Journal of Nanomaterials & Molecular NanotechnologyISSN: 2324-8777

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Research Article, J Nanomater Mol Nanotechnol Vol: 5 Issue: 3

Survivability of Polyethylene Degrading Microbes in the Presence of Titania Nanoparticles

Salma Alvi1*, Ishtiaq A Qazi2, Anwar Baig M3, Saadia Andleeb4, Aashifa Yaqoob5 and Ch. Tahir Mehmood6
1Institute of Environmental Sciences and Engineering (IESE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan
2Department of Environmental Sciences, Forman Christian College (A Chartered University), Lahore 54600, Pakistan
3Institute of Environmental Sciences and Engineering (IESE), National University of Science and Technology (NUST), Islamabad, Pakistan
4Atta ur Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan
5TB Control Program (NTP), Ministry of National Health Services Regulation& Coordination. Government of Pakistan, Islamabad, Pakistan
6Institute of Environmental Sciences (IESE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology (NUST), Islamabad, Pakistan
Corresponding author : Salma Alvi
MS (Environmental Sciences), Institute of Environmental Sciences and Engineering (IESE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan
Tel: +92 3125022100
E-mail: [email protected]
Received: February 17, 2016 Accepted: May 05, 2016 Published: May 10,2016
Citation: Alvi S, Qazi IA, Baig AM, Andleeb S, Yaqoob A, et al. (2016) Survivability of Polyethylene Degrading Microbes in the Presence of Titania Nanoparticles. J Nanomater Mol Nanotechnol 5:3. doi:10.4172/2324-8777.1000185


Survivability of Polyethylene Degrading Microbes in the Presence of Titania Nanoparticles

The bacterial degradation of Polyethylene, not unlike the other plastic materials, is generally very slow. The presence of titania (TiO2) nanoparticles (TNPs) faster photocatalytic degradation of polyethylene can take place which can help in accelerating the bacterial degradation; in the presence of TNPs, polyethylene is initially photo catalytically degraded to smaller pieces which allows the bacteria initially degrade polyethylene to smaller size that allow the TNPs to act more effectively. But, TNPs also have a germicidal effect and it is important to find out the safe concentration of TNPs with minimal toxicity and good photocatalytic activity. For identification of the polyethylene degrading microbial strains from the soil, from samples collected from the local plastic dumpsite, were subjected to gram staining, biochemical tests and 16s rRNA gene sequencing, and then tested for polyethylene degradation under different TNP concentrations. In this work it has been established, through Optical Density (OD), Colony counting (CFU measurement), Scanning Electron Microscopy (SEM) and measurement of the Carbonyl Index (CI) using FTIR, that up to a concentration of 1%, the TNPs have a positive effect on polymer degradation. Results show that biodegradation of polyethylene in the presence of TNPs was much stronger than the degradation by TNPs alone or biodegradation in the absence of TNPs. The results could play an important role in the development of environment friendly shopping bags and other polyethylene based products.

Keywords: Titania nanoparticles; Polyethylene; Pseudomonas aeruginosa; Colony forming unit; Optical density; SEM; FTIR


Titania nanoparticles; Polyethylene; Pseudomonas aeruginosa; Colony forming unit; Optical density; SEM; FTIR


Long chain monomers of ethylene constitute a polymer commonly known as polyethylene. The worldwide consumption of polyethylene is increasing at a rate of 12% on annual basis and global generation rate of polymer is about 140 million tons per annum. Polyethylene, in common with other plastics has lower tendency to degrade in the environment naturally so it has a potential to harm the environment in a variety of ways [1]. The polymer backbone in plastics is mainly composed of carbon and is utilized by microbes as an energy source, thus, bacterial degradation of plastic does occur in nature, albeit very slowly. The process of polyethylene degradation by bacteria starts by the discharge of enzymes which convert the polymer into monomer(s) by breaking larger chains into smaller ones that can then be metabolized by the microbes [2] like Pseudomonas sp., Streptococcus sp., Staphylcoccus sp., Micrococcus sp. and Moraxella sp.; and fungi [3,4] Aspergillus niger, Aspergillus glaucus, Actinomycetes sp. and Saccharomonospora genus. Microbes native to plastic waste dump sites including Pseudomonas sp., E. Coli, Klebsiella and bacillus sp. have however, been reported to be fairly efficient at degrading polyethylene [5].
Plastic degradation in the environment also takes place through interaction with sun light. The photons of UV component of solar radiation, in particular, have sufficient energy to break the polymer chain bonds, and plastic material, left in the open, tends to discolor and become brittle over time. The process can be accelerated by the addition of a photocatalytic agent such as titanium dioxide TiO2, commonly known as ‘titania’. Thus, incorporation of titania nanoparticles (TNPs) in polyethylene has proved to be very effective in this context [6,7]. When the TNPs used have been doped with suitable metals, the photocatalytic degradation occurs very fast even under visible region of the light spectrum.
Photocatalytic toxicity of TNPs to microbes is well known [8]. However, there are indications that nanoparticles in the dark either facilitate degradation process or have little negative effects on bacterial population [9]. This study was aimed at testing the hypothesis that polyethylene films containing TNPs, initially broken down into small fragments would be more effectively bacterially degraded. As such, the sequential degradation process was expected to be faster than either bacterial or photocatalytic degradation alone.
The methodology followed for this purpose was to isolate a dominant polyethylene degrading bacteria from a waste site; acclimatize the strain for polyethylene degradation in laboratory; separately test toxicity level of TNPs for the isolated strain; study bacterial growth with polyethylene as food source in the absence and presence of TNPs (at tolerant concentrations as determined above); and compare these results with photocatalytic degradation by TNPs alone. A number of characterization techniques and methodologies were used in this context including XRD, SEM and EDS for material characteristics, morphological, biochemical and gene sequencing for bacterial characterization and SEM and carbonyl index utilizing FTIR for degraded polyethylene analysis. Details of the experiments and discussion of the results is given below.

Materials and Methods

Titanium Dioxide (GPR, BDH Chemicals Ltd. England) was used for the synthesis of pure TiO2 nanoparticles. Solidified agar petri plates were prepared with the help of Nutrient Agar (Merck, VM 100650 943). Xylene (Merck, Germany) to dissolve PE beads
Preparation of polyethylene powder
Polyethylene beads (purchased from the local market) were converted into powdered form by boiling with xylene at 120°C and stirring 300 rpm to get high surface area material for effective biodegradation. To evaporate the solvent, the material was left in open for 3 days. The powdered polyethylene was consecutively washed with 70% ethanol to remove xylene residues and subsequently dried in oven at 40-50°C for 24 hours. The powder was used as the key carbon source for the biodegradation process [9,10].
Synthesis of TNPs
TNPs were synthesized using Liquid Impregnation (LI) method [7,8,11]. Precisely, 6 g of titania (general purpose reagent, purity> 99%) was added in 240 mL distilled water and stirred vigorously to obtain homogenous solution at 325 rpm on magnetic stirrer (model: Stuart SB162). After 48 hours of stirring, the solution was sonicated at room temperature for 40 minutes. Sonication was done using JAC Ultrasonic 1505, JINWOO. After complete precipitation, the slurry was allowed to settle overnight. The pH of TNPs was found to be 6.47. The final solution was dried at 105oC for 12 hours in hot air oven. Finally, the dried material was powdered in mortar pestle and then calcined in muffle furnace (NEY M-525 series II) at 500°C for 5 hours.
Characterization of Titania nanoparticles
X ray diffraction (XRD) analysis: The crystal structure of the powdered TNPs was determined by the X-ray diffraction spectroscopy (XRD) technique. The XRD pattern of TiO2 was attained under CuKα radiation with wavelength of 0.1540 nm in the position 2Ɵ, and scanned in the range of 20°-80° with a fixed step of 0.5° using STOE, Scintag Theta-Theta X-ray Diffractometer system and the average crystallite size was determined by Scherrer's calculator using X’Pert Highscore (plus) software [8,11].
Scanning electron microscopy (SEM): The surface morphology of TNPs was determined by SEM (JSM-6490A, JEOL) with an accelerating voltage of 20 kV [12,13].
Electron dispersion microscopy (EDS): Energy dispersive spectroscopy (EDS) measurements were performed on a Compact Detector Unit (CDU) incorporated into SEM to find out elemental composition. The EDS spectrum was obtained at an acceleration voltage of 20 kV and collected for 50s [14].
Sample collection from plastic waste disposal sites
Six samples were collected from three waste disposal sites (two from each site) containing small pieces of polyethylene. Samples were collected from a depth of 5-8 cm using spatula, packed in air tight polybags and brought to the laboratory.
Moisture content (%) and pH measurement of waste samples
After moving back to the lab, small portions of soil samples were placed on filter paper and the initially weighed and subsequently, samples were dried at 110°C in an oven overnight and weighed again a number of times until a constantly same weight was achieved. Moisture of soil samples was calculated by applying the following formula obtained from AWPA.
Here, ‘MC’ is moisture present in soil sample, ‘W’ is the initial weight right after sampling and w is the stable weight obtained after continuous oven drying. pH was calculated by using pH meter by Electrometric approach using assembled glass electrode [15].
Isolation and identification of dominant microbial culture
A composite sample was prepared by mixing the viable waste samples which was then suspended in distilled water with gentle shaking followed by filtration. This solution was used for further experimentation. Microbial isolation from collected soil samples was done using serial dilution, spreading and streaking methods. After isolation of dominant strains visually, further identification was performed by morphological, biochemical and molecular characterization.
Morphological characterization
A. Colony morphology: Streaking was performed on nutrient agar repeatedly to observe single colony at each step of purification in terms of color (from naked eye and under microscope), shape, margin, size, texture, elevation and pigmentation.
B. Cell morphology: The microbial culture was smeared on a clean glass slide and subsequently air-dried and heat fixed. Gram straining was performed to check cell morphology of isolated strain regarding peptidoglycan to test strain either positive or negative [16].
C. Motility test: Hanging drop technique was used for motility test. A drop of autoclaved distilled water was placed on the cover slip, and fresh bacterial suspension was prepared with the help of a sterile toothpick. Then the cover slip was carefully inverted over the depression and the drop was allowed to hang from the cover slip into the slide cavity. Subsequently, the slide was observed under the microscope at 100X to visualize the motility of the bacteria.
Biochemical characterization: For biochemical identification of the bacterial strain API 20E (Analytical Profile Index 20 Enterobacteria) kits were used [17]. Banded by results from this experiment, dominant microbial culture was also streaked on cetrimide agar to confirm identification of Pseudomonas aeruginosa.
Molecular characterization: 16S rRNA Gene Sequencing was done by following the standard protocol and strain was sent to Genome Analysis Department Macrogen Inc. Korea and after gene sequencing, nucleotide sequence was submitted to gene bank of NCBI (National Center for Biotechnology Information) for accession number.
Optimization of microbial tolerance for Titania nanoparticles: Pure culture was revived on agar plates to get fresh culture after incubation. The maximum concentration of titania nanoparticles, tolerated by pure culture, was tested by inoculating pure culture (300 μl) into 250-ml Erlenmeyer flasks placed in orbital shaker at optimum pH of (7.0 ± 0.4) and temperature (37°C) and constant shaking (160 rpm). Different concentrations of titania nanoparticles were added with increasing trend from 0.05%, 0.1%, 0.25%, 0.5%, 1% and 1.5% respectively. After an interval of 24 hours, samples were collected to measure optical density (OD) at 600 nm and colony forming unit per milliliter (CFU/ml) and mean generation time (tgen) of microbial colonies was calculated using following formula;
Here, Xt = higher CFU/ml value, Xo = lower CFU/ml value
Comparative polyethylene biodegradation with Titania nanoparticles: For the degradation experiment, 200 ml of mineral salt media (pH 7.0 ± 0.04) was added to 250-mL Erlenmeyer flasks holding PE powder at a concentration of 20 mg/ml. 600 μl of active inoculum was introduced to flasks. The experiment got started with controls lacking TNPs and containing 1% of titania nanoparticles, respectively. The flasks were kept in incubator at 37°C with constant shaking (150 rpm). The trends were monitored for bacterial growth by measuring the OD at 600 nm after regular intervals of 1 day. The λ max was also determined for monitoring changes in the broth due to polymer dissolution. Degraded products were recovered from the media after 5th day of incubation.
Retrieval of degraded products: Degraded powder samples were collected from the media after passing through filter paper and following filtrate evaporation. The remaining material was gathered, and centrifuged at 2000 rpm for 40 min to get rid of the microbial biomass. The supernatant was placed in an oven to dry at 60°C for 24 hours to remove moisture and the resulting dry sample was collected and examined by SEM and FTIR, taking pure PE powder as the control.
A. Scanning electron microscopy (SEM): Scanning Electron Microscope was used to obtain the high resolution images. The focused beam of electrons produces images which provide us information like appearance, form, and size, elemental composition of polyethylene powder before and after degradation [13].
B. Fourier Transform Infrared (FT-IR) Spectroscopy: The degraded samples recovered, from the media when the experiment was completed, were examined by FTIR spectra [12] and different peaks relative to CH2 deformation, formation, bending, and stretching and carbonyl bond were compared with reference (control). Perkin Elmer FTIR Spectrophotometer was used to document FTIR spectra in potassium bromide (KBr). Carbonyl index (CI) was also calculated using C-O (1720, 1735 cm-1) and C-H (1465 cm-1) bond intensities from FTIR spectra. Following formula was used for CI calculation;

Results and Discussion

Characterization of TNPs
X-ray diffraction (XRD) analysis of TNPs: Objects are attributed to nano size when their dimensions lie within range of 100 nm. XRD determines the crystalline phase of the nanoparticles by analyzing diffraction of X-rays. Average crystallite size of nanoparticles was determined by using Scherer formula (Table 1) [8].
Table 1: X-ray Diffractometer Results of Pure TNPs.
L = Average particle size
k = 0.891, a shape factor of spherical particles
λ = 0.1542, wavelength of X-Rays
β = Full width of a diffraction line at half of maximum intensity (FWHM)
θ = Diffraction angle of crystal phase
Crystallite size was calculated as 42.51 nm and lattice strain 0.0039. Peaks of XRD results reveal that nanoparticles have crystalline structure (see Figure 1). Strong diffraction peaks at 25.271º and 47.980º confirm that synthesized TNPs are in anatase phase using diffraction angle 20º-80º [18]. Sample was compared with card no. JCPDS 01-089-4921. The crystal structure was found to be tetragonal which was in agreement of that reported in literature.
Figure 1: XRD Pattern of Pure TNPs.
Scanning electron microscopic (SEM) images: The surface morphology of TNPs was examined by JEOL JSM-6460 SEM at 20,000 magnifications. The image of the pure titania shows that particles are spherical in shape and distributed in the range of 50.00-72.11 nm (Figure 2). Image of undoped TiO2 (Figure 2) confirms the presence of a porous, sponge like structure of high roughness and complexity. Such structure indicates the high surface area which has been proven to be extremely efficient for the photo catalytic degradation purposes [19].
Figure 2: SEM Image of TNPs.
Energy dispersive spectroscopy (EDS): EDS examined elemental composition of pure TiO2 nanoparticles. Figure 3 and Table 2 show the presence of Ti and O elements in the representative sample. It varies from point to point showing diverse composition of the prepared NPs that confirms the SEM results. It also confirms that nanoparticles contain oxygen and titanium only.
Table 2: EDS Results of TNPs.
Figure 3: EDS Analysis of TNPs.
Physical characteristics of the waste dump soil
A broad range of moisture can allow microbes to survive. In this study, the moisture content in collected waste soil samples were found between ranges of 61.24-67.77%. The maximum microbial biomass has been determined in soils of pretty high moisture content and the optimal level for the actions of aerobes mostly is a 50-75% of the soil moisture carrying capability [20].
In this study the pH of soil sample was optimized for culturing bacterial strains. Before growing microbes in artificial media, measurement of pH is a main factor to provide microbes a survivable environment. For the present study, pH was acclimatized in collected waste soil samples. Four samples at pH of 6.9. 7.2, 7.4 and 7.6 were taken as suitable for the optimal growth of the microbial strains. Bacteria can bear pH from 4 to 10 during soil reactions, but the recorded suitable pH for the common microbes lies between basic and neutral [21].
Identification of isolated strain
For identification of the isolated strain, results were evaluated from morphological, biochemical characterization and gene identification as well.
Morphological characterization: Isolated bacterial strain was studied for form, color, opacity, elevation, margin and surface. The isolated strain was assigned name as SA1 in Tables 3 and 4 representing colony and cell morphology of isolated bacteria respectively.
Table 3: Colony and Cell Morphology of Bacteria Isolated from Waste Soil.
Table 4: Comparative Microbial Growth and Generation Time with Different Concentrations of TNPs.
The strain SA1 turned out to be gram negative when observed under microscope after gram staining. SA1 was bacilli, appeared in pairs and was motile when observed under microscope at 100X
Biochemical characterization: Biochemical characterization was performed using API20E and results were determined through analyzing the codes in API 20E software. The biochemical Characterization indicated the SA1 as Pseudomonas aeruginosa. Selected bacterial isolate was capable of catalase and oxidase production Results of present study correspond to numerous studies where indigenous polyethylene degrading bacterial strains were isolated from plastic waste dumpsite. Microbes found as PE biodegraders and associated microbes have reported to be identified as Pseudomonas sp., Bacillus sp., Staphylococcus sp., Aspergillus nidulans, Aspergillus flavus and Streptomyces sp. [1]. It has been reported that polyethylene degradation by Pseudomonas sp. AKS2 is relatively fast as it can degrade 5 ± 1% of the starting material in 45 days without prior oxidation [22].
Molecular characterization and Gene sequencing of strain SA1: Gene sequencing of SA1 was performed at Genome Analysis Department Macrogen Inc. Korea. The strains were screened and noise was removed manually. Strain was identified through BLAST search available at National Center for Biotechnology Information (NCBI) databases revealing up to 100% similarity to Pseudomonas aeruginosa. Furthermore, nucleotide sequence was assigned accession number as KU198667.
Optimization of microbial tolerance for Titania nanoparticles (TNPs)
Microbial growth profiling concerning of Optical density (OD): Determination of the maximum concentration level of TNPs that could allow the maximum growth of microbes was required in order to study its effect upon polyethylene biodegradation.
Of the six concentrations 0.05%, 0.1%, 0.25%, 0.5%, 1% and 1.5% (w/v) taken to perform assay, microbial culture exhibited a progressive decrease in bacterial OD (at 600 nm) with the addition of nanoparticles at increasing concentrations (Figure 4). This increase became detrimental in concentration of 1.5%. However, the concentration of 1% was found to moderately affect the bacterial growth trend because all growth phases were successfully completed with regressive growth. Hence, 1% was selected as the optimum concentration for further experimentations.
Figure 4: Tolerance Level of the Polymer-Degrading Microbes against TNPs Concentration.
Colony forming unit CFU/ml and generation time calculation: The CFU counts confirmed the above observations with reduced values when the cultures were grown with gradual increase of TNPs as compared to control (Figure 5). Distinct microbial colonies that could be counted were observed at the 7th dilution factor so it was then used to count CFU/ml.
Figure 5: Comparative Growth Profiling of Microbes in the Presence and Absence of TNPs.
As in case of OD, till the concentration of 0.1% TNPs, there was no significant effect on the growth of microbes. Even at concentration of 1%, a gradual decrease was noted in log and lag phase of microbial cycle but microbial colonies were growing productively till 48 hours. A deadly decrease in microbial growth was observed with concentration of 1.5%. So, the concentration of 1% was taken to continue with furthur experimentation. Mean generation time for isolated strain is reported in Table 4. Microbial colonies showed reverting growth with increasing concentrations of TNPs. Control exhibited maximum generaton per hour in the absence of TNPs while with 1.5% concentration of TNPs, k value showed decay of isolated strain during log phase. With concentration of 1%, generation per hour was 13.9 and generation time was 4.3 which were most optimal to use in biodegradation of polyethylene when compared to lowest and highest concentration of TNPs. So, final experiment was carried out to analyze effect of TNPs on performance of polyethylene degrading microbes.
Polyethylene biodegradation analysis in the presence and absence of Titania nanoparticles (TNPs)
During the process of biodegradation, optical density (OD) and λmax were assessed comparatively using spectrophotometer and reported in Figures 6 and 7.
Figure 6: Comparative Microbial Growth Trend in the Presence and Absence of TNPs.
Figure 7: Microbial Counts CFU/ml in the Presence and Absence of TNP’s.
As expected, the TNPs do exhibit bactericidal and antibacterial effects above a certain concentration (1.5% w/v in our case). Below this concentration, however, there is continuous growth of bacteria in nutrient broth and minimal media with polyethylene serving as the sole carbon source. Optical density at 600 nm was found with shorter lag phase and early log phase in the presence of TNPs as compared to control in the absence of TNPs.
In the case of the λmax of the media, control was found to be constant at 212 nm for the first 2 days, which thereafter shifted to 243 nm after 3 days, finally attaining a value of 242 nm after 4 days. The shift in λmax suggests that there are changes taking place in the polymer backbone between 2 to 4 days of incubation as a result of microbial action. On the other hand, in the presence of TNPs, a λmax shift from 211 nm to 219 nm was observed within 1 day, suggesting rapid changes occurring in the polymer backbone during the log phase. The value of λmax showed slight decrease during the stationary phase, suggesting no significant changes in the chemical structure of PE during this period.
CFU/ml showed similar trend like OD and λmax in the presence and absence of TNPs showed viability of P. aeruginosa in degradation experiment. The CFU/ml calculation revealed that the introduction of polyethylene to microbes has supported their growth rate, indicating the acceptance of the polymer as a nutrient. CFU/ml was initially noted to be 84×105 CFU/ml and 102×105 CFU/ml in absence and presence of TNPs after 24 hours of incubation. Growth trend was sustained throughout incubation period with regressive growth in the presence of TNPs.
Moreover, CFU/ml is showing more subsided growth curve than OD. But in both graphs, the presence of TNPs shows less deadly effect on log phase rather than stationary phase where a slight decline can be observed in Figure 7.
Polyethylene degradation analysis
Scanning electron microscopy (SEM) analysis: To analyze morphological changes in the polyethylene powder before and after degradation, SEM characterization was conducted as done by other workers [23,24]. Before degradation, there is a smooth and clear surface. After biodegradation, surface is comparatively much rougher with some holes also appearing after nano-degradation. But after nano-biodegradation, a much higher degree of degradation can be observed in Figure 8.
Figure 8: SEM images of polyethylene powder before Degradation (a), after Microbial Degradation (b), Nanodegradation (c) and Nanobiodegradation.
Fourier transforms infrared spectroscopy FTIR: FTIR (Figure 9) indicate the characteristic peaks associated with the polyethylene organic polymer. The reduction in C-H bond intensity and an increase in C-O bond in the degraded polymer, as compared to the nascent material, as represented by other workers [25] is also obvious in our case.
Figure 9: FT-IR Spectra of PE Degraded by Microbes in the Presence and Absence of TNP’s.
Carbonyl Index CI: An obvious reduction in CI was observed after incubation of samples [26]. After degradation, CI value was reduced to 1.10 in samples exposed to Pseudomonas aeruginosa, and to 1.05 in samples with TNPs as compared to pure polyethylene as control. Maximum reduction (0.99) was observed in sample degraded by both TNPs and P. aeruginosa as presented in Figure 10. The Carbonyl Index is a good means to study the degree of oxidation that has occurred as in the process C-H bond are changed to C-O. In our case, nascent material has CI value of 1.10 which decreased progressively for biodegraded (only) and nano degraded (only) to 0.99 for synergistic nano biodegradation.
Figure 10: Change in Carbonyl Index in the Presence and Absence of TNPs.


It has previously been reported [9] that the native microbes, of a plastic dumpsite, show more potential for conversion of polymer into carbon dioxide and water. Similarly the presence of nano material such as concentration of 0.01% NanoBarium Titanate (NBT) nanoparticles has previously been reported to enhance LDPE biodegradation [27]. Fullerene-60 and Super Magnetic Iron Oxide Nanoparticles (SPION) have also been reported to support microbial growth trend in a progressive way [28]. In support of these observations our work indicates that P. aeruginosa, isolated form a local dumpsite, can effectively degrade polyethylene at 1% concentration of TNPs. We have found that the presence of TNPs in polyethylene, to a certain concentration can be beneficial for the degradation of polymer. In this case, the biodegradation is supported by photocatalytic degradation with improvement in the net degree of polyethylene degradation. Thus, these findings can be used to develop environment friendly plastic shopping bags and other products.


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