Chitin nanocrystals and nanofibers as nano-sized fillers into thermoplastic starch-based biocomposites processed by melt-mixing

Chemical Engineering Journal 256 (2014) 356–364

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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej

Chitin nanocrystals and nanofibers as nano-sized fillers into thermoplastic starch-based biocomposites processed by melt-mixing Asier M. Salaberria, Jalel Labidi ⇑, Susana C.M. Fernandes ⇑ Biorefinery Processes Research Group, Department of Chemical and Environmental Engineering, Polytechnic School, University of the Basque Country (UPV/EHU), Pza. Europa 1, 20018 Donostia-San Sebastian, Spain

h i g h l i g h t s  Chitin nanofillers were incorporated in thermoplastic starch matrix by melt-mixing.  Starch nanocomposites showed better mechanical properties than starch matrix.  Starch nanocomposites presented good thermal stability.  Materials with nanofibers showed better properties than those with nanocrystals.  These nanocomposites contribute to a breakthrough in chitin applications.

a r t i c l e

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Article history: Received 7 May 2014 Received in revised form 30 June 2014 Accepted 2 July 2014 Available online 8 July 2014 Keywords: Chitin Nanocrystals Nanofibers Starch Nano-biocomposites Melt-mixing

a b s t r a c t Chitin nano-size fillers, i.e. nanocrystals (CHNC) and nanofibers (CHNF), were incorporated in thermoplastic starch matrix via melt-mixing. The two types of thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) were characterized and compared in terms of morphology, chemical and crystal structure, thermal and mechanical properties, and water resistance. In general, all thermoplastic starch-based nano-biocomposites showed better thermal stability, mechanical properties, and storage modulus than thermoplastic starch matrix without chitin nano-size fillers. This can be linked to the good dispersion of the nano-size fillers in the matrix, resulting from their chemical similarity, and also to the strong nano-size fillers–matrix adhesion by hydrogen bonding interactions. The results showed that the final properties of the nano-biocomposites were dependent of the concentration and type of chitin nanosize filler introduced in the thermoplastic starch matrix. In general, the thermoplastic starch-based nanobiocomposites prepared with chitin nanofibers showed better thermal and mechanical properties and storage modulus than those prepared with chitin nanocrystals. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Chitin is the second most abundant carbohydrate and the most abundant amino-carbohydrate on earth, broadly available as shellfish (crustaceans and mollusks) wastes. It is a linear polysaccharide consisting of b(1 ? 4) linked N-acetyl-D-glucosamine units (Fig. 1a) [1]. In crustaceans exoskeleton, chitin occurs as micro/nanofibrils [2,3] consisting of crystalline and amorphous domains [4] that can be isolated into different nanoforms (nano-whiskers/-crystals [4–9] and nanofibers [9–14]). ⇑ Corresponding authors. Tel.: +34 94 301 71 78; fax: +34 94 301 71 30 (J. Labidi). Tel.: +34 94 301 85 40; fax: +34 94 301 71 30 (S.C.M. Fernandes). E-mail addresses: [email protected] (J. Labidi), [email protected] (S.C.M. Fernandes). http://dx.doi.org/10.1016/j.cej.2014.07.009 1385-8947/Ó 2014 Elsevier B.V. All rights reserved.

The low density, large surface, hydrophilicity, chemical reactivity, and biodegradable and biocompatible nature make chitin nanoforms unique candidates as reinforcing nano-size fillers in polymer nanocomposites [4]. The use of chitin whiskers in reinforcing thermoplastic nanocomposites was first reported by Paillet and Dufresne in 2001 [15] where chitin whiskers were employed as nano-size fillers in poly(styrene-co-butyl acrylate) matrix. Thereafter, various studies have been done using chitin nano-size fillers in different polymeric matrixes, such as poly(caprolactone) [16], natural rubber [7,17,18], soy protein isolate [19], poly(vinyl alcohol) [20–22], chitosan [23,24] and starch [25]. Despite these previous studies, contrarily to cellulose nanowhiskers and starch nanoparticles, the use of chitin nanoforms in nano-biocomposites has not already been widely investigated [26], in particular, their use as nano-sized fillers into starch-based nano-biocomposites.

