*3.2. Effect of Various Plasticizers on Microbulbifer* sp*. SOL66 Cell*

Before comparing biodegradability, a toxicity test on bacteria *Microbulbifer* sp. SOL66 was conducted with all 8 plasticizers. If a plasticizer that prevents cell growth is used, the biodegradation process using microorganisms is disrupted. So, compatibility between the plasticizer and *Microbulbifer* sp. SOL66 was confirmed. Although the toxicity of a plasticizer does not impede all biodegradation processes, confirmation of toxicity can provide a certain reference point for evaluating the suitability of the plasticizer for PHB, which can be easily degraded by *Microbulbifer* sp. SOL66. As a result of the toxicity test with various plasticizers, the effect of each plasticizer addition on the growth of *Microbulbifer* sp. SOL66 was varied (Figure 1).

In all cases, the growth of *Microbulbifer* sp. SOL66 was reduced when a plasticizer was added. However, in the case of triacetin, the cell growth gradually increased as the concentration increased. Except for this, the addition of bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) sebacate, lauric acid, tributyl citrate, and tributyl 2-acetylcitrate showed a generally decreasing tendency as the concentration increased. When L-Linalool and geraniol, which are natural monoterpenoid plasticizers, were added, *Microbulbifer* sp. SOL66 did not grow at all when more than 8 mM and 4 mM were added, respectively. However, this does not mean that plasticizers that reduce or inhibit microbial growth are

not good when added to PHB. It suggests that plasticizers may adversely affect cell growth when applied directly to cells. The impact of toxicity as a molecule might be different from the role inside of the polymer.

**Figure 1.** Growth tendency with the addition of various plasticizers. The cell growth pattern of *Microbulbifer* sp. SOL66 was expressed as optical density with the addition of plasticizers at concentrations of 1 mM, 2 mM, 4 mM, and 8 mM, lauric acid\* was added at 0.16 mM, 0.32 mM, 0.64 mM, and 1.28 mM due to solubility issues.

#### *3.3. Thermal Properties Analyzed by DSC*

To confirm the change in chemical properties by the addition of plasticizer, DSC analysis was performed to measure the glass transition temperature (*Tg*), melting temperature (*Tm*), and crystallization temperature (*Tc*). In general, it is known that adding a plasticizer creates free space between the polymers, thereby reducing the *Tg* value and softening the physical properties by allowing the components of the polymer to move freely [13,47,48]. The DSC result showed that the *Tg* value of plasticized PHB film is low compared with non-plasticized PHB, except for PHB film containing 20% of L-Linalool (Table 2). It can be supposed that the addition of various kinds of plasticizers increased the softness of PHB. Not only the *Tg* value but also the *Tm* value and *Tc* value changed according to the addition of the various plasticizers, but the crystallinity value (*Xc*) derived from the enthalpy change at melting temperature (Δ*Hm*) is more important in analyzing the properties. *Xc* value was calculated using Equation (1) [23,29,30].

$$X\_c(\%) = \frac{\Delta H\_m}{\Delta H\_m 100\%} \times \frac{100}{W\_{PHB}} (\%) \tag{1}$$


**Table 2.** Thermal properties of plasticized PHB analyzed by using DSC.

The melting enthalpy of a 100% crystalline 3-hydroxybutyric acid (Δ*Hm* 100%) was reported to be 146 J/g and *WPHB* means the weight fraction of PHB in the plasticized PHB [49]. By using this, *Xc* values of plasticized PHB were calculated. The crystallinity of non-plasticized PHB was 43.1%, whereas the crystallinity of plasticized PHB was increased, except when 20% of bis(2-ethylhexyl) sebacate and 10% of L-Linalool were added to PHB. This is because adding a plasticizer improves the mobility of the polymer chain and promotes crystallization [44].

#### *3.4. Effect of Various Plasticizers on the Biodegradability of PHB by Microbulbifer* sp*. SOL66*

After confirming the toxicity of various plasticizers as molecules themselves, the effect of plasticizers inside of PHB on the growth of cells was compared. In addition, the biodegradability of plasticized PHB was compared to screen the optimal plasticizer for better quality PHB that can be degraded by *Microbulbifer* sp. SOL66. Eight kinds of plasticizers were added to PHB film with 10% and 20% concentrations, respectively. After the sterilization process, plasticized PHB films were cultured together with *Microbulbifer* sp. SOL66 in a 37 ◦C shaking incubator for two days. After cultivation, the growth expressed as optical density was first measured to confirm the effect of the plasticizer contained in the PHB film on the *Microbulbifer* sp. SOL66 cells. In the case of culturing the PHB films containing 10% of the plasticizers, growth itself was reduced compared to the case of culturing the PHB film without the plasticizer as a whole (Figure 2a). However, there was no case where growth did not occur. On the other hand, even when the PHB films containing 20% of the plasticizers were cultured, the growth pattern was similar depending on the type of added plasticizer, but in the case of culture of the PHB film containing 20% of geraniol, *Microbulbifer* sp. SOL66 did not grow (Figure 2b). The result of completely inhibiting the growth of *Microbulbifer* sp. SOL66 when 20% of geraniol was included in the film was similar to the previous toxicity test, suggesting geraniol is not a suitable plasticizer for PHB when the evaluation of PHB degradation is conducted with *Microbulbifer* sp. SOL66., but the growth pattern when the plasticizer was included in the film was different from that when the plasticizer was directly applied to microbial cells.

**Figure 2.** The growth pattern of *Microbulbifer* sp. SOL66 cultured with (**a**) 10% plasticized and (**b**) 20% plasticized PHB films.

As a result of analyzing the degree of degradation of the PHB film containing various plasticizers through GC-MS, when most plasticizers were added, the degree of degradation tended to decrease. In the case of degradation of PHB film containing 10% of plasticizers, the biodegradability of tributyl citrate and tributyl 2-acetylcitrate was similar to that of the film without plasticizers (Figure 3a). When 20% of plasticizers were included, the biodegradability of triacetin and tributyl citrate was comparable to that of the nonplasticized PHB (Figure 3b). When 20% of triacetin was added, the degree of degradation was higher than that of the non-plasticized PHB, but in the case of 10%, the degree of degradation was not very high. On the other hand, in the case of tributyl citrate, since it showed excellent robustness of biodegradability in both 10% and 20% plasticized PHB, it could be concluded that tributyl citrate was suitable as a plasticizer to be added to PHB, which is greatly degraded with *Microbulbifer* sp. SOL66, suggesting the toxic effect of plasticizer itself and degradation of plasticized PHB is different.

**Figure 3.** The comparison of degradation of (**a**) 10% plasticized and (**b**) 20% plasticized PHB films by *Microbulbifer* sp. SOL66.

#### *3.5. Comparison of Other Properties with Using Various Analytical Instruments*

As PHB with tributyl citrate showed higher degradation than PHB without tributyl citrate, the surface change with the addition of 10% and 20% of tributyl citrate after two days of degradation by *Microbulbifer* sp. SOL66 was compared using SEM. Since the surfaces of the non-plasticized PHB film and the plasticized PHB film before degradation were similar, the surface area characteristics of the film could be confirmed by checking the surface after degradation. The SEM result showed that the non-plasticized PHB film looked as if degradation had progressed as the cracked cross-section was split at an angle, and the cross-sectional portion where the tributyl citrate-added film was degraded was curved rather than angled (Figure 4). Through the comparison of the parts where the degradation has progressed in this way, it was possible to visually compare the difference in the physical properties of the surface.

**Figure 4.** Representative images of surface changes observed by scanning electron microscopy. Comparison of differences in surface changes after two days of degradation by *Microbulbifer* sp. SOL66 through observation of 500 and 1000 magnifications.

Next, the change in molecular weight according to the addition of tributyl citrate before and after degradation by *Microbulbifer* sp. SOL66 was compared. As indicated in (Table 3), there was little difference between the number average molecular weight (*Mn*) values of plasticized and non-plasticized PHB films before degradation by *Microbulbifer* sp. SOL66. However, there was a slight difference after 2 days of degradation. When tributyl citrate was added, the number average molecular weight decreased more than when it was not added. Conversely, polydispersity index (PDI) values after degradation were higher when tributyl citrate was added. Through the difference in molecular weight change, it was found that adding tributyl citrate to the PHB film could cause a change in degradation by *Microbulbifer* sp. SOL66.


**Table 3.** Comparison of molecular weight change before and after degradation by *Microbulbifer* sp. SOL66 according to the addition of 0%, 10%, and 20% of tributyl citrate.

To find out the reason for high microbial degradation under the influence of plasticizers, XRD spectra of non-plasticized PHB film and PHB film plasticized with tributyl citrate were presented and compared (Figure 5a). The shape of the spectra was similar with or without plasticizer addition. In all three spectra, two strong peaks were commonly observed at 2θ = 13.5 and 2θ = 16.9, which are assigned with (020) and (110) diffraction planes. These peaks correspond to orthorhombic α-form crystals of PHB [50]. However, when a plasticizer was added, the intensities of these two peaks were slightly changed, meaning that crystal structures were slightly changed.

**Figure 5.** Changes in various properties according to the addition of tributyl citrate. Comparison of (**a**) XRD spectra and (**b**) thermo gravimetric analysis (TGA) result. Mechanical properties change expressed as (**c**) tensile strength (MPa), Young's modulus (MPa), and (**d**) relative elongation at break (%) measured using a universal testing machine (UTM).

Next, the pattern of thermal degradation according to the presence or absence of the plasticizer was compared through the TGA results (Figure 5b). The pattern of abruptly decreasing weight near 250 ◦C was similar, but when tributyl citrate as a plasticizer was added, the weight was already greatly reduced before entering the rapidly decreasing section [29]. It was confirmed that the stability to heat decreased according to the concentration of the plasticizer added. Additionally, the higher the concentration of tributyl citrate, the lower the stability was. This is because plasticizer loss occurs when a high temperature is applied [27].

Lastly, the change in mechanical properties expressed as tensile strength, Young's modulus, and relative elongation at break were compared through the universal testing machine results. When tributyl citrate as a plasticizer was added, tensile strength and Young's modulus were decreased by more than half (Figure 5c). However, the decrease in both values according to the increase in the concentration of the plasticizer was not very large. On the other hand, there was a large difference in the length of the film stretched (Figure 5d). When the elongation of the film without the addition of tributyl citrate was set to 100, and a relative comparison was conducted, the elongation was nearly doubled when 10% of tributyl citrate was included in PHB film, and it was almost tripled when 20% of tributyl citrate was included. This is because the addition of tributyl citrate as a plasticizer reduced the intermolecular interactions of PHB [51].
