Effect of WS2 Inorganic Nanotubes on Isothermal Crystallization Behavior and Kinetics of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)

Article Effect of WS2 Inorganic Nanotubes on Isothermal Crystallization Behavior and Kinetics of Poly(3-Hydroxybutyrate-co-3-hydroxyvalerate)

Tyler Silverman 1, Mohammed Naffakh 1,* ID , Carlos Marco 2 and Gary Ellis 2 ID

1 Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid (ETSII-UPM), José Gutiérrez Abascal 2, 28006 Madrid, Spain; [email protected] 2 Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain; [email protected] (C.M.); [email protected] (G.E.) * Correspondence: [email protected]; Tel.: +34-913-363-164

Received: 15 December 2017; Accepted: 6 February 2018; Published: 9 February 2018

Abstract: Nanocomposites of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and tungsten disulfide inorganic nanotubes (INT-WS2) were prepared by blending in solution, and the effects of INT-WS2 on the isothermal crystallization behavior and kinetics of PHBV were investigated for the first time. The isothermal crystallization process was studied in detail using various techniques, with emphasis on the role of INT-WS2 concentration. Differential scanning calorimetry (DSC) and polarized optical microscopy (POM) showed that, in the nucleation-controlled regime, crystallization rates of PHBV in the nanocomposites are influenced by the INT-WS2 loading. Our results demonstrated that low loadings of INT-WS2 (0.1–1.0 wt %) increased the crystallization rates of PHBV, reducing the fold surface free energy by up to 24%. This is ascribed to the high nucleation efficiency of INT-WS2 on the crystallization of PHBV. These observations facilitate a deeper understanding of the structure-property relationships in PHBV biopolymer nanocomposites and are useful for their practical applications.

Keywords: inorganic nanotubes; PHBV; nanomaterials; morphology; crystallization kinetics

1. Introduction Over recent years, bio-based products have attracted increasing interest due to escalating environmental concerns and diminishing fossil resources [1]. Consequently, there is and has been a growing demand and interest, in both academic and industrial realms, to investigate biopolymers (i.e., polymeric material of non-fossil, biological origin) and develop strategies that can implement them for societal needs. Biopolymers can be broadly classiﬁed into two main categories: agropolymers, such as starch and other carbohydrates, proteins, etc., and biodegradable polymers, such as polyhydroxyalkanoates (PHAs), poly(lactic acid), etc. Biosynthetic polymers such as PHAs are linear, aliphatic polyesters that are produced by a microbial process in a sugar-based medium, where in certain bacteria they act as carbon (energy) storage banks [2]. A family of these materials from over 150 different monomers can be obtained with incredibly diverse properties [3]. Of these, polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), and their copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), are commonly used matrices in bio and eco-composites [4]. Whilst PHB exhibits high stiffness and crystallinity, the incorporation of 3-hydroxyvalerate (HV) groups in a random copolymer with 3-hydroxybutyrate (HB) is a strategy used to increase the ﬂexibility and processing capabilities of the polymer, reducing stiffness, melting point and crystallinity of the copolymer on increasing the HV content [3]. Despite improved thermal and mechanical properties, PHBV still presents some disadvantages, which include a narrow processing

Polymers 2018, 10, 166; doi:10.3390/polym10020166 www.mdpi.com/journal/polymers Polymers 2018, 10, 166 2 of 14 window, a slow crystallization rate, and low values of strain-at-break, along with a higher cost when compared with petroleum-based synthetic polymers [5]. To solve the aforementioned limitations, methods such as physical blending or chemical structure design combined with processing conditions can be applied to improve the performance of PHA products [3,6]. Nanocomposite strategies have been suggested to overcome the inherent shortcomings of biopolymer-based materials, and nano-biocomposites obtained by introducing nanofillers into biopolymers result in very promising materials, manifesting improved thermal and mechanical properties whilst maintaining material biodegradability, without introducing toxicity [6]. These find applications mainly in packaging, agriculture, and biomedical or hygiene devices, and represent an emerging alternative towards environmentally benign and economically viable chemical production [7]. Depending on the processing conditions from the melt into the solid state, biopolymeric materials may partially crystallize into a semicrystalline morphology that affects the aforementioned relevant properties. For this reason, numerous studies have been undertaken to characterize the crystallization behavior and to control the crystallinity, the crystallization kinetics, the spherulitic superstructure, or the crystal polymorphism, employing calorimetry and optical microscopy techniques [3,8]. In particular, calorimetry enables quantification of transition temperatures and enthalpies in isothermal and non-isothermal modes. Lorenzo et al. [8] have suggested a methodology for the minimization of possible errors associated with data manipulation in the measurement and analysis of conventional experimental DSC data. Isothermal crystallization experiments performed by DSC showed an increase in the crystallization kinetics of polycaprolactone (PCL) with increases in carbon nanotubes content as a consequence of the supernucleation effect [9]. Making use of fast scanning chip calorimeters and combining both approaches allowed them to shed further light on fundamental details of the polymer-crystallization process [10]. Furthermore, systematic studies of nucleation, crystallization, melting, and reorganization are made possible for a large number of polymers. In particular, promising research serving as a conceptual study to quantitatively approach the link between the condition of cooling the melt of crystallizable polymers, the formation of crystal nuclei and the cold-crystallization behavior have been successfully developed [11]. The enhancement of biodegradability, biocompatibility, thermal conductivity and mechanical proprieties of biopolymeric materials can be achieved by adding nanofillers [12–15]. On the other hand, the use of layered transition metal dichalcogenide nanofillers such as tungsten and molybdenum disulfide (WS2, MoS2) inorganic fullerenes (IFs) and inorganic nanotubes (INTs) [16,17] is expected to produce advanced nanocomposite materials [18,19]. As well as unique electronic and mechanochemical behavior, these novel nanomaterials show remarkable properties like high impact resistance and flexibility under tensile stress, excellent tribological behavior, superior fracture resistance to shockwaves, and simple and relatively inexpensive fabrication methods [20]. Recently, the incorporation of WS2 in polymer systems has demonstrated a range opportunities for many new applications. For example, IF-WS2 nanoparticles were used to produce advanced nylon-6 nanocomposites [21]. In particular, it was shown that introducing IF-WS2 nanoparticles into nylon-6 provoked a strong nucleation effect which induced changes in the crystal growth process. In the same way, the addition of low WS2 loadings strongly increased the crystallization rate of PHBV [22]. For these systems, drawing induced during melt crystallization process has been shown to vary the crystalline structure (i.e., from α to β) leading to improved mechanical properties in melt-spun bio-based PHBV fibers [23]. Similarly, WS2 nanotubes (INT-WS2) have been shown to improve the thermal, mechanical and tribological properties of biopolymers like poly(3-hydroxybutyrate) (PHB) [24] and poly(L-lactic acid) (PLLA) [25], and the bio-applied polymer poly(ether ether ketone) (PEEK) [26]. Additionally, from an environmental viewpoint, INT-WS2 have demonstrated much lower cytotoxicity than other nanoparticle fillers, such as silica or carbon black [27] and have shown promise with respect to biocompatibility in the case of salivary gland cells [28]. The present work continues progress in this field and is centered on the incorporation of INT-WS2 as nanoreinforcements to improve the processability and performance of PHBV. Scanning electron Polymers 2018, 10, 166 3 of 14

