**1. Introduction**

World plastic production has been increasing every year and reached 368 million tons in 2019 [1]. Among this plastic, 79% is dumped in landfills or the environment, where it takes from around 20 to 600 years to degrade [2]. In addition, the incineration of plastic waste causes serious emissions of greenhouse gases, which accelerates global warming and abnormal climate change [3]. According to the sixth assessment report of the Intergovernmental Panel on Climate Change (IPCC), the usage of petroleum-based plastic materials is still increasing, and the report warned that this will lead to irreversible climate change within around 10 years [4]. As a result, much attention is being paid to the development and use of alternative eco-friendly plastic materials, such as biodegradable plastic from renewable resources.

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that is regarded as a source of alternative materials because it has similar physical properties to petroleum-based plastic [5]. PHA is biosynthesized and accumulated by many bacteria in their cytoplasm as carbon storage materials when they encounter harsh growth conditions in the presence of excess carbon sources, which are classified into two groups, based on the numbers of carbon

**Citation:** Jeon, J.-M.; Park, S.-J.; Son, Y.-S.; Yang, Y.-H.; Yoon, J.-J. Bioconversion of Mixed Alkanes to Polyhydroxyalkanoate by *Pseudomonas resinovornas*: Upcycling of Pyrolysis Oil from Waste-Plastic. *Polymers* **2022**, *14*, 2624. https:// doi.org/10.3390/polym14132624

Academic Editor: Adriana Kovalcik

Received: 8 June 2022 Accepted: 22 June 2022 Published: 28 June 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/).

atoms composed of monomers, with different material properties: short-chain-length PHA (scl-PHA) composed of monomers with 3 to 5 carbon atoms and medium-chain-length PHA (mcl-PHA) composed of 6 to 14 carbon atoms [6]. Among such materials, mcl-PHA has numerous industrial applications, such as coating materials, pressure-delicate glues, and polymer-binding agents in organic solvent-free paints, and also it can be used in a series of biomedical applications [7]. It is produced by PHA-accumulating bacteria such as *Pseudomonas* species, and it has various physicochemical properties depending on the monomer composition and ratio [8,9]. The overall cost of the PHA production process is still expensive; therefore, there have been many studies that have attempted to use various carbon materials such as organic waste, seaweed biomass, animal fat, chitin, and oil waste [5,10]. Among them, pyrolysis oil based on plastic waste also has the potential for use as a carbon source for the production of PHA by micro-organisms. Pyrolysis oil contains aliphatic and aromatic hydrocarbon compounds depending on the catalyst type, plastic waste type, and cracking conditions [11]. The aliphatic hydrocarbon compounds from pyrolysis of plastic waste are predominantly composed of olefins (C20+), and these can be converted to low-carbon alkane or alkene compounds via hydrocracking [12].

PHA production based on alkanes has been studied since the 1980s, and it was discovered that many hydrocarbon-degrading bacteria can degrade and utilize various alkanes as a carbon source to grow and accumulate PHA in vivo [13]. Most alkanes can be utilized through the β-oxidation pathway with conversion to the carboxylic acid formed by alkane monooxygenase and then utilized to acyl-CoA, which can be used as a monomer for PHA accumulation [14]. Therefore, numerous studies have demonstrated the production of PHA from single alkanes by various *Pseudomonas* species, and n-octane was shown to be an economical carbon source for the production of mcl-PHA by *P. oleovorans* [15]. Since then, the maturation of plastic-waste-based pyrolysis oil conversion technology has drawn attention to the use of organic resources containing large amounts of alkane compounds. However, its uses have been limited to applications such as heating oil due to problems such as the fact that it must be additionally purified with a single compound for use in the chemical process. In the case of conversion to biodegradable plastic materials, polyethylene (PE) pyrolysis wax contains a low level of n-octane, and it was demonstrated that *P. oleovorans* is not suitable for producing mcl-PHA from PE pyrolysis wax as a sole carbon source [16]. There are differences in the efficiency of conversion of various alkanes to PHA monomers depending on the affinity with polymerase, and the portion of each alkane in the monomer composition of the produced PHA may be different. Consequently, each alkane affects the monomer composition of the produced PHA, which is an important factor for determining its physical properties [16]. Therefore, if waste oil or plastic pyrolysis oil, which contain mixtures of alkanes, are used for PHA production, it is necessary to find and evaluate a suitable PHA-producing strain for application to the alkane mixture resources.

In this study, *Pseudomonas* species were evaluated for production of mcl-PHA using alkane mixtures containing n-octane, n-decane, and n-dodecane as a sole carbon source, and the optimal condition for increasing PHA production was determined. In addition, the relationship between the ratio of various alkanes and the composition of the produced PHA monomer was determined.

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

#### *2.1. Micro-Organism and Culture Conditions*

*P. fluorescens* (ATCC 42821), *P. putida* (ATCC 1751), *P. resinovorans* (ATCC 12498), and *P. stutzeri* (ATCC 1066) were used to screen for producers of mcl-PHA from alkanes as a carbon source. All the strains and cultures were incubated with a working volume of 50 mL in a 250 mL flask at 30 ◦C for 48 h, and the initial pH was set to 7. Cell growth was monitored by measuring optical density at 600 nm (OD600). All the strains used in this study were precultured in LB medium at 30 ◦C for 24 h; then, 1% (*v*/*v*) of cultured cells were used for inoculation for further study. All components were sterilized via autoclaving for at least 20 min at 121 ◦C. As a preculture, 1% (*v*/*v*) frozen stock was incubated overnight

at 30 ◦C in a shaking incubator in 14 mL round bottom tubes with 5 mL of Luria–Bertani medium broth (LB) (Difco, Detroit, MI, USA). To screen for the optimal strain, each strain was cultured in glucose-free M9 media containing either 10% n-octane, 10% n-decane, or 10% n-dodecane as a sole carbon source at 30 ◦C for 48 h. To compare cell growth and mcl-PHA production, *P. resinovorans* was cultured in LB medium, cultured in glucose free M9 media with various carbon sources, including 2% (*w*/*w*) of glucose, 10% (*v*/*v*) of mixed alkane (n-octane, n-decane, and n-dodecane of the same volume), and 10% of each alkane.

The amount of nitrogen and phosphorus needed to maximize PHA production was determined using various concentrations in the medium and the determined mixed alkane. The effect of nitrogen and phosphorus concentration (0 to 1.0% and 0 to 5%, respectively) on *P. resinovorans* growth, PHA accumulation, and its monomer composition was studied. Finally, *P. resinovorans* was cultured in a 7 L bioreactor (GF Fermentech, Cheongju, Korea) with a working volume of 3 L at the optimized culture condition. It was operated at 30 ◦C for 72 h, with a stirring speed of 300 rpm and 3 V/min of gas flow for aeration.

#### *2.2. mcl-PHA Recovery from Biomass*

Methyl ethyl ketone (MEK), methanol, and chloroform were used as extraction and purification solvents for mcl-PHA recovery. MEK was added to the lyophilized cell and the mixture maintained at 60 ◦C for 6 h in sealed screw-top test tubes. Then, the tubes were briefly vortexed and incubated at room temperature. After mixing, the tubes were centrifuged for 10 min at 2500× *g*. The supernatant was transferred to a sealed screw-top tube, and then 3 volumes of methanol were added to remove the lipids remaining in the solvent. The PHA was dissolved with chloroform by heating at 100 ◦C in a heat block for 4 h. The tubes were incubated at room temperature until the solvent evaporated.
