the typical Py-GC/MS chromatogram of PLAPHB-CNCs obtained by pyrolysing the film at 1000  C for 0.5 s. The pyrolysis of all PLA based films is characterized by the presence of two peaks with very similar mass spectra (m/z ¼ 32, 43, 45 and 56) in which the peak at 17 min corresponds to meso-lactide and the peak at 18 min with the highest signal intensity in all samples to (L) and/or (D)-lactide [32]. Films with PHB showed the broad peak Fig. 5. Optical micrographs (20) of PLA, PLA-PHB and nanocomposite films and their EDF-z profiles. M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 Fig. 6. Infrared spectra (2000e1200 cm 147 1 ) of PLA, PLA-PHB and nanocomposite films before and after different time of incubation under composting conditions. of crotonic acid (6.7 min, m/z ¼ 39, 41, 68, 69, and 86) [12,36], while nanocomposte films showed a peak at 22.5 min assigned to thermal degradation products of the cellulose structure (m/z ¼ 55, 69, 87 and 103) [47]. The groups of small peaks appearing at retention times between 19 min and 22 min were assigned to the thermal degradation products of PLA with the characteristic series of signals at m/z ¼ 56 þ (n  72) attributed to PLA degradation products such as dimers (n ¼ 2) and trimers (n ¼ 3) [32]. In general, the intensity of peaks decreased with composting time. However, the mesolactide intensity showed a lower decrease with respect to the (D,L)lactide equivalent. The ratio meso-lactide:lactide has been used as a semi-quantitative sign of the degradation of PLA [12,32,36,48,49]. Fig 7(b) shows the reduction of (D,L)-lactide with respect to mesolactide after the pyrolysis of the recovered samples. No significant differences were observed between PLA and PLA-PHB blend until 7 days in composting, but after 10 days, the PLA relationship [lactide/ meso-lactidet¼10 days/lactide/meso-lactidet¼0 days] highly decreased. Nanocomposites showed similar reduction in 3 days of composting, but higher times revealed higher reduction for unfunctionalized nanocomposites (PLA-CNC and PLA-PHB-CNC). The estimated reduction of the (D,L)-lactide form with respect to the meso-lactide followed a similar tendency that the disintegrability test. In this sense, PLA-CNC and PLA-PHB-CNC showed the highest degradation rate suggestive of the polymer shortening by the hydrolysis 148 M.P. Arrieta et al. / Polymer Degradation and Stability 107 (2014) 139e149 Fig. 7. a) Py-GC/MS chromatogram of PLA-PHB-CNCs nanocomposite film, b) mesolactide:lactide ratio loss of PLA, PLA-PHB and nanocomposite films. resulted in a higher amount of lactic acid. It is known that microorganisms prefer the L-lactide form of lactic acid rather than the Dform, thus there was a higher enzymatic degradation of L-lactide influencing the formation of a higher amount of meso-lactide form during the pyrolysis test [48]. 4. Conclusions PLA-PHB based nanocomposite films reinforced with synthesized CNC and functionalized CNCs intended for food packaging were developed and characterized. A reduction of the viscosity molecular weight of PLA, by approximately 5%, occurred due to thermal processing reaching higher reduction with the presence of CNC (10%) and CNCs (15%). Higher detriment of the viscosity molecular weight was observed for PHB after processing in PLA-PHB. Conversely, in the case of PLA-PHB-CNC and PLA-PHB-CNCs, the addition of nanocellulose improve the thermal stability leading to a lesser reduction of PHB viscosity molecular weight and practically unaffected the viscosity molecular weight of PLA. The combination of PHB and the better dispersed CNCs demonstrated the reinforcing effect increase simultaneously the Young modulus and elongation at break, with comparable tensile strength to those of neat PLA. PHB and functionalized CNCs showed a slight UV blocking effect on the virtually transparent PLA matrix. Although the addition of PHB led to a decrease in PLA high transparency, it did not compromise the ultimate optical properties due to the low film thickness achieved. The presence of PHB increased the crystallinity of PLA and its nucleation effect reduced the polymer chains mobility enhancing the oxygen barrier performance of final PLA-PHB blend films while the wettability was reduced. Moreover, functionalized CNCs, which increases the polymer-nanoparticle interfacial adhesion, also reduced the oxygen transmission at the same time as it decreased the surface adhesive forces improving the water resistance. Finally, CNC based nanocomposites showed the highest rate of disintegration in compost, while the surface hydrolysis of functionalized CNCs nanocomposite films started somewhat later and the presence of crystalline PHB delayed the disintegration process. The results of this research suggest that the novel combination of PLA-PHB blends and functionalized CNCs opens a new perspective for their industrial application as short-term food packaging. Acknowledgements This research was supported by the Ministry of Science and Innovation of Spain (MAT2011-28468-C02-01 and MAT201128468-C02-02). M.P. Arrieta thanks Generalitat Valenciana (Spain) for Santiago Grisolía Fellowship (GRISOLIA/2011/007) and cnica de Vale ncia for the Development Support Universitat Polite Programme PAID-00-12 (SP20120120). The Authors acknowledge Gesenu S.p.a. for compost supply. Authors gratefully thank Prof.
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