*2.5. Chemical and Mechanical Analysis*

DSC analysis of the plasticized PHB films and non-plasticized PHB films was performed using the Discovery DSC instrument (TA Instruments, New Castle, DE, USA) at temperatures ranging from −80 ◦C to 200 ◦C, while the heating and cooling rate was 10 ◦C/min in an N2 atmosphere [23–25].

To analyze the crystalline/amorphous characteristics of plasticized and non-plasticized PHB films, XRD analysis was performed using an X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) with a Cu Kα (λ = 1.54 Å) source, operating at 40 kV and 30 mA as the applied voltage and current, respectively. The 2θ range was from 3◦ to 60◦ at a scanning rate of 2◦/min [25–28].

Thermogravimetric analysis (TGA) was performed using a TGA N-1000 (Scinco, Seoul, Korea). Samples were heated from 30 ◦C to 700 ◦C, at a heating rate of 10 ◦C/min under nitrogen flow (20 mL/min) [29–31].

Mechanical properties of plasticized and non-plasticized PHB films were measured using an EZ-SX universal testing machine (Shimadzu, Kyoto, Japan) [32–34]. Before measurement, PHB films were cut into a rectangular sample of size 60 mm × 10 mm (length × width). Thickness, which depends on the presence and amount of plasticizer, was measured using an 1111–100 A mini digital caliper (Insize, Loganville, GA, USA). Data were collected at a strain rate of 20 mm/min. After measurement, tensile strength, young's modulus, and elongation at break were calculated from a plot formed by TRAPEZIUM X software.

#### *2.6. SEM Analysis*

Scanning electron microscopy (SEM) analysis was performed to analyze the surface changes in the plasticized and non-plasticized PHB films after degradation by *Microbulbifer* sp. SOL66. Recovered PHB films after 2 days of degradation by *Microbulbifer* sp. SOL66 were washed and lyophilized before SEM analysis. The lyophilized films were then coated with gold dust at 5 mA for 300 s, and back-scatter electron images were obtained using a TM4000Plus SEM instrument (Hitachi, Tokyo, Japan) at a voltage of 5 kV [35].

#### *2.7. GPC Analysis*

Gel permeation chromatography (GPC) analysis was performed to detect the molecular weight changes in plasticized and non-plasticized PHB films after degradation by *Microbulbifer* sp. SOL66. The sample preparation process was carried out with the same method as our previous research [21]. A high-performance liquid chromatography (HPLC) apparatus was used for measuring molecular weight change, consisting of a loop injector (Rheodyne 7725i), an isocratic pump with dual heads (YL9112), column oven (YL9131), columns (Shodex, K-805, 8.0 mm I.D. × 300 mm; Shodex, K-804, 8.0 mm I.D. × 300 mm), and RI detector (YL9170). Sixty microliters of the sample from which bubbles were removed were injected. Only twenty microliters of injected samples were used as analytes for analysis in the injector part. The mobile phase was chloroform, and this flow rate was maintained at 1.0 mL/min. The temperature was maintained at 40 ◦C for analysis. The data were analyzed using YL-Clarity software for a single YL HPLC instrument (YOUNG IN Chromass, Anyang, Republic of Korea). The molecular weight was calculated with polystyrene standards ranging from 5000 to 2,000,000 g/mol [35].

#### **3. Results**

#### *3.1. Comparison of Properties of Various Plasticizers*

Eight kinds of plasticizers containing bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) sebacate, lauric acid, triacetin, tributyl citrate, tributyl 2-acetylcitrate, L-Linalool, and geraniol which were often used for increasing properties of conventional plastics and bioplastics were included in the PHB film. As listed in (Table 1), all of the plasticizers that have been already reported for plasticizing PHB have very complex molecular structures and various molecular weights. Bis(2-ethylhexyl) adipate is the most commonly used plasticizer [36] because it can improve the flexibility of plastics and it has been regarded as a safe plasticizer. However, there is a precedent in which hepatic, brain, and cardiac injuries were found in rats when exposed to a large amount of this substance [37]. Although bis(2-ethylhexyl) sebacate is relatively less dangerous than bis(2-ethylhexyl) adipate, it is still not a recommended plasticizer because it is still dangerous [38,39]. Additionally, lauric acid can be used as a plasticizer because it forms a hydrogen bond when incorporated into the plastic, decreasing the force of each plastic molecule, resulting in a decrease in glass transition temperature [40,41]. Triacetin is a well-known plasticizer for PHB due to its improved biodegradability of PHB [42,43]. Tributyl citrate and tributyl 2-acetylcitrate are widely

used as plasticizers in bioplastics such as PHB due to their high biocompatibility [27,44,45]. L-Linalool and geraniol are natural monoterpenoids, which are components of essential oils derived from plants, and have a unique scent. Since it can be obtained from nature rather than chemically synthesized, it has the advantage of being environmentally friendly [46]. Since all plasticizers have their strengths and weaknesses, both physical properties and biodegradability were considered to select the most appropriate plasticizer for PHB.


**Table 1.** Molecular properties and applied references of plasticizers used in this study.
