Rapid Biodegradation of Low-Density Polyethylene (LDPE) Without Pre-Treatment by Bacillus Strains Isolated from Garbage Dumpsites

Rapid Biodegradation of Low-Density Polyethylene (LDPE) Without Pre-Treatment by Bacillus Strains Isolated from Garbage Dumpsites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rapid Biodegradation of Low-Density Polyethylene (LDPE) Without Pre-Treatment by Bacillus Strains Isolated from Garbage Dumpsites Fahmina Akhtar, Maisha Farzana, Abida Nurun Nahar, Dholon Kumar Paul This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9075950/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The recalcitrant Low-Density Polyethylene (LDPE) is a multifaceted polymer. Therefore, it has slowly gained popularity for ease of access while simultaneously becoming a threat to nature. Investigation of diverse bacteria and exploring their natural ability to degrade LDPE has become a pressing matter in countries like Bangladesh, where thoughtless utilization and indiscriminate disposal of plastics are practiced. This study examined the potential of bacteria to degrade the untreated low-density polyethylene (LDPE). Bacteria were isolated from partially degraded plastic wastes collected from three open garbage dump sites in Dhaka, Bangladesh. Incubation of untreated plastic film inoculated with isolated bacteria resulted in significant weight loss (16%-26%). Both the SEM and Optical Microscope images at 2000x, and 1000x magnifications respectively, revealed surface topology modifications in forms of erosion, cracks, grooves, and minor holes. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the transformation of recalcitrant LDPE film.16S rRNA gene sequencing identified the potential bacteria as Bacillus pumilus strain FMA1, Bacillus aerius strain FMA2, Bacillus sp. (in: firmicutes) strain FMA3, Bacillus cereus strain FMA4, and Bacillus atrophaeus strain FMA5. A significant finding was the detection of Bacillus aerius strain FMA2 and Bacillus atrophaeus strain FMA5, which have no previous LDPE degradation records. More importantly the Bacillus aerius strain FMA2 showed the best degradation potential among the five isolates. A Phylogenetic Tree constructed using the DNA sequence data signified the evolutionary relationship among the bacterial species. This research reveals the prevalence of Bacillus species as soil bacterium in Dhaka city along with their inherent biodegradation capabilities, emphasizing an eco-friendly management of emerging plastic pollution. Biodegradation LDPE Untreated Bacillus species 16S rRNA FTIR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Plastic wastes, persistent all around the world in a wide range of environments-from Mount Everest to Mariana Trench as well as in atmosphere [1], have become a global crisis. The significant portion of plastic wastes is primarily disposable low-density polyethylene (LDPE) and mainly used as single-use packaging products [2]. In 2023, 21.4% of 413.8 Mt of globally produced plastic corresponded to polyethylene (PE) [3]. LDPE is highly resistant to environmental degradation, eventually breaking down into microplastics (MPs, < 5 mm) [4] has emerged as a critical environmental issue. Thoughtless utilization, indiscriminate disposal or “throwaway culture”, inadequate waste management infrastructure, and informal waste sector are the main reasons for placing Bangladesh as one of the top plastic polluted countries. In addition this pollution is on the rise as urbanization escalates, with Dhaka (the capital city), generating most of the waste. This city generates around 14 to 15 million single-use polyethylene bags which are immediately discarded in garbage dump sites [5]. Despite a bustling plastic recycling industry, only 10% of daily plastic waste is recycled [6]. There are only two proper landfills inside Dhaka city: the Matuail and Aminbazar landfills which are insufficient to meet the growing plastic consumption [7]. In addition, nearly 95% of landfills in Bangladesh are unsanitary or open dumps, therefore, a significant contributor to plastic pollution. The impacts of this pollution are mammoth, contaminating soil and water sources, hampering ecosystems and biodiversity, entering the food chain as microplastics and ultimately posing health risk to humans, clogging drains and waterways resulting in urban flooding etc. In addition, plastic pollution is affecting the economy by negatively impacting tourism, fisheries, and other sectors depending on pure water and healthy ecosystems. Conventional plastic degradation methods generate a substantial amount of greenhouse gases such as methane and carbon dioxide [8]. Among the sources of greenhouse gas emissions, approximately 5% can be attributed to landfills alone [9]. Moreover, leachate from the deposited waste can seep into the groundwater or surface water, subsequently endangering both the water quality and human and animal lives [8]. A study on the health hazards in Bangladesh observed that people living close to landfills are more prone to developing bronchial diseases, pneumonia, diarrhea, headache, appetite loss and itching among many other complications [7]. Vector borne diseases are also accelerated by the insect and animal infestations near landfills. Since habitable land is a finite resource, landfills should only be considered a temporary solution to the ever-increasing dilemma. That being the case, the introduction of more environmentally friendly plastic waste disposal methods like biodegradation by means of applying microorganisms is inevitable, because it can consume less energy and reduce the release of harmful chemicals. Unfortunately, such comprehensive evaluation of LDPE biodegradation by bacteria isolated from the soils of Bangladesh is still lacking. Through our research, we attempted to address this pressing matter by designing a valid experiment that provides an efficient and sustainable solution to plastic waste management in our country. Particularly, our objectives included a thorough evaluation of biodegradation of LDPE by soil microorganisms without any sort of pre-treatment because any sort of pre-treatment would be an artifact and definitely obstruse the inherent metabolic capabilities of microorganisms, successful identification of microorganisms responsible for biodegradation, determination of chemical bond modifications through biodegradation, observation of plastic surface topology changed after biodegradation and to provide a sustainable plastic waste management system for Bangladesh. 2. Materials and Methods Collection of sample and isolation of bacteria Partially degraded plastic samples buried approximately 1 meter deep into the soil were collected from three different open garbage dump sites in Dhaka City- Shahjahanpur Railway Colony, Mirpur, and Jashohara Railway [Figure 1 ]. Aseptic collection of plastic samples was ensured by the use of sterile forceps and gloves while picking up the plastics. The collected samples were brought to the laboratory within an hour in airtight zip lock bags for further processing. Parts of the plastic samples, measuring approximately 2 sq. inches, which showed partial degradation were thinly cut using sterile scissors and forceps after careful inspection. Each of the samples were mixed separately with 0.9% saline in different test tubes. Serial dilution up to 10 − 6 dilution factor was done for each of the samples. Among them, dilution factors 10 − 1 , 10 − 5 and 10 − 6 were cultured on Trypticase Soy Agar (TSA) media using spread plate technique. 10 − 1 dilution of each sample was also cultured in Nutrient Agar (NA) media to get an idea regarding the unique microbiota present on each of the sample and its surrounding soil. After 24 hours of incubation, morphologically distinct colonies were selected from plates of 10 − 5 and 10 − 6 dilutions and sub-cultured on Nutrient Agar (NA) plates to get pure isolates. After incubation, five morphologically distinct colonies were selected and labelled: 1, 2, 3, 4, and 5. Preparation of Low-Density Polyethylene (LDPE) films for incubation For this experiment we used polyethylene film eventually collected from commercially available plastic wrap as it is a kind of LDPE and the most widely used plastic lately. 1cm 2 of plastic film (from a new unused pack) was cut with sterile scissors, sprayed with 70% ethanol[11] under laminar air flow while wearing gloves to ensure zero contamination. Initially selected bacterial colonies were inoculated into Nutrient Broth (NB) and incubated overnight at 37 o C for 24 hours. 1ml inoculum was collected from overnight incubated Nutrient Broth. It was then inoculated into Minimal Salt Media (MSM): (g/l: K 2 HPO 4 1.0, KH 2 PO 4 0.2, NaCl 1.0, CaCl 2 .2H 2 O 0.002, H 3 BO 3 0.005, NH 4 (SO 4 ) 2 1.0, MgSO 4 .7H 2 O 0.5, CuSO 4 .5H 2 O 0.001, ZnSO 4 .H 2 O 0.001, MnSO 4 .H 2 O 0.001, Fe 2 (SO 4 ) 3 .6H 2 O 0.01, Agar 15) using spread plate technique [12]. The prepared plastic films were placed in the middle of the plates [Figure 2 ] and three replicas for each sample were incubated for 30 days at 37 o C. Gram Staining Gram staining of the isolated bacteria was conducted to distinguish between the gram positive and gram-negative bacteria, also to observe their morphology. 24 hours fresh bacterial culture was smeared onto a glass slide with a few drops of distilled water using a loop. After allowing it to air dry, it was heat fixed by holding over a flame. First, a few drops of the primary dye (crystal violet) were added and kept for 1 minute. After that the slide was washed with distilled water. This was followed by a few drops of Gram's iodine which was kept for 45 seconds before washing with distilled water. The slide was then washed with 95% ethanol for 20 seconds. Finally a few drops of safranin were added which was kept for 1 minute and later washed with distilled water. The slide was allowed to air dry. Finally the dried slide was observed under a compound microscope at 100x magnification with immersion oil to confirm both the morphology and the type of bacteria. Bacterial DNA Extraction DNA of the selected isolates was extracted using the Boil Extraction Method. At first, fresh culture was inoculated in Luria Bertani Broth (LB) and incubated for 24 hours at 37 o C in a shaker incubator at 135 rpm. The next day, for each isolate, 700µl of the broth was transferred in respective Eppendorf tubes for centrifugation at 13000 rpm for 10 minutes. The supernatants were then carefully discarded and 300 µL of PBS (Phosphate Buffer Saline) was added and mixed with slow re-pipetting motions for washing the pellets. The mixtures were re-centrifuged at 13000 rpm for 5 minutes. The supernatants were discarded, 200 µL of the TE (Tris-EDTA) buffer was added, and the mixtures were subjected to boiling at 100°C for 15 minutes inside a water-bath. After this, the mixtures were immediately cooled in a freezer at -20°C for 10 minutes and were then subjected to a final round of centrifugation (10 minutes at 13000 rpm). The debris was discarded this time and the supernatants were collected in fresh Eppendorf tubes. The extracted DNA collections were securely stored at -20°C until further use. Polymerase Chain Reaction DNA of the selected colonies was extracted and PCR was performed later to assess the bacterial diversity among the samples. Extracted DNA was amplified using 27F (5'AGAGTTTGATCCTGGCTCAG-3') as forward primer and 1492R (5'-CGGTTACCT TGTTACGACTT-3') as reverse primer [13]. The PCR was carried out in a thermal cycler and the conditions were: 95°C for 5 min for initial denaturation, 95°C for 30 seconds for denaturation, 52°C for 45 seconds for primer annealing, 72°C for 1min 30 seconds for strand elongation and 72°C for 10 minutes for final extension. The PCR was run 30 cycles and the final product was stored at -20°C for further analysis. Agarose gel electrophoresis using 0.8% agarose was done to assess the presence of DNA. 16S rRNA Sequencing 16S rRNA sequencing was carried out at International Centre for Diarrheal Disease Research, Bangladesh (ICDDR’B). DNA Baser Assembler software was used to retrieve the FASTA file of the received data. The achieved sequences were then added in the genetic sequence database “GenBank” to obtain the organism’s name and accession number. Weight Loss Measurement After 1 month of incubation, the plates were taken out of the incubator and the LDPE films were washed with 2% Sodium dodecyl sulfate (SDS). It was then placed on a filter paper and allowed to dry at 60 o C overnight [14]. The films were then weighed on a 6-digit precision balance. The percentage weight loss was measured using the following formula: Percentage of weight loss = [(Initial weight – Final weight) / Initial weight] * 100 [14] Microscopy Scanning Electron Microscopy and Optical Polarizing Microscopy both were employed to confirm the plastic biodegradation. These microscopic analyses were carried out at Bangladesh University of Engineering and Technology (BUET). 