Characterization of low-density polyethylene (LDPE) films degraded using bacteria strains isolated from oil-contaminated soil

ABSTRACT This study assessed the low-density polyethylene (LDPE) film degradation potential of microorganisms isolated from oil-contaminated soil and also analyzed the morphological and chemical composition of LDPE films after the biodegradation period. The bacteria strains isolated from oil-contaminated soil were standardized and used to degrade the pretreated LDPE films in mineral salt media. Thereafter, they were incubated for 78 days at 37°C in an incubator shaker, and the degraded LDPE films were analyzed quantitatively and qualitatively (using scanning electron microscope (SEM) images and energy dispersal x-ray (EDX)). Isolates A32 and BTT4 amongst other bacteria isolates showed the highest LDPE film degradation activity, with a weight reduction of 71.80% and 89.72% respectively, and were identified using the 16S rRNA sequencing technique. The EDX results showed that LDPE film incubated with A32 has the highest reduction in carbon and nitrogen (23.8% and 44.9% respectively) when compared with the Control. However, LDPE film incubated with BTT4 had an increase in calcium and chlorine (139% and 40% respectively), when compared with the control. Similarly, the SEM images showed the appearance of pinholes, cracks and particles on the surfaces of LDPE films incubated with A32 and BTT4 contrary to the controls. A32 and BTT4 were identified as Proteus mirabilis (Accession number: MN124173.1) and Proteus mirabilis (Accession number: KY027145.1) respectively. Proteus mirabilis showed viable plastic biodegradation potentials and may be useful in the management of plastic waste, leading to a reduction in global plastic waste and a clean environment. GRAPHICAL ABSTRACT


Introduction
Plastics are cheap durable polymer materials consisting of synthetic and semi-synthetic compounds mostly derived from petrochemicals and are commonly used in every household and everyday activities [1].Polyethylene plastics make up a larger percentage of synthetic polymer production all over the world, of which a bulk contains lowdensity polyethylene (LDPE) plastics [2,3].It has become increasingly difficult to do without plastics in our everyday life, due to their characteristic nature, and flexibility.They are known to be odorless, transparent, highly flexible even at low temperature, high impermeability by water vapor, and chemically inert at room temperature.Some of its properties are due to its degree of crystallinity which ranges between 50% and 60% [4].LDPE is prepared by polymerization of ethylene at high pressure and is utilized for diverse purposes such as packaging material for foods, snacks, medical products, handy bag, agricultural products, electronics and mechanical products [2,45].They are persistent and not easily degraded in the environment, due to its high molecular weight, long chain carbon atoms, degree of crystallinity and hydrophobic nature [46].
Used plastics are usually managed through recycling and landfilling, while a reasonable portion is disposed unconsciously and indiscriminately in the environment.Exposure of such materials to photo-degradation deteriorates the quality of the material by degrading them into micro-plastics, which further contaminate the environment [78].These microplastics enter into the food chain/web and are consumed by terrestrial and/ or marine animals.Accumulation of micro-plastics in their digestive system can cause harm and eventually death, hence micro-plastics tend to pose a serious environmental threat [5,[9][10][11].
Indiscriminate dumping and accumulation of used LDPE can lead to environmental pollution, and cause hazards such as blockage of waterways and drainages.The presence of LDPE in soil can hinder the amount of moisture, nutrients, air, minerals and organisms within the soil, as well as obstructing plant growth, hence reducing soil quality and productivity [5].Environmental pollution resulting from LDPE waste poses a threat to flora and fauna, hence the need for such LDPE to be degraded.LDPE can be cleaned off from the environment using light (photo-degradation), heat (thermal degradation), and biological (biodegradation), of which biological method is eco-friendlier [4].Microorganisms are increasingly being used as agents of biodegradation, because they utilize contaminants as their source of carbon and energy, therefore metabolizing them into smaller compounds, including CO 2 , H 2 O and humus, which are ecofriendly end products [1012].Due to the presence of long carbon chain in plastics as well as LDPE, their degradation is dependent on the morphology and chemical structure of the plastic [4].Several bacteria have been used previously for the degradation of LDPE plastics.In a study by Begum et al., in which polythene bag was degraded using Desulfotomaculum nigrificans and Pseudomonas alcaligenes isolated from plastic-contaminated soil for 30 days.They observed that there was an increase in weight loss of the polythene bag with an increase in the incubation period [13].Mixed culture of Pseudomonas aeruginosa and Brevibacterium sp. was used to degrade LLDPE plastic for 30 days, and it resulted in about 2-7% weight reduction of the LLDPE plastics [14].Indigenous bacterial isolates isolated from hydrocarbon-contaminated soils have been previously used for the degradation of LDPE plastic by incubating them for 56 days.At the end of the incubation period, it resulted in a 25% weight reduction in the LDPE plastics [15].
The impact of environmental pollution from plastics is enormous, since they are not easily degraded, they persist in the environment and in turn affect human health.Hence, the management and treatment of LDPE in an ecofriendly manner is a global concern.This study is aimed at assessing the efficiency of LDPE degradation using novel microorganisms isolated from oil-contaminated soil, as well as characterizing the LDPE films after the degradation period by analyzing the morphological and chemical composition.

