*2.3. Design of Experiments and Mixture Analysis*

To develop a strategy for optimizing cell growth and PHA production, a mixture analysis model of three alkanes (n-octane, n-decane, and n-dodecane) was developed and populated using a standard mixture-analysis methodology and the Minitab V19 program. For the design of mixture-analysis experiments to populate the model, we used a simplex lattice method. The degree of the lattice for this mixture analysis was 2; therefore, the experimental design contained the set of all 10 combinations. All experiments were performed using 50 mL cultures with 20% total alkane content, and the cultures were cultured at 30 ◦C for 48 h in duplicate. To plot mixture contours, a mixture regression using the model-fitting method was applied with full quadratic component terms initially included. In the data analysis, the coefficients with p value below 0.1 were used as parameters.

### *2.4. Characterization of Obtained mcl-PHA*

The quantity and composition of PHA were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), using a slight modification of a method described previously [17]. For analysis, the microbial culture after the completion of growth was centrifuged at 10,000× *g* for 30 min, washed with deionized water two times, and suspended in 1 mL of water. The suspended samples were subjected to lyophilization, and the freeze-dried cells from each experiment were subjected to methanolysis. A weighed sample was placed in a Teflon-stoppered glass vial, and 1 mL chloroform and 1 mL methanol/sulfuric acid (85:15 *v*/*v*) were added to the vial. The samples were incubated at 100 ◦C for 2 h, cooled to room temperature, and incubated on ice for 10 min. After adding 1 mL of ice-cold water, the samples were mixed thoroughly using a vortex for 1 min and then centrifuged at 2000× *g*. The organic phase (bottom) was carefully extracted using a pipette and was moved to clean borosilicate glass tubes. A 2 μL portion of the organic phase of these samples was then injected into a gas chromatograph (6090N, Agilent Technologies, Santa Clara, CA, USA) using a flame ionization detector (FID) and a 30 m × 250 μm DB-FFAP capillary column with hydrogen as the carrier gas. The inlet of the gas chromatograph was maintained at 250 ◦C, and the oven was held at 80 ◦C for 5 min,

heated to 220 ◦C at 20 ◦C min−1, and then held at 220 ◦C for 5 min. Peak detection was performed using a flame ionization detector, which was maintained at 300 ◦C. The fatty acid content was analyzed via GC–MS chromatography (Perkin Elmer Clarus 500, Waltham, MA, USA) according to the modified method previously reported in [17]. About 1 uL of methanolized sample was injected into the Clarus 680 GC-MS equipped with triple axis detector carrying Elite 5 ms column (30 mm length × 0.25 mm internal diameter × 0.25 mm film) at a split ratio of 10:1 with column flow 1.0 mL min<sup>−</sup>1. The injector temperature was set at 280 ◦C while the oven and column temperatures were programmed as 10 ◦C for 1 min, then increased to 130 ◦C at 11 ◦C min−1, held for 2 min, and increased to 310 ◦C at 10 ◦C min<sup>−</sup>1, and held for 10 min. Helium was used as carrier gas at 47.3 mL min−<sup>1</sup> and 0.40 bar pressure. Mass spectra were acquired at 1250 scan speed using electron-impact energy of 70 eV at 200Uc ion source and 280 ◦C interface temperatures, respectively. Complete instrument control was available through TurboMass™ driver. NIST/EPA/NIH library was used to predict the methylated PHAs and their corresponding mass ion. Statistical analysis was carried out through one-way ANOVA, where *p* < 0.05 was considered to be statistically significant.

To study the melting behavior of synthesized polymer, differential scanning calorimetry (DSC) analysis were performed by Discovery DSC (TA Instruments, Bellefonte, PA, USA) in the temperature range from −80 to +100 ◦C. The glass transition temperature (Tg) was determined at a heating rate of 20 ◦C/min. In this study, Tg was taken as the midpoint of the step-transition. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity (Mw/Mn) were determined by gel permeation chromatography (GPC) conducted in THF solution at 35 ◦C and a flow rate of 1 mL/min. A 10 μL sample in THF at a concentration of 1% *w*/*v* was injected. Polystyrene standards with narrow molecular-mass distribution were used to generate a calibration curve.

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

#### *3.1. Screening of Alkane-Based PHA-Producing Strains*

After the discovery in 1983 that *P. oleovorans* could produce mcl-PHA using n-octane, various *Pseudomonas* species were found to be capable of PHA production using alkane compounds [18–20]. However, few studies have reported on the use of various alkane compounds as a carbon source because most studies were focused on the use of n-octane. The composition of pyrolysis oil from polyethylene-based waste plastics is determined by its process conditions, and C8- to C22-saturated hydrocarbons are predominantly included in the case of pyrolysis oil received from the Korea Institute of Industrial Technology (Supplementary Figure S1). To find a suitable strain that is capable of mcl-PHA production using alkanes, *P. fluorescens*, *P. resinovorans*, *P. stutzeri*, and *P. putida* were selected and cultured in minimal media with 10 (*v*/*v*)% of either n-octane, n-decane, or n-dodecane as the sole carbon source. Each species showed different growth activity, and *P. resinovorans* showed better growth than the others (Figure 1). In addition, *P. resinovorans* was cultured in range of 1 to 50% of n-octane to investigate the relationship between the concentration of alkane and growth. The highest cell growth (1.34 g/L), PHA production (0.31 g/L), and PHA content (approximately 20 (*w*/*w*)%) were produced with 20 (*v*/*v*)% of n-octane (Figure 2). Meanwhile, with more than 30% n-octane in the medium, the cell dry weight (CDW), PHA amount, and PHA content decreased sharply to 0.68 g/L, 0.09 g/L, and 13.45%, respectively, which is attributed to the oxygen rate being rapidly reduced as the oil and liquid ratio increased [21].

**Figure 1.** Cell growth of *Pseudomonas* species using n-octane, n-decane, and n-dodecane as a sole carbon source. Each alkane is present as 10% (*v*/*v*) in minimal medium.

**Figure 2.** Cell growth and PHA production by *P. resinovorans* in the range of n-octane.

*P. resinovorans* was selected for the recycling of pyrolysis oil, which contains various olefin compounds, because it is able to utilize alkanes and produces more mcl-PHA than the other *Pseudomonas* species, including *P. oleovorans*. In addition, to maximize the production of mcl-PHA, the optimum concentration of alkanes in the medium was determined to be 20%.
