*3.1. Morphology*

Figure 1 shows the morphology of the here-obtained MCM-41 powder. Figure 1a,b present the SEM images of the MCM-41 powders with and without eugenol, respectively. One can observe that the silica particles presented a spherical shape with a mean size of around 100 nm, where the incorporation of eugenol slightly reduced their particle size. Therefore, the incorporation of eugenol did not alter the morphology of the mesoporous MCM-41 type nanoparticles. TEM was carried out in order to further ascertain the morphology of the MCM-41 particles. Figure 1c confirmed the spherical shape of the MCM-41 particle without eugenol, showing that their mean size was 96.1 ± 3.8 nm. A similar morphology can be observed in Figure 1d for the MCM-41 powder with eugenol, having a mean diameter of 88.6 ± 2.1 nm. Similar results were reported by Ribes et al. [40] in which the immobilization of eugenol and thymol on the surface of MCM-41 did not affect the integrity of the mesoporous silica

particles. Also, Ruiz-Rico et al. [41] observed that the appearance of fumed silica, amorphous silica, and MCM-41 particles did not change after functionalization with thymol. Indeed, MCM-41 has been widely used as a model material in the context of porosity characterization owing to its peculiar features, such as high surface area, large pore volume, low toxicity, high chemical and thermal stability, and versatile chemical modifiable surface. It has been reported that the pore structure is organized in the form of hexagonal arrays of uniform tubular channels of controlled width [47,48]. As a result, mesoporous silica nanoparticles are excellent candidates for reference adsorbents for standardizing adsorption measurements and methods for characterization of porous solids due to their regular pore structure, high stability, and also convenient method of synthesis [49,50].

**Figure 1.** Scanning electron microscopy (SEM) images of: (**a**) Mobil Composition of Matter (MCM-41); (**b**) MCM-41 with eugenol. Scale markers of 1 μm. Transmission electron microscopy (TEM) images of: (**c**) MCM-41 and (**d**) MCM-41 with eugenol. Scale markers of 100 nm.

Figure 2 shows the resultant electrospun mats obtained from the neat PHBV solution and the different solutions of PHBV/MCM-41 with eugenol. One can observe that, in all cases, the electrospinning process generated a mat composed of non-woven fibers with a similar morphology. Table 1 summarizes the mean diameters of the electrospun fibers. The neat PHBV fibers without MCM-41, processed in the same conditions, presented a mean diameter of 0.89 ± 0.30 μm. It can be observed that the mean diameters of the electrospun fibers varied in the 0.6–0.7 μm range when the silica particles were incorporated. However, one can observe that the electrospun fibers with the highest particle contents, that is, 15 and 20 wt.-% MCM-41, presented certain cross-linking or fibers coalescence. This can be related to difficulties encountered during the fiber formation more likely due to a phenomenon of particle aggregation in the electrospinning process. Indeed, it is known that high nano-sized filler contents habitually lead to the formation of beaded regions in the electrospun fibers [51,52].

**Figure 2.** Scanning electron microscopy (SEM) images of the electrospun fibers of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV)/Mobil Composition of Matter (MCM)-41 with eugenol: (**a**) Neat PHBV; (**b**) 2.5 wt.-% MCM-41 + eugenol; (**c**) 5 wt.-% MCM-41 + eugenol; (**d**) 7.5 wt.-% MCM-41 + eugenol; (**e**) 10 wt.-% MCM-41 + eugenol; (**f**) 15 wt.-% MCM-41 + eugenol; (**g**) 20 wt.-% MCM-41 + eugenol. Scale markers of 10 μm.

**Table 1.** Mean diameters of the electrospun fibers of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV)/Mobil Composition of Matter (MCM)-41 with eugenol.