microscopy (SEM) observations revealed that an excellent dispersion of highly efficient INT-WS2 nucleating agents was achieved, leading to composites with substantially enhanced thermal and mechanical properties [29]. However, to date the influence of the nanofiller on the crystallization behavior and kinetics of PHBV under isothermal conditions has not been investigated. Here, this process is studied in detail using differential scanning calorimetry (DSC) and polarized optical microscopy (POM) techniques, with particular emphasis on the role of INT-WS2 concentration. The research reported provides a better understanding of the structure-property relationship of PHBV biopolymer nanocomposites, with an outlook towards extending their practical applications.

2. Experimental Section

2.1. Materials and Processing The PHBV biopolymer employed, containing 2.0 wt % hydroxyvalerate (HV) with a reported Mw = 410 kg mol−1, was obtained in powder form from Goodfellow Cambridge, Ltd. (Huntingdon, UK) and used as received. The tungsten disulfide inorganic nanotubes (INT-WS2) were provided by NanoMaterials, Ltd. (Yavne, Israel) and used without chemical modification. Several formulations of PHBV/INT-WS2 (0.1, 0.5 and 1.0 wt %) nanocomposites were prepared [29]. The nanofiller was dispersed in a solution of PHBV in chloroform (HPLC grade, Sigma-Aldrich Química SL, Madrid, Spain), which was subsequently precipitated in methanol (HPLC grade, Sigma-Aldrich Química SL, Madrid, Spain), then filtered and dried in a vacuum oven at 50 ◦C for 24 h.

2.2. Characterization Techniques

2.2.1. Differential Scanning Calorimetry (DSC) The isothermal crystallization and melting behavior of the new nanocomposites were studied using a Perkin Elmer DSC7/Pyris differential scanning calorimeter (Perkin-Elmer España SL, Madrid, Spain). The instrument was calibrated for temperature and heat flow using high purity indium and zinc standard. A tau lag calibration of the instrument for different heating rates was performed using indium. The experimental and theoretical procedures used in this study are similar to those employed in our previous publications for PLLA/INT-WS2 [25] and nylon-6/IF-WS2 [21]. In this case, samples of 6–11 mg were placed in sealed 40 µL aluminum pans under a flowing nitrogen atmosphere. Before cooling, the samples were maintained for 5 min at 180 ◦C to erase any prior thermo-mechanical history and to assure maximum thermal stability of the components as well as the reproducibility of the results. Then the molten samples were cooled at fastest achievable rate of ◦ −1 64 C min to specific isothermal crystallization temperatures (Tc) and maintained until crystallization was completed (i.e., complete return to baseline), and the heat evolved during crystallization was recorded as a function of time at selected Tc. Pyris DSC7 kinetic software was used to obtain partial areas from the data points of the exotherm, corresponding to a given degree of the total crystalline transformation. The crystallinity of PHBV in the samples was determined, after normalizing for −1 filler content, using a value of ∆Hm for 100% crystalline PHBV (low HV content) of 146 J g [3,30]. The isothermal crystallization step was followed by a heating step up to 180 ◦C at a rate of 5 ◦C min−1, and melting temperatures (Tm) were taken as the peak maxima of the melting endotherms.

2.2.2. Polarized Optical Microscopy (POM) Polarized optical microscopy (POM) was used to investigate the spherulitic morphology of neat PHBV and the PHBV/INT-WS2 nanocomposites employing a Mettler FP-80HT (Mettler-Toledo SAE, Barcelona, Spain) hot stage on a Reichert Zetopan Pol polarizing microscope equipped with a Nikon FX35A 35 mm SLR camera. The isothermal crystallization cycles consisted in a 5-min hold period at ◦ ◦ −1 ◦ 180 C followed by rapid cooling at 20 C min to speciﬁc crystallization temperatures, Tc = 110 C, Polymers 2018, 10, 166 4 of 14

