**1. Introduction**

In recent years, people have become more aware of environmental pollution and have made efforts to avoid the continuous use of non-degradable conventional plastics [1,2]. Bioplastics are a suitable alternative to disposable products and can be produced and degraded by microorganisms [3]. There are many kinds of bioplastics such as polylactic acid (PLA), poly(butylene adipate-*co*-terephthalate) (PBAT), and polyhydroxybutyrate (PHB) that can substitute non-degradable conventional plastics [4,5]. Since PLA and PBAT have superior properties, they are already being used little by little in various industrial fields such as packaging and agriculture industries [6,7].

In contrast to PLA and PBAT, which are not produced directly by microorganisms, PHB has the advantage that it can be produced directly through fermentation. Several microorganisms have been reported to have PHB accumulation capability, since PHB is a metabolite in the butanoate metabolism of microorganisms [8]. Moreover, PHB accumulators also express PHB depolymerase and can easily degrade PHB compared with PLA and PBAT. However, compared to PLA and PBAT, which are easily observed in daily life, PHB is not frequently observed. The main reason is that PHB is brittle: it does not stretch well and is easily torn, compared with other kinds of bioplastics [9–12].

**Citation:** Cho, J.Y.; Kim, S.H.; Jung, H.J.; Cho, D.H.; Kim, B.C.; Bhatia, S.K.; Ahn, J.; Jeon, J.-M.; Yoon, J.-J.; Lee, J.; et al. Finding a Benign Plasticizer to Enhance the Microbial Degradation of Polyhydroxybutyrate (PHB) Evaluated by PHB Degrader *Microbulbifer* sp. SOL66. *Polymers* **2022**, *14*, 3625. https://doi.org/ 10.3390/polym14173625

Academic Editor: Helena Felgueiras

Received: 29 July 2022 Accepted: 29 August 2022 Published: 1 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

There are many ways to strengthen PHB properties, but the most commonly used method is to use a plasticizer. A plasticizer is an additive added to change the physical properties of a material. When a plasticizer is added to a polymer such as plastic, the attractive force between the polymer chains is reduced, thereby increasing its flexibility [13]. Many kinds of plasticizers have already been added to non-degradable conventional plastics [14]. Since the addition of plasticizers greatly affects the physical properties of plastics, research on adding various plasticizers to bioplastics is being actively conducted [15,16]. Because most plasticizers are chemical substances with complex structures that are not related to microorganisms, there is a possibility that some plasticizers may adversely affect microorganisms [17]. In this case, the advantages of bioplastics may be weakened by the effect of plasticizers on microorganisms or their biodegradability. Therefore, carrying out research into finding a plasticizer that has good compatibility with bioplastic-degrading microorganisms is important. When carrying out such research, there are limitations of time and place when conducting all kinds of experiments in natural environments such as soil or ocean.

Therefore, in this study, plasticized PHB was prepared by adding various kinds of plasticizers to PHB, and their properties were compared through various analytical instruments such as differential scanning calorimetry (DSC), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and universal testing machine (UTM). The effect of various plasticizers on the *Microbulbifer* sp. SOL66 (already been proven as a superior PHB degrading strain in our laboratory) was studied [18,19]. PHB degradation was also evaluated by studying the difference in the weight loss of PHB added with various kinds of plasticizers. This result suggests which plasticizer is suitable or unsuitable for use in PHB. By using *Microbulbifer* sp. SOL66 that can almost completely degrade PHB in 2–3 days, a benign plasticizer with good properties and good biodegradability could be selected very quickly, while overcoming the limitations of time and place.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

All chemicals used in this study were of analytical grade. PHB pellets (Average MW: 550,000 g/mol) were obtained from Goodfellow Cambridge Ltd. (Huntingdon, UK). Chloroform and ethanol were obtained from Fisher Scientific (Hampton, NH, USA). Bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) sebacate, lauric acid, triacetin, tributyl citrate, tributyl 2-acetylcitrate, l-Linalool, and geraniol were obtained from Sigma-Aldrich (St. Louis, MO, USA).