TEM was also performed in order to evaluate the distribution of the MCM-41 particles inside the electrospun fibers. The detailed morphologies of the electrospun mats of PHBV/MCM-41 with eugenol, at different particle contents, are shown in Figure 3. One can observe that at low contents, that is, from 2.5 wt.-% to 7.5 wt.-% MCM-41 with eugenol, the functionalized silica nanoparticles were relatively well distributed inside the electrospun fibers. However, for higher filler contents, the MCM-41 particles were mainly agglomerated in certain regions of the fibers. This fact supports the above-described morphology during the SEM analysis by which the silica nanoparticles interconnected the fibers in the electrospun mats. A similar morphology was recently reported, for instance, by Cherpinski et al. [53] in PHB fibers containing palladium nanoparticles (PdNPs).

The morphology of the electrospun materials was also analyzed by SEM in order to ascertain the effect of the film-forming process on the PHBV fibers. Figure 4 shows the SEM images at both the cross-section and surface of the electrospun PHBV materials containing different amounts of MCM-41 with eugenol. The surface cryo-fractures of the electrospun materials, shown in the left column, revealed the formation of a continuous film with much reduced porosity. This process has been ascribed to a process of fibers coalescence that occurs during annealing, that is, at a temperature below the polymer's Tm [54]. In the case of the electrospun films having the highest particle contents, that is, 15 and 20 wt.-% MCM-41 with eugenol, the films presented a higher porosity and also certain plastic deformation. This observation can be related to the above-described fiber morphology and, more importantly, to the presence of high loadings of eugenol that could plasticize the PHBV matrix and/or migrate during the annealing process. In the top view of the electrospun films, shown in the

right column, one can clearly observe that the film sample containing 20 wt.-% MCM-41 presented higher porosity on its surface. This morphology confirms that contents above 15 wt.-% MCM-41 with eugenol are not optimal to be processed by electrospinning and thermally post-treatment at 160 ◦C. Similar findings were concluded when electrospun mats of PHBV with ~20 mol.-% HV were post-treated at higher temperatures than optimal, resulting in an increased porosity due to partial polymer melting and/or degradation [55].

**Figure 3.** Transmission electron microscopy (TEM) images of the electrospun fibers of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV)/Mobil Composition of Matter (MCM)-41 with eugenol: (**a**) 2.5 wt.-% MCM-41 + eugenol; (**b**) 5 wt.-% MCM-41 + eugenol; (**c**) 7.5 wt.-% MCM-41 + eugenol; (**d**) 10 wt.-% MCM-41 + eugenol; (**e**) 15 wt.-% MCM-41 + eugenol; (**f**) 20 wt.-% MCM-41 + eugenol. Scale markers of 200 nm.

Figure 5 shows the visual aspect of the resulting annealed electrospun PHBV films containing MCM-41 with eugenol. Although the contact transparency of the films was similar in all the samples, the films with the highest particle contents, that is, 15 and 20 wt.-% MCM-41 with eugenol, developed a yellow color. A similar yellowing and, in some cases, browning was previously observed by Muratore et al. [56] after the incorporation of eugenol into commercial paper prepared by grafting of this EO onto cellulose at 120–180 ◦C. This effect was ascribed to the intrinsic eugenol color, which is a pale yellow oily liquid, as well as secondary reactions and/or by-products due to thermal oxidation and chain scission of the substrate favored by high temperatures and prolonged time. Therefore, the incorporation of up to 10 wt.-% MCM-41 with eugenol successfully allows the production of contact transparent films of PHBV.

**Figure 4.** Scanning electron microscopy (SEM) images of the films cross-section (left) and top view (right) of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV)/Mobil Composition of Matter (MCM)-41 with eugenol: (**<sup>a</sup>**,**b**) 2.5 wt.-% MCM-41 + eugenol; (**<sup>c</sup>**,**d**) 5 wt.-% MCM-41 + eugenol; (**<sup>e</sup>**,**f**) 7.5 wt.-% MCM-41 + eugenol; (**g**,**h**) 10 wt.-% MCM-41 + eugenol; (**k**,**j**) 15 wt.-% MCM-41 + eugenol; (**k**,**l**) 20 wt.-% MCM-41 + eugenol. Scale markers of 50 μm and 100 μm.

**Figure 5.** Visual aspect of the electrospun films of poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) (PHBV)/Mobil Composition of Matter (MCM)-41 with eugenol.
