*3.2. PHA Production by P. resinovorans Using Various Types of Carbon*

Microbial metabolism for the accumulation of PHA occurs in two different ways: (1) short-chain carbon sources are usually utilized by biosynthesis for accumulation of PHA by the phaABC pathway, through acetyl-CoA to acetoacetyl-CoA by phaA (thiolase), while phaB (oxidoreductase) and phaC (polymerase) are mainly involved in intracellular processes; and (2) long-chain carbon sources, such as fatty acids or alkanes, are utilized through the β-oxidation pathway, and various acyl-CoA transferases convert to intermetabolites, including the mcl-PHA precursor [17]. *P. resinovorans* has both metabolisms for PHA accumulation; therefore, there is a need to evaluate the PHA production of different carbon sources. *P. resinovorans* was cultured using 2% of glucose, 20 (*v*/*v*)% of each alkane (n-octane, n-decane, or n-dodecane), and 20 (*v*/*v*)% of mixed alkane to compare PHA production and the monomer composition in accordance with the carbon

source (Table 1). With glucose as the carbon source, 1.56 g/L of PHA was produced that contained 1.47% 3-hydroxyhexanoate (3HHx), 10.60% 3-hydroxyoctanoate (3HO), and 87% 3-hydroxyoctanoate (3HD). Among the carbon sources with 20% of a single alkane (n-octane/n-decane/n-docane), the best cell growth and PHA production were found using n-octane as the sole carbon source. This produced a CDW of 1.74 g/L, PHA of 0.37 g/L, and PHA content of 22.4% that contained 4.59% of 3HHx, 86.05% of 3HO, and 9.36% of 3HD. When the mixed alkane was used, 0.69 g/L of PHA was produced that contained 11.77% of 3HHx, 74.39% of 3HO, and 13.84% of 3HD. Although cell growth was not the highest, the mixed alkane showed an approximately 50% increase in PHA production compared with using n-octane.

**Table 1.** Cell growth, PHA production, and monomer composition by *P. resinovorans* in various carbon sources.


3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate.

#### *3.3. Mixture Analysis of Alkanes (n-octane, n-decane, and n-dodecane) as a Carbon Source for mcl-PHA Production*

To investigate the effect of the three alkanes on cell growth, PHA production, and PHA content, *P. resinovorans* was grown as 10 culture compositions based on the mixture analysis model, as described in "Materials and methods" (Table 2). For each culture, we measured the cell growth, amount of PHA, and PHA content. The results of the statistical analysis were shown by contour plots and the predicted highest cell growth, PHA titer, and PHA content using *P. resinovorans* were determined (Figure 3).


**Table 2.** Monomer composition of 10 conditions by mixture analysis.

3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate.

Contour plots for cell growth predicted the best ratio to be when n-octane, n-decane, and n-dodecane are 10.36%, 5.48%, and 4.5%, respectively, which produced CDW of 5.28 g/L (Figure 3A). In the case of 9.15% of n-octane, 6.44% of n-decane, and 4.29% of n-dodecane, the PHA titer was 2.39 g/L of PHA (Figure 3B). In addition, the PHA content calculated by the amount of PHA contained in the cell was predicted to be a maximum of 52.33% content (*w*/*v*) when n-octane, n-decane, and n-dodecane are 8.63%, 6.86%, and 4.50%, respectively (Figure 3C). The optimal alkane composition for total PHA production predicted by the model is a combination of n-octane, n-decane, and n-dodecane, rather than pure alkane or a combination of two different alkanes [16,22,23]. The reason why a

combination of alkane compounds in the medium is beneficial is not currently clarified, but is estimated to be due to the better activities of alkane hydroxylase of *P. resinovorans* when the mixed alkane exists [24,25].

**Figure 3.** Mixture contour plots of mixed alkane composition for PHA production. The optimal composition ratio between n-octane, n-decane, and n-dodecane was determined as 20% (*v*/*v*) as a total concentration. (**A**): CDW (g/L), (**B**): PHA (g/L), and (**C**): PHA content (%) of different mixed alkane compositions.

#### *3.4. Jar-Scale Fermentation for mcl-PHA Production in Media-Optimized Conditions*

Carbon, nitrogen, and phosphorus are essential nutrients for growth, and their limitation can trigger mcl-PHA production in micro-organisms. However, the specific molecular mechanisms that drive this synthesis in *Pseudomonas* species under unfavorable growth conditions remain poorly understood. Therefore, it is necessary to determine the optimal concentration of nitrogen and phosphorus, because mcl-PHA production is related to their concentration when alkanes are used as carbon sources. To determine the optimal conditions, nitrogen concentrations were observed from 0% to 1.0% and phosphorus concentrations from 0% to 3.0% (Figure 4). When the concentration of nitrogen in the culture medium increased from 0 to 0.05%, PHA production tended to increase accordingly. The maximized PHA produced in 0.05% nitrogen was 1.31 g/L, but in the medium where the nitrogen concentration was more than 0.1%, PHA production decreased (Figure 4A). When the concentration of phosphorus in the medium increased from 0 to 1%, the production of PHA increased as well (Figure 4B). The maximum PHA production was 1.14 g/L at a concentration of 1% phosphorus. However, PHA production tended to decrease when the concentration of phosphorus was more than 2%. These results suggest that the optimal phosphorus concentration in the medium is 1.0%.

Using the optimized alkane combination and nitrogen and phosphorus concentration, *P. resinovorans* was cultured in a 7 L jar fermenter (3 L of working volume). The CDW, amount of PHA, and PHA content reached 3.5 g/L, 2.1 g/L, and 60%, respectively (Figure 5). Previous reports focused on *P. oleovorans* for the production of mcl-PHA with n-octane, and these show production of up to 16.8 g/L of PHA by fed-batch culture, while n-decane and n-dodecane were not considered because of low PHA production (Table 3). Compared with other *Pseudomonas* species, it was proven that use of *P. resinovorans* is more effective for production of mcl-PHA when mixed alkane is used as a carbon source.


**Table 3.** mcl-PHA production using various alkanes by Pseudomonas species.

CDW: final cell dry weight, PHA formed: final concentration of PHA, 3HB: 3-hydroxybutyrate, 3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HN: 3-hydroxynonanoate, 3HD: 3-hydroxydecanoate, 3HUD: 3-hydroxyundecanoate, 3HDD: 3-hydroxydodecanoate and 3HTD: 3-hydroxytetradecanoate.

#### *3.5. Physical Properties of Produced mcl-PHA by P. resinovorans*

The thermal properties of the produced mcl-PHA were determined by means of DSC (Table 4 and Supplementary Figure S2). The Tg of the samples ranged from −43.44 to −34.29, which can be regarded as values typical for this type of PHA. The sample also showed a Tm in the range of 39.62 with a ΔHm of 13.2 J/g. In addition, these values are typical for mcl-PHAs and indicate classical rubber- to latex-like characteristics [26]. The Mw, Mn, and

polydispersity (Mw/Mn) were similar for the copolyesters, despite the differences in their monomer compositions. The Mn and Mw were 267,649 and 630,526 Da, respectively.

**Table 4.** Physical properties of mcl-PHA from *P. resinovorans* using mixed alkane.


In addition, Z average (Mz) and Z + 1 average (Mz + 1) molar mass were determined as 1,110,977 and 1,729,603 Da, respectively. The polydispersity index (PDI) was calculated (PDI = Mw/Mn) as 2.36, more or less similar to that of the PHA value of mcl-PHAs biosynthesized by other known *Pseudomonas* species.

**Figure 4.** Effects of nitrogen and phosphorus limitation on PHA production. (**A**): CDW, PHA titer, and PHA content (%) in range of 0 to 1% nitrogen concentration. (**B**): CDW, PHA titer, and PHA content (%) in range of 0 to 3% phosphate concentration.

**Figure 5.** PHA production by *P. resinovorans* in optimized condition. Batch culture was performed in 7 L scale jar fermenter (3 L as working volume) with optimized culture medium.

#### **4. Conclusions**

Recent studies have shown that the use of renewable carbon sources can produce a significant reduction in actual production costs. A very interesting research topic would be for industrially produced waste oil or oil produced by pyrolysis of waste plastics to be used as a fermentation substrate for production of PHA, which is a promising bioplastic. Therefore, it is important to find PHA-producing micro-organisms and establish culture conditions in accordance with the carbon composition of waste oil or oil from waste plastics.

In this study, the ability of *P. resinovorans* to produce PHA from alkanes (n-octane, n-decane, and n-dodecane), especially mixed alkane, was identified, and a model was built to improve mcl-PHA production. Compared with other *Pseudomonas* species, *P. resinovorans* was much more effective in utilizing mixed alkane, and it can be an attractive microbial host for production of PHA. Although the regulation of PHA production based on alkane utilization has not been discovered, the results of this study are meaningful for use as basic data for the application of various renewable carbon resources containing alkanes.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14132624/s1, Figure S1: Pyrolysis oil of waste-plastic and its composition, Figure S2: DSC analysis of purified PHA produced by *P. resinovorans* using mixed alkane.

**Author Contributions:** J.-M.J.: Writing—Original Draft, Investigation, Data Curation. S.-J.P.: Methodology, Investigation, Data Curation. Y.-S.S.: Data Curation. Y.-H.Y.: Writing—Review and Editing, Methodology. J.-J.Y.: Conceptualization, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT or MSIT; NRF-2020R1A2C2102381) and the Korea Institute of Industrial Technology (KITECH) through Development of eco-friendly production system technology for a total periodic resource cycle (21-0140).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
