*2.4. Statistical Analysis*

The test data were evaluated through analysis of variance (ANOVA) using STATGRAPHICS Centurion XVI v 16.1.03 from StatPoint Technologies, Inc. (Warrenton, VA, USA). Fisher's least significant difference (LSD) was used at the 95% confidence level (*p* < 0.05). Mean values and standard deviations were also calculated.

#### **3. Results and Discussion**

#### *3.1. Morphology and Optical Properties*

## 3.1.1. Optical Appearance

Figure 1 shows the contact transparency pictures of the electrospun PHB fibers, Figure 1a–c, as well as of their respective annealed films, Figure 1d–f. From these pictures, it can be observed that all the electrospun fiber mats were completely opaque due to the ultrathin size of the fibers that generate a significant level of porosity and hence refract the light very strongly [21]. On the other hand, the annealed films presented an improved contact transparency, specially the sample with CTAB. Due to the presence of the PdNPs, the films developed an expected dark color.

**Figure 1.** Contact transparency pictures of the electrospun poly(3-hydroxybutyrate) (PHB) fibers containing palladium nanoparticles (PdNPs) and their respective annealed films: (**a**) PHB/PdNP fibers; (**b**) PHB/PdNP/hexadecyltrimethylammonium bromide (CTAB) fibers; (**c**) PHB/PdNP/tetraethyl orthosilicate (TEOS) fibers; (**d**) PHB/PdNP film, (**e**) PHB/PdNP/CTAB film, (**f**) PHB/PdNP/TEOS film.

#### 3.1.2. Morphology of Electrospun PHB Materials

The morphology of the electrospun fibers and their annealed films were studied by SEM. Representative images of all the electrospun samples are gathered in Figure 2. The images were taken from the surface and cross-sections of the obtained fibers and films. As shown in Figure 2a, the diameter of the neat electrospun PHB fibers was distributed primarily in the range of 200–600 nm, presenting a smooth and bead-free morphology. In particular, the mean diameter was found to be at 350 ± 147 nm. One can observe that the presence of the PdNPs led to a fraction with increased fiber diameter and also resulted in a spindle-type beads formation. This effect can be observed in Figure 2d,g, corresponding to the electrospun PHB/PdNP and CTAB-containing PHB/PdNP fibers, respectively. This observation may sugges<sup>t</sup> that, in some fibrilar regions, partial agglomeration of the PdNPs may occur. Indeed, some degree of agglomeration is a common phenomenon in composites due to the large surface area and high total surface energy associated with nanoparticles incorporated into polymer matrices that makes them amenable to clustering [22]. The nanofibers diameter was previously reported to decrease when the surfactant concentration increased in the electrospinning solution [23]. Interestingly, the TEOS-containing PHB/PdNP fibers, shown in Figure 2j, presented a significant fraction of the fibers with reduced fiber diameter. This effect has been previously described for other electrospun materials and it has been particularly attributed to both the expected decrease in surface tension and an increase in conductivity, which in turn produce an increase in the stretching forces in the jet and consequently decreases the fiber diameter [24–26].

From the SEM images of the fibers cross-sections, one can observe that Figure 2b,e, corresponding to the neat PHB and PHB/PdNP fibers, respectively, showed similar morphologies. In particular, both electrospun mats presented cross-sections with relatively high porosity and low compaction. Alternatively, the cross-section of the surfactant-containing PHB/PdNP fibers, included in Figure 2h,k, were seen to be more compacted since the adhesion among the fibers in the layered structure was higher. For all layers, it was also possible to perceive some particles aggregation that may not be necessarily related to the presence of the PdNPs but, more probably, to additives such as the nucleating agen<sup>t</sup> boron nitride, originally included in the biopolymer by the manufacturer.