Polymers 2018, 10, x FOR PEER REVIEW 4 of 14 ◦ ◦ 112 C and 122 C. Samples were maintained at Tc for enough time to allow the monitorization of the 112 °C and 122 °C. Samples were maintained at Tc for enough time to allow the monitorization of the spherulitic growth process. spherulitic growth process. 3. Results 3. Results 3.1. Isothermal Crystallization 3.1. Isothermal Crystallization The physical and mechanical properties of semicrystalline polymers depend on the morphology, The physical and mechanical properties of semicrystalline polymers depend on the the crystalline structure and the degree of crystallinity. Much effort has been devoted to study morphology, the crystalline structure and the degree of crystallinity. Much effort has been devoted the isothermal crystallization kinetics of new PHBV/INT-WS2 bionanocomposites, with a view to study the isothermal crystallization kinetics of new PHBV/INT-WS2 bionanocomposites, with a to control the crystallization rate, degree of crystallinity and, consequently, its morphology and view to control the crystallization rate, degree of crystallinity and, consequently, its morphology and properties. In this respect, the isothermal melt crystallization kinetics of neat PHBV and its properties. In this respect, the isothermal melt crystallization kinetics of neat PHBV and its nanocompositesnanocomposites was was investigated investigated with with DSC DSC overover aa widewide range of crystallization crystallization temperatures temperatures from from ◦ ◦ 94 94C °C to to 130 130C. °C. The The curves curves in in Figure Figure1 1indicate indicate the the total total timetime forfor the complete crystallization crystallization process process at theat the above-mentioned above-mentioned temperatures temperatures and and areare truncatedtruncated at the time when when no no further further crys crystallizationtallization waswas evident evident by by DSC. DSC. Figure Figure1a 1a shows shows that that at at higher higher TTcc more time is is required required to to fully fully crystallize crystallize the the purepure PHBV PHBV sample. sample. At lower At lowerTc the curvesTc the shifted curves to shifted shorter to times, shorter indicating times, increased indicating crystallization increased ratescrystallization directly proportional rates directly to the proportional isothermal crystallizationto the isothermal temperature crystallization employed. temperature The crystallization employed. behaviorThe crystallization of the nanocomposites behavior of with the temperature nanocomposites was similar, with temperature Figure1b–d. was These similar, results Figure are consistent 1b–d. withThese the theory results of are crystallization consistent kinetics, with the implying theory of that crystallization as the supercooling kinetics, (i.e., implying the difference that asbetween the thesupercooling melting and (i.e. crystallization the difference temperatures) between the melting increases, and thecrystallization crystallization temperature rate acceleratess) increases, and the the crystallizationcrystallization exotherm rate accelerates becomes and sharper, the crystallization controlled in exotherm turn by the becomes evolution sharper of the, controlled number ofin crystalturn nucleiby the formed evolution during of crystallization the number of process crystal of nuclei the biopolymer formed during matrix. crystallization In previous process studies of on the the nucleationbiopolymer behavior matrix of. In PLLA previous reported studies by on Androsch the nucleation et al. [ 11behavior], it was of shown PLLA reported that isothermal by Androsch formation et of crystalal. [11],nuclei it was atshown high that supercooling isothermal of formation the melt canof crystal be quantiﬁed nuclei at byhigh the supercooling analysis of crystallizationof the melt can at elevatedbe quantified temperature. by the analysis of crystallization at elevated temperature.

110110 ºC ºC 126 ºC 108108 ºC ºC 124 ºC

106 106ºC ºC 122 ºC

104 ºC104 ºC 120 ºC

102 ºC102 ºC 118 ºC

Endotherm up up Endotherm (a.u.)

Endotherm up up Endotherm (a.u.) Endotherm up up Endotherm (a.u.) - 100 ºC

- 100 ºC -

98 ºC98 ºC 116 ºC Heat Flow Heat Heat Flow Heat 96 ºC96 ºC (a)(a) Flow Heat (c)

0 010 1020 2030 30 40 40 50 50 60 60 70 70 80 80 9090 100100 110110 120120 0 10 20 30 40 50 60 70 80 90 100 110 TimeTime (min) (min) Time (min)

128 ºC 126 ºC 112112 ºC ºC

110110 ºC ºC 124 ºC

108 ºC108 ºC 122 ºC

Endotherm up up (a.u.) Endotherm

Endotherm Up Up (a.u.) Endotherm Endotherm Up Up (a.u.) Endotherm

106 ºC106 ºC - - - 120 ºC

104 ºC104 ºC 118 ºC

Heat Flow Heat Heat Flow Heat Heat Flow Heat (b)(b) (d)

0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 8080 9090 0 10 20 30 40 50 60 70 80 90 TimeTime (min) (min) Time (min)

FigureFigure 1. 1.Differential Differential scanningscanning calorimetry calorimetry (DSC (DSC)) thermograms thermograms of isothermal of isothermal crystallization crystallization of (a) of (a)PHBV PHBV;; (b) poly(3-hydroxybutyrate-poly(3-hydroxybutyrate-coco--3-hydroxyvalerate)/3-hydroxyvalerate)/ tungsten tungsten disulf disulﬁdeide inorganic inorganic nanotubes nanotubes

(PHBV/INT-WS(PHBV/INT-WS2)2) (0.1 (0.1 wtwt. %); %); ( c()c) PHBV/INT-WS PHBV/INT-WS22 (0.5(0.5 wt. wt % %)) and and ( (dd)) PHBV/INT PHBV/INT-WS-WS2 (1.02 (1.0 wt. wt %) %) obtainedobtained at theat the indicated indicated crystallization crystallization temperatures. temperatures.

Polymers 2018, 10, 166 5 of 14

By comparing Figure1a with Figure1b–d, it is clear that at the Tc the exothermic peaks for the nanocomposites are in all cases sharper than those for pure PHBV indicating that the INT-WS2 accelerates the crystallization process of the polymer in the nanocomposites. The reduction in the time to reach overall crystallization can be employed to describe this acceleration process. For example, the PHBV copolymer without INT-WS2 fully crystallized after approximately 120 min ◦ at Tc = 110 C, whereas for the 0.5 wt % nanocomposite material it took less than 4 min at Tc = 116 ◦C (i.e., at a temperature of 4 ◦C higher than that of the pure polymer). From the data it can be seen that the incorporation of low INT-WS2 weight-fractions in PHBV nanocomposite allows the crystallization to take place at higher temperatures and over larger intervals (96–110 ◦C for PHBV, ◦ ◦ ◦ 116–126 C for PHBV/INT-WS2 (0.1 wt %), 104–112 C PHBV/INT-WS2 (0.5 wt %) and 118–128 C for PHBV/INT-WS2 (1.0 wt %). The relative crystallinity, X(t), can be deﬁned by the following expression:

R t dH(t) dt ∆H X(t) = 0 dt = t t ( ) (1) R ∞ dH t ∆H∞ 0 dt dt where dH/dt is the heat ﬂow rate; ∆Ht is the heat generated at time t; ∆H∞ is the total heat generated to the end of the crystallization process. The classical Avrami Equation (2) was employed to describe the isothermal crystallization kinetics:

X(t) = 1 − exp(−ktn) (2) where n is the Avrami exponent, which is a constant that depends on the nucleation mechanism and type of crystal growth, and k is the Avrami rate constant associated with nucleation and growth rate parameters. Equation (2) can be rewritten as:

log[− ln(1 − X(t))] = log k + n log t (3) and the values of log [−ln(1 − X(t))] plotted versus log t, allowing the Avrami exponent n and the crystallization rate constant k to be calculated from the slope and intercept of the linear fit, respectively. It is rare that the Avarmi equation can be used to describe the entire crystallization process, but is widely accepted as valid at the early stage of crystallization, as previously reported in our studies for nylon-6/IF-WS2 [21] and PLLA/INT-WS2 [25]. Linear regressions of these straight lines at low levels of crystalline transformation (5–40%) yielded the Avrami exponents (n) shown in Table1. As an example, Figure2 illustrates the Avrami plots for both neat PHBV and PHBV/INT-WS 2 (1.0 wt %) at different crystallization temperatures and the data are represented in Table1. The average values obtained for n varied with the INT-WS2 concentration: n ≈ 3.0 for neat PHBV, n ≈ 3.3 for PHBV/INT-WS2 (0.1 wt %), n ≈ 4.2 for PHBV/INT-WS2 (0.5 wt %) and n ≈ 4.4 for PHBV/INT-WS2 (1.0 wt %). According to the ideal case of the Avrami equation for nucleated crystallization with three-dimensional crystal growth, the value of the exponent should be n = 3 [31]. A value of n = 4 indicates ideal three-dimensional growth with a linear increase in nucleation sites over time due to heterogeneous nucleation, which was expected for the binary composite materials with the addition of nanoparticles. However, the ideal state was not achieved during the crystallization process probably due to athermal crystallization and/or imperfections within the polymer network (entanglements, single chains transversing multiple lamellae, etc.) as well as secondary crystallization processes, mixed nucleation modes and the change in material density [3]. Moreover, even some experimental factors such as an error introduced in the determination of the onset of crystallization or induction time, the establishment of the baseline and incomplete isothermal crystallization data, the effect of the cooling rate from the melt to the isothermal crystallization temperature and the conversion range employed for the fitting can lead to non-integer values of n [8]. In addition, the supernucleation effect of the nanotubes could also be connected to the variation of the n exponents [9]. The values of n reported in the literature for PHBV systems are Polymers 2018, 10, 166 6 of 14 Polymers 2018, 10, x FOR PEER REVIEW 6 of 14 dispersed,P(3HB-co-3HV) ranging (6.6 from mol 1.7 % toHV). 4 [3 Chan]. Liu et al.al. [[3233]], also however, obtained obtainedn values n ofvalues 2.0–2.2 across for P(3HB- a rangeco -3HV)of HV (6.6contents mol % of HV). 2.35 Chan–2.7, etand al. [33 Saad], however, et al. [34 obtained] obtainedn values an exponentacross a range of 3.8 of HV for contents thin P(3HB) of 2.35–2.7, films. andMeanwhile Saad et al., Xu [34 et] obtainedal. [35] applied an exponent the Avrami of 3.8 for equation thin P(3HB) to IR films. data Meanwhile,for isothermal Xu etcrystalli al. [35]zation applied of theP(3HB) Avrami and equation Nodax to (P(3HB IR data-co for-3HHx)) isothermal and crystallization obtained an ofn value P(3HB) of and 1.72 Nodax and (P(3HB- 2.08 respectively,co-3HHx)) andindicating obtained that an a nheterogeneousvalue of 1.72 nucleation and 2.08 respectively, mechanism indicatingexists in this that case. a heterogeneous Nonetheless, the nucleation increase mechanismof the Avrami exists exponent in this case. compared Nonetheless, to the neat the increase sample ofdid the justify Avrami the exponent fact that comparedthe initial tonucleation the neat samplestage was did enhanced justify the factby the that nanoparticles the initial nucleation. With the stage linearized was enhanced Avrami by plots, the nanoparticles. the intercept With value the is linearizedln(k) and the Avrami overall plots, rate the constant intercept (k) value is easily is ln( determined.k) and the overall rate constant (k) is easily determined.

0.0 (a) -0.5

-1.0 )]

X -1.5

- ln(1

- -2.0 ln[

-2.5

-3.0

-3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 ln t (min)

0.0 (b) -0.5

-1.0

)] X

- -1.5 ln(1

- -2.0 ln[

-2.5

-3.0

-3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 ln t (min)

Figure 2. Avrami plots of the crystallization of (a) PHBV and (b) PHBV/INT-WS2 (1.0 wt %) as a Figure 2. Avrami plots of the crystallization of (a) PHBV and (b) PHBV/INT-WS2 (1.0 wt. %) as a function of the crystallization temperature (Tc). function of the crystallization temperature (Tc).

Polymers 2018, 10, 166 7 of 14

Table 1. Isothermal crystallization parameters of PHBV and PHBV/INT-WS2 nanocomposites. Tc = crystallization temperature, τ0.5 = the time needed to reach 50% crystalline transformation, n = Avrami exponent, kn = overall rate constant, Tm = melting temperature and σe = fold surface free energy. (*) The main error arises from baseline selection during data processing. We estimate the

following errors: ±1 J/g for (∆Ht) and ±100 for ∆(τ0.5).

INT-WS 2 T (◦C) τ (min) n k × 105 T /T (◦C) σ (erg cm−2) Content (wt %) c 0.5 n m1 m2 e 96 2.4 4.4 ± 0.1 15.65 149.6/160.0 98 3.3 3.8 ± 0.1 7.31 148.5/159.5 100 4.9 3.3 ± 0.1 3.71 147.5/158.0 102 6.9 2.9 ± 0.2 2.51 147.1/157.3 0 75 ± 3 104 9.5 2.7 ± 0.1 1.5 146.9/156.8 106 15.1 2.5 ± 0.1 0.77 145.7/154.9 108 26.3 2.3 ± 0.1 0.42 144.7/153.6 110 49.3 2.0 ± 0.2 0.28 145.2/155.8 104 3 4.0 ± 0.1 8.49 145.2/155.8 106 5.2 3.6 ± 0.1 1.83 144.2/154.4 0.1 108 9.3 3.3 ± 0.2 0.43 144.1/153.7 57 ± 2 110 19 3.0 ± 0.1 0.1 144.4/153.0 112 37.2 2.7 ± 0.1 0.05 144.8/152.3 116 1.4 5.2 ± 0.1 145.23 156.7/165.2 118 2.2 4.6 ± 0.2 17.5 154.6/165.2 120 4.1 4.2 ± 0.1 1.84 153.6/161.2 0.5 71 ± 3 122 8.6 3.9 ± 0.1 0.16 153.3/160.0 124 19.5 3.7 ± 0.2 0.01 153 126 47.6 3.8 ± 0.2 0.003 152.5 118 2.3 4.6 ± 0.1 14.37 152.4/160.8 120 3.8 4.3 ± 0.1 2.29 152.7/160.3 122 6.3 4.1 ± 0.1 0.34 153.1/160.0 1 58 ± 2 124 10.8 4.1 ± 0.2 0.04 153.8/160.3 126 19 3.9 ± 0.2 0.01 154 128 34.9 4.0 ± 0.2 0.005 154.7