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(b) OH O

O

OH

HO

(a)

OH

HO

O

HO

Amylose

COCH3 OH

O

n

OH

HN

O O

O

HO

O HO

NH COCH3

O

O OH

n

OH OH

HO

O

O

OH OH

OH O

O

O

HO OH

O HO

OH

O O OH

O HO OH

Amylopectin

n

Fig. 1. Chemical structures of chitin (a) and starch (b, amylase and amylopectin structures).

Starch is an agro-sourced polysaccharide that can be found in different vegetable sources such as wheat, corn, rice, potato and cassava [27]. It consists of D-glucose units and has two main biomacromolecules, i.e. amylose (sparsely branched carbohydrate based on a-(1 ? 4) links) and amylopectin (multi-branched carbohydrate based on a-(1 ? 4) and a-(1 ? 6) bonds) (Fig. 1b) [28]. Generally, amylopectin represents 70–75% of the starch and amylose the 20–25%. In the last years, starch has received considerable attention because of its wide availability, low cost, non-toxicity and biodegradability. Nonetheless, starch has to be enhanced in terms of it mechanical properties, water sensitivity and long-term stability [29]. To improve this weakness, starch in its granular structure, can be processed by gelatinization/melting process to be converted into a melted state knowing as ‘thermoplastic starch’ and used in multiphase systems (blends and composites). Different methods have been used to process starch, including solution casting, internal mixing, extrusion, injection molding and compression molding; and different polymers and nanoparticles have been used in the preparation of the multiphase systems [28,29]. To the best of our knowledge, only one study dealing with the preparation and characterization of starch-based composites reinforced with chitin nanoparticles has been published [25]. These starch-based materials were prepared by casting-evaporation technique and characterized in terms of morphology, structural, thermal and mechanical properties. Regarding the current state-of-the-art, we investigated the role of different chitin nano-size fillers (nanocrystals and nanofibers) on the final properties of thermoplastic starch-based nano-biocomposites processed by melt-mixing. The chemical and crystal structure, the morphology, and the thermal and mechanical properties were evaluated and compared. The use of both chitin nanocrystals and nanofibers as fillers resulted in substantial improvements in the mechanical properties of the thermoplastic starch. Nonetheless, thermoplastic starch-based nano-biocomposites prepared with chitin nanofibers displayed better results than those processed with chitin nanocrystals. 2. Materials and methods

(Chile). Corn starch (S, P99.0%), glycerol (99.5%), anhydrous NaOH pellets, HCl (37% w/w), acetone and ethanol were purchased from Sigma–Aldrich. All reagents were used as received without further purification. 2.2. Extraction of a-chitin from lobster wastes

a-Chitin was extracted from lobster wastes according to previous approaches [30]. Summarizing, the proteins were first removed using a 2 M NaOH solution for 24 h at 25 ± 5 °C under vigorous stirring; subsequently, the minerals (CaCO3) were isolated in HCl (2 M) for 3 h at 25 ± 5 °C; and finally, the pigments and lipids were extracted using acetone followed by ethanol for 6 h under reflux at 30 ± 5 °C. The obtained a-chitin was filtered and washed with distilled water. The resulting a-chitin was dried at 60 ± 5 °C overnight in an oven. The degree of N-acetylation was found to be 96% by 13C NMR. 2.3. Isolation of a-chitin nano-size fillers 2.3.1. Nanocrystals: acid-hydrolysis of a-chitin a-Chitin nanocrystals (CHNC) were isolated by acid-hydrolysis of the obtained a-chitin using 3 M HCl at 100 ± 5 °C for 90 min under vigorous stirring and refluxing, aiming to hydrolyze the amorphous regions of the chitin. The ratio of HCl to chitin powder was 30 mL g1 [5]. After acid-hydrolysis, the obtained suspension was dispersed in distilled water and washed by centrifugation. The suspension was then transferred to dialysis membranes (SpectraPor 12 000–14 000 MWCO regenerated cellulose dialysis membranes from Spectrum Laboratories) and dialyzed for 5 days, changing deionized water every 12 h. To disintegrate remaining chitin nanocrystal aggregates and to obtain a stable and homogeneous suspension, the sample was subjected to ultrasonic treatment for 10 min (Vibracell 75043 from Bioblock Scientific). Finally, a-chitin nanocrystals were filtered to get a final suspension around 4 wt% and stored at 4 °C before used. The resulting CHNC presented rod-like morphology with average diameter of 60 nm and length of 300 nm. The degree of acetylation of the CHNC was estimated to be 92% by 13C NMR spectroscopy and crystallinity index to be 89% using Focher et al. method [31].