1000x magnification of the treated LDPE films was used for visualization under Optical Polarizing Microscope. The resulting pictures were analyzed to observe initial changes in surface topography [11]. For SEM images a Zeiss Sigma 300 VP (Jena, Germany) microscope was used. The images were taken at 2000x magnification for detailed surface analysis and for investigation of the morphological changes occurring during degradation [15]. Fourier Transform Infrared Spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) is an important method to detect the functional groups present on the sample surface when treated to bacterial isolates. It was performed at Bangladesh University of Engineering and Technology (BUET) using the Shimadzu IRSpirit spectrophotometer. The data were collected at a wavelength range from 4000 to 400 cm -1 . “Origin 2022” software was used to plot the graph using the resulting data to obtain the peak heights. Phylogenetic Tree A phylogenetic tree was generated using the MEGA 12 software to figure out the evolutionary relationship and genetic diversity of the bacteria capable of plastic degradation. 3. Results Gram Staining and Bacterial Colony Morphology Bacteria grew on nutrient agar media for 24 hours leading to the development of unique morphological features. Their color, transparency, shape, surface appearance, and elevation are summarized in Table 1 . Gram staining results demonstrated that all of the colonies isolated were Gram-positive bacteria. Almost all of the colonies showed that bacteria were rod-shaped which is expected as rod-shaped bacteria are ubiquitous in the environment, particularly in the soil. Table 1 Colony morphology Isolate Colony Morphology Shape Gram Stain Color Translucency Surface Appearance Elevation 1 Pale-yellow Slightly translucent Smooth Umbonate Bacilli Purple (positive) 2 Dark yellow Opaque Glistening Convex Bacilli Purple (positive) 3 Yellow Opaque Wrinkled Raised Bacilli Purple (positive) 4 Off-white Opaque Rough Raised Bacilli Purple (positive) 5 Off-white Translucent Wrinkled Raised Bacilli Purple (positive) Weight loss analysis The loss in dry weights of the microbially treated LDPE films after incubating them for 30 days in MSM medium are calculated and summarized in Table 2 . The value of percentage weight loss is mean ± standard deviation. Table 2 Loss in Dry Weights of LDPE films seen in plates incubated with different bacterial colonies. Sample Initial Weight, mg Mean Final Weight, mg Weight loss (%) 1 1.00 0.833 16.67± .06 2 1.00 0.767 23.33±.06 3 1.00 0.833 16.67±.06 4 1.00 0.733 26.67±.06 5 1.00 0.833 16.67±.06 Polymerase Chain Reaction ( PCR )and 16S rRNA sequencing analysis After the PCR, an agarose gel electrophoresis was conducted at 0.8% concentration. A 1kb plus ladder was loaded into a well. Figure 3 shows the PCR results. All wells showed clear PCR bands with adequate DNA in it. 16S rRNA sequencing of the PCR products of the isolates revealed them to be Bacillus pumilus strain FMA1 , Bacillus aerius strain FMA2 , Bacillus sp. (in: firmicutes) strain FMA3 , Bacillus cereus strain FMA4 and Bacillus atrophaeus strain FMA5. Table 3 Identified Bacterial Isolates. Colony isolate NCBI Accession Number Organism 1 OR186211 Bacillus pumilus strain FMA1 2 OR186212 Bacillus aerius strain FMA2 3 OR186213 Bacillus sp. (in: firmicutes) strain FMA3 4 OR186214 Bacillus cereus strain FMA4 5 OR186215 Bacillus atrophaeus strain FMA5 Optical Microscope observation In order to get a wide overview and the initial physical changes in the surface topography of the untreated LDPE films incubated with bacterial isolates, they were subjected to optical magnification at 1000x. Figure 4 shows changes in the surface topography of the LDPE films in comparison to the negative control (NC). Scanning Electron Microscopy Scanning Electron Microscopy revealed major disruptions on the surface of the plastic samples. Cavities, holes, erosions, and cracks are present on all five untreated LDPE films incubated with bacterial isolates. In contrast to a smooth surface on the negative control film. Figure 5 demonstrates the results of the Scanning Electron Microscopy. FTIR analysis The FTIR method is important for analyzing the functional changes in LDPE films. Figure 6 shows FTIR graphs of LDPE films incubated with five bacterial isolates (1, 2, 3, 4 and 5) and negative control of LDPE (NC) film. All of the changes recorded in the FTIR graphs are considered with NC as a reference graph. The peaks in the graphs indicate the vibration of a specific chemical bond which are labelled in all graphs. The examined prominent peaks along with the functional groups supported by references are provided in Fig. 6 . The differences in transmittance (%T) of each of bacterial sample incubated with LDPE relative to the negative control (NC) were calculated and is presented in Fig. 7 . Table 4 The Interpretation of Change in Transmittance Related to Biodegradation of LDPE Film Incubated with Five Bacterial Samples Compared to Negative Control. 717–718 cm − 1 (Rocking Deformation) Bacillus pumilus strain FMA1 Bacillus aerius strain FMA2 Bacillus sp. (in: firmicutes) strain FMA3 Bacillus cereus strain FMA4 Bacillus atrophaeus strain FMA5 Relation with Biodegradation Higher transmittance Higher transmittance Higher transmittance Higher transmittance Lower transmittance Any kind of change is a spectral signature of the physical and chemical breakdown of the main PE hydrocarbon chains during biodegradation. [16] 1248–1375 cm − 1 (CH 3 Symmetric Deformation Lower transmittance Higher transmittance Lower transmittance Lower transmittance Lower transmittance Higher transmittance indicates a reduction in the number of these C-H bonds, meaning the polymer chains were breaking down during the incubation. [17] Whereas a lower transmittance suggests the formation of new functional groups, such as carbonyl (𝐶=𝑂) and hydroxyl (𝑂−𝐻) groups, which are often built as part of the oxidation and degradation.[18] 1463–1468 cm − 1 (CH 2 Bending Vibration) Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance is an indicator that the vibrations of the 𝐶𝐻2 groups were changing. This happened because the overall crystallinity of the material was likely decreasing as it was broken down. Which is a spectroscopic sign that the LDPE was undergoing degradation. [19] 1650–1800 cm − 1 (Carbonyl Region) Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance means the decrease in the concentration of these specific functional groups, showing that the degradation was progressing beyond just initial oxidation and into the assimilation phase during the period of incubation. [20] 1710–1750 cm − 1 (C = O Peak) Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance The observed changes signify that the bacteria were utilizing the oxidized portions of the LDPE polymer chains (specifically the carbonyl groups) as a carbon source for their metabolic activities during incubation. [21] 2849–2851 cm − 1 (CH 2 Symmetric Stretching) Higher transmittance Higher transmittance Higher transmittance Lower transmittance Lower transmittance Higher transmittance is a direct sign of polymer degradation. It means the 𝐶𝐻2 bonds were breaking down. This could be a cause of microbial activity or other processes like oxidation that broke the polymer chains. On the other hand, lower transmittance is less common in simple biodegradation. It can indicate that the material was becoming more complex by incorporating other compounds that contain C-H bonds. For example, if the LDPE were blended with another material or if degradation was occurring in a way that led to the formation of new, smaller hydrocarbons that were still being measured by the peak. [22] 2917–2920 cm − 1 (CH 2 Asymmetric Stretching) Lower transmittance Higher transmittance Lower transmittance Lower transmittance Lower transmittance Higher transmittance at 2917–2920 cm⁻¹ is a key indicator of polymer degradation. It signifies the cleavage and breakdown of the main LDPE polymer backbone's C-H bonds, leading to shorter chains and the formation of new functional groups like carbonyl (C = O) and hydroxyl (O-H), which appear at different wavelengths. A Lower transmittance might indicate an error or it can suggest for a pretreatment of the LDPE otherwise the bacteria were unable to start oxidation within 1 month period. [23] 3200–3600 cm − 1 (O-H Region) Higher transmittance Higher transmittance Higher transmittance Higher transmittance Higher transmittance The bacterial activity was effectively altering or consuming the oxygen-containing functional groups on the LDPE surface as part of the biodegradation process. [24] Phylogenetic Tree analysis The Phylogenetic tree was constructed using the Mega 12 software which shows the relationship among the genera and species (Fig. 8 ). 