Another common way to estimate the overall rate constant, also k, is by the well-ﬁtting logarithmic representation of the following expression [36]:

ln 2 kn = n (4) (τ0.5) where τ0.5 is the time needed to reach 50% crystalline transformation. Values of kn obtained for both neat PHBV and its nanocomposites are represented in Figure3, where the effect of the crystallization temperature (Tc) and INT-WS2 concentration on the overall crystallization rate can be observed ◦ ◦ (Table1). By increasing Tc from 96 C to 110 C, the crystallization of neat PHBV becomes hindered and kn decreases due to the excessive mobility of the polymer chains that reduces the development of nucleation sites at the higher temperature. A similar phenomenon takes place for PHBV/INT-WS2 ◦ (1.0 wt %) in the range of 118–128 C. The values of kn for the nanocomposites were found to be higher −5 ◦ in all cases than those for PHBV. Whilst PHBV presented a value of kn ≈ 2.51 × 10 at Tc = 102 C, ◦ in the case of the nanocomposites, the values of kn of around the same order were obtained at 120 C for a concentration of 1.0 wt % of INT-WS2. The results show that INT-WS2 nanoparticles are effective nucleating agents for PHBV, promoting the nucleation of the crystallization of the polymer chains at small nanoﬁller loadings, without altering its crystal structure [29]. Additionally, the presence of increasing content of INT-WS2 also improves the crystallinity of PHBV, as can be seen in Figure4. Polymers 2018,, 10,, 166x FOR PEER REVIEW 8 of 14 Polymers 2018, 10, x FOR PEER REVIEW 8 of 14 0 0 PHBV -2 PHBVPHBV/INT-WS2 (0.1 wt.%) -2 PHBV/INTPHBV/INT-WS2-WS2 (0.1 (0.5 wt.%) wt.%) -4 PHBV/INTPHBV/INT-WS2-WS2 (0.5 (1.0 wt.%) wt.%) -4 y = -0.6588x + 63.564 R² = 0.9854 PHBV/INT-WS2 (1.0 wt.%) -6 y = -0.6588x + 63.564

R² = 0.9854 )

n -6 -

) y = -0.2838x + 22.946 n - -8 R² = 0.995 y = -0.2838x + 22.946

(min -8 R² = 0.995

y = -0.9472x + 107.55 n (min R² = 0.9976 -10 0.8 y = -1.1255x + 128.64 y = -0.9472x + 107.55 n R² = 0.9976 0.7 0.8 R² = 0.9934 ln k ln -10 y = -1.1255x + 128.64 0.7 R² = 0.9934 ln k ln 0.6

-12 ) 0.6 1

- 0.5

-12 ) 1

- 0.5

0.4 (min

0.4

0.5 (min

-14 τ 0.3

0.5 1/

τ 0.3

-14 0.2 1/ 0.2 0.1 -16 0.1 -16 0.0 950.0 100 105 110 115 120 125 130 135 95 100 105 110 115 120 125 130 135 Crystallization temperature (ºC) Crystallization temperature (ºC) -18-18 9595 100100 105105 110110 115115 120 125125 130130 135135 CrystallizationCrystallization temperature temperature (ºC) (ºC)

FigureFigure 3. Logarithmic3.Logarithmic Logarithmic plots plots plots of ofof the thethe global globalglobal rate raterate constant constant ( (kk)) of of PHBV/INTPHBV/INT PHBV/INT-WS-WS-WS2 2nanocomposites 2nanocompositesnanocomposites as as a asa afunction functionfunction of of of the the the crystallization crystallization crystallization temperature temperature temperature ( (T (TTc);cc);); the the the inset inset inset represents represents the the the inverse inverse inverse of of crystallization crystallization crystallization

half-timehalfhalf-time-time (1/ (1/ττ0.50.5τ)0.5 )as) as asa a functiona functionfunction of ofof T Tc. c..

70 70

60 60

50 50

40 40

30 Crystallinity (%) Crystallinity

Crystallinity (%) Crystallinity 20 PHBV 20 PHBVPHBV/INT-WS2 (0.1 wt.%) 10 PHBV/INTPHBV/INT-WS2-WS2 (0.1 (0.5 wt.%) wt.%) 10 PHBV/INTPHBV/INT-WS2-WS2 (0.5 (1.0 wt.%) wt.%) PHBV/INT-WS2 (1.0 wt.%) 0 0 95 100 105 110 115 120 125 130 135 95 100 105 110 115 120 125 130 135 Crystallization temperature (ºC) Crystallization temperature (ºC) Figure 4. Variation of the crystallinity of isothermal crystallization of PHBV/INT-WS 2 Figure 4. Variation of the crystallinity of isothermal crystallization of PHBV/INT-WS2 nanocomposites Figurenanocomposites 4. Variation as a function of the of crystallinitythe crystallization of isothermaltemperature ( crystallizationTc). of PHBV/INT-WS2 as a function of the crystallization temperature (Tc). nanocomposites as a function of the crystallization temperature (Tc). Hereafter, the melting behavior of PHBV/INT-WS2 will be presented in order to understand the Hereafter, the melting behavior of PHBV/INT-WS2 will be presented in order to understand dependenceHereafter, ofthe the melting double behaviormelting temperatures of PHBV/INT of- WSPHBV2 will with be composition. presented in Figure order 5to shows understand the DSC the the dependence of the double melting temperatures of PHBV with composition. Figure5 shows dependencemelting curvesof the double obtained melting in this temperatures study after of PHBV isothermal with composition. crystallization Figure at different 5 shows Tthec forDSC thePHBV DSC/IN meltingT-WS2 curves(1.0 wt. obtained%), where in a clear this studydouble after melting isothermal behavior crystallizationwas observed and at differentthe resultsT forc for melting curves obtained in this study after isothermal crystallization at different Tc for PHBV/INT-WSall samples are2 (1.0 summarized wt %), where in Table a clear 1. doubleNo clear melting difference behavior was observed was observed in the andevolution the results of the for PHBV/INT-WS2 (1.0 wt. %), where a clear double melting behavior was observed and the results for all samplesdouble melting are summarized temperature in with Table INT1. No-WS clear2 due difference the large wasshift observedof the crystallization in the evolution interval of theof PHBV double all samples are summarized in Table 1. No clear difference was observed in the evolution of the meltingto higher temperature temperature with and INT-WS direct2 duetemperature the large comparisons shift of the crystallization were not feasible interval (i.e., of the PHBV measurable to higher double melting temperature with INT-WS2 due the large shift of the crystallization interval of PHBV temperaturetemperature and range direct for temperature neat PHBV comparisonswas 96–110 ° wereC, whereas not feasible for the (i.e., 1.0 thewt. measurable % nanocomposite, temperature the to higher temperature and direct temperature comparisons were not feasible (i.e., the measurable rangemeasurable for neat temperature PHBV was range 96–110 was◦ C,118 whereas–128 °C). for the 1.0 wt % nanocomposite, the measurable temperature range for neat PHBV was 96–110 °C, whereas for the 1.0 wt. % nanocomposite, the temperature range was 118–128 ◦C). measurable temperature range was 118–128 °C).

Polymers 2018, 10, x 166 FOR PEER REVIEW 9 of 14

128 ºC

126 ºC

124 ºC

Endotherm Up Up Endotherm (a.u.) 122 ºC -

120 ºC

Heat Flow Heat 118 ºC Tm1 Tm2

110 120 130 140 150 160 170 180 Temperature (ºC)

Figure 5. Melting DSC thermograms of PHBV/INT-WS2 (1.0 wt. %) nanocomposite obtained at a Figure 5. Melting DSC thermograms of PHBV/INT-WS2 (1.0 wt %) nanocomposite obtained at a −1 heating rate of 5 °C◦C min − 1afterafter isothermal isothermal crystallization crystallization at at the the indicated indicated temperatures. temperatures.

3.2. Crystallization Activation Energy 3.2. Crystallization Activation Energy For further insight into the crystallization behavior of PHBV and its nanocomposites, the free For further insight into the crystallization behavior of PHBV and its nanocomposites, the free energy of folding (σe) was calculated using the Lauritzen and Hoffman (L–H) model [37,38], energy of folding (σe) was calculated using the Lauritzen and Hoffman (L–H) model [37,38], previously previously adopted to calculate the isothermal crystallization activation energy of nylon-6/IF-WS2 adopted to calculate the isothermal crystallization activation energy of nylon-6/IF-WS2 [21] and [21] and PLLA/INT-WS2 [25]. In this approximation, σe represents the energy required to fold the PLLA/INT-WS2 [25]. In this approximation, σe represents the energy required to fold the polymer polymer chains during crystallization. In agreement with the kinetic theory of crystallization and chains during crystallization. In agreement with the kinetic theory of crystallization and independent independent regime type, the crystallization rate (G) can be expressed as: regime type, the crystallization rate (G) can be expressed as: U * K ∗ K g(III) GG0 exp[]exp[]U g(III) (5) G = G0 exp[− R() TTfTT] exp[− ] (5) R(Tccc− T0 ) f Tc∆T where G0 is a temperature independent pre-exponential term, U* is the activation energy required where G0 is a temperature independent pre-exponential term, U* is the activation energy required for chain movement (2.45 × 1066 cal kmol−−1),1 T0 is the temperature at which there is no chain motion for chain movement (2.45 × 10 cal kmol ), T0 is the temperature at which there is no chain motion 0 (usually T0 = Tg − 51.6 K), R is the universal gas constant, ΔT is the undercooling, or 푇0푚 − Tc where (usually T0 = Tg − 51.6 K), R is the universal gas constant, ∆T is the undercooling, or Tm − Tc where 0 푇푚0 is the equilibrium melting temperature, f is a correction factor that accounts for the variation of Tm is the equilibrium melting temperature, f is a correction factor that accounts for the variation of 0 0 the equilibrium melting enthalpy (Δ퐻푚0 ) with temperature, defined as 2Tc/(Tc + 푇푚0), and Kg is the the equilibrium melting enthalpy (∆Hm) with temperature, defined as 2Tc/(Tc + Tm), and Kg is the nucleatinucleationon constant for Regime III [3, [3,39], which can be expressed by:by: 0 4bT0uem 0 4b0σuσeTm KgIII()= 0 (6) Kg(III) 0 (6) kHkBmB∆Hm where k is the Boltzmann constant, 1.38 × 10−−2626 kJ K−1,− b10 = 7.2 nm and corresponds to the thickness of where k is the Boltzmann constant, 1.38 × 10 kJ K , b0 = 7.2 nm and corresponds to the thickness e u aof single a single crystalline crystalline monolayer monolayer added added during during growth growth [3], [3 ], and and σσ eandand σσ uareare the the basal basal and and lateral interfacial free free energies energies of of the the crystallite, crystallite, respectively. respectively. The The logarithmic logarithmic representation representation of the of first the term first c termof Equation of Equation (5) versus (5) versus 1/fTc ∆1/TfTisΔ presentedT is presented in Figure in Figure6 for all6 for the all samples the samples analyzed, analy andzed the, and linear the linear fits observed support unique regime behavior. The values of Kg(III) were calculated from the fits observed support unique regime behavior. The values of Kg(III) were calculated from the slopes of slopetheses plots. of these plots.

PolymersPolymers2018 2018,,10 10,, 166x FOR PEER REVIEW 1010 ofof 1414

) y = -20.854x + 75.476 0 R² = 0.9774

T y = -11.113x + 39.921 -

c 0 R² = 0.9733

/R(T -2 *

-4 y = -19.511x + 82.08 R² = 0.9985

ln G + U G+ ln -6 PHBV PHBV/INT-WS2 (0.1 wt.%) y = -27.546x + 112.86 -8 R² = 0.9961 PHBV/INT-WS2 (0.5 wt.%)) -10 PHBV/INT-WS2 (1.0 wt.%) -12 3.0 3.5 4.0 4.5 5.0 1/(fT ΔT) 105 c

FigureFigure 6.6Logarithmic. Logarithmic plots plots of Lauritzen of Lauritzen and Hoffman and (L–H) Hoffman equation (L– forH) PHBV/INT-WS equation for2 nanocomposites. PHBV/INT-WS2 nanocomposites.

Although the literature values found for ∆Hm vary considerable since they are fundamentally conditionedAlthough by the the literature determination values methodfound for [3 Δ],H them vary inﬂuence considerable of this since experimental they are fundamentally variability in theconditioned comparative by the analysis determination of the valuesmethod of[3] the, the interfacial influence of free this energies experimental can be variability eliminated in the by applyingcomparative the analysis Hoffman of approximation the values of the [38 interfacial], which determines free energies the can interfacial be eliminated free energy by applying using thethe followingHoffman expression: approximation [38], which determines the interfacial free energy using the following expression: 0 p σu = α∆Hm a0b0 (7) 0 where α = 0.24 (for high melting polyesters)um and Haba0b0 =00 38.01 Å [40] that corresponds to the chain(7) cross-section in the PHBV crystal. Thus, the basal interfacial free energy can be derived from the followingwhere α = equation: 0.24 (for high melting polyesters) and a0b0 = 38.01 Å [40] that corresponds to the chain cross-section in the PHBV crystal. Thus, the basalkBK interfacial( ) free energy can be derived from the = g√III following equation: σe 0 (8) 4b0Tmα a0b0

Under these considerations and based on ourkKB previous g() III studies of the isothermal crystallization  e  (8) of polymer/WS2 systems [21,25], the values of σe obtained0 for PHBV, PHBV/INT-WS2 (0.1 wt %), 4b0 Tm a 0 b 0 −2 PHBV/INT-WS2 (0.5 wt %) and PHBV/INT-WS2 (1.0 wt %) are around 75, 57, 71 and 58 erg cm , respectively.Under these There considerations is a clear trend and for based decreasing on our valuesprevious of σstudiese as the of INT-WS the isothermal2 content crystallization is increased. Fromof polymer/WS these results,2 systems we can [ conclude21,25], the that values between of σ arounde obtained 6–24% for lowerPHBV, energy PHBV/INT is required-WS2 (0.1 to generate wt. %), crystallinePHBV/INT nuclei-WS2 (0.5 of PHBV, wt. % which) and PHBV/INT in turn also-WS promotes2 (1.0 wt. the % formation) are around of new75, 57, PHBV 71 and crystal 58 erg surfaces. cm−2, Thisrespectively. excellent matchingThere is a suggests clear trend that for PHBV decreasing crystals mightvalues grow of σeon as thethe INT-WS INT-WS2 surface2 content by is an incre epitaxialased. mechanismFrom these in results, absence we of can the chemical conclude interaction that between between around the 6 INT-WS–24% lower2 and energyPHBV. More is required in-depth to experimentsgenerate crystalline will be conductednuclei of PHBV to verify, which this in proposal turn also in promotes a future study. the formation of new PHBV crystal surfaces. This excellent matching suggests that PHBV crystals might grow on the INT-WS2 surface 3.3. Spherulitic Growth Analysis from POM Observation by an epitaxial mechanism in absence of the chemical interaction between the INT-WS2 and PHBV. MoreOptical in-depth light experiments microscopy will images be conducted of crystals to verify produced this proposal during in the a future isothermal study. crystallization of PHBV and its nanocomposite containing INT-WS2 were obtained. As an example, Figures7 and3.3. 8Spherulitic shows the Growth spherulitic Analysis morphology from POM of Observation neat PHBV and PHBV/INT-WS 2 (1.0 wt %) isothermally ◦ ◦ crystallizedOptical at light 100 microscopyC and 122 C,images respectively. of crystals produced during the isothermal crystallization of PHBV and its nanocomposite containing INT-WS2 were obtained. As an example, Figures 7 and 8 shows the spherulitic morphology of neat PHBV and PHBV/INT-WS2 (1.0 wt. %) isothermally crystallized at 100 °C and 122 °C, respectively.

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PHBV PHBV/INT-WS2 (1.0 wt.%)

(a) (b) (g) (h)

(e) (f) (k) (l)

FigureFigure 7 7.. POMPOM images images of PHBV/INT of PHBV/INT-WS-WS2 nanocomposites2 nanocomposites.. PHBV: PHBV:(a–f) are ( aimages–f) are taken images at 8 taken min; 12 at ◦ min8 min;; 23 12 min min;; 30 23 min; min; 3037 min; min 37 and min after and after 60 mi 60n min rapidly rapidly cooled cooled to to 80 80 °C,C, resp respectively;ectively; and PHBV/INTPHBV/INT-WS-WS2 2(1.0(1.0 wt. wt % %):): (g (g––l)l )are are images images taken taken at at 4 4 min min;; 5 5 min min;; 6 6 min min;; 7 7 min min,, 8 8 min min and and 10 10 min, min, respectively.respectively. Scale bar 100 μm.µm.

TheThe mainly mainly circular circular superstructures superstructures are are indicative indicative of the of formation the formation of a central of a nucleus central followed nucleus followedby radial, by outward radial, outward growth, asgrowth, expected. as expected. In Figure In7 eFigure crystal 7e diameters crystal diameters of approximately of approximately 130 µm 130were μm observed were observed after 37 afte minr 37 and min inand Figure in Figure7k crystal 7k crystal diameters diameters of 140of 140µ mμm were were seenseen after only only 88 min. min. This This observation observation further further confirms confirms the the acceleration acceleration of ofthe the crystallization crystallization process process of PHBV of PHBV by 2 incorporationby incorporation of INTof INT-WS-WS . Another2. Another effect effect observed observed in in the the nucleated nucleated systems systems was was an an average decreasedecrease in in crystal crystal diameters diameters of of the the neighboring neighboring spherulites spherulites before before impingement. impingement. This This was was due due to toa highera higher density density of of nucleation nucleation sites sites and and faster faster crystal crystal formation formation compared compared to to the the neat neat copolymer. copolymer. Assuming linear, constantconstant growthgrowth rates,rates, a a linear linear slope slope of of the the growth growth diameter diameter with with respect respect to timeto time for 2 forthe the four four samples samples clearly clearly indicated indicated that that crystal crystal growth growth is is faster faster in in the the samplessamples containingcontaining INT-WSINT-WS2 thanthan in in the the pure pure PHBV PHBV sample sample (Figure (Figure 88).). The The crystal crystal growth growth rates rates for forneat neat PHBV PHBV are are0.54 0.54and and0.92 −1 −1 ◦ ◦ μm0.92 minµm min at Tc =at 110Tc °=C 110and C112and °C, 112 respectively.C, respectively. The crystal The crystalgrowth growth rates for rates the 0.1, for the0.5 0.1,and 0.51.0 andwt. %1.0 nanocomposites wt % nanocomposites are 2.61, are 4.48, 2.61, and 4.48, 8.62 and μm 8.62min−µ1,m which min− is1, sixteen which istimes sixteen faster times for the faster 1.0 forwt. the % sample1.0 wt % compared sample compared to the neat to thepolymer neat polymer at the two at the different two different Tc values.Tc values. Another Another way to way interpret to interpret this figurethis figure is by iscomparing by comparing the time the required time required for the for spherulites the spherulites to reach to a reachcertain a certaindiameter. diameter. To reach To a ◦ diameterreach a diameter of, for example, of, for example, 35 μm at 35Tc µ= m122 at °TC,c inorganic= 122 C, inorganicnanotube fractions nanotube of fractions 1.0 wt. % of, a 1.0 0.5 wt wt. %, % a, and0.5 wt0.1 %,wt. and % in 0.1 PHBV, wt % needed in PHBV, approximately needed approximately 4, 8, and 13 4, min, 8, and respectively. 13 min, respectively. However, a However,neat PHBV a ◦ ◦ sampleneat PHBV requires sample around requires 38 and around 65 min 38 for and Tc 65 = 110 min °C for andTc =112 110 °C,C respectively.and 112 C, This respectively. is another This clear is indicationanother clear of indication the increase of thein the increase crystallization in the crystallization rate of PHBV rate of due PHBV to the due incorporation to the incorporation of the well-dispersed INT-WS2 nanofiller. In particular, the increasing slope of the growth rate of the of the well-dispersed INT-WS2 nanofiller. In particular, the increasing slope of the growth rate of the spherulite radii as a function of INT-WS2 concentration may be due to the increased nucleation sites spherulite radii as a function of INT-WS2 concentration may be due to the increased nucleation sites availableavailable thus thus facilitating accele acceleratedrated growth in either case. Mo Morere research would be required.

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140

y = 2.5231x + 2.8141 y = 4.3089x + 2.9703 R² = 0.9971 120 R² = 0.9882

) 100 y = 8.79x - 1.2

m R² = 0.9796 y = 0.5229x + 2.2741

μ R² = 0.999 80

y = 0.9027x + 0.3399 PHBV Tc = 110ºC

Spherulite radii ( radii Spherulite 40 R² = 0.9974 PHBV Tc = 112ºC PHBV/INT-WS2 (0.1 wt.%) Tc = 122ºC 20 PHBV/INT-WS2 (0.5 wt.%) Tc = 122ºC PHBV/INT-WS2 (1.0 wt.%) Tc = 122ºC 0 0 20 40 60 80 100 120 140 160 180 Time (min)

Figure 8. Isothermal spherulitic radii of PHBV/INT-WS2 nanocomposites obtained at the indicated Figure 8. Isothermal spherulitic radii of PHBV/INT-WS2 nanocomposites obtained at the indicated crystallizationcrystallization temperatures. temperatures.

4. Conclusions 4. Conclusions

From the melt-crystallization measurements, it was shown that INT-WS2 strongly affect the From the melt-crystallization measurements, it was shown that INT-WS2 strongly affect the crystallization of PHBV polymer. INT-WS2 accelerate the crystallization process of PHBV in the crystallization of PHBV polymer. INT-WS2 accelerate the crystallization process of PHBV in the nanocomposites and shift the crystallization temperature/interval for PHBV to higher temperatures nanocomposites and shift the crystallization temperature/interval for PHBV to higher temperatures with increasing INT-WS2 content. The experimental data fitted very well to the Avrami kinetic with increasing INT-WS2 content. The experimental data fitted very well to the Avrami kinetic model. In particular, it was found that the value of the Avrami exponent n for PHBV/INT-WS2 model. In particular, it was found that the value of the Avrami exponent n for PHBV/INT-WS2 nanocomposites increased compared to that for neat PHBV, and the analysis of the activation energy nanocomposites increased compared to that for neat PHBV, and the analysis of the activation energy of crystallization regime III using the Lauritzen and Hoffman (L–H) model showed that for the of crystallization regime III using the Lauritzen and Hoffman (L–H) model showed that for the PHBV/INT-WS2 nanocomposites, the fold surface free energy (σe) of PHBV chains decreased with PHBV/INT-WS2 nanocomposites, the fold surface free energy (σe) of PHBV chains decreased with increasing INT-WS2 content. Similarly, the variation of the spherulitic radii of PHBV with increasing INT-WS2 content. Similarly, the variation of the spherulitic radii of PHBV with crystallization crystallization time and concentration of nanofiller, calculated from the POM micrographs, also time and concentration of nanofiller, calculated from the POM micrographs, also supports the supports the spherulite growth-accelerating effect of PHBV. While the crystallization occurred at spherulite growth-accelerating effect of PHBV. While the crystallization occurred at much higher much higher temperatures with the incorporation of INT-WS2, the direct comparisons of temperatures with the incorporation of INT-WS2, the direct comparisons of crystallization rates were crystallization rates were not quantifiable at equivalent temperatures because of the acceleration not quantifiable at equivalent temperatures because of the acceleration effect of the doped materials. effect of the doped materials. This new knowledge obtained from the crystallization kinetics of the This new knowledge obtained from the crystallization kinetics of the PHBV biopolymer and its PHBV biopolymer and its nanocomposites can provide an essential benchmark for optimizing the nanocomposites can provide an essential benchmark for optimizing the design and processing of design and processing of PHBV-based thermoplastic materials with desirable properties. PHBV-based thermoplastic materials with desirable properties.

Acknowledgments:Acknowledgments:This This work work was was supported supported by by the the Spanish Spanish Ministry Ministry Economy Economy and and Competitivity Competitivity (MINECO), (MINECO), ProjectsProject MAT2013-41021-Ps MAT2013-41021-P and and MAT2017-84691-P. MAT2017-84691-P Mohammed. Mohammed Naffakh Naffakh would would also also like like to to acknowledge acknowledge the the MINECOMINECO for for a ‘Rama ‘Ramónón y Cajal’y Cajal’ Senior Senior Research Research Fellowship. Fellowship.

AuthorAuthor Contributions: Contributions:Mohammed Mohammed Naffakh Naffakh conceivedconceived and designed the the work; work; T Tyleryler Silverman Silverman performed performed the the DSC experiments; Carlos Marco performed the POM experiments; All authors contributed to the scientific DSC experiments; Carlos Marco performed the POM experiments; All authors contributed to the scientific discussion and Mohammed Naffakh, Tyler Silverman and Gary Ellis wrote the paper. discussion and Mohammed Naffakh, Tyler Silverman and Gary Ellis wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.

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