2.1. Materials Cervimunida johni lobster (known as yellow lobster) wastes in the form of powder were kindly supplied by Antartic Seafood S.A.

2.3.2. Nanofibers: high pressure homogenization of a-chitin a-Chitin nanofibers (CHNF) were obtained using a dynamic high pressure homogenizing (GEA Niro Soavi S.p.A, Italy) following

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Salaberria et al. approach [14]. In short, extracted dry a-chitin was first dispersed in distilled water at 1 wt% and mechanically pretreated, and then, the suspension passed through the dynamic high pressure homogenizing operating at 1000 bar. Obtained a-chitin nanofibers were filtered to get a final suspension around 4 wt% and stored at 4 °C before used. CHNF presented long and highly entangled nano-size fibrils morphology with average widths of 90 nm and lengths of 5 lm. The degree of N-acetylation was found to be 97% by 13C NMR and crystallinity index to be 85% using Focher et al. method [31]. 2.4. Processing of thermoplastic starch-based nano-biocomposites The thermoplastic starch-based nano-biocomposites were processed by melt-mixing and molding techniques. Briefly, different amounts of CHNF or CHNC (5, 10, 15 and 20 wt%, relative to dry starch mass) were first dispersed in a solution containing distilled water (25%) and glycerol (30%) and ultrasonicated for 10 min before adding starch granules (45%). The mixtures were then extruded twice using a HAAKE MiniLab micro compounder (Thermo Electron Corporation) at 120 ± 2 °C and 60 rpm. Finally, the thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF, Table 1) were molding in an injection molding machine (HAAKE Minijet II, Thermo scientific) according to the standard norm ISO 527-2-5A (2012). Before characterization, all composites were kept in a conditioning cabinet at 50% relative humidity (RH) at 25 °C for a week to ensure the stabilization of their water content.

SEM micrographs of thermoplastic starch-based nano-biocomposites’ fractured cross-section were obtained using a Carl Zeiss Ultra Plus field emission scanning electron microscope (FE-SEM). Samples were prepared applying a thin gold layer. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was used to analyze the chemical structure of the resulting thermoplastic starch-based nano-biocomposites. ATR-FTIR spectra were recorded on a Nicolet Nexus 670 equipped with a KRS-5 crystal of refractive index 2.4 and using an incidence angle of 45°. The spectra were taken in a transmittance mode in the wavenumber range of 750–4000 cm1, with resolution of 4 cm1 and after 128 scan accumulations. X-ray diffraction patterns were measured with a Philips X’pert Pro automatic diffractometer using Cu-Ka radiation (operating at 40 kV and 40 mA) over the angular range of 5–40°. Table 1 Samples identification and CHNC and CHNF content in the thermoplastic starch-based nano-biocomposites; and thermogravimetric features of thermoplastic starch matrix (S), a-chitin nanocrystals (CHNC) and nanofibers (CHNF), and thermoplastic starchbased nano-biocomposites (S/CHNC and S/CHNF).

S CHNC S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20 CHNF S/CHNF5 S/CHNF10 S/CHNF15 S/CHNF20

Thermogravimetric analysis (TGA) assays were carried out in a TGA/SDTA 851 Mettler Toledo instrument. Samples were heated at a constant rate of 10 °C min1 from room temperature to 900 °C under a nitrogen atmosphere of 20 mL min1. The thermal decomposition temperature was taken as the onset of significant (P0.5%) weight loss, after the initial moisture loss. 2.7. Mechanical properties Tensile tests were performed under ambient conditions using a Material Testing Systems (MTS Insight 10) device using a load cell of 10 kN and a deformation rate of 3 mm min1. Tensile strength, tensile modulus, and elongation at break were calculated using MTS TestWorks 4 software. The results presented are an average of 10 determinations. 2.8. Dynamic mechanical analysis (DMA) DMA measurements were performed in an Eplexor 100 N analyzer (Gabo equipment), working in tension mode at 1 Hz with a pre-load of 0.05 N (maximum deformation of 0.02). The temperature was raised from 100 to 150 °C, and measurements were carried out with a heating rate of 2 °C min1. The results presented for each sample (25  10  1.2 mm, prepared in an injection molding machine) are an average of at list three measurements. 2.9. Water uptake

2.5. Morphological, chemical and crystal characterization

Sample

2.6. Thermal stability

Chitin nano-size fillers

Thermogravimetric featuresa

Nanocrystals (wt%)

Nanofibers (wt%)

Tdi (°C)

Tdm (°C)

– – 5 10 15 20 – – – – –

– – – – – – – 5 10 15 20

252 242 237 230 223 222 247 257 259 262 263

285 294 281 273 274 270 299 286 288 289 288

a Where Tdi is the initial degradation temperature and Tdm is the maximum degradation temperature.

Thermoplastic starch-based nano-biocomposites specimens (17  10  3.3 mm) were completely immersed in distilled water at room temperature to assess their water uptake by swelling. After 5, 15, 30, 45, 60, 75, 90, 150 and 210 min the samples were taken out, their surfaces wiped dry, weighed and reimmersed. The mass gain at each time (W, water uptake) was calculated as:

W ð%Þ ¼

Wt  W0  100 W0

where W0 is the sample’s initial mass and Wt is the sample’s mass after immersion time, t. 3. Results and discussion 3.1. Morphological and structural analysis Two types of thermoplastic starch nano-biocomposites using achitin nanocrystals (S/CHNC) and a-chitin nanofibers (S/CHNF) were successfully processed by melt-mixing approach. One of the advantages in using starch as matrix and chitin as nano-size fillers is their similar polysaccharide chemical structure (Fig. 1), which could benefit the nanofiller-matrix interactions by intermolecular hydrogen bonding. FE-SEM images revealed this strong interfacial adhesion between the two polysaccharides. Fig. 2 shows that both chitin nano-size fillers were effectively incorporated and well dispersed within thermoplastic starch matrix, forming a rough fibril matrix compared to the homogeneous one typically obtained in the absence of chitin nano-size fillers. As shown in Fig. 2 the fragile fractured surfaces of S/CHNC15 and S/CHNF15 exhibit completely different morphologies. S/CHNC15 displays a total impregnation with random oriented nanocrystals without entanglement between them, showing a rather smooth homogeneous surface compared with S/CHNF15, which exhibits a web-like structure within the thermoplastic starch matrix.

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Fig. 2. Normal camera images and FE-SEM (fragile fractured zones) images of thermoplastic starch matrix (S), and thermoplastic starch-based nano-biocomposites containing 15 wt% of a-chitin nanocrystals (S/CHNC15) or nanofibers (S/CHNF15). The magnifications selected for FE-SEM images were 1000  (top) and 30 000  (bottom).

(a) S/CHNF

Transmittance (%)

S/CHNC

1 659

νC=O 4000

3500

1 659

νC=O

1 624 δ NH 1 557

3000

1500

1000

4000

3500

1 624 δ NH 1 557

3000

Wavenumbers (cm-1)

1500

1000

Wavenumbers (cm-1)

(b) S/CHNF

S/CHNC

S

S

S/CHNC5

S/CHNF5 S/CHNC10

S/CHNF10 S/CHNC20

S/CHNF20

CHNC, C.I.= 89 % 10

20

30

40

50

60

CHNF, C.I.= 85 % 70

Diffraction angle 2θ ( )

10

20

30

40

50

60

70

Diffraction angle 2θ ( )

Fig. 3. ATR-FTIR spectra (a) and X-ray diffractograms (b) of thermoplastic starch matrix (S), a-chitin nanocrystals (CHNC) and nanofibers (CHNF), and thermoplastic starchbased nano-biocomposites (S/CHNC and S/CHNF).

Fig. 3a shows the ATR-FTIR spectra of S, CHNC, CHNF and thermoplastic starch nano-biocomposites containing 5, 10, 15 and 20 wt% of each chitin nano-size filler. Starch, CHNC and CHNF showed characteristic spectra corresponding to their polysaccharide chemical structure, including the intense bands at 1005– 1060 cm1, which were ascribed to the CO stretching (mCO) of the COH, CH2OH and COC groups in the pyranose rings of starch and

chitin [17,33]. The incorporation of both CHNC and CHNF within starch matrix was confirmed by the detection of a band at 1557 cm1, ascribed to NH bending (mNH, amide II), and the bands at 1624 and 1659 cm1, corresponding to the carbonyl stretching (mC@O, amide I) [17,33]. This latter signal was overlapped with the absorption band at 1650 cm1 that was assigned to water associated with starch [32]. However, this band was gradually hidden

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with the incorporation of chitin fillers into the starch matrix. The intensities of the mNH and mC@O bands of both thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) were gradually increasing with the incorporation of chitin nano-size fillers, indicating a homogeneous integration into the S matrix, as also implied by the FE-SEM imaging (Fig. 2). Thermoplastic starch showed typical B and V-type X-ray diffraction patterns with characteristic peaks at 2h 16.8° and 2h 12.9°, 19.7° and 22.4°, respectively (Fig. 3b) [34,35]. As shown in Fig. 3b, all X-ray diffractograms of the thermoplastic starch-based nano-biocomposites displayed typical diffraction peaks of thermoplastic starch and diffraction peaks at 2h 9.5°, 19.5°, 20.9° and 23.4°, which are typical of a-chitin nanoforms [14,36]. Interestingly, the increase of CHNC content seemed to favor the retrogradation process, i.e. an increase in the overall crystallinity of the system resulting from the nucleating effect of CHNC. This phenomenon was already early recognized in the literature for the thermoplastic starch-based materials prepared with cellulose nanocrystals [23,37].

additional reason may be the accumulation of glycerol on the surface of chitin nanocrystals. The accumulation of plasticizer in the chitin/amylopectin interfacial zone improves the ability of amylopectin chains to crystallize leading to the formation of possible crystalline zone around the fibrils [39]. Such phenomenon also occurred when cellulose whiskers/nanocrystals were incorporated in thermoplastic starch matrix [40]. In contrast, when CHNF were incorporated into the thermoplastic starch matrix, the nano-biocomposites showed slight improvement of thermal stability (up to 4 °C, Table 1). Actually, the incorporation of CHNF increased the thermal stability of the thermoplastic starch matrix, that was ascribed to the: (i) better thermal stability of CHNF compared to CHNC; (ii) strong interactions between the surface OH and residual NH2 groups of CHNF and the OH groups of starch molecules; and (iii) minor accumulation of glycerol on the surface of chitin nanofibers due to their web-like structure. 3.3. Mechanical properties of the biocomposites

3.2. Thermal properties Thermogravimetric analysis (TGA) of thermoplastic starch, CHNC, CHNF and thermoplastic starch-based nano-biocomposites was carried out to evaluate their thermal stability and degradation profiles (Fig. 4). Thermoplastic starch (S) and all thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) showed two mass losses, at around 100 and 200 °C, related to the volatilization of water and glycerol, respectively. CHNC and CHNF only showed the mass loss, before the onset temperature, related to the water volatilization. In nitrogen atmosphere, TGA tracing of thermoplastic starch exhibited one weight-loss-step with a maximum degradation step at 285 °C, while a-chitin nano-size fillers showed at 294 and 299 °C, for CHNC and CHNF, respectively (Table 1). The maximum degradation rates were assigned to the degradation of the polysaccharides [14,37,38]. The incorporation of CHNC into thermoplastic starch matrix resulted in a decrease in the thermal stability of the resulting nano-biocomposites compared with thermoplastic starch matrix. The maximum degradation temperatures of the S/CHNC range from 281 to 270 °C for S/CHNC5 and S/CHNC20, respectively (Table 1). This decrease in onset temperature (up to 15 °C) with an increase in CHNC content may be due to the decrease in flexibility of amylopectin chains in the presence of crystalline chitin. An

The effect of both chitin nano-size fillers and their content on the large strain behavior of thermoplastic starch and thermoplastic starch-based nano-biocomposites were analyzed up to their failure. The Young’s modulus, the tensile strength, and the elongation at break were determined from the stress–strain curves (Figs. 5 and 6). Tension assays shown in Fig. 5 for the thermoplastic starch matrix displayed a nonlinear stress–strain curve, in which the tension increases gradually in the beginning followed by an extremely slow increase until break (reaching high elongation values (85%), and modest Young’s modulus (85 MPa) and tensile strength (4.4 MPa)). Regarding the thermoplastic starch-based nano-biocomposites, as shown in Fig. 5, the incorporation and increase in the CHNC or CHNF content led to a considerable increase in the mechanical properties, by the incidence of important changes in the stress– strain curves, indicating a more fragile behavior. Young’s modulus and tensile strength increased with increasing CHNC and CHNF content (Fig. 6). For instance, for S/CHNC nano-biocomposites with 5 and 20 wt% of CHNC, Young’s modulus increased from 222 to 390 MPa, whereas the tensile strength increased from 7.1 to 10.8 MPa, respectively. Better results were found with the S/CHNF nano-biocomposites, in which Young’s modulus increased from

100

TGA

90

dTGA

80

S CHNC S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20

Weight loss (%)

70 60 50 200

Temperature (ºC)

30 20 10

dTGA S CHNF S/CHNF5 S/CHNF10 S/CHNF15 S/CHNF20

70

400

40

TGA

80

Weight loss (%)

90

100

60 50 200

40

400

Temperature (ºC)

30 20 10

S/CHNC

0

S/CHNF

0 200

400

Temperature (ºC)

600

800

200

400

600

800

Temperature (ºC)

Fig. 4. Thermogravimetric curves (TGA) and derivative curves (dTGA) of thermoplastic starch matrix (S), a-chitin nanocrystals (CHNC) and nanofibers (CHNF) and thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) containing 5, 10, 15 and 20 wt% of chitin nano-size fillers.

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15

S S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20

S S/CHNF5 S/CHNF10 S/CHNF15 S/CHNF20

S/CHNF Tensile strength (MPa)

Tensile strength (MPa)

S/CHNC

10

5

10

5

0

0 0

20

40

60

80

100

0

20

40

60

80

100

Elongation at break (%)

Elongation at break (%)

Fig. 5. Stress–strain diagram of thermoplastic starch matrix (S) and thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) containing 5, 10, 15 and 20 wt% of chitin nano-size fillers.

500

100

80

16

400 350 300 250 200 150

Elongation at break (%)

Tensile stregth (MPa)

14 12 10 8 6 4

0

S/

S/

60

40

20

0

CH S N C H C5 NC S/ CH 10 NC S/ CH 15 NC 2 S/ CH 0 NF S/ CH 5 NF S/ CH 10 NF S/ CH 1 5 NF 20

0

CH S N C H C5 NC S/ CH 10 NC S/ CH 1 5 NC 2 S/ CH 0 NF S/ CH 5 NF S/ CH 10 NF S/ CH 15 NF 20

50

2

S/

100

S/

Young modulus (MPa)

450

CH S N C H C5 NC S/ CH 10 NC S/ CH 15 NC 2 S/ CH 0 N S/ CH F5 NF S/ CH 1 0 NF S/ CH 1 5 NF 20

18

S/

20

550

S/

600

Fig. 6. Young’s modulus, tensile strength and elongation at break of thermoplastic starch (S) and thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) containing 5, 10, 15 and 20 wt% of chitin nano-size fillers.

330 to 520 MPa and the tensile strength increased from 9.9 to 15.0 MPa in the nano-biocomposites with 5 and 20 wt%, respectively. Consequently, the incorporation of CHNC or CHNF in the thermoplastic starch matrix caused a considerable decrease in the elongation at break in the nano-biocomposites. In general, the mechanical performance of the resulting thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) is the result of: (i) the formation of a rigid network of the CHNC or CHNF (due to the interaction among the chitin nano-size fillers by intra- and intermolecular hydrogen bonds); (ii) the mutual entanglement between the chitin nano-size fillers and the starch matrix; and (iii) the efficient stress transfer from the matrix to the chitin nano-size fillers [25,26]. In addition, the mechanical behavior of the S/CHNC can also be the result of an increase in the crystallinity of the system resulting from the nucleating of the CHNC. In the case of S/CHNF, the mechanical properties can be essentially attributed to the percolation effect because of the resulting long and highly entangled nano-size fibrils with high aspect ratio (L/ d = 60) leading to stronger networks (Fig. 2). These results are in good agreement with previous work related to the reinforcement of composite materials with cellulose nanosize fillers and chitin nanocrystals [25,26,41].