#### *2.2. Toxicity Test of Various Plasticizers to the Growth of Microbulbifer* sp*. SOL66*

To confirm the effect of various plasticizers on microbial cells, growth measurements were conducted under the effect of different concentrations of plasticizers. Bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) sebacate, triacetin, tributyl citrate, tributyl 2-acetylcitrate, l-Linalool, and geraniol were used in concentrations from 0.25 mM to 16 mM. Only lauric acid was used in concentrations from 0.02 mM to 1.28 mM due to solubility. *Microbulbifer* sp. SOL66 cells were precultured in 5 mL marine broth (MB; Kisanbio, Seoul, Republic of Korea) in a shaking incubator at 37 ◦C, 200 rpm, for 24 h. Cells taken from the preculture were used to inoculate into the main culture process. In this process, pipetting robot (Integra, Le Locle, Switzerland) was used for the dilution of each plasticizer. Before the experiment, all kinds of plasticizers were dissolved in the HPLC grade ethanol to give a final concentration of 1.6 M of each plasticizer, except lauric acid. It cannot be dissolved significantly so a stock solution of 128 mM was prepared. All of the stock solutions were sterilized by using a filter (pore size, 0.2 μm). A 96-well cell culture plate (Thermo Fisher Scientific, MA, USA) was used for the culture process. MB liquid media 200 μL were inoculated with 2% of *Microbulbifer* sp. SOL66 in all the spotted wells, except for the first column. In the first column, 400 μL of MB liquid media containing 4% of *Microbulbifer* sp. SOL66 were spotted. After 4 μL of each plasticizer was added to the first column, a 96-well cell culture

plate was set on the pipetting robot instrument. Then, according to the dilution sequence pre-arranged on this instrument, the solution was diluted two-fold while passing to the next column sequentially. In the dilution process, the mixing process through pipetting 5 times was also included for each dilution. After that, it was incubated in a shaking incubator at 37 ◦C, 200 rpm, for 24 h. The optical density after incubation was measured at 595 nm using a 96-well microplate reader (Tecan, Mannedorf, Switzerland) [20].

#### *2.3. Degradation of PHB Containing Various Plasticizers under the Liquid Condition*

For the liquid culture degradation test, PHB films with a thickness of 0.04 mm containing various plasticizers were prepared with 10% (*w*/*w*) and 20% (*w*/*w*) of concentrations. PHB pellets were placed in a glass bottle, into which 50 mL of chloroform was added. Then, each plasticizer was added to 10% and 20% of the total mass. The bottles were heated at 60 ◦C in a water bath until the contents were completely dissolved. Heated chloroform containing plasticized PHB was poured into a glass Petri dish in a fume hood, and a film was made by evaporating all of the solvents. Next, all films were cut by 20 mg each and then sterilized by immersing in 70% ethanol and drying in UV light. Then, each film was cultivated with the precultured *Microbulbifer* sp. SOL66 in 5 mL of MB liquid media [21]. After 2 days of cultivation at 37 ◦C, 200 rpm, optical density was measured with the same process described in the previous section. Degraded PHB films were recovered and washed with distilled water to remove impurities containing cell debris. In the case of time-dependent analysis, PHB films degraded by *Microbulbifer* sp. SOL66 after 1, 2, and 3 days of cultivation were recovered and treated with the same procedure. Recovered films were analyzed using a GC-MS instrument to measure weight loss.

#### *2.4. GC-MS Analysis*

The degree of degradation was calculated with GC-MS data. Before using the GC-MS instrument, fatty acid methyl ester (FAME) derivatization was conducted for preparing the GC-MS sample [21,22]. Then, 1 mL of methanol/sulfuric acid (85:15 *v*/*v*) and 1 mL of chloroform were mixed in each glass vial containing lyophilized PHB films. Additionally, each vial was sealed with a screw cap and Teflon tape and was heated using a heating block at 100 ◦C for 2 h for FAME derivatization. After the heating process, the samples were subsequently cooled at a low temperature for approximately 10 min, 1 mL of HPLC-grade water was added to each vial for making layer separation, and the samples were gently vortexed for 1 min. The bottom layer (chloroform) was transferred to a new 1.5 mL e-tube containing sodium sulfate anhydrous to remove water. Each sample was filtered (pore size, 0.2 μm) into a clean borosilicate glass tube. The prepared sample was analyzed by GC-MS (Perkin Elmer, Waltham, MA, USA) equipped with a fused silica capillary column (Elite-5 ms, 30 m × 0.25 mm i.d. × 0.25 μm) and subjected to a linear temperature gradient for analysis (40 ◦C for 1 min, increased at 15 ◦C/min to 120 ◦C and held for 2 min, and then increased at 10 ◦C/min to 280 ◦C and held for 10 min). The injector port temperature was 250 ◦C. Mass spectra were obtained by electron impact ionization at 70 eV, and scan spectra were obtained within the range of 45–450 m/z. Selected ion monitoring was used for the detection and fragmentation analysis of the major products. A standard curve was constructed using each kind of film to quantify the degraded PHB films. When a plasticizer was added to each film, the degree of degradation was calculated by considering the ratio.