In the SEM images of the films cross-sections, shown in Figure 2c,f,i,l, it can be observed that the electrospun fibers fused and interconnected among each other after the annealing treatment at 160 ◦C, successfully leading to the packing of the fiber mat into a continuous film. Among the here-prepared films, the neat PHB film showed a more uniform, smooth, and homogeneous surface. After the addition of the PdNPs and surfactants, the films became more heterogeneous, rougher, and also presented some cavities. This morphology change can be then related to the presence of the PdNPs, which more likely interfered in the fibers coalescence process.

#### 3.1.3. Dispersion of PdNPs

In order to provide a more resolved information about the dispersion of the PdNPs into the PHB biopolymer matrix, TEM was performed on the nanocomposite fibers and films. The distribution of the PdNPs inside the electrospun fibers are illustrated in the TEM images included in Figure 3a,d,g. From the TEM image included in Figure 3a, one can clearly discern that the PdNPs were mainly agglomerated in certain regions of the submicron PHB fibers. One can also observe that the addition of both surfactants, i.e., CTAB and TEOS, as respectively shown in Figure 3d,g, successfully improved the PdNPs dispersion in the PHB fibers. Dispersion and distribution was, however, seen higher in the CTAB-containing sample.

**Figure 2.** Scanning electron microscopy (SEM) images taken on the surface views and cross-sections of the electrospun poly(3-hydroxybutyrate) (PHB) fibers containing palladium nanoparticles (PdNPs) and their respective annealed films: (**a**) Surface view of the neat PHB fibers; (**b**) Cross-section of the neat PHB fibers; (**c**) Cross-section of the neat PHB film; (**d**) Surface view of the PHB/PdNP fibers; (**e**) Cross-section of the PHB/PdNP fibers; (**f**) Cross-section of the PHB/PdNP film; (**g**) Surface view of the PHB/PdNP/hexadecyltrimethylammonium bromide (CTAB) fibers; (**h**) Cross-section of the PHB/PdNP/CTAB fibers; (**i**) Cross-section of the PHB/PdNP/CTAB film; (**j**) Surface view of the PHB/PdNP/tetraethyl orthosilicate (TEOS) fibers; (**k**) Cross-section of the PHB/PdNP/TEOS fibers; (**l**) Cross-section of the PHB/PdNP/TEOS film.

In relation to the annealed films, the PHB/PdNP film without surfactant, shown in Figure 3b,c, still presented a clear aggregation of the particles. As opposite, Figure 3e,f, for the CTAB-containing film sample, and Figure 3h,i, for the TEOS-containing film sample, clearly showed that the PdNPs were evenly and relatively well distributed in the PHB films without forming agglomerates. In the case of the TEOS-containing film sample, the nanoparticle dispersion was less uniform when compared to the sample prepared with CTAB surfactant due to the absence of large grey dark areas. In both PHB films, very small nanoparticles of approximately 5 ± 2 nm can be seen, being homogeneously dispersed along the biopolymer matrix. The present results are in agreemen<sup>t</sup> with the results showed by Shaukat et al. [27], where PdNPs were incorporated into polyamide 6 (PA6)/clay nanocomposites and the nanoparticles were largely separated from each other and oriented in all possible directions in the polymer matrix. However, due to the absence of surfactant, some particles still agglomerated into clusters of bigger sizes that increased at the PA6 interfaces with the nanoclays.

**Figure 3.** Transmission electron microscopy (TEM) images of the electrospun poly(3-hydroxybutyrate) (PHB) fibers containing palladium nanoparticles (PdNPs) and their respective annealed films: (**a**) PHB/PdNP fibers; (**b**,**<sup>c</sup>**) PHB/PdNP film; (**d**) PHB/PdNP/ hexadecyltrimethylammonium bromide (CTAB) fibers; (**<sup>e</sup>**,**f**) PHB/PdNP/CTAB film; (**g**) PHB/PdNP/tetraethyl orthosilicate (TEOS) fibers; (**h**,**i**) PHB/PdNP/TEOS film.