4. Discussion In Bangladesh thousands of tons of solid waste generated across the country on a daily basis. Rapid urbanization and economic growth are responsible for this waste surge particularly in urban cities. Per capita municipal urban waste generation now stands around 0.5 Kg per day and is projected to more than double by 2041[25]. Organic wastes made up the majority of the municipal solid waste. Organic waste management is challenging because of being wet and heavy. Meanwhile the plastic waste has been rising with increased use of packaging and disposal products -around 14 to 15 million polyethylene bags are discarded after their first use every day in Dhaka [26]. There is a lack of proper landfills ( scientifically designed with engineered barriers, environment controls, and operational practices for safe isolation of solid wastes). For instance there are only two sprawling landfills dealing with the huge waste loads in Dhaka, therefore, rapidly running out of spaces. In addition, Dhaka is unequivocally one of the most densely populated large cities in the world, having limited land available for managing or treating the wastes. As a result, a significant portion of waste is managed through unrecorded and unsanitary open dumping. Therefore, a sustainable solution is required, and microbial biodegradation is such a process which would minimize the environmental impact by breaking down plastics into beneficial substances. Though there has been extensive research conducting microbial biodegradation of plastics including Low Density Polyethylene (LDPE) worldwide for the last few decades. But there is a lack of research that focuses on biodegradation in Bangladesh. Hence, the need for this study was warranted. Inevitably in our study, we chose to test on plastic samples collected from open dumpsites in Dhaka city. All the sample sites [Figure 1 ] were highly concentrated with partially degraded plastic wastes which provided an ideal environment to collect and test the soil bacteria that were responsible for long-term degradation. While the isolated colonies exhibited notable morphological distinctions when observed without magnification, subsequent examination through gram staining revealed the presence of exclusively Gram-positive bacilli [Table 1 ]. Following incubation with untreated LDPE film as their sole carbon source, visible growth in the MSM media confirmed the successful degradation of LDPE by the inoculated bacteria [12]. Subsequently, the rate of degradation was measured through the most widely used primary methods like- calculation of dry weight loss of incubated LDPE films, analysis of surface morphology, and assessment of change in chemical structure. Calculation of the percent weight loss of incubated LDPE is the fundamental step for assessing the extent of biodegradation. The variation in weight loss is influenced by several factors (i.e. type of both plastics and bacteria, the pre-treatment of the plastic, incubation time, environmental conditions etc.). Unsurprisingly, greater loss in dry weight is suggested to be evident with a longer incubation time along with high incubation temperature or pre-treatment [27], [28], [29]. A weight loss of 1.75% after 30 days[30] and 10.7% after 60 days [28] of incubation with Bacillus sp. were also reported in some studies. Another study showed 18.9% weight loss after 180 days of incubation with Bacillus pumilus [31], whereas our findings showed a minimum weight loss of 16.67±.06 % only after 30 days. To previous studies also demonstrated the LDPE degrading abilities of Bacillus cereus , reporting a 6.33%[13] and 20.28%[32] weight loss after 45 days and 112 days of incubation respectively. The variation in weight loss and incubation period in our study with these studies is monumental as we have observed 26.67±.06 % weight loss after only 30 day of incubation [Table 2 ]. In our study, substantial loss in dry weights (16–26%) were recorded of LDPE without any pre-treatment and only for one month of incubation. With the highest being from sample “4 ( Bacillus cereus strain FMA4 )” while sample 1, 2, 3, and 5 demonstrated a near similar level of degradation. This indicates the significant biodegradation potential of Bacillus spp. The structural changes occurring due to induced degradation is an important parameter for measuring the degree of degradation in a molecular level [33]. FTIR has been used in numerous degradation studies to determine the decrease in native bonds that provides evidence of the polymer being fragmented into shorter chains. Additionally, generation of new or loss of functional groups further indicates the degradation process [34]. In a complete biodegradation pathway, the polymer is oxidized by the oxygen in the air to form carbonyl groups which then forms carboxylic groups, ultimately, the intermediates undergo β-oxidation and enter the citric cycle which form CO 2 and H₂O [35]. Native bonds in polyethylene correspond to the following peaks: 2918 cm -1 (CH 2 asymmetric stretching), 2851 cm -1 (CH 2 symmetric stretching), 1468 cm -1 (bending deformation), 1373 cm -1 (CH 3 symmetric deformation) and 718 cm -1 (rocking deformation). These peaks are seen in the FTIR for the control LDPE (NC) [Figure 6 ] [36]. In the FTIR spectroscopy of all LDPE samples incubated with bacterial colonies, there is both an increase and decrease in the percentage of transmittance at these peaks while comparing with the NC [Figure 7 ] indicating that less of that native bond is present. To specify the relationship of these changes along with biodegradation ability of each of the bacterial sample is provided in Table 4 . A new peak around 1600 cm -1 is seen for 2, 3, 4 and 5 that corresponds to absorbance of aromatic C = C bonds. Absorbance of this bond has been said to be indicative of biodegradation in previous studies [11]. The further analysis includes the study of surface morphology. We did both optical microscopy and Scanning Electron Microscopy (SEM). Optical microscopy offered quick and easy overall conditions showing cracks, minute holes, and other disruptions along with bacterial adhesion on the surface of all the samples in comparison to the control [Figure 4 ]. This colonization indicates bacterial attack leading to biodegradation [33]. The photographs also showed the adhesion to be scattered and mostly concentrated around the fissures. These findings correspond with studies that suggest bacterial susceptibility in the amorphous region of the LDPE samples [34]. On the other hand SEM provided more detailed micro-features including cracks, disintegration, irregular surfaces, fissures, erosions [Figure 5 ] compared to control LDPE films characterized by smooth and uniform surface, no noticeable surface defects or mechanical failure [11]. All of these outcomes confirm the degradation capacity of the bacterial isolates [37]. 16S rRNA gene phylogenetic analysis and BLAST based comparative homology analysis for 16S rRNA gene sequences (∼1000 bp) of PE degrading isolates revealed that in our study all the five LDPE degraders belong to the phylum Firmicutes in class Bacillus. Phylogenetic analysis based on the Neighbor-Joining (NJ) tree, and Maximum Likelihood (ML) indicated that all isolates belonged to a monophyletic cluster that was distinct from the other polyethylene degrading bacteria known to Pseudomonas plecoglossicida , Rhodococcus pyridinivorans , Rhodococcus ruber , Lactobacillus acidophilus , and Lysinibacillus xylanilyticus . It means that they have the same genetic composition, and also their independent evolution for polyethylene-degrading ability. In the Bacillus clade, B. pumilus (FMA1), B. aerius (FMA2) and Bacillus sp. (FMA3) were clustered in Clade A, with very low bootstrap value of 1% and branch length 0.003, and closer to B. mexicanus and B. licheniformis , both known as a polymer degrader. B. atrophaeus (FMA5) was clustered to B. halotolerans in Clade B having bootstrap values of 0.999% and branch length of 0.0025, whereas Lysinibacillus xylanilyticus grouped as a separate clade C with the B. cereus (FMA4) having also boostrap value 0.999% and branch length 0.0438 showed functional convergence while being taxonomically divergent species among all samples investigated below sub-family. Other described PE-degrading genera, Pseudomonas plecoglossicida (Proteobacteria), Rhodococcus pyridinivorans and R. ruber (Actinobacteria), and Lactobacillus acidophilus ( Firmicutes ) within distinct, consistently supported branches like the bootstrap value of 99.9 to 97.5% and branch length of 0.038 to 0.052. The high bootstrap values of > 0.98 and different branch lengths 0.002–0.052 in this tree suggested that LDPE-degrading capability is phylogenetically stable for the bacterial isolates but has diverse origins relative to evolutionary origins. These results indicate that polyethylene degradation has emerged independently in several bacterial lineages as a result of convergent functional adaptation to withstand the high percentage of plastic wastes in our soil environment, which supports our previous results particularly the dry weight loss after incubation. Additionally, to the best of our knowledge, we are reporting Bacillus atrophaeus strain FMA5 and B acillus aerius strain FMA2 , as an LDPE degrading bacteria for the very first time. Based on our findings, it can be concluded that the Bacillus species exhibit a remarkable capacity to effectively biodegrade low-density polyethylene (LDPE). Notably, it is worth mentioning that the bacterial isolates utilized for our study that yielded insightful observations upon examination, were sourced from all the selected sampling sites. 6. Summary and Conclusion The findings of this study decisively demonstrate the inherent capacity of the soil bacteria isolated from open dumpsites in Dhaka, Bangladesh to initiate and sustain the degradation of low-density polyethylene (LDPE) films without the need for thermal or chemical pretreatment. The Quantitative analysis, evidenced by a significant percentage of dry weight loss over the short one-month incubation period (e.g., a mean loss of more than 20%), while most of the other studies were carried out for a longer duration established the bio-assimilation of LDPE constituents. This quantitative evidence was strongly corroborated by Fourier-Transform Infrared Spectroscopy (FTIR), which revealed characteristic chemical alterations on the polymer surface. The increase in the percentage of transmittance at several peaks (e.g., 2918 cm − 1 , 2851 cm − 1 , 1468 cm − 1 , 1373 cm − 1 , 718 cm − 1 ) indicated the less presence of the native bonds. On the other hand the emergence and intensification of new bands within the carbonyl (C = O) stretching region (approx. 1715 cm − 1 ) and the hydroxyl (O-H) region (approx. 3400 cm − 1 ) confirm the initial oxidative steps of biodegradation, which are critical for increasing hydrophilicity and making the polymer backbone accessible to enzymatic attack. The visual analyses of surface topography provided compelling evidence for the physical and biological processes involved. Both the Optical microscopy and Scanning Electron Microscopy (SEM) images revealed profound structural changes, including the cracks, visible pitting, fissure development, and erosion marks indicative of microbial colonization and enzymatic activity. This physical alteration confirms the effective interaction between the bacteria and the hydrophobic LDPE surface. Furthermore, the phylogenetic analysis successfully identified the prevalence of the key plastic degrading species responsible (e.g., member of the Bacillus genera), thereby directly linking the observed degradation phenomena to specific, characterized bacterial strains. In addition the phylogenetic analysis identified two novel strains of Bacillus designated as FMA2 (B acillus aerius) and FMA5 ( Bacillus atrophaeus). Sequences of these strains were deposited in the NCBI GenBank Database (accession numbers provided in Table 3 ). Collectively, these results confirm a mechanism involving microbial adhesion, oxidative cleavage of the long alkane chains, and subsequent bio-assimilation of the resulting low-molecular-weight fragments. In summary, this research successfully established a clear link between the identified bacterial strains and quantitative, chemical, and morphological changes in the LDPE film, validating the hypothesis of direct, non-pretreated biodegradation potential. While the observed weight loss and spectral shifts strongly suggest polymer degradation, future studies must incorporate advanced techniques such as Gel Permeation Chromatography (GPC) to conclusively quantify the reduction in average molecular weight. Moving forward, the isolated strains represent a high-value candidate for optimizing bioremediation processes. Further research should focus on optimizing culture conditions, building a microbial consortia between the bacterial strains mentioned in the paper along with other highly degrading microorganisms, and identifying and isolating the specific depolymerase enzymes responsible for the observed oxidative cleavage to enhance the rate and efficiency of LDPE waste management to ensure the cleaning of the current mammoth load of plastic pollution not only in Bangladesh but also worldwide. Declarations Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution F.A. conceptualized the study, designed the methodology, supervised the research, and provided project administration. M.F. and A.N.N. performed the experimental investigations, including the isolation of bacterial strains, 16S rRNA sequencing, and the incubation assays. D.K.P. conducted the formal data analysis and contributed significantly to the interpretation of the physical and chemical characterization results (FTIR and SEM). F.A. and M.F. wrote the main manuscript text. D.K.P. contributed to the writing, review, and editing of the manuscript. All authors reviewed and approved the final manuscript. Acknowledgement We are grateful to Dr. Muhammad Rakibul Islam, Professor, Department of Physics, Bangladesh University of Engineering and Technology for the support in the FTIR analyses and also for opti-cal microscopy. Data Availability The metagenomic raw data have been deposited at NCBI GenBank (Accession no. : OR186211, OR186212, OR186213, OR186214, OR186215). All other data are available from the corresponding author upon request. References D. M. Wafaa, M. W. Sadik, H. F. Eissa, and K. Tonbol, “Biodegradation of low-density polyethylene LDPE by marine bacterial strains Gordonia alkanivorans PBM1 and PSW1 isolated from Mediterranean Sea, Alexandria, Egypt,” Sci Rep , vol. 15, no. 1, p. 16769, 2025, doi: 10.1038/s41598-025-96811-z. I. Varyan et al. , “The Use of Natural Rubber as an Initiator of LDPE Biodegradation in Soil,” Polymers (Basel) , vol. 17, no. 21, 2025, doi: 10.3390/polym17212885. M. L. Oliveira, G. M. Miranda, and D. S. 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Available: https://api.semanticscholar.org/CorpusID:95040097 M. Bhatia, A. Girdhar, A. Tiwari, and A. Nayarisseri, “Implications of a novel Pseudomonas species on low density polyethylene biodegradation: an in vitro to in silico approach,” Springerplus , vol. 3, no. 1, p. 497, 2014, doi: 10.1186/2193-1801-3-497. R. Ingavale and P. Raut, “Comparative biodegradation studies of LDPE and HDPE using bacillus weihenstephanensis isolated from garbage soil,” Nature Environment and Pollution Technology , vol. 17, pp. 649–655, Jan. 2018. Y. Otake, T. Kobayashi, H. Asabe, N. Murakami, and K. Ono, “Biodegradation of low-density polyethylene, polystyrene, polyvinyl chloride, and urea formaldehyde resin buried under soil for over 32 years,” J Appl Polym Sci , vol. 56, no. 13, pp. 1789–1796, 1995, doi: https://doi.org/10.1002/app.1995.070561309. J. P. Harrison, C. Boardman, K. O’Callaghan, A.-M. Delort, and J. Song, “Biodegradability standards for carrier bags and plastic films in aquatic environments: a critical review,” R Soc Open Sci , vol. 5, no. 5, p. 171792, May 2018, doi: 10.1098/rsos.171792. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9075950","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607548597,"identity":"615d2037-f753-4c84-9d9d-89bac8c0e580","order_by":0,"name":"Fahmina 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17:54:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9075950/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9075950/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104887325,"identity":"553f1142-85ab-4ae2-b8a0-a5b64726d1fd","added_by":"auto","created_at":"2026-03-18 10:11:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193079,"visible":true,"origin":"","legend":"\u003cp\u003eSample locations in Dhaka, Bangladesh [10]\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/4266347de40b5a5b2d3cbcd8.jpg"},{"id":104887405,"identity":"3fc57bea-d1a2-45d1-9434-6ec2293cedc5","added_by":"auto","created_at":"2026-03-18 10:11:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51276,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of LDPE film for incubation.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/9699488040d6f33ccc9d14f7.jpg"},{"id":104887392,"identity":"dfb122dc-9d7d-4665-8a31-4444c3d6da11","added_by":"auto","created_at":"2026-03-18 10:11:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84918,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCR bands seen after gel run were done at 0.8% alongside a 1kb+ ladder.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/82ecff05516369f9a47b3e97.jpg"},{"id":104887394,"identity":"ce420957-d854-4731-a215-bd41ec7411d1","added_by":"auto","created_at":"2026-03-18 10:11:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192927,"visible":true,"origin":"","legend":"\u003cp\u003eOptical microscopy (1000x) revealing the surface changes of non-pretreated LDPE with bacterial isolates (\u003cstrong\u003e1, 2, 3, 4 \u003c/strong\u003eand \u003cstrong\u003e5\u003c/strong\u003e) and LDPE film without inoculated bacteria (NC) after 1 month of incubation. \u003cstrong\u003ea.\u003c/strong\u003eFilm incubated with \u003cstrong\u003eFMA1\u003c/strong\u003e revealed a prominent crack that is marked with an arrow, minute holes which are circled, and surface disruptions that have been indicated with a box. \u003cstrong\u003eb.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA2\u003c/strong\u003e only showed a slight crack (arrow). \u003cstrong\u003ec.\u003c/strong\u003e Film showed cracks (arrows), minute holes (circles), and disruption (box) incubated with \u003cstrong\u003eFMA3\u003c/strong\u003e. \u003cstrong\u003ed.\u003c/strong\u003eMajor disruption (box), a slight crack (arrow) and few holes (circles) were seen in film after incubation with \u003cstrong\u003eFMA4\u003c/strong\u003e. \u003cstrong\u003ee.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA5 \u003c/strong\u003ehad cracks (arrows) and minute holes (circle).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/22e7ce03fa5ef7599dae02dd.jpg"},{"id":104887413,"identity":"b1d240af-cd5d-40df-9ace-35bd2524422a","added_by":"auto","created_at":"2026-03-18 10:11:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":185908,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope images of untreated LDPE with bacterial isolates (\u003cstrong\u003e1, 2, 3, 4 \u003c/strong\u003eand \u003cstrong\u003e5\u003c/strong\u003e) and negative control LDPE film (NC) after 1 month of incubation.\u003cstrong\u003e \u003c/strong\u003eNC shows no disruptions on its surface. \u003cstrong\u003ea.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA1\u003c/strong\u003e shows cavities on the plastic surface. \u003cstrong\u003eb.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA2\u003c/strong\u003e shows cracks and erosions on the plastic surface. \u003cstrong\u003ec.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA3\u003c/strong\u003eshows large cracks. \u003cstrong\u003ed.\u003c/strong\u003e Various cracks and erosions are seen on the surface of the plastic film incubated with \u003cstrong\u003eFMA4\u003c/strong\u003e. \u003cstrong\u003ee.\u003c/strong\u003e Film incubated with \u003cstrong\u003eFMA5 \u003c/strong\u003eshows multiple cavities and cracks.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/2027df0586a921158953cab1.jpg"},{"id":104887521,"identity":"15a7a45e-9319-49ae-abdd-4d8be75ac93e","added_by":"auto","created_at":"2026-03-18 10:11:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":118031,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR graph of incubated LDPE for 1 month with (Samples 1, 2, 3, 4 and 5) and incubated LDPE without bacteria (Negative Control, NC). \u003cstrong\u003ea.\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e showed greater percentage in transmission at 1468 cm\u003csup\u003e-1\u003c/sup\u003e and 1373 cm\u003csup\u003e-1\u003c/sup\u003e compared to the peaks in NC. \u003cstrong\u003eb.\u003c/strong\u003e \u003cstrong\u003e2\u003c/strong\u003e showed greater percentage in transmission as seen at the following wavelengths compared to NC: 718 cm\u003csup\u003e-1\u003c/sup\u003e, 1373 cm-\u003csup\u003e1\u003c/sup\u003e, 1468 cm\u003csup\u003e-1\u003c/sup\u003e, 2851 cm\u003csup\u003e-1\u003c/sup\u003e and 2918 cm\u003csup\u003e-1\u003c/sup\u003e. A new peak at 1600 cm\u003csup\u003e-1\u003c/sup\u003e was also observed. \u003cstrong\u003ec.\u003c/strong\u003e \u003cstrong\u003e3\u003c/strong\u003e had a noticeably higher percentage in transmission for the peaks at wavelengths 1468 cm\u003csup\u003e-1\u003c/sup\u003e, 1373 cm\u003csup\u003e-1\u003c/sup\u003e and 718 cm\u003csup\u003e-1\u003c/sup\u003e compared to NC. A new peak around 1600 cm\u003csup\u003e-1\u003c/sup\u003e was seen. \u003cstrong\u003ed.\u003c/strong\u003e \u003cstrong\u003e4\u003c/strong\u003e displayed the most drastic visual change with less steep curves than NC, correlating with the greater weight loss percentage. Greater percentage in transmission was seen for peaks at wavelengths 718 cm\u003csup\u003e-1\u003c/sup\u003e, 1373 cm\u003csup\u003e-1\u003c/sup\u003e, 1468 cm\u003csup\u003e-1\u003c/sup\u003e and 2918 cm\u003csup\u003e-1\u003c/sup\u003e. A new peak at 1600 cm\u003csup\u003e-1\u003c/sup\u003e was also observed. \u003cstrong\u003ee.\u003c/strong\u003e \u003cstrong\u003e5\u003c/strong\u003e showed a higher percentage in transmission for peaks at wavelengths 1468 cm\u003csup\u003e-1\u003c/sup\u003e and 1373 cm\u003csup\u003e-1\u003c/sup\u003e compared to NC while there was a new peak at 1600 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/216ec38f994eee1021da1ce0.jpg"},{"id":104887355,"identity":"d87a80d1-0896-42ae-8d64-172e127f95ac","added_by":"auto","created_at":"2026-03-18 10:11:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74681,"visible":true,"origin":"","legend":"\u003cp\u003eRelative change in intensity in LDPE films incubated with five bacterial isolates Vs. Negative Control (LDPE film incubated without bacteria). Bars above the zero line (+): Indicate higher transmittance compared to the control. Bars below the zero line (-): Indicate lower transmittance compared to the control.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/fbe5e549846377b09d0d17ad.jpg"},{"id":104887403,"identity":"d190aa5d-7c99-4f22-8cd9-b18029a250d9","added_by":"auto","created_at":"2026-03-18 10:11:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":78510,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic tree of the 16s rRNA gene sequences from the polyethylene degrading isolates (FMA1- FMA5) and related type species. The phylogenetic tree showed that these isolates, which are classified under the \u003cem\u003eFirmicutes\u003c/em\u003ephylum and the \u003cem\u003eBacillus \u003c/em\u003eclass, fell into the one distinct monophyletic cluster separate from other polyethylene degraders such as \u003cem\u003ePseudomonas plecoglossicida, Rhodococcus pyridinivorans, Rhodococcus ruber, Lactobacillus acidophillus, \u003c/em\u003eand \u003cem\u003eLysinibacillus xylanilyticus\u003c/em\u003e. The boostrap values from 1000 iterations are showed at the branches. \u003cem\u003eB. pumilus\u003c/em\u003e FMA1, \u003cem\u003eB. aerius\u003c/em\u003e FMA2, \u003cem\u003eBacillus sp\u003c/em\u003e. FMA3, and \u003cem\u003eB.\u003c/em\u003e \u003cem\u003eatrophaeus\u003c/em\u003e FMA5 were grouped under two clades with low bootstrap values, indicative of functional convergence. The PE degrading ability has independently evolved in multiple bacterial lineages, suggestive of convergent functional evolution. Bars, 0.005 and 0.002, represent 0.5% and 0.2% nucleotide substitutions, respectively.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/095891a4eebadefb1731614c.jpg"},{"id":105904147,"identity":"f0ecfc7a-1bde-4ce6-965f-65f4ad93991f","added_by":"auto","created_at":"2026-04-01 10:04:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2056096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9075950/v1/ab792f08-a00c-4f22-9580-b4c5bc32f7b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rapid Biodegradation of Low-Density Polyethylene (LDPE) Without Pre-Treatment by Bacillus Strains Isolated from Garbage Dumpsites","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlastic wastes, persistent all around the world in a wide range of environments-from Mount Everest to Mariana Trench as well as in atmosphere [1], have become a global crisis. The significant portion of plastic wastes is primarily disposable low-density polyethylene (LDPE) and mainly used as single-use packaging products [2]. In 2023, 21.4% of 413.8 Mt of globally produced plastic corresponded to polyethylene (PE) [3]. LDPE is highly resistant to environmental degradation, eventually breaking down into microplastics (MPs, \u0026lt;\u0026thinsp;5 mm) [4] has emerged as a critical environmental issue. Thoughtless utilization, indiscriminate disposal or \u0026ldquo;throwaway culture\u0026rdquo;, inadequate waste management infrastructure, and informal waste sector are the main reasons for placing Bangladesh as one of the top plastic polluted countries. In addition this pollution is on the rise as urbanization escalates, with Dhaka (the capital city), generating most of the waste. This city generates around 14 to 15\u0026nbsp;million single-use polyethylene bags which are immediately discarded in garbage dump sites [5]. Despite a bustling plastic recycling industry, only 10% of daily plastic waste is recycled [6]. There are only two proper landfills inside Dhaka city: the Matuail and Aminbazar landfills which are insufficient to meet the growing plastic consumption [7]. In addition, nearly 95% of landfills in Bangladesh are unsanitary or open dumps, therefore, a significant contributor to plastic pollution. The impacts of this pollution are mammoth, contaminating soil and water sources, hampering ecosystems and biodiversity, entering the food chain as microplastics and ultimately posing health risk to humans, clogging drains and waterways resulting in urban flooding etc. In addition, plastic pollution is affecting the economy by negatively impacting tourism, fisheries, and other sectors depending on pure water and healthy ecosystems.\u003c/p\u003e \u003cp\u003eConventional plastic degradation methods generate a substantial amount of greenhouse gases such as methane and carbon dioxide [8]. Among the sources of greenhouse gas emissions, approximately 5% can be attributed to landfills alone [9]. Moreover, leachate from the deposited waste can seep into the groundwater or surface water, subsequently endangering both the water quality and human and animal lives [8]. A study on the health hazards in Bangladesh observed that people living close to landfills are more prone to developing bronchial diseases, pneumonia, diarrhea, headache, appetite loss and itching among many other complications [7]. Vector borne diseases are also accelerated by the insect and animal infestations near landfills. Since habitable land is a finite resource, landfills should only be considered a temporary solution to the ever-increasing dilemma. That being the case, the introduction of more environmentally friendly plastic waste disposal methods like biodegradation by means of applying microorganisms is inevitable, because it can consume less energy and reduce the release of harmful chemicals.\u003c/p\u003e \u003cp\u003eUnfortunately, such comprehensive evaluation of LDPE biodegradation by bacteria isolated from the soils of Bangladesh is still lacking. Through our research, we attempted to address this pressing matter by designing a valid experiment that provides an efficient and sustainable solution to plastic waste management in our country. Particularly, our objectives included a thorough evaluation of biodegradation of LDPE by soil microorganisms without any sort of pre-treatment because any sort of pre-treatment would be an artifact and definitely obstruse the inherent metabolic capabilities of microorganisms, successful identification of microorganisms responsible for biodegradation, determination of chemical bond modifications through biodegradation, observation of plastic surface topology changed after biodegradation and to provide a sustainable plastic waste management system for Bangladesh.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cem\u003eCollection of sample and isolation of bacteria\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePartially degraded plastic samples buried approximately 1 meter deep into the soil were collected from three different open garbage dump sites in Dhaka City- Shahjahanpur Railway Colony, Mirpur, and Jashohara Railway [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]. Aseptic collection of plastic samples was ensured by the use of sterile forceps and gloves while picking up the plastics. The collected samples were brought to the laboratory within an hour in airtight zip lock bags for further processing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eParts of the plastic samples, measuring approximately 2 sq. inches, which showed partial degradation were thinly cut using sterile scissors and forceps after careful inspection. Each of the samples were mixed separately with 0.9% saline in different test tubes. Serial dilution up to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e dilution factor was done for each of the samples. Among them, dilution factors 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e were cultured on Trypticase Soy Agar (TSA) media using spread plate technique. 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dilution of each sample was also cultured in Nutrient Agar (NA) media to get an idea regarding the unique microbiota present on each of the sample and its surrounding soil. After 24 hours of incubation, morphologically distinct colonies were selected from plates of 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e dilutions and sub-cultured on Nutrient Agar (NA) plates to get pure isolates. After incubation, five morphologically distinct colonies were selected and labelled: 1, 2, 3, 4, and 5.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePreparation of Low-Density Polyethylene (LDPE) films for incubation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor this experiment we used polyethylene film eventually collected from commercially available plastic wrap as it is a kind of LDPE and the most widely used plastic lately. 1cm\u003csup\u003e2\u003c/sup\u003e of plastic film (from a new unused pack) was cut with sterile scissors, sprayed with 70% ethanol[11] under laminar air flow while wearing gloves to ensure zero contamination. Initially selected bacterial colonies were inoculated into Nutrient Broth (NB) and incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours. 1ml inoculum was collected from overnight incubated Nutrient Broth. It was then inoculated into Minimal Salt Media (MSM): (g/l: K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1.0, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.2, NaCl 1.0, CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO 0.002, H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e 0.005, NH\u003csub\u003e4\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e 1.0, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 0.5, CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO 0.001, ZnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO 0.001, MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO 0.001, Fe\u003csub\u003e2\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO 0.01, Agar 15) using spread plate technique [12]. The prepared plastic films were placed in the middle of the plates [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e] and three replicas for each sample were incubated for 30 days at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eGram Staining\u003c/em\u003e \u003c/p\u003e \u003cp\u003eGram staining of the isolated bacteria was conducted to distinguish between the gram positive and gram-negative bacteria, also to observe their morphology.\u003c/p\u003e \u003cp\u003e24 hours fresh bacterial culture was smeared onto a glass slide with a few drops of distilled water using a loop. After allowing it to air dry, it was heat fixed by holding over a flame. First, a few drops of the primary dye (crystal violet) were added and kept for 1 minute. After that the slide was washed with distilled water. This was followed by a few drops of Gram's iodine which was kept for 45 seconds before washing with distilled water. The slide was then washed with 95% ethanol for 20 seconds. Finally a few drops of safranin were added which was kept for 1 minute and later washed with distilled water. The slide was allowed to air dry. Finally the dried slide was observed under a compound microscope at 100x magnification with immersion oil to confirm both the morphology and the type of bacteria.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacterial DNA Extraction\u003c/em\u003e \u003c/p\u003e \u003cp\u003eDNA of the selected isolates was extracted using the Boil Extraction Method. At first, fresh culture was inoculated in Luria Bertani Broth (LB) and incubated for 24 hours at 37\u003csup\u003eo\u003c/sup\u003eC in a shaker incubator at 135 rpm. The next day, for each isolate, 700\u0026micro;l of the broth was transferred in respective Eppendorf tubes for centrifugation at 13000 rpm for 10 minutes. The supernatants were then carefully discarded and 300 \u0026micro;L of PBS (Phosphate Buffer Saline) was added and mixed with slow re-pipetting motions for washing the pellets. The mixtures were re-centrifuged at 13000 rpm for 5 minutes. The supernatants were discarded, 200 \u0026micro;L of the TE (Tris-EDTA) buffer was added, and the mixtures were subjected to boiling at 100\u0026deg;C for 15 minutes inside a water-bath. After this, the mixtures were immediately cooled in a freezer at -20\u0026deg;C for 10 minutes and were then subjected to a final round of centrifugation (10 minutes at 13000 rpm). The debris was discarded this time and the supernatants were collected in fresh Eppendorf tubes. The extracted DNA collections were securely stored at -20\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePolymerase Chain Reaction\u003c/em\u003e \u003c/p\u003e \u003cp\u003eDNA of the selected colonies was extracted and PCR was performed later to assess the bacterial diversity among the samples. Extracted DNA was amplified using 27F (5'AGAGTTTGATCCTGGCTCAG-3') as forward primer and 1492R (5'-CGGTTACCT TGTTACGACTT-3') as reverse primer [13]. The PCR was carried out in a thermal cycler and the conditions were: 95\u0026deg;C for 5 min for initial denaturation, 95\u0026deg;C for 30 seconds for denaturation, 52\u0026deg;C for 45 seconds for primer annealing, 72\u0026deg;C for 1min 30 seconds for strand elongation and 72\u0026deg;C for 10 minutes for final extension. The PCR was run 30 cycles and the final product was stored at -20\u0026deg;C for further analysis. Agarose gel electrophoresis using 0.8% agarose was done to assess the presence of DNA.\u003c/p\u003e\n\u003ch3\u003e16S rRNA Sequencing\u003c/h3\u003e\n\u003cp\u003e16S rRNA sequencing was carried out at International Centre for Diarrheal Disease Research, Bangladesh (ICDDR\u0026rsquo;B). DNA Baser Assembler software was used to retrieve the FASTA file of the received data. The achieved sequences were then added in the genetic sequence database \u0026ldquo;GenBank\u0026rdquo; to obtain the organism\u0026rsquo;s name and accession number.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWeight Loss Measurement\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAfter 1 month of incubation, the plates were taken out of the incubator and the LDPE films were washed with 2% Sodium dodecyl sulfate (SDS). It was then placed on a filter paper and allowed to dry at 60\u003csup\u003eo\u003c/sup\u003eC overnight [14]. The films were then weighed on a 6-digit precision balance. The percentage weight loss was measured using the following formula:\u003c/p\u003e \u003cp\u003ePercentage of weight loss = [(Initial weight \u0026ndash; Final weight) / Initial weight] * 100 [14]\u003c/p\u003e \u003cp\u003e \u003cem\u003eMicroscopy\u003c/em\u003e \u003c/p\u003e \u003cp\u003eScanning Electron Microscopy and Optical Polarizing Microscopy both were employed to confirm the plastic biodegradation. These microscopic analyses were carried out at Bangladesh University of Engineering and Technology (BUET).\u003c/p\u003e \u003cp\u003e1000x magnification of the treated LDPE films was used for visualization under Optical Polarizing Microscope. The resulting pictures were analyzed to observe initial changes in surface topography [11].\u003c/p\u003e \u003cp\u003eFor SEM images a Zeiss Sigma 300 VP (Jena, Germany) microscope was used. The images were taken at 2000x magnification for detailed surface analysis and for investigation of the morphological changes occurring during degradation [15].\u003c/p\u003e \u003cp\u003e \u003cem\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFourier transform infrared spectroscopy (FTIR) is an important method to detect the functional groups present on the sample surface when treated to bacterial isolates. It was performed at Bangladesh University of Engineering and Technology (BUET) using the Shimadzu IRSpirit spectrophotometer. The data were collected at a wavelength range from 4000 to 400 cm\u003csup\u003e-1\u003c/sup\u003e. \u0026ldquo;Origin 2022\u0026rdquo; software was used to plot the graph using the resulting data to obtain the peak heights.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhylogenetic Tree\u003c/em\u003e \u003c/p\u003e \u003cp\u003eA phylogenetic tree was generated using the MEGA 12 software to figure out the evolutionary relationship and genetic diversity of the bacteria capable of plastic degradation.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cem\u003eGram Staining and Bacterial Colony Morphology\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBacteria grew on nutrient agar media for 24 hours leading to the development of unique morphological features. Their color, transparency, shape, surface appearance, and elevation are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Gram staining results demonstrated that all of the colonies isolated were Gram-positive bacteria. Almost all of the colonies showed that bacteria were rod-shaped which is expected as rod-shaped bacteria are ubiquitous in the environment, particularly in the soil.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eColony morphology\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eColony Morphology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eShape\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGram Stain\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTranslucency\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSurface Appearance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eElevation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePale-yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlightly translucent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUmbonate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePurple (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDark yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOpaque\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlistening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConvex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePurple (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOpaque\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWrinkled\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaised\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePurple (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOff-white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOpaque\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaised\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePurple (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOff-white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTranslucent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWrinkled\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaised\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePurple (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eWeight loss analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe loss in dry weights of the microbially treated LDPE films after incubating them for 30 days in MSM medium are calculated and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The value of percentage weight loss is mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLoss in Dry Weights of LDPE films seen in plates incubated with different bacterial colonies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial Weight, mg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean Final Weight, mg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeight loss (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026plusmn; .06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.33\u0026plusmn;.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026plusmn;.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.733\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.67\u0026plusmn;.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026plusmn;.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePolymerase Chain Reaction\u003c/em\u003e (\u003cem\u003ePCR )and 16S rRNA sequencing analysis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAfter the PCR, an agarose gel electrophoresis was conducted at 0.8% concentration. A 1kb plus ladder was loaded into a well. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the PCR results. All wells showed clear PCR bands with adequate DNA in it.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e16S rRNA sequencing of the PCR products of the isolates revealed them to be \u003cem\u003eBacillus pumilus\u003c/em\u003e strain \u003cb\u003eFMA1\u003c/b\u003e, \u003cem\u003eBacillus aerius\u003c/em\u003e strain \u003cb\u003eFMA2\u003c/b\u003e, \u003cem\u003eBacillus\u003c/em\u003e sp. (in: firmicutes) strain \u003cb\u003eFMA3\u003c/b\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e strain \u003cb\u003eFMA4\u003c/b\u003e and \u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain \u003cb\u003eFMA5.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentified Bacterial Isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColony isolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNCBI Accession Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR186211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus pumilus\u003c/em\u003e strain \u003cb\u003eFMA1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR186212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus aerius\u003c/em\u003e strain \u003cb\u003eFMA2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR186213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e sp. (in: firmicutes) strain \u003cb\u003eFMA3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR186214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e strain \u003cb\u003eFMA4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR186215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain \u003cb\u003eFMA5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cem\u003eOptical Microscope observation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn order to get a wide overview and the initial physical changes in the surface topography of the untreated LDPE films incubated with bacterial isolates, they were subjected to optical magnification at 1000x. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows changes in the surface topography of the LDPE films in comparison to the negative control (NC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eScanning Electron Microscopy\u003c/em\u003e \u003c/p\u003e \u003cp\u003eScanning Electron Microscopy revealed major disruptions on the surface of the plastic samples. Cavities, holes, erosions, and cracks are present on all five untreated LDPE films incubated with bacterial isolates. In contrast to a smooth surface on the negative control film. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrates the results of the Scanning Electron Microscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFTIR analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe FTIR method is important for analyzing the functional changes in LDPE films. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows FTIR graphs of LDPE films incubated with five bacterial isolates (1, 2, 3, 4 and 5) and negative control of LDPE (NC) film. All of the changes recorded in the FTIR graphs are considered with NC as a reference graph. The peaks in the graphs indicate the vibration of a specific chemical bond which are labelled in all graphs. The examined prominent peaks along with the functional groups supported by references are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe differences in transmittance (%T) of each of bacterial sample incubated with LDPE relative to the negative control (NC) were calculated and is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe Interpretation of Change in Transmittance Related to Biodegradation of LDPE Film Incubated with Five Bacterial Samples Compared to Negative Control.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e717\u0026ndash;718 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(Rocking Deformation)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus pumilus\u003c/em\u003e strain FMA1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus aerius\u003c/em\u003e strain FMA2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e sp. (in: firmicutes) strain FMA3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e strain FMA4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain FMA5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRelation with Biodegradation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAny kind of change is a spectral signature of the physical and chemical breakdown of the main PE hydrocarbon chains during biodegradation. [16]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1248\u0026ndash;1375 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(CH\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eSymmetric Deformation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigher transmittance indicates a reduction in the number of these C-H bonds, meaning the polymer chains were breaking down during the incubation. [17] Whereas a lower transmittance suggests the formation of new functional groups, such as carbonyl (\u0026#119862;=\u0026#119874;) and hydroxyl (\u0026#119874;\u0026minus;\u0026#119867;) groups, which are often built as part of the oxidation and degradation.[18]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1463\u0026ndash;1468 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(CH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eBending Vibration)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigher transmittance is an indicator that the vibrations of the \u0026#119862;\u0026#119867;2 groups were changing. This happened because the overall crystallinity of the material was likely decreasing as it was broken down. Which is a spectroscopic sign that the LDPE was undergoing degradation. [19]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1650\u0026ndash;1800 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(Carbonyl Region)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigher transmittance means the decrease in the concentration of these specific functional groups, showing that the degradation was progressing beyond just initial oxidation and into the assimilation phase during the period of incubation. [20]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1710\u0026ndash;1750 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(C\u0026thinsp;=\u0026thinsp;O Peak)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThe observed changes signify that the bacteria were utilizing the oxidized portions of the LDPE polymer chains (specifically the carbonyl groups) as a carbon source for their metabolic activities during incubation. [21]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2849\u0026ndash;2851 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(CH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eSymmetric Stretching)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigher transmittance is a direct sign of polymer degradation. It means the \u0026#119862;\u0026#119867;2 bonds were breaking down. This could be a cause of microbial activity or other processes like oxidation that broke the polymer chains. On the other hand, lower transmittance is less common in simple biodegradation. It can indicate that the material was becoming more complex by incorporating other compounds that contain C-H bonds. For example, if the LDPE were blended with another material or if degradation was occurring in a way that led to the formation of new, smaller hydrocarbons that were still being measured by the peak. [22]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2917\u0026ndash;2920 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(CH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eAsymmetric Stretching)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLower transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigher transmittance at 2917\u0026ndash;2920 cm⁻\u0026sup1; is a key indicator of polymer degradation. It signifies the cleavage and breakdown of the main LDPE polymer backbone's C-H bonds, leading to shorter chains and the formation of new functional groups like carbonyl (C\u0026thinsp;=\u0026thinsp;O) and hydroxyl (O-H), which appear at different wavelengths. A Lower transmittance might indicate an error or it can suggest for a pretreatment of the LDPE otherwise the bacteria were unable to start oxidation within 1 month period. [23]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3200\u0026ndash;3600 cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(O-H Region)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHigher transmittance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThe bacterial activity was effectively altering or consuming the oxygen-containing functional groups on the LDPE surface as part of the biodegradation process.\u0026nbsp;[24]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePhylogenetic Tree analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe Phylogenetic tree was constructed using the Mega 12 software which shows the relationship among the genera and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn Bangladesh thousands of tons of solid waste generated across the country on a daily basis. Rapid urbanization and economic growth are responsible for this waste surge particularly in urban cities. Per capita municipal urban waste generation now stands around 0.5 Kg per day and is projected to more than double by 2041[25]. Organic wastes made up the majority of the municipal solid waste. Organic waste management is challenging because of being wet and heavy. Meanwhile the plastic waste has been rising with increased use of packaging and disposal products -around 14 to 15\u0026nbsp;million polyethylene bags are discarded after their first use every day in Dhaka [26]. There is a lack of proper landfills ( scientifically designed with engineered barriers, environment controls, and operational practices for safe isolation of solid wastes). For instance there are only two sprawling landfills dealing with the huge waste loads in Dhaka, therefore, rapidly running out of spaces. In addition, Dhaka is unequivocally one of the most densely populated large cities in the world, having limited land available for managing or treating the wastes. As a result, a significant portion of waste is managed through unrecorded and unsanitary open dumping. Therefore, a sustainable solution is required, and microbial biodegradation is such a process which would minimize the environmental impact by breaking down plastics into beneficial substances. Though there has been extensive research conducting microbial biodegradation of plastics including Low Density Polyethylene (LDPE) worldwide for the last few decades. But there is a lack of research that focuses on biodegradation in Bangladesh. Hence, the need for this study was warranted. Inevitably in our study, we chose to test on plastic samples collected from open dumpsites in Dhaka city.\u003c/p\u003e \u003cp\u003eAll the sample sites [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e] were highly concentrated with partially degraded plastic wastes which provided an ideal environment to collect and test the soil bacteria that were responsible for long-term degradation. While the isolated colonies exhibited notable morphological distinctions when observed without magnification, subsequent examination through gram staining revealed the presence of exclusively Gram-positive bacilli [Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]. Following incubation with untreated LDPE film as their sole carbon source, visible growth in the MSM media confirmed the successful degradation of LDPE by the inoculated bacteria [12]. Subsequently, the rate of degradation was measured through the most widely used primary methods like- calculation of dry weight loss of incubated LDPE films, analysis of surface morphology, and assessment of change in chemical structure.\u003c/p\u003e \u003cp\u003eCalculation of the percent weight loss of incubated LDPE is the fundamental step for assessing the extent of biodegradation. The variation in weight loss is influenced by several factors (i.e. type of both plastics and bacteria, the pre-treatment of the plastic, incubation time, environmental conditions etc.). Unsurprisingly, greater loss in dry weight is suggested to be evident with a longer incubation time along with high incubation temperature or pre-treatment [27], [28], [29]. A weight loss of 1.75% after 30 days[30] and 10.7% after 60 days [28] of incubation with \u003cem\u003eBacillus\u003c/em\u003e sp. were also reported in some studies. Another study showed 18.9% weight loss after 180 days of incubation with \u003cem\u003eBacillus pumilus\u003c/em\u003e [31], whereas our findings showed a minimum weight loss of 16.67\u0026plusmn;.06 % only after 30 days. To previous studies also demonstrated the LDPE degrading abilities of \u003cem\u003eBacillus cereus\u003c/em\u003e, reporting a 6.33%[13] and 20.28%[32] weight loss after 45 days and 112 days of incubation respectively. The variation in weight loss and incubation period in our study with these studies is monumental as we have observed 26.67\u0026plusmn;.06 % weight loss after only 30 day of incubation [Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. In our study, substantial loss in dry weights (16\u0026ndash;26%) were recorded of LDPE without any pre-treatment and only for one month of incubation. With the highest being from sample \u0026ldquo;4 (\u003cem\u003eBacillus cereus\u003c/em\u003e strain \u003cb\u003eFMA4\u003c/b\u003e )\u0026rdquo; while sample 1, 2, 3, and 5 demonstrated a near similar level of degradation. This indicates the significant biodegradation potential of \u003cem\u003eBacillus spp.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe structural changes occurring due to induced degradation is an important parameter for measuring the degree of degradation in a molecular level [33]. FTIR has been used in numerous degradation studies to determine the decrease in native bonds that provides evidence of the polymer being fragmented into shorter chains. Additionally, generation of new or loss of functional groups further indicates the degradation process [34]. In a complete biodegradation pathway, the polymer is oxidized by the oxygen in the air to form carbonyl groups which then forms carboxylic groups, ultimately, the intermediates undergo β-oxidation and enter the citric cycle which form CO\u003csub\u003e2\u003c/sub\u003e and H₂O [35]. Native bonds in polyethylene correspond to the following peaks: 2918 cm\u003csup\u003e-1\u003c/sup\u003e (CH\u003csub\u003e2\u003c/sub\u003e asymmetric stretching), 2851 cm\u003csup\u003e-1\u003c/sup\u003e (CH\u003csub\u003e2\u003c/sub\u003e symmetric stretching), 1468 cm\u003csup\u003e-1\u003c/sup\u003e (bending deformation), 1373 cm\u003csup\u003e-1\u003c/sup\u003e (CH\u003csub\u003e3\u003c/sub\u003e symmetric deformation) and 718 cm\u003csup\u003e-1\u003c/sup\u003e (rocking deformation). These peaks are seen in the FTIR for the control LDPE (NC) [Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e] [36]. In the FTIR spectroscopy of all LDPE samples incubated with bacterial colonies, there is both an increase and decrease in the percentage of transmittance at these peaks while comparing with the NC [Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e] indicating that less of that native bond is present. To specify the relationship of these changes along with biodegradation ability of each of the bacterial sample is provided in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A new peak around 1600 cm\u003csup\u003e-1\u003c/sup\u003e is seen for 2, 3, 4 and 5 that corresponds to absorbance of aromatic C\u0026thinsp;=\u0026thinsp;C bonds. Absorbance of this bond has been said to be indicative of biodegradation in previous studies [11].\u003c/p\u003e \u003cp\u003eThe further analysis includes the study of surface morphology. We did both optical microscopy and Scanning Electron Microscopy (SEM). Optical microscopy offered quick and easy overall conditions showing cracks, minute holes, and other disruptions along with bacterial adhesion on the surface of all the samples in comparison to the control [Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e]. This colonization indicates bacterial attack leading to biodegradation [33]. The photographs also showed the adhesion to be scattered and mostly concentrated around the fissures. These findings correspond with studies that suggest bacterial susceptibility in the amorphous region of the LDPE samples [34]. On the other hand SEM provided more detailed micro-features including cracks, disintegration, irregular surfaces, fissures, erosions [Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e] compared to control LDPE films characterized by smooth and uniform surface, no noticeable surface defects or mechanical failure [11]. All of these outcomes confirm the degradation capacity of the bacterial isolates [37].\u003c/p\u003e \u003cp\u003e16S rRNA gene phylogenetic analysis and BLAST based comparative homology analysis for 16S rRNA gene sequences (\u0026sim;1000 bp) of PE degrading isolates revealed that in our study all the five LDPE degraders belong to the phylum Firmicutes in class \u003cem\u003eBacillus.\u003c/em\u003e Phylogenetic analysis based on the Neighbor-Joining (NJ) tree, and Maximum Likelihood (ML) indicated that all isolates belonged to a monophyletic cluster that was distinct from the other polyethylene degrading bacteria known to \u003cem\u003ePseudomonas plecoglossicida\u003c/em\u003e, \u003cem\u003eRhodococcus pyridinivorans\u003c/em\u003e, \u003cem\u003eRhodococcus ruber\u003c/em\u003e, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, and \u003cem\u003eLysinibacillus xylanilyticus\u003c/em\u003e. It means that they have the same genetic composition, and also their independent evolution for polyethylene-degrading ability. In the \u003cem\u003eBacillus\u003c/em\u003e clade, \u003cem\u003eB. pumilus\u003c/em\u003e (FMA1), \u003cem\u003eB. aerius\u003c/em\u003e (FMA2) and \u003cem\u003eBacillus sp.\u003c/em\u003e (FMA3) were clustered in Clade A, with very low bootstrap value of 1% and branch length 0.003, and closer to \u003cem\u003eB. mexicanus\u003c/em\u003e and \u003cem\u003eB. licheniformis\u003c/em\u003e, both known as a polymer degrader. \u003cem\u003eB. atrophaeus\u003c/em\u003e (FMA5) was clustered to \u003cem\u003eB. halotolerans\u003c/em\u003e in Clade B having bootstrap values of 0.999% and branch length of 0.0025, whereas \u003cem\u003eLysinibacillus xylanilyticus\u003c/em\u003e grouped as a separate clade C with the \u003cem\u003eB. cereus\u003c/em\u003e (FMA4) having also boostrap value 0.999% and branch length 0.0438 showed functional convergence while being taxonomically divergent species among all samples investigated below sub-family. Other described PE-degrading genera, \u003cem\u003ePseudomonas plecoglossicida\u003c/em\u003e (Proteobacteria), \u003cem\u003eRhodococcus pyridinivorans\u003c/em\u003e and \u003cem\u003eR. ruber\u003c/em\u003e (Actinobacteria), and \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e (\u003cem\u003eFirmicutes\u003c/em\u003e) within distinct, consistently supported branches like the bootstrap value of 99.9 to 97.5% and branch length of 0.038 to 0.052. The high bootstrap values of \u0026gt;\u0026thinsp;0.98 and different branch lengths 0.002\u0026ndash;0.052 in this tree suggested that LDPE-degrading capability is phylogenetically stable for the bacterial isolates but has diverse origins relative to evolutionary origins. These results indicate that polyethylene degradation has emerged independently in several bacterial lineages as a result of convergent functional adaptation to withstand the high percentage of plastic wastes in our soil environment, which supports our previous results particularly the dry weight loss after incubation. Additionally, to the best of our knowledge, we are reporting \u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain \u003cb\u003eFMA5\u003c/b\u003e and B\u003cem\u003eacillus aerius\u003c/em\u003e strain \u003cb\u003eFMA2\u003c/b\u003e, as an LDPE degrading bacteria for the very first time. Based on our findings, it can be concluded that the \u003cem\u003eBacillus\u003c/em\u003e species exhibit a remarkable capacity to effectively biodegrade low-density polyethylene (LDPE). Notably, it is worth mentioning that the bacterial isolates utilized for our study that yielded insightful observations upon examination, were sourced from all the selected sampling sites.\u003c/p\u003e"},{"header":"6. Summary and Conclusion","content":"\u003cp\u003eThe findings of this study decisively demonstrate the inherent capacity of the soil bacteria isolated from open dumpsites in Dhaka, Bangladesh to initiate and sustain the degradation of low-density polyethylene (LDPE) films without the need for thermal or chemical pretreatment. The Quantitative analysis, evidenced by a significant percentage of dry weight loss over the short one-month incubation period (e.g., a mean loss of more than 20%), while most of the other studies were carried out for a longer duration established the bio-assimilation of LDPE constituents. This quantitative evidence was strongly corroborated by Fourier-Transform Infrared Spectroscopy (FTIR), which revealed characteristic chemical alterations on the polymer surface. The increase in the percentage of transmittance at several peaks (e.g., 2918 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2851 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1468 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1373 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 718 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) indicated the less presence of the native bonds. On the other hand the emergence and intensification of new bands within the carbonyl (C\u0026thinsp;=\u0026thinsp;O) stretching region (approx. 1715 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the hydroxyl (O-H) region (approx. 3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) confirm the initial oxidative steps of biodegradation, which are critical for increasing hydrophilicity and making the polymer backbone accessible to enzymatic attack.\u003c/p\u003e \u003cp\u003eThe visual analyses of surface topography provided compelling evidence for the physical and biological processes involved. Both the Optical microscopy and Scanning Electron Microscopy (SEM) images revealed profound structural changes, including the cracks, visible pitting, fissure development, and erosion marks indicative of microbial colonization and enzymatic activity. This physical alteration confirms the effective interaction between the bacteria and the hydrophobic LDPE surface. Furthermore, the phylogenetic analysis successfully identified the prevalence of the key plastic degrading species responsible (e.g., member of the \u003cem\u003eBacillus\u003c/em\u003e genera), thereby directly linking the observed degradation phenomena to specific, characterized bacterial strains. In addition the phylogenetic analysis identified two novel strains of \u003cem\u003eBacillus\u003c/em\u003e designated as FMA2 (B\u003cem\u003eacillus aerius) and\u003c/em\u003e FMA5 (\u003cem\u003eBacillus atrophaeus).\u003c/em\u003e Sequences of these strains were deposited in the NCBI GenBank Database (accession numbers provided in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Collectively, these results confirm a mechanism involving microbial adhesion, oxidative cleavage of the long alkane chains, and subsequent bio-assimilation of the resulting low-molecular-weight fragments.\u003c/p\u003e \u003cp\u003eIn summary, this research successfully established a clear link between the identified bacterial strains and quantitative, chemical, and morphological changes in the LDPE film, validating the hypothesis of direct, non-pretreated biodegradation potential. While the observed weight loss and spectral shifts strongly suggest polymer degradation, future studies must incorporate advanced techniques such as Gel Permeation Chromatography (GPC) to conclusively quantify the reduction in average molecular weight. Moving forward, the isolated strains represent a high-value candidate for optimizing bioremediation processes. Further research should focus on optimizing culture conditions, building a microbial consortia between the bacterial strains mentioned in the paper along with other highly degrading microorganisms, and identifying and isolating the specific depolymerase enzymes responsible for the observed oxidative cleavage to enhance the rate and efficiency of LDPE waste management to ensure the cleaning of the current mammoth load of plastic pollution not only in Bangladesh but also worldwide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests\u003c/h2\u003e \u003cp\u003e☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.A. conceptualized the study, designed the methodology, supervised the research, and provided project administration. M.F. and A.N.N. performed the experimental investigations, including the isolation of bacterial strains, 16S rRNA sequencing, and the incubation assays. D.K.P. conducted the formal data analysis and contributed significantly to the interpretation of the physical and chemical characterization results (FTIR and SEM). F.A. and M.F. wrote the main manuscript text. D.K.P. contributed to the writing, review, and editing of the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to Dr. Muhammad Rakibul Islam, Professor, Department of Physics, Bangladesh University of Engineering and Technology for the support in the FTIR analyses and also for opti-cal microscopy.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe metagenomic raw data have been deposited at NCBI GenBank (Accession no. : OR186211, OR186212, OR186213, OR186214, OR186215). All other data are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD. M. Wafaa, M. W. Sadik, H. F. Eissa, and K. Tonbol, \u0026ldquo;Biodegradation of low-density polyethylene LDPE by marine bacterial strains Gordonia alkanivorans PBM1 and PSW1 isolated from Mediterranean Sea, Alexandria, Egypt,\u0026rdquo; \u003cem\u003eSci Rep\u003c/em\u003e, vol. 15, no. 1, p. 16769, 2025, doi: 10.1038/s41598-025-96811-z.\u003c/li\u003e\n\u003cli\u003eI. Varyan \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;The Use of Natural Rubber as an Initiator of LDPE Biodegradation in Soil,\u0026rdquo; \u003cem\u003ePolymers (Basel)\u003c/em\u003e, vol. 17, no. 21, 2025, doi: 10.3390/polym17212885.\u003c/li\u003e\n\u003cli\u003eM. L. Oliveira, G. M. Miranda, and D. S. 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Ono, \u0026ldquo;Biodegradation of low-density polyethylene, polystyrene, polyvinyl chloride, and urea formaldehyde resin buried under soil for over 32 years,\u0026rdquo; \u003cem\u003eJ Appl Polym Sci\u003c/em\u003e, vol. 56, no. 13, pp. 1789\u0026ndash;1796, 1995, doi: https://doi.org/10.1002/app.1995.070561309.\u003c/li\u003e\n\u003cli\u003eJ. P. Harrison, C. Boardman, K. O\u0026rsquo;Callaghan, A.-M. Delort, and J. Song, \u0026ldquo;Biodegradability standards for carrier bags and plastic films in aquatic environments: a critical review,\u0026rdquo; \u003cem\u003eR Soc Open Sci\u003c/em\u003e, vol. 5, no. 5, p. 171792, May 2018, doi: 10.1098/rsos.171792.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biodegradation, LDPE, Untreated, Bacillus species, 16S rRNA, FTIR","lastPublishedDoi":"10.21203/rs.3.rs-9075950/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9075950/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe recalcitrant Low-Density Polyethylene (LDPE) is a multifaceted polymer. Therefore, it has slowly gained popularity for ease of access while simultaneously becoming a threat to nature. Investigation of diverse bacteria and exploring their natural ability to degrade LDPE has become a pressing matter in countries like Bangladesh, where thoughtless utilization and indiscriminate disposal of plastics are practiced. This study examined the potential of bacteria to degrade the untreated low-density polyethylene (LDPE). Bacteria were isolated from partially degraded plastic wastes collected from three open garbage dump sites in Dhaka, Bangladesh. Incubation of untreated plastