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Microbiology and Biotechnology Letters

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Environmental Microbiology (EM)  |  Biodegradation and Bioremediation

Microbiol. Biotechnol. Lett. 2024; 52(2): 189-194

https://doi.org/10.48022/mbl.2404.04005

Received: April 4, 2024; Revised: May 21, 2024; Accepted: June 7, 2024

Biodegradation of Low-Density Polyethylene by Acinetobacter guillouiae PL211 Isolated from the Waste Treatment Facility

Ye-Jin Kim1,2,3, Jang-Sub Lee1,2,3, Jeong-Ann Park4, Hyun-Ouk Kim1,2, Kwang Suk Lim1,2, and Suk-Jin Ha1,2,3*

1Department of Bioengineering and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
2Department of Biohealth-Machinery Convergence Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea
3Institute of Fermentation and Brewing, Kangwon National University, Chuncheon 24341, Republic of Korea
4Department of Environmental Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea

Correspondence to :
Suk Jin Ha,       sjha@kangwon.ac.kr

Abstract

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Plastics are consistently produced owing to their practicality and convenience. Unmanaged plastics enter the oceans, where they adversely impact marine life, and their degradation into nano-plastics due to sunlight and weathering is of concern for all living beings. Nano-plastics affect humans via the food chain, emphasizing the necessity for effective solutions. Microbial biodegradation has been suggested as a solution, offering the advantages of minimal environmental impact and the utilization of decomposition byproducts in microbial metabolic pathways. In this study, fifty-seven bacterial strains were isolated and identified from a waste-treatment facility. Cultivation in a minimum medium with low-density polyethylene (LDPE) beads as the sole carbon source resulted in the selection of the LDPE-degrading strain Acinetobacter guillouiae PL211. The selected strain was cultured at high cell density with LDPE as a carbon source, and Fourier transform infrared (FT-IR) analysis confirmed chemical changes on the LDPE bead’s surface. Field-emission scanning electron microscopy (FE-SEM) analysis revealed substantial biodegradation of the LDPE surface. These results demonstrated the capability of A. guillouiae PL211 to biodegrade LDPE beads. This discovery demonstrates the potential of an environmentally friendly process to addressing polyethylene waste issues.

Keywords: Low-density polyethylene, biodegradation, Acinetobacter guillouiae, FE-SEM, FT-IR

Body

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Synthetic plastics first emerged in the early 20th century, driven by their practicality and convenience, leading to versatile applications [1−4]. Consequently, the production volume of plastics has consistently increased, surging 260-fold from the 1950s to the present day in 2022 [5]. With the rise in plastic production and use, the proportion of plastics in municipal solid waste (MSW) has increased tenfold, from <1% in the 1960s to over 10% in the 2000s [2]. Unmanaged plastics find their way to the sea through coastal areas and rivers and are degraded into smaller-sized nano-plastics due to sunlight and weathering effects [6]. This process poses a threat, causing physical harm and toxicity to aquatic organisms, with potential repercussions on humans through the food chain [6−9]. Studies have revealed that humans consistently ingest microplastics, raising concerns about their impact on human health, including inflammation and lung damage, necessitating urgent measures to address this critical issue [10].

Petroleum-derived plastics can be categorized based on their skeletal structures into heteroatomic backbone plastics and carbon-carbon backbone plastics [11]. Among carbon-carbon backbone plastics, polyethylene (PE) is the most commonly utilized. PE is classified into high-density polyethylene (HDPE), medium-density polyethylene (MDPE), and low-density polyethylene (LDPE) [11]. Among these, LDPE is widely used in packaging materials and is the most abundantly produced [12]. The structural characteristics of PE impart high hydrophobicity, and its natural decomposition can take over a century [13−15]. Consequently, PE waste has been known as a significant contributor to environmental pollution among various polymers [7, 16, 17].

Microbial biodegradation of PE is proposed to resolve issues related to plastic waste [18]. This approach offers advantages due to its minimal environmental side effects, and the assimilation of chemical byproducts generated during the decomposition of PE by the microbes [18−20]. Microbial enzymes break down PE polymers into oligomers and monomers [19]. Microbial enzymes can be broadly categorized into intracellular enzymes that function within the cell and extracellular enzymes that operate outside the cell [19, 21]. Extracellular enzymes have been more extensively studied, as plastics are too large to pass through the cell membrane [21]. Various microorganisms, including bacteria, fungi, and algae, have been discovered to biodegrade PE utilizing extracellular enzymes [21−24]. However, the molecular mechanism of PE biodegradation remains unclear, necessitating further research in this area [20].