3.4. Dynamic mechanical analysis Fig. 7 shows the storage modulus (E0 ) and tan delta profiles as a function of temperature for thermoplastic starch matrix and thermoplastic starch-based nano-biocomposites filled with different amounts of CHNC or CHNF. As expected, the storage modulus (related to the materials stiffness) of all starch-based nano-biocomposites was higher than thermoplastic starch matrix, and increased with the incorporation of both CHNC and CHNF. Nonetheless, starch-based nano-biocomposites filled with CHNF showed higher storage modulus values than those prepared with CHNC. These results were in good agreement with the tensile test assays referred before (Fig. 6). Chang et al. [25] also verified a positive effect on the storage modulus with the introduction of chitin nanoparticles on glycerol plasticized-potato starch matrices. The tan delta curves (Fig. 7, gray profiles) for thermoplastic starch and thermoplastic starch-based nano-biocomposites revealed the biphasic nature of the materials. The tan delta is sensitive to the molecular motion and its peak represents the glass transition temperature (Tg). The lower transition (named b) was due to a glycerol-rich phase (also known as starch-poor phase); whereas the higher transition (named a) was attributed to the starch-rich phase

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A.M. Salaberria et al. / Chemical Engineering Journal 256 (2014) 356–364 0,8 16000 14000

0,8 16000

S S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20

S/CHNC

S S/CHNF5 S/CHNF10 S/CHNF15 S/CHNF20

14000 0,6

12000

0,6

12000

10000

E’

6000

0,4

8000

E’

Tan delta

6000

0,2

4000

0,2

4000

2000

Tan Delta

Tan delta

8000

Tan Delta

10000 0,4

E' (MPa)

E' (MPa)

S/CHNF

2000 0,0

0

-90

-60

-30

0

30

60

90

120

0,0

0

-2000

-2000

150

-90

-60

-30

Temperature (ºC)

0

30

60

90

120

150

Temperature (ºC)

Fig. 7. Temperature dependence of storage modulus (black profiles) and of tan delta (gray profiles) of thermoplastic starch matrix (S) and thermoplastic starch-based nanobiocomposites (S/CHNC and S/CHNF) containing 5, 10, 15 and 20 wt% of chitin nano-size fillers.

dispersed in the thermoplastic starch matrix [39]. The increase of the Tg in starch-rich domains in the presence of CHNC or CHNF could be explain by the strong affinity between the OH groups of starch molecules and the surface OH and residual NH2 groups of chitin nano-size fillers. This affinity could result in a limited molecular motion of starch molecules in contact with the chitin nanosize fillers affecting the flexibility of the thermoplastic starch matrix (as demonstrated before).

Table 2 Glass transition temperatures related with transitions of glycerol-rich (Tg,b) and starch-rich (Tg,a) domains of thermoplastic starch-based nano-biocomposites. Sample

Tg,b (°C)

Tg,a (°C)

S S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20 S/CHNF5 S/CHNF10 S/CHNF15 S/CHNF20

48.1 47.6 47.3 46.0 47.4 45.2 46.4 48.7 47.2

68.6 64.6 75.0 74.1 75.7 74.8 73.6 73.5 74.8

3.5. Water uptake The mass gain of water as a function of immersion time for all thermoplastic starch-based nano-biocomposites filled with CHNC or CHNF is shown in Fig. 8. The immersion time of samples in water was limited to 210 min because after this time samples started to break down. Interestingly, S/CHNC and S/CHNF nano-biocomposites absorbed water following different trends. In the case of the S/CHNC nano-biocomposites, all samples showed higher extent of water absorption than thermoplastic starch matrix; while the water absorption capacity of the S/CHNF nano-biocomposites was dependent of the percentage of CHNF in the thermoplastic starch matrix. S/CHNF nano-biocomposites filled with 15% and 20% of CHNF showed higher resistance to water absorption than

[39]. The glass transition temperatures associated with the two phases (Tg,b and Tg,a) of the materials are listed in Table 2. The lower transition (Tg,b) of the nano-biocomposites was not affected by the incorporation of the different concentrations of CHNC or CHNF. Nonetheless, the Tg of the starch-rich fraction (Tg,a), related to the glass transition of thermoplastic starch, shifted to higher temperatures (ca. 7 °C) in the presence of both chitin nano-size fillers. This behavior revealed that CHNC and CHNF interacted with starch-rich domains better than with glycerol-rich domains when

100

100

S S/CHNC5 S/CHNC10 S/CHNC15 S/CHNC20

80

Water uptake (%)

Water uptake (%)

80

S S+CHNF5 S+CHNF10 S+CHNF15 S+CHNF20

S/CHNC

60

40

20

S/CHNF

60

40

20

0 0

30

60

90

120

Time (min)

150

180

210

0 0

30

60

90

120

150

180

210

Time (min)

Fig. 8. Water uptake of thermoplastic starch matrix (S) and thermoplastic starch-based nano-biocomposites (S/CHNC and S/CHNF) containing 5, 10, 15 and 20 wt% of chitin nano-size fillers, as a function of time.

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thermoplastic starch matrix, whereas those filled with 5% and 10% of CHNF exhibited lower resistance than thermoplastic starch matrix (Fig. 8). Usually the incorporation of polysaccharide nanofillers in starch-based matrices improves the moisture resistance of the final materials, because these nanoentities represent a resistance for the diffusion of water molecules along the nanofilledmatrix [26]. Contrarily, in this study, the presence of the residual NH2 groups, that present more affinity for water than OH groups, at the surface of the nanocrystals/fibers of chitin facilitated water diffusion through the matrices. Nonetheless, in the case of S/ CHNF15 and S/CHNF20 nano-biocomposites, the presence of a significant amount of CHNF, with a web-like morphology, in the thermoplastic starch matrix was more important than the presence of the surface residual NH2 groups. Consequently, geometrical impedance for water molecules diffusion through thermoplastic starch matrix was created by this network structure, and an improvement of moisture resistance was observed.

4. Conclusion Thermoplastic starch-based nano-biocomposites with good thermal stability, mechanical strength and storage modulus were successfully processed by the introduction of chitin nanocrystals or nanofibers via melt-mixing approach into thermoplastic starch matrix. The introduction of chitin nano-size fillers and their good dispersion into thermoplastic starch matrix were confirmed by ATR-FTIR and FE-SEM analysis. The improvement in the mechanical properties and storage modulus, and the good thermal stability of the resulting nano-biocomposites may be linked not only to the good nano-size fillers dispersion in the matrix, but also to the strong nano-size fillers– matrix adhesion by hydrogen bonding interactions. The mechanical property of the nano-biocomposites can be adjusted as a function of the nano-size fillers load, and as a function of the nano-size fillers morphology. CHNF displayed better mechanical properties than CHNC because of their high aspect ratio and entangled nano-size fibrils. The water sensitivity of the thermoplastic starch-based nano-biocomposites can be controlled by the nature and amount of chitin nano-size fillers introduced in the thermoplastic starch matrix. Acknowledgments The authors thankful for the financial support from the Department of Education, Universities and Investigation of the Basque Government through project IT672-13. The authors also acknowledge the technical and human support of General Research Services (SGIker) from the UPV/EHU for X-ray and 13C NMR analysis. References [1] C. Peniche, W. Argüelles-Monal, F.M. Goycoolea, Chitin and chitosan: mayor sources properties and applications, in: A. Gandini, M.N. Belgacem (Eds.), Polymers and Composites from Renewable Resources, Elsevier, Amsterdam, 2008, pp. 517–542. [2] C.K.S. Pillai, W. Paul, C.P. Shrama, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (2009) 641–678. [3] P. Chen, A.Y. Lin, J. Mckittrick, M.A. Meyers, Structure and mechanical properties of crab exoskeletons, Acta Biomater. 4 (2008) 587–596. [4] J.B. Zeng, Y.S. He, S.L. Li, Y.Z. Wang, Chitin whiskers: an overview, Biomacromolecules 13 (2012) 1–11. [5] J.D. Goodrich, W.T. Winter, a-Chitin nanocrystals prepared from shrimp shells and their specific surface area measurement, Biomacromolecules 8 (2007) 252–257. [6] N. Butchosa, C. Brown, P.T. Larsson, L.A. Berglund, V. Bulone, Q. Zhou, Nanocomposites of bacterial cellulose nanofibers and chitin nanocrystals: fabrication, characterization and bactericidal activity, Green Chem. 15 (2013) 3404–3413.

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