Chemicals
All chemicals used in this study were of high purity and analytical grade.Nutrient agar and 70% (v/v) ethanol were purchased from HiMEDIA Laboratories, India, while other chemicals were purchased from BDH (Poole's, England) and Sigma-Aldrich (Germany).

Isolation and screening of microorganism
Soil samples were collected in sterile sample bags at the depth of 5-15 cm from a transformer oil spillage site in Ile-Ife, Osun State, Nigeria, and taken immediately to the laboratory for microbial analysis.A stock solution was prepared by introducing 1 g of the soil sample in 10 mL distilled water, thereafter, serial dilutions were carried out by introducing 1 mL of the stock in 9 mL distilled water to make a concentration of 10 −1 .This was further diluted till a concentration of 10 -6 was reached.A concentration of 0.1 mL of 10 -4 concentration was plated on nutrient agar and incubated at 37°C for 18-24 hrs.Colonies obtained were sub-cultured to obtain pure isolates which were used for downstream analysis.

Pretreatment and preparation of LDPE films
The LDPE films used in this study were food grade lowdensity polyethylene (Ziploc bags) of about 18µ thickness, cut to an approximate size of 3 × 3 cm and a weight of 0.1000 g using an analytical balance were used.The polyethylene films were washed with deionized water and sterilized for about 3 h using 70% (v/v) ethanol (HiMEDIA Laboratories, India) and air dried at 60°C.These were then used as carbon source for the aforementioned microorganisms in the biodegradation experiment.

Inoculum preparation for biodegradation
Bacterial isolates were standardized to uniform inoculum size by inoculating each isolate into the nutrient broth and incubated at 37°C for 24 h, and then centrifuged at 3000 rpm for 15 min.The cell pellets were adjusted to 0.5 McFarland Standard at a wavelength of 590 nm and used as the starting culture for other analyses [12].

LDPE biodegradation experiment
The sterilized LDPE films were introduced into 100 mL of sterilized Mineral salt medium (1 g K 2 HPO 4 , 1 g KH 2 PO 4 , 1 g NH 4 NO 3 , 0.02 g CaCl 2 , 0.05 g FeCl 3 , 0.2 g MgSO 4 and 1 g glucose), each inoculated with 15% standardized culture and incubated in an incubator shaker at 200 rpm for 78 days at 30°C alongside with a control containing no bacteria isolate (Control NG) while maintaining another control by not subjecting the sterilized LDPE films to any growth condition (Control).
At the end of 78 days, the LDPE films were removed from the incubator shaker, washed with deionized water and immersed in 2% (v/v) sodium dodecyl solution for one hour to free the LDPE film from the metabolic protein of the cultured media.Thereafter, the LDPE films were rinsed with distilled water, oven-dried overnight at 60°C, and weighed to determine weight loss.They were then stored in a sterile clean air-tight container for further analysis [16].

Percentage LDPE weight reduction
After 78 days incubation period, the LDPE films were dried and weighed using an analytical balance.The percentage weight reduction of the LDPE films after degradation was calculated according to Samanta et al. [8]: where W i is the initial weight of plastics before degradation; W f is the final weight of the plastics after the degradation period.Samples with a high percentage LDPE weight reduction were then subjected to chemical analysis.

Chemical characterization of degraded LDPE films
The surface morphology characterization and elemental composition of the unincubated sterilized LDPE film (Control), incubated sterilized LDPE film without bacteria isolate (Control NG) and incubated sterilized LDPE films with bacteria isolates were carried out using Field Emission Scanning electron microscope (FE-SEM) (JEOL JSM-7600F) coupled with an Energy Dispersive X-ray Spectroscopy (EDX).