In this study, bacterial strains were screened from two types of soil and one type of plastic collected from a waste treatment facility in Gangwon-do, South Korea. The screened bacterial strains were cultured on LB agar medium (LB broth 25 g l-1, agar 15 g l-1) and NB agar medium (nutrient broth 8 g l-1, agar 15 g l-1). The inoculated LB and NB agar media were incubated at 30℃ for 48 hours. Fifty-seven bacterial strains were isolated from single colonies growing on the media and stored. The genomic DNA of the fifty-seven bacterial strains was extracted using the i-Genomic BYF DNA Extraction Mini Kit (17361, iNtRON Biotechnology). The 16S ribosomal RNA (16S rRNA) region was amplified from the extracted genomic DNA using Premix TaqTM and primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’). The amplified DNA was sequenced through Next Generation Sequencing (NGS) analysis by Macrogen. The 16S rRNA sequences were analyzed using MegaBLAST at the National Center for Biotechnology Information (NCBI). Tweenty strains were identified as Pseudomonas spp., whereas twelve bacterial strains were identified as Acinetobacter spp. Other bacterial strains, including Aeromonas sp., Bacillus subtilis, Enterobacter sp., and Klebsiella sp., were also identified. Acinetobacter spp. have been reported to biodegrade polyethylene. Acinetobacter sp. isolated from agricultural film recycling plant wastewater sludge is reported to reduce the weight of LDPE film by 15 ± 0.85% over 90 days, transforming the LDPE film into a more hydrophilic form [25]. Therefore, the Acinetobacter sp. strains isolated in this study may exhibit LDPE biodegradation capabilities.

Fifty-seven bacterial strains were evaluated for their LDPE biodegradation capabilities. These strains were inoculated into MS medium containing K2HPO4 2.27 g l-1, KH2PO4 0.95 g l-1, (NH4)2SO4 0.67 g l-1, metal solution 2 ml l-1 (Na2EDTA·2H2O 6.37 g l-1, ZnSO4·7H2O 1.0 g l-1, CaCl2·2H2O 0.5 g l-1, FeSO4·7H2O 2.5 g l-1, NaMoO4· 2H2O 0.1 g l-1, CuSO4·5H2O 0.1 g l-1, CoCl2· 6H2O 0.2 g l-1, MnSO4·H2O 0.52 g l-1 and MgSO4·7H2O 60.0 g l-1) with LDPE beads 20 g l-1 (obtained from Sigma-Aldrich, US: 427772) as the sole carbon source. The flask culture conditions were 30℃ and 200 rpm for 91 days. Uninoculated culture medium with LDPE beads was maintained under similar conditions as a negative control. The LDPE beads were recovered after incubation, and the bacterial cell were eliminated from the beads by sodium dodecyl sulfate (2%). The recovered LDPE beads were then washed twice with sterile water and thoroughly dried at 60℃ in a drying oven. The surfaces of the LDPE beads were characterized using field-emission scanning electron microscopy (FE-SEM, JSM-7900F, JEOL) to confirm the LDPE biodegradation capabilities of the test bacterial strains. Finally, the samples were analyzed at an acceleration voltage of 5.0 kV. The LDPE bead from the uninoculated control was observed at 500x magnification to have a smooth surface with no apparent defects (Fig. 1A). Among the fifty seven bacterial strains, one exhibited biodegradation of LDPE beads and were identified as Acinetobacter guillouiae PL211. Observations at 500x magnification revealed irregular cracks and defects on the surface of LDPE treated with A. guillouiae PL211 (Fig. 1B). In a previous study, LDPE strips treated with A. calcoaceticus exhibited morphological changes in surface after incubating for four months [26]. Similar to our studies, this report showed the presence of cavities and pits. Surface erosion was believed to be due to the enzymatic biodegradation of LDPE by bacterial strains.

Figure 1.FE-SEM micrographs of LDPE bead incubated for 91 days with bacterial strains. (A) Uninoculated control at 60 × and 500 × magnification, (B) A. guillouiae PL211 treated LDPE at 70 × and 500 × magnification.

For further investigation, A. guillouiae PL211 was incubated in MS medium added with LDPE beads at an initial 10 optical density (OD600 nm), and LDPE biodegradation was monitored at weekly intervals. During cultivation, the surface of the LDPE beads may be chemical changed because of the bacterial enzymes. The white colored LDPE beads were getting changed to light yellow color at the end of incubation which suggested that cells might be more attached to LDPE beads because of chemical change to hydrophilic. The functional group changes on the LDPE beads surface were analyzed using Fouriertransform infrared spectroscopy (FT-IR; ALPHA II, Bruker). LDPE beads from the uninoculated MS medium were used as negative controls. FT-IR spectra were recorded in the range of 4000−390 cm-1 for each sample. Compared to the negative control without bacterial treatment, LDPE beads treated with A. guillouiae PL211 showed new peaks within two weeks cultivation (Fig. 2A). In A. guillouiae PL211-treated LDPE, new peaks appeared at 3300, 1630, and 1540 cm-1 wavenumbers, representing O-H (hydroxyl group), C=C (alkane group), and N-O (nitro group), respectively (Fig. 2B and Fig. 2C). Similar formation of hydroxyl and alkane groups has been reported in previous reports about PE degradation mediated by bacteria [27−29]. The emergence of these new peaks suggests a chemical change in the characteristics of LDPE bead surface. In particular, such alterations in functional groups occur upon oxidation of PE, which can be catalyzed by extracellularly secreated enzymes such as alkane hydrolase, leading to the introduction of oxygen atoms into the chemical structure of PE [29, 30]. This process is considered a pivotal step in the PE biodegradation [31]. Hydroxyl groups are essential for the formation of carbonyl groups, which can be converted to esters for final cleavage by lipases or esterases [31]. Also, the introduction of oxygen functionalities results in a shift from nonpolar to polar characteristics in the LDPE beads [29]. This change not only provides reactive sites for enzymatic action but also increases instability [29]. This implies that the introduction of oxygen functionalities may enhance LDPE biodegradation through improved enzyme accessibility and reactivity. In addition, the formation of N-O (nitro group) is a unique change that is not usually observed. In a previous report, it was hypothesized that nitric oxide synthesized by microorganism permeates through the outer membrane and spreads to the interface [27].

Figure 2.FT-IR spectra of LDPE beads after 4 weeks of incubation with high cell density culture of A. guillouiae PL211 (A) 4000– 390 cm-1, (B) 3500–3000 cm-1 and (C) 1700–1500 cm-1 wavenumbers. Negative control is LDPE bead incubated for 4 weeks without bacterial strain treatment.

The surface biodegradation of the LDPE beads recovered at weekly intervals was analyzed using FE-SEM. Significant changes were observed in the LDPE beads treated with A. guillouiae PL211 after incubation for two weeks. The surface of LDPE beads treated with A. guillouiae PL211 exhibited irregular cracks, defects, and cavities, whereas the LDPE beads from the negative control had a flatten surface with no observable changes (Fig. 3). These results confirmed the capability of A. guillouiae PL211 to noticeably biodegrade LDPE beads within two weeks.

Figure 3.FE-SEM micrographs of LDPE beads incubated for 4 weeks with high cell density culture of A. guillouiae PL211. (A) without bacterial treatment for 4 weeks; with A. guillouiae PL211 after (B) 1 week, (C) 2 weeks, (D) 3 weeks, and (E) 4 weeks at 2000 × magnification.

In conclusion, 57 bacterial strains were screened from waste treatment establishment. The screened strains were cultured with LDPE beads as the sole carbon source in MS medium. Following incubation with A. guillouiae PL211, FE-SEM analysis showed the strain’s ability to biodegrade LDPE. Changes of chemical structural on the LDPE beads surface were revealed when the biodegradation was monitored at weekly using FTIR analysis. The introduction of oxygen functionalities changed the LDPE surface from nonpolar to polar, supplying reactive sites for enzymatic reaction. Moreover, FE-SEM analysis showed noticeable defects on the surface of LDPE bead. These results demonstrated the capability of A. guillouiae PL211 to biodegrade LDPE beads which might be used to tackle the issues associated with polyethylene waste.

Acknowledgments

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This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF).

Author Contributions

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Ye-Jin Kim contributed to the study conception, design, material preparation and data collection. Jang-Sub Lee contributed to material preparation. Jeong-Ann Park, Hyun-Ouk Kim, Kwang Suk Lim contributed to the study conception and design. Suk-Jin Ha contributed to review and editing of the manuscript.

Funding

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This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2022R1A4A1019201).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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