Industrial Crops and Products 67 (2015) 439–447

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Industrial Crops and Products

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Processing of PLA nanocomposites with cellulose nanocrystalsextracted from Posidonia oceanica waste: Innovative reuse ofcoastal plant

E. Fortunati a,∗, F. Luzia, D. Pugliaa, R. Petruccia, J.M. Kennya,b, L. Torrea

a University of Perugia, Civil and Environmental Engineering Department, UdR INSTM, Strada di Pentima 4, 05100 Terni, Italyb Institute of Polymer Science and Technology, ICTP-CSIC, Juan de la Cierva 3, 28006, Madrid, Spain

a r t i c l e i n f o

Article history:Received 13 October 2014Received in revised form 20 January 2015Accepted 31 January 2015Available online 14 February 2015

Keywords:PlantWaste revalorizationCellulose nanocrystalsPoly(lactic acid)Nanocomposites

a b s t r a c t

Poly(lactic acid) (PLA) nanocomposite films, reinforced with cellulose nanocrystals (CNC) extracted fromPosidonia oceanica plant waste, were produced by solvent casting and their morphological, mechani-cal, thermal, optical and migration properties were studied. Cellulose nanocrystals were successfullyextracted through an optimized chemical treatment, followed by sulphuric acid hydrolysis. The nanocrys-tals were added to the neat polymer at two different weight percentages (1 and 3%wt) using a commercialsurfactant to increase the dispersion of CNC in the biodegradable matrix. All the nanocomposites keptthe optical transparency of the PLA matrix, while morphological investigations underlined the rougherfracture surfaces of the CNC based systems and a more porous structure of the PLA matrix, induced bythe addition of surfactant modified s-CNC. The surfactant favours the cellulose nanocrystal dispersion inthe polymer matrix, remarkably enhancing the nucleation effect for matrix crystallization and producingits plasticization. The migration levels for all the studied nanocomposites were well below the legislativelimits required for their use as food packaging materials. The successful production of biodegradablenanocomposites incorporating cellulosic sources from biomass waste suggests the possibility of usingthese new bio-nanocomposites in industrial applications.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

In the last few years, biodegradable polymers have been investi-gated and considered as an alternative to non-degradable matrices,in order to develop new environmental friendly materials able toaddress the problems generated by plastic waste. Poly(lactic acid)(PLA) is one of the most important organic candidates for food pack-aging (Averous, 2004); this polymer is a thermoplastic polyesterderived from renewable resources, such as the fermentation ofstarch and other polysaccharides and represents a valid alterna-tive to petrochemical-derived products (Fortunati et al., 2012).Bio-based polymers have a lower negative environmental impactthan traditional plastics. Products realised when using a PLA matrixare biodegradable, compostable and may be dumped in ideal con-ditions since they completely disappear in less than one month(Jonoobi et al., 2010; Oksman et al., 2003). In last few years, PLA isbecoming more and more popular due to its superior transparency,

∗ Corresponding author. Tel.: +39 744492921; fax: +39 744492950.E-mail address: elena.fortunati@unipg.it (E. Fortunati).

high mechanical properties and easy processability with respectto other green polymers (Arrieta et al., 2014a,b). PLA is an eco-nomically feasible material (Auras et al., 2004) and one of the mostinteresting biomaterials approved by the US Food and Drug Admin-istration (FDA) as a food contact substance; it is used as packagingfor some short shelf-life applications (Arrieta et al., 2013; Hwanget al., 2012) and it is also used in rigid and flexible food packagingapplications (Boonyawan et al., 2011). Furthermore, PLA is usedto produce disposable cutlery (plates, salad cups, drinking cups,lids and drinking straws), film packaging and bags (Auras et al.,2005). However, when compared to equivalent petroleum basedpolymers used in food applications, PLA suffers the limitationsof lower water permeability (necessary for fresh food packaging),poor oxygen barrier characteristics and relatively poor thermal andmechanical properties (Petersen et al., 2001). Therefore, the devel-opment of nanocomposites represents a valid method to increasethe physical properties of biodegradable polymers, without affect-ing their transparency (Fortunati et al., 2012a). Among the manynanoparticles adopted as nano-reinforcements for bio-based poly-mers, cellulose nanocrystals are gaining more and more interest asthey represent a feasible method for increasing the properties of a

http://dx.doi.org/10.1016/j.indcrop.2015.01.0750926-6690/© 2015 Elsevier B.V. All rights reserved.

440 E. Fortunati et al. / Industrial Crops and Products 67 (2015) 439–447

biomaterial suitable for food packaging (Sturcová et al., 2005). Cel-lulose nanocrystals (CNC) can be obtained in the form of rigid rodmonocrystalline domains with diameters ranging from 1 to 100 nmand from 10 to 100 nm in length (Matos Ruiz et al., 2000). In general,the nanocrystal aspect ratio (diameter/length) can vary from 1:1 to1:100 and the dimensions of the CNC depend on the raw materialutilized for their extraction and the intensity of the chemical pro-cess for their production (Cranston and Gray, 2006; Fortunati et al.,2013a,b). CNC have a crystalline structure (Fortunati et al., 2012b)and an elastic modulus ranging at around 150 GPa (Cavaille et al.,2000). However, cellulose nanocrystals are very difficult to use in ananocomposite approach with water insoluble polymers like PLA,because their high ability to produce strong hydrogen bonding.Therefore, CNC have to be transferred from water to an appropri-ate solvent in order to produce and process composite systems. Inthis context, physical modification by commercial surfactant wasfound to be a possible strategy to obtain a better dispersion of CNCin an organic solvent and, consequently, in the used polymer matrix(Petersson et al., 2007).

Posidonia oceanica is a Mediterranean alga that appears as rawmaterial with balls shape (Aegagropili) in many beaches. Somestudies have focused on the potential use of the fibrous wastesof P. oceanica, for example, they have been used as a sourceof fillers for potato starch-based films with interesting results(Khiari et al., 2011). Moreover, fibres from P. oceanica were hot-pressed with wheat gluten protein as a binder-matrix material toobtain polymer composites based on fully renewable resources(Ferrero et al., 2013). The use of these fibres as a reinforce-ment in a polyethylene/maleic anhydride grafted polyethylenematrix was also reported (Puglia et al., 2014). Other research hasfocused on the possibility of extracting high-grade cellulose (97% �-cellulose) from P. oceanica using H2O2 and organic peracids, (Colettiet al., 2013) or on the production of carboxymethyl cellulose frombleached cellulose pulp (Aguir and M’Henni, 2006). Few examplesare reported in literature regarding the extraction and characteri-zation of cellulose crystallites from algae or marine plant (Hanley

et al., 1997; Hua et al., 2014; Revol, 1982; Saritha et al., 2013)while Bettaieb et al. (2014) reported about the preparation andcharacterization of cellulose nanostructures (both nanocrystals andnanofibrils) from P. oceanica.

Based on this background, the extraction of CNC from P.oceanica by acid hydrolysis was performed and their novel inte-gration in a biodegradable PLA matrix for the production ofbio-nanocomposites has been proposed in this work.

P. oceanica is characterized by relatively high amounts ofethanol/toluene extractives (10.7%), ashes (12%) and 40% of cellu-lose content. Moreover, lignin content is relative high (Khiari et al.,2010). Therefore, the extraction of high crystalline cellulose rep-resents a relevant challenge for P. oceanica waste. As a result, theresearch reported in this paper focuses on the optimization of theextraction of CNC from P. oceanica and their use for the develop-ment of fully compostable and biodegradable PLA matrix films forfood packaging applications. The final goal of the research was toassess the production of a polymer film with good mechanical prop-erties whilst at the same time maintaining the optical transparencyand thermal stability of the neat polymer. In order to increase thedispersion of cellulose nanocrystals in the PLA matrix, the surfaceof the nanocrystals was modified using a commercial surfactant,according to previous results obtained by Fortunati et al. (2012).Thereafter, the morphological, thermal, mechanical and migrationproperties of the PLA based nanocomposites containing surfactantmodified s-CNC was compared with the results obtained with theunmodified CNC based PLA formulations.

2. Experimental

2.1. Materials

Poly(lactic acid) (PLA) in the form of fibres (specific gravity of1.25 g cm−3) was supplied by MiniFibres, Inc. (USA). P. oceanicawaste raw material was collected by Aitex (Alcoy, Alicante, Spain).All the chemical reagents were supplied by Sigma–Aldrich® andused as received.

Fig. 1. (a) Visual image of Posidonia oceanica raw material, (b) FESEM investigation and diameter distribution of unbleached fibres, (c) visual observation of bleached fibres,(d) morphological investigation and diameter distribution of bleached fibres.

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2.2. Fibre chemical pre-treatment

P. oceanica raw material (Fig. 1a) was washed and rinsed severaltimes in distilled water in order to eliminate sand and other soilcontaminants, after which, it was dried in an oven at 80 ◦C for 24 h.Then they were chopped into 5–10 mm fragments and a de-waxingstep in a toluene/ethanol mixture (2:1 volume/volume) for 6 h wascarried out followed by a filtration and a washing procedure withethanol for 30 min.

Two bleaching treatments were applied for cellulose extraction.The fibres were treated with a 0.7% wt/v NaClO2 solution and thenthe fibres were boiled for 2 h (fibre/liquor ratio 1:50). Thereafter thepH of the solution was lowered to ca. 4 by adding acetic acid. Then,a 5%wt/v sodium bisulphate solution treatment was carried out(Chattopadhyay and Sarkar, 1946; Fortunati et al., 2014a, 2013a,b).The obtained holocellulose was finally treated with a 17.5%wt/vNaOH solution. After filtration and washing, the obtained materialwas dried at 60 ◦C in a vacuum oven.

2.3. Cellulose nanocrystal extraction and modification

Cellulose nanocrystals (CNC) were prepared from bleached P.oceanica fibres by means of acid hydrolysis (64%(wt/wt) of sul-phuric acid at 45 ◦C for 30 min) following the recipe used byCranston and Gray (2006). The resulting cellulose nanocrystalaqueous suspension, obtained after centrifugation, dialysis andultrasonic treatment was approximately 0.3% (wt/wt) and the yieldwas ca. 14%.

In order to increase the dispersion of the CNC in the polymermatrix and to enhance the final properties of the nanocompositesystems, the obtained cellulose nanocrystals were modified witha surfactant, (Beycostat A B09 - CECCA S.A.) (Heux et al., 2000),which was an acid phosphate ester of ethoxylatednonylphenol. Themodified CNC (s-CNC) were prepared adding the surfactant in theproportion of 1/4 (wt/wt) directly to the CNC suspension.

Finally, before freeze drying, the pH of both cellulose nanocrys-tal suspensions, unmodified and surfactant modified, was raised toapproximately 9 by using a 0.25%wt NaOH solution, aimed at assur-ing the thermal stability of the produced nanocrystals (Peterssonet al., 2007; Fortunati et al., 2012b).

2.4. Characterization of bleached fibres and cellulose nanocrystals

The morphology of the unbleached P. oceanica fibres extractedfrom the raw material and of the bleached fibres, prepared as previ-ously reported (Fortunati et al., 2014a), was investigated by meansof field emission scanning electron microscopy (FESEM, Supra 25-Zeiss). In order to determine the fibre diameter distribution, FESEMmicrographs of both unbleached and bleached fibres were analysedwith NIS-Elements BR (Nikon) software. Sixty measurements wereperformed to offer a representation of the diameter distribution.

The length and diameter of cellulose nanocrystals were deter-mined using an atomic force microscope (AFM) from DigitalInstruments having a NanoScope III controller with a MultiModehead (Veeco) and an integrated silicon tip/cantilever. AFM phaseand height images were obtained operating in a tapping mode.The diameter of the nanocrystals was measured in the AFM heightimages, assuming a cylindrical shape of nanocrystals.

Thermogravimetric measurements (TGA) of unbleached andbleached P. oceanica fibres and of CNC extracted from bleachedfibres were performed with a Seiko Exstar 6300 TG analyser withheating scans from 30 to 900 ◦C at 10 ◦C min−1 in a nitrogen atmo-sphere.

Fourier infrared (FT-IR) spectra of P. oceanica unbleached,bleached fibres and CNC embedded in KBr discs were recorded

by a Jasco FT-IR 615 instrument in the 400–4000 cm−1 range, intransmission mode.

2.5. PLA nanocomposite film preparation

PLA and PLA nanocomposite films were prepared by meansof solvent casting. PLA (0.75 g) was dissolved in 25 mL of CHCl3with stirring at room temperature (RT). The obtained solution wascast onto a Petri dish and then dried for 24 h at RT. PLA basednanocomposites containing 1%wt and 3%wt of unmodified (CNC)and surfactant modified (s-CNC) cellulose nanocrystals were man-ufactured. After freeze-drying procedure, the organic solvent wasadded to unbleached CNC or s-CNC and exposed to sonication(Vibracell, 750 W) for 1 min in an ice bath. Furthermore, for thepreparation of the PLA nanocomposite films, a specific amount ofCNC or s-CNC suspension was added to the previously preparedpolymer solution and then cast at RT. Films with a thickness ofapproximately 50 �m were obtained for each formulation.

2.6. Characterization of PLA nanocomposite films

The transparency of the PLA based nanocomposite was inves-tigated by absorption measurements. A PerkinElmer Instruments(Lambda 35) UV–vis spectrophotometer, working at a wavelengthinterval ranging from 250 to 900 nm, was used in order to investi-gate the optical properties of the produced composites.

The microstructure of the PLA nanocomposite fractured surfaceswas investigated by scanning electron microscope, FESEM, aftergold sputtering of the surfaces.

Differential scanning calorimeter (DSC, Mettler Toledo 822/e)investigations were done from −25 to 210 ◦C, at 10 ◦C min−1,applying two heating and one cooling scan. Melting and cold crys-tallization temperatures and enthalpies (Tm, Tcc and �Hm, �Hcc)were determined from the first and second heating scan, whilecrystallization phenomena were analyzed during the cooling scan.The glass transition temperature (Tg) was registered for each scan.Three samples were used to characterize each formulation.

Thermogravimetric analysis (TGA - Seiko Exstar 6300) from30 to 600 ◦C at 10 ◦C min−1 under a nitrogen atmosphere wasperformed for each sample. Neat PLA and PLA nanocompositesmechanical properties were evaluated through tensile tests per-formed on rectangular samples (50 × 10 mm2), according to the UNIISO 527 testing standards, with a load cell of 500 N, a crossheadspeed of 1 mm min−1 and an initial gauge length of 25 mm. Thespecimens were dried in a vacuum oven at 40 ◦C for 72 h before thetest. The elastic modulus (E), the yield stress and strain (�Y, �Y), thetensile strength (�b) and elongation at break (�b) were calculatedfrom the resulting stress–strain curves.

2.7. Overall migration tests of PLA nanocomposites

Taking into account the possible application of the produced PLAnanocomposites for fresh-food packaging, overall migration testswere run in triplicate in simulant A (10% (v/v) ethanol/water solu-tion) and alternative simulant D2 (isooctane). Rectangular strips of10 cm2 in 10 mL of food simulants were used. Samples were kept inthe ethanol solution in a controlled atmosphere at 40 ◦C for 10 days,according to European Commission Regulation EU 10/2011, whilesamples in isooctane were kept at 20 ◦C for 2 days, according toEuropean Standard EN 1186-1/2002. At the end of the experiment,films were removed and the simulants evaporated. The residueswere weighed with an analytical balance with ±0.01 mg precisionand the migration value in �g kg−1 of each simulant was deter-mined.

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Fig. 2. (a) Photograph and (b) AFM investigation of extracted cellulose nanocrystals. (c) Thermal and (d) chemical properties of unbleached fibres, bleached materials andCNC from Posidonia oceanica.

3. Results and discussion

3.1. Characterization of bleached fibres and cellulosenanocrystals from P. oceanica

The dimension and microstructure of unbleached and bleachedfibres were investigated by visual observation and FESEM investi-gation, while the morphological features of cellulose nanocrystals(CNC) extracted by means of the hydrolysis procedure were ana-lysed with AFM. The results are summarized in Figs. 1 and 2.Fig. 1a,c give an indication of the appearance of the unbleachedand bleached fibres. The mean diameter of the unbleached fibresobtained from the raw material was 84 ± 26 �m (Fig. 1b). Afterthe application of alkali and bleaching pre-treatments, a whiten-ing process was observed as a consequence of the elimination oflignin based components (Fig. 1c). Moreover, the obtained fibresappeared separated into micro-sized filaments, showing a diame-ter reduction with respect to that of the initial ones, with a meanvalue diameter of 7 ± 2 �m (Fig. 1d). The bleached fibres showeda smooth and clean surface and evident defibrillation occurredwith respect to the unbleached structures, justifying the observeddiameter reduction (consequence of the removal of the non-fibrouscomponents from the fibre surface). After the chemical treatment,the obtained microfibres were hydrolyzed with sulphuric acid inorder to produce cellulose nanocrystals.

Fig. 2a shows a white powder which is the CNC after the freeze-drying process. The AFM image (Fig. 2b) highlights the effectivenessof the acid hydrolysis treatment (yield of the reaction: 14%), con-firming the fact that the aqueous suspension contains individualcellulose nanocrystals with an acicular structure, (180 ± 28) nm inlength a diameter of (4.9 ± 1.3) with a consequent aspect/ratio of36.7. In a recent published article, Bettaieb et al. (2014) have sim-ilarly found that CNC from P. oceanica balls exhibited an averagediameter of 8 nm and a length of 276 nm with an aspect ratio of

34.6, confirming our results. Moreover, the here measured dimen-sions confirmed us that CNC extracted from P. oceanica plant can becompared, from the morphological point of view, with nanocrystalsobtained from other plant fibers, so it is expected that they couldhave an analogous efficient reinforcing effect in a polymeric matrix,as discussed by Bras et al. (2011). The thermogravimetric analysis ofthe unbleached fibres, bleached fibres, and of the hydrolysed cel-lulose nanocrystals from P. oceanica was also performed and therelated results are reported in Fig. 2c. The FT-IR spectra of the samematerials are reported in Fig. 2d. As already observed by Puglia et al.(2014), the first step of the thermal degradation of the unbleachedfibres is characterized by moisture removal at a temperature below150 ◦C, while above 150 ◦C and up to 450 ◦C, the chemical bonds ofthe macromolecules break up to release the volatile compounds.Then, the hemicellulose and cellulose start to break up and releasemore volatiles, with main degradation peaks observed at 294 ◦Cand 335 ◦C, respectively. After bleaching, the derivative curve (DTG)peak attributed to moisture uptake at low temperatures is reduced,while a much narrower main degradation peak of the bleached P.oceanica fibres appears, indicating a material with a more definedcomposition. The shift of the main peak to a higher temperature(356 ◦C for bleached fibres with respect to 335 ◦C for the unbleachedones) indicates a treated fibre increased thermal stability due tothe reduced amount of non-cellulosic material and the presence ofhigh crystalline cellulosic components. As experimentally shown,the peak which occurred due to hemicellulose decomposition dis-appears, indicating its effective removal from the unbleached fibresdue to the bleaching treatment. The results of the thermal degra-dation of CNC extracted from bleached fibres are also reportedin Fig. 2c. From the comparison of the decomposition tempera-tures related to the main degradation peaks (291 ◦C for CNC withrespect to the initial 335 ◦C for unbleached fibres and 356 ◦C forthe bleached fibres), reduced thermal stability of the acid treatedcrystals obtained from bleached fibres is observed and this can be

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Fig. 3. Visual observation (a) and transmittance results (b) of PLA and PLA nanocomposites.

attributed to the presence of sulphate groups remaining from acidhydrolysis (Wang et al., 2007). Moreover, the reduction in the ther-mal stability of CNC may be the result of an increase in the amount ofshort cellulose chains, due to the high specific surface area and freechain end of the nanocrystals obtained by hydrolysis (Kargarzadehet al., 2012).

A FT-IR analysis of the P. oceanica fibres was performed(Fig. 2d). The spectral bands at 3175–3490 cm−1 are assigned toO H stretching intra-molecular hydrogen bonds for cellulose I,2850–2970 cm−1 to C H stretching and the signal at 1511 cm−1

is associated to the aromatic ring present in lignin (Cocozza et al.,2011; Coletti et al., 2013; Izquierdo et al., 2012; Salapare Iii et al.,2013). The FT–IR spectra of bleached fibres and CNC from P. oceanicaconfirmed the results already observed in the thermogravimet-ric analysis: after chemical treatment, the band at 1511 cm−1 wasabsent, confirming the removal of the lignin fraction. In the caseof CNC, the signals at 1167, 1112, 1061, 1023, 893 cm−1, assignedto asymmetric C O C bridge stretching, anhydroglucose ringasymmetric stretching, C O stretching, in-plane C H deforma-tion and C H deformation of cellulose, respectively, changed inintensity. Moreover, the signal at 1377 cm−1, related to the bend-ing vibration of the C O bonds in the polysaccharide aromaticrings, was more evident in the CNC spectrum. It should also beunderlined that the signals at 1430 and 1420 cm−1 (CH2 scissoringmotion), 1334 cm−1 (O H in plane bending), 1316 cm−1 (CH2 wag-ging), 1158 cm−1 (C C ring stretching band), 1061 cm−1 (C O Cpyranose ring stretching vibration) and 893 cm−1 (associated to cel-lulosic �-glycosidic linkages) were more evident in the case of theCNC, confirming the cellulosic I nature of the produced CNC (Nelsonand O’Connor, 1964a,b).

3.2. Characterization of PLA nanocomposites

3.2.1. Transparency and morphological propertiesFig. 3 shows the UV–vis characterization and photos of PLA and

PLA nanocomposite films. PLA is a transparent polymer (transmit-tance of 95% at a wavelength of 700 nm). The transparency studiesshowed that the optical nature of PLA was not influenced by cel-lulose nanocrystal presence, at least up to the highest contentof cellulose studied (3%wt), due to the nano-scaled dispersion ofCNC. However, PLA 1s-CNC and PLA 3s-CNC formulations showedan absorption band centred at 280 nm due to the presence of thesurfactant modified cellulose (Fortunati et al., 2014b). The colour

distribution in the film suggests that the CNC nanocrystals wereuniformly distributed confirming the good CNC dispersion obtainedduring the processing of the PLA nanocomposites.

The fractured surfaces of neat PLA and PLA nanocompositeswere investigated by means of FESEM (Fig. 4), in order to evaluatethe influence of cellulose nanocrystal addition and the effects oftheir modification on the PLA microstructure. A relatively smoothfractured surface was observed for the neat PLA film sample. Arougher fractured surface was instead detected for the unmod-ified CNC based nanocomposites as it is possible to see in theinsert at higher magnification (Fig. 4); the rougher surfaces under-line a more brittle nature of CNC based formulations respect tothe PLA neat film. Moreover, the fractured surface of PLA 3CNCshows higher roughness due to the higher content of CNC respectof PLA 1CNC. Different behaviour was detected for surfactant mod-ified CNC based formulations, which showed a porous structureinduced by the presence of the surfactant and which was more evi-dent in the case of the 3%wt s-CNC based composite. PLA 1s-CNCfracture surface shows a great evidence of uniform dispersion ofmodified cellulose nanocrystals and plastic deformation.

3.2.2. Thermal propertiesThe effects of unmodified CNC and surfactant modified s-CNC

on the thermal properties of the PLA matrix were investigatedusing differential scanning calorimeter (DSC) and thermogravimet-ric (TGA) measurements.

The DSC thermal properties are summarized in Table 1, whilethe thermograms for the cooling and the second heating scan arereported in Fig. 5a and b, respectively. During the cooling scan(Fig. 5a and Table 1), no significant changes were observed inthe glass transition temperature (Tg) of neat PLA and PLA CNCbased formulations, in agreement with a previous published work(Fortunati et al., 2012b). However, a shift to lower Tg values wasobserved in the PLA 1s-CNC system (about 15 ◦C), while a lessintense signal, which was difficult to detect, was registered forthe PLA 3s-CNC formulation. This effect may be attributed to thepresence of the surfactant that acts as a plasticizer of the polymermatrix, reducing the glass transition temperature. These resultsalso confirmed the FESEM investigations, that revealed a moreporous and less brittle fractured surface of the s-CNC based formu-lations. Moreover, a clear increase in the enthalpies related to thecrystallization phenomena was detected during the cooling of thesurfactant modified based nanocomposites. The enhancement of

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Fig. 4. FESEM investigation of fracture surfaces of PLA and PLA nanocomposites.

crystallization enthalpies clearly indicates that the addition of cel-lulose nanocrystals favours PLA crystallization and this behaviourwas more marked in the case of the PLA 3s-CNC system, whichshowed the highest value of crystallization enthalpy.

The behaviour during the second heating scan (Fig. 5b andTable 1) confirms the positive effect of introducing cellulosenanocrystals on the thermal properties of the PLA matrix. Thedouble melting behaviour, with one peak at around 163 ◦C andthe second one at 166 ◦C, observed for neat PLA and for thePLA 1CNC nanocomposite, can be attributed to the formation ofsmall and imperfect crystals during rapid cooling that change intomore stable crystals through melting and recrystallization at low

heating rates or to lamellar species with different perfectiondegrees (Bitinis et al., 2013; Arrieta et al., 2014a,b). Multi-peakmelting was also observed for the other nanocomposites, whichshowed a less intense peak at around 145 ◦C, 150 ◦C and 147 ◦Cfor PLA 1s-CNC, PLA 3CNC and PLA 3s-CNC, respectively, with themain peaks at around 157 ◦C, 161 ◦C and 157 ◦C, respectively. Thepresence of less intense peaks at lower temperatures underlinesthe difference in the capability to re-crystallize of reinforced PLA,more evident in the case of s-CNC based formulations and in thecase of the highest content of cellulose (3%wt). Furthermore, areduction in the cold crystallization temperatures was observedfor the nanocomposites, with a more visible effect in the case of

Table 1Thermal properties of PLA and PLA nanocomposites.

Formulations Cooling scan

Tg (◦C) �Hc (J g−1) Tc (◦C)

PLA 49.9 ± 7.4 1.1 ± 0.2 89.1 ± 0.2PLA 1CNC 50.8 ± 1.1 1.5 ± 0.5 92.7 ± 1.3PLA 1s-CNC 35.9 ± 0.2 8.4 ± 0.9 84.0 ± 0.1PLA 3CNC 47.4 ± 1.4 2.5 ± 0.1 89.7 ± 0.7PLA 3s-CNC 35.7 ± 1.8 88.0 ± 1.3

Second heating scan

Tg (◦C) �Hcc (J g−1) Tcc (◦C) �Hm (J g−1) Tm (◦C)

PLA 52.2 ± 7.0 31.2 ± 4.0 118.6 ± 4.8 37.4 ± 3.9 163.1 ± 2.1PLA 1CNC 54.3 ± 1.2 34.8 ± 3.1 111.5 ± 2.2 37.6 ± 3.2 164.1 ± 1.9PLA 1s-CNC 40.6 ± 0.3 22.0 ± 3.5 91.5 ± 0.5 39.3 ± 2.8 157.2 ± 0.2PLA 3CNC 50.9 ± 0.2 31.9 ± 0.1 99.4 ± 0.1 39.3 ± 2.8 161.0 ± 0.4PLA 3s-CNC 40.4 ± 2.6 157.1 ± 1.6

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Fig. 5. DSC thermograms: (a) cooling scan, (b) second heating scan and (c) derivativeDTG curves of PLA and PLA nanocomposites.

PLA 1sCNC, which showed a shift to lower temperatures at about30 ◦C. Therefore, the ability to re-crystallize increased in filled PLAand especially in the case of s-CNC based composites. Finally, thePLA 3s-CNC presented completely different behaviour during thesecond heating scan. In fact, this formulation does not show a peakrelated to cold crystallization, underlining the fact that the pres-ence of a higher content of surfactant modified cellulose (3%wts-CNC) strongly affects the thermal behaviour of the PLA matrix.The surfactant favours the dispersion of the filler in the polymermatrix and the nucleation effect was remarkably enhanced whena more homogeneous s-CNC dispersion was achieved (Fortunatiet al., 2012b).

The thermal behaviour of PLA based nanocomposites was alsoevaluated by thermogravimetric analysis and the effects of CNCand s-CNC on the PLA properties were analysed. The derivativecurves of the obtained nanocomposites are reported in Fig. 4c. PLAdecomposes in a single step process with an initial degradationtemperature (Tonset) of 270 ◦C and maximum degradation rate tem-perature (Tmax) centred at 332 ◦C. When the CNC were added tothe PLA, the Tmax decreased while increasing the filler fraction andthis reduced thermal stability can easily be attributed to the lowerthermal stability of the cellulose nanocrystals, which are character-ized as already reported, by a maximum degradation rate at aboutaround 291 ◦C (Fig. 2c). The temperatures for the maximum degra-dation rate were 323 ◦C and 313 ◦C for PLA 1CNC and PLA 3CNC,respectively. The s-CNC based nanocomposites showed multi-stepdegradation, due to presence of the surfactant. The first peak isrelated to the degradation of the PLA, while the second one can beattributed to the degradation of the surfactant, as reported in a pre-vious study by Fortunati et al. (Fortunati et al., 2012b). In the caseof the surfactant modified cellulose based formulations, the max-imum degradation of PLA 1s-CNC and PLA 3s-CNC was at 329 ◦Cand 313 ◦C, respectively, with no relevant changes with respect tothe thermal behaviour of the composites with unmodified CNC.

3.2.3. Mechanical behaviourPLA and PLA nanocomposite mechanical properties were eval-

uated by tensile tests and the results are summarized in Table 2. Aspreviously reported (Fortunati et al., 2014a, 2012b), the additionof unmodified CNC produces an increase in Young’s modulus andstrength at break values with respect to the PLA matrix, enhancingthe reinforcement effects induced by the cellulose crystals. Thiseffect is more evident in the case of the 3%wt based formulationthat showed the highest Young’s modulus (1930 MPa). Moreover,a decrease in the �B of the CNC based nanocomposite with respectto the PLA matrix was observed, with a more pronounced effectfor the PLA 3CNC system, confirming the increase in brittlenessinduced by the addition of unmodified CNC. Cellulose nanocrystals,in fact, could cause substantial local stress concentration (Fortunatiet al., 2014a). The surfactant modified s-CNC based formulationspresented a different behaviour. There was an evident decrease inthe Young’s modulus values for the PLA 1s-CNC and PLA 3s-CNCwith a reduction of about 18% and 34%, respectively, in comparisonto the PLA matrix.

Moreover, a relevant increase in the elongation at break wasdetected for PLA 1s-CNC, underlining the positive effect of s-CNCon the plasticization of the PLA matrix. Focusing on the elongationat break values obtained for all PLA based formulations, it is rele-vant to observe that the films show an evident ductile behaviour.The main reason of this behaviour is related to the processingmethodology selected for this work, as previously reported. Rhimet al. (2006), in fact, demonstrated that PLA processed by sol-vent casting shows a ductile behaviour with very high elongationat break due to the residual solvent present in the material thatact as a plasticizer. This effect is maintained and amplified in thenanocomposites, especially for PLA 1s-CNC formulation, and couldbe ascribed to a more efficient dispersion of the functionalized cel-lulose nanocrystals, that guarantees a better mechanical response(Arrieta et al., 2014a,b). Moreover, a high level of deformation atbreak was also obtained for the PLA 3s-CNC (100.9%), confirmingthe positive effect of the modification on the mechanical responseof the nanocomposites. The data obtained through tensile testingvalidated the plasticization effect induced by the s-CNC discussed inthe thermal and morphological sections, suggesting the possibilityof modulating the mechanical properties according to applicationrequests, introducing unmodified CNC or surfactant modified s-CNC to the biodegradable polymer matrix.

446 E. Fortunati et al. / Industrial Crops and Products 67 (2015) 439–447

Table 2Mechanical properties of PLA and PLA nanocomposites.

Formulations �y (MPa) �y (%) �b (MPa) �b (%) EYoung (MPa)

PLA 14.7 ± 5.1 2.3 ± 0.5 16.5 ± 3.1 277.9 ± 33.6 1205 ± 100PLA 1CNC 31.3 ± 1.6 3.0 ± 0.5 27.1 ± 6.3 200.1 ± 53.3 1330 ± 90PLA 1s-CNC 22.9 ± 4.2 2.6 ± 0.6 22.9 ± 1.2 286.1 ± 25.2 993 ± 190PLA 3CNC 42.1 ± 2.0 2.7 ± 0.6 21.9 ± 3.5 100.9 ± 27.9 1930 ± 170PLA 3s-CNC 25.7 ± 4.8 3.1 ± 0.1 20.2 ± 1.9 210.4 ± 48.1 800 ± 100

Fig. 6. Overall migration in 10%(v/v) ethanol and isooctane for PLA and PLAnanocomposites.

3.2.4. Overall migration studiesOverall migration tests with food simulants were carried out

and the results are shown in Fig. 6. The idea was to demonstratethe possible practical application of the produced nanocompositefilms in the food packaging field.

After 10 days of incubation at 40 ◦C in ethanol 10%(v/v), themaximum migration level was 0.11 mg kg−1 of simulant measuredfor the PLA 3CNC formulation. This value is much lower thanthe migration limits for food contact materials, 60 mg kg−1 simu-lant, established by the current European legislation (CommissionRegulation EU 10/2011).

Analyzing the migration values in isooctane, there was an evi-dent effect on the cellulose nanocrystal amount with increasedmigration values for 3%wt CNC based formulations. In this case(isooctane simulant), a maximum level of 0.09 mg kg−1 wasmeasured for the highest percentage of cellulose nanocrystals(PLA 3CNC) but similar high values were also detected for thePLA 3s-CNC formulation. The higher migration values detected for3%wt CNC or s-CNC based formulations can be ascribed to themorphology of these systems, that showed a porous structure (asdiscussed in the morphological section) and are also related to theplasticising effect induced by the presence of the surfactant thatfacilitated the diffusion processes.

However, it should be pointed out that all the migration val-ues for the studied systems are well below the overall migrationlegislative limits, suggesting the possibility to use the proposedformulations for food-packaging.

4. Conclusions

Poly(lactic acid) (PLA) based nanocomposite films reinforcedwith cellulose nanocrystals (CNC) and functionalized cellulose (s-CNC), proposed for industrial application for food packaging, weresuccessfully developed and characterized.

Acicular cellulose nanocrystals (CNC) with dimensions rangingaround 180 nm in length and 5 nm in diameter on average wereisolated by acid hydrolysis from P. oceanica plant wastes with a14% yield and were added to the neat PLA polymer at two differentweight percentages (1–3%wt). In order to increase the dispersion of

the CNC in the biodegradable matrix, the surface of the nanocrystalswas modified by using a commercial surfactant.

All the nanocomposites maintained the optical transparency ofthe PLA matrix, while morphological investigations underlined arougher fractured surface for the CNC based systems and a moreporous structure induced by the surfactant modified nanocelluloses-CNC. The presence of the surfactant on the nanocrystal sur-face favoured their dispersion in the polymer matrix. Data frommechanical tests confirmed the plasticization effect induced bys-CNC, suggesting the possibility of modulating mechanical prop-erties according to application requests, by introducing unmodifiedCNC or surfactant modified s-CNC in a biodegradable polymermatrix. Finally, the migration levels of all the studied nanocompos-ites tested with two food simulants were well below the Europeanlegislative limits.

The present research suggests the possibility of revalorizingwaste biomass to produce high performance, sustainable and low-cost nanocomposite formulations, with perspective applications infood packaging systems.

Acknowledgements

The authors acknowledge the financial support of the SEAMAT-TER European project: Revalorisation of coastal algae wastes intextile nonwoven industry with applications in building noise iso-lation, LIFE11 ENV/E/000600, Funding Program: LIFE+. Call 2011.We also wish to acknowledge the “Materials + Technologies” Groupfrom the University of the Basque Country UPV/EHU, Spain and inparticular Prof. Aitor Arbelaiz for atomic force microscopy investi-gations.

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