16s rRNA gene sequence homology analysis of the isolates
The bacteria isolates with good degradation activity were cultured in a nutrient broth at 37 • C for 24 h, and centrifuged at 4722 rpm for 5 min.The pellet was washed with phosphate buffered saline (PBS) and re-suspended in PBS for DNA extraction.Genomic DNA of the isolates were extracted using the ZymoBIOMICS™ DNA MiniPrep kit (Zymo Research Corp., USA) according to the manufacturer's instructions, as previously used by Onajobi et al. [17] and prepared in a 96 well plate for cycle sequencing.PCR amplification of the 16S rDNA gene was carried out using the universal primers 27F (AGAGTTTGATCMTGGCT-CAG) and 1525R (AAGGAGGTGWTCCARCCGCA).The reaction consists of 2.5 µL of 10X PCR buffer, 1 µL of 25 mM MgCl 2 , 1 µL each of forward primer and reverse primer, 1 µL of DMSO, 2 µL of 2.5 mM DNTPs, 0.1 µL of 5 µg/µL Taq DNA polymerase, and 3 µL of 10 ng/µL DNA.The total reaction volume was made up to 25 µL using 13.4 uL Nuclease free water.The PCR cycling was carried out at 94°C for 5 min for initial denaturation, annealing at 56°C for 30 s, elongation at 72°C for 45 s for 36 cycles with final elongation at 72°C for 7 min.The amplicons were then visualized on ethidium bromide-stained 1.5% agarose gel.The PCR amplicons were purified using Ethanol/EDTA precipitation method and sequenced using ABI-PRISM 3500 genetic analyzer (Applied Biosystems, USA).
Bioedit software was used for opening and editing the sequences, and the sequence identification was carried out using GenBank's Basic Local Alignment Search Tool (BLAST) algorithm on National Centre for Biotechnology and Information (NCBI) database.

Bacteria isolation and percentage weight reduction in LDPE degradation
Five potential LDPE bio-degraders named ATT1, ATT2, A32, BTT2 and BTT4 were isolated from the soil sample, of which isolates BTT4 and A32 had the highest percentage weight reduction of 89.72% and 71.80% respectively of LDPE films, the lowest being ATT2 having 22.02% LDPE weight reduction has shown in Figure 1.This revealed high utilization and degradation of the LDPE films by the bacteria isolates.

EDX analysis result
Figures 2 (a-d) show the EDX spectra of the Control, Control NG, LDPE film incubated with A32 isolate for 78 days and LDPE film incubated with BTT4 isolate for 78 days.In Figures 2(a and b), there were no significant changes in the chemical compositions of the elements of both the Control and Control NG despite subjecting Control NG to the same growth condition as A32 and BTT4.However, obvious elemental changes were observed in both A32 and BTT4 in contrast to both controls, thus indicating the effect of the bacteria isolates in the degradation of the LDPE films.
The percentage elemental compositions of LDPE films incubated with A32 and BTT4 in comparison with the Control showed that A32 and BTT4 had percentage increase in sulfur, calcium and chlorine when compared with the Control, contrary to carbon and nitrogen which showed a high utilization by A32 and BTT4.Nevertheless, A32 had the highest carbon and nitrogen utilization of 23.8% and 44.9% respectively when compared with BTT4 having 4.0% and 40.7% carbon and nitrogen utilization respectively.Similarly, a reverse elemental utilization in terms of oxygen and sodium was observed in the study as A32 had a 10% and 22.2% increase in oxygen and sodium content respectively while isolate BTT4 had a decrease of 24.0% and 22.2% oxygen and sodium respectively (Table 1).

Scanning electron microscope (SEM) analysis result
The changes in the surface morphology of the LDPE films were analyzed using SEM.Figures 3(a-h) typify

Discussion
Bacteria and fungi such as Pseudomonas, Brevibaccillus, Bacillus, Fusarium, Aspergillus, etc. have been commonly implicated in the degradation of both lowand high-density polyethylene [1819].In our study, Proteus mirabilis showed a higher ability to degrade LDPE films, as compared with other bacteria isolates tested.All the bacteria isolates showed the ability to degrade LDPE films after 78 days (eleven weeks) of incubation with a weight reduction ranging from 22% to 89%.This study is in agreement with studies of Ali and Wahab [20], in which Proteus mirabilis isolated from sea view soil, Karachi, was used to degrade plastics up to 26.4% after 30 days of biodegradation period using a shake flask method.Proteus mirabilis have been previously reported to degrade petroleum hydrocarbons, herbicides, and pesticides [21], and PAHs (pyrene, phenanthrene, and fluoranthene), heterocyclic aromatics (di-benzothiophene) and aliphatic hydrocarbons [22].The difference in the weight loss of the LDPE films by the microorganisms may be due to the difference in the utilization of the LDPE films by the microorganisms, as well as the difference in the strain of the microorganisms.As microorganisms differ in their genetic composition, their enzymatic activities also differ [23], hence the degree of degradation of LDPE films is dependent on the microbial strain and chemical composition and structural arrangement of the LDPE films and plastics [24].
The weight loss of plastic materials as a result of biodegradation can be attributed to loss of quality and integrity of the polymer [25].
Quantitative reduction in the composition of carbon and nitrogen in the LDPE films incubated with the Proteus mirabilis strains shows their ability to utilize LDPE films as their source of carbon and nitrogen, this finding is similar to studies of Kunlere et al. [25].However, an inverse relationship in the carbon composition result of LDPE film degraded using Proteus mirabilis (Accession number: MN124173.1)and LDPE degraded film obtained using Proteus mirabilis (Accession number: KY027145.1)was observed.This may be due to differences in the bacteria strain hence affecting their degradation and carbon utilization potentials.The variation in the elemental composition of the LDPE obtained by both Proteus mirabilis strains validates the degradation of the LDPE film.In the LDPE film degraded by Proteus mirabilis (Accession number: KY027145.1),elements like calcium (over 100% increase in composition), sulfur and chlorine are increased with a corresponding increase in elements such as calcium, chlorine, sodium and sulfur observed in the LDPE film degraded by Proteus mirabilis (Accession number: MN124173.1).
The degradation potential of the bacteria isolates was further elucidated by SEM as the control samples (whether incubated or unincubated) had appearances of smooth surface having no pits, cracks or any particles on its surfaces (Figures 3a-d) thus suggesting no observable degradation.However, the presence of pores, pinholes and visible cracks on films incubated with Proteus mirabilis brought about an increase in porosity and fragility in the morphology of the material [26].This therefore indicates the bacteria potential in degrading polyethylene [27].The presence of cracks, holes or particles has been reported as key features in the microbial degradation of LDPE films [52628] as several cracks were observed on the surfaces of LDPE films incubated with strains of Proteus mirabilis.However, films treated with Proteus mirabilis (Accession number: KY027145.1)were subjected to more degradation contrast to Proteus mirabilis (Accession number: MN124173.1).The adherence of microorganisms to the surface of plastic materials is an important phase in the degradation of plastics.This attribute differs from one microorganism to another since the microorganisms differ in their characteristics, gene and enzyme production [20], hence the difference in the morphology of the plastics after the degradation period.

Conclusion
In the present study, Proteus mirabilis strains isolated from oil-contaminated soils were found useful in the biodegradation of polyethylene films owing to the growth of the microorganisms in the growth media coupled with reduced LDPE weight.The SEM and EDX analysis showed changes in the surface morphology of the LDPE films incubated with bacteria isolate, revealing the presence of cracks, pinholes and particles pores and cracks as well as quantitative decrease in carbon and nitrogen in the degraded LDPE films.This implies effective utilization of the LDPE films as their source of carbon and nitrogen by the microorganisms.Proteus mirabilis strains which have been scarcely used in the degradation of LDPE films, show significant potential in reducing the environmental pollution hazard arising from plastic.

Figure 1 .
Figure 1.Percentage weight reduction in LDPE film samples.

Figure 2 .
Figure 2. EDX result of (a) unincubated LDPE film, (b) LDPE film incubated without microorganism for 78 days, (c) LDPE film incubated with A32 isolate for 78 days and (d) LDPE film incubated with BTT4 isolate for 78 days.
the micrograph of Control, Control NG, LDPE film incubated with A32 isolate for 78 days and LDPE film incubated with BTT4 isolate for 78 days at different scale bar.The Control NG LDPE film which was incubated without microorganisms as shown in Figures3(c and d) shows sponge-like morphology with the presence of pores and leaf-like particles contrary to the Control (Figures3a and b).The variation in morphology of the Control NG and Control possibly indicates the influence of the growth condition Control NG was subjected to.In Figures3(e) and 3(g), pinhole morphology, visible cracks and particles stacked upon one another were observed on the surface of the LDPE film with the presence of visible pores of different sizes.

3. 3 .
16s rRNA sequencing of the isolates Isolate A32 and BTT4 having good LDPE degradation activity were identified as Proteus mirabilis with accession number MN124173.1 and KY027145.1 respectively, having a percentage relatedness of 95.31% and 98.40% respectively with sequence data in NCBI database.

Figure 3 .
Figure 3. SEM micrograph at different scale bar of (a, b) unincubated LDPE film, (c, d) LDPE film incubated without microorganism for 78 days, (e, f) LDPE film incubated with A32 isolate for 78 days and (g, h) LDPE film incubated with BTT4 isolate for 78 days.

Table 1 .
Percentage differences in the elemental composition of LDPE films incubated with each bacteria isolates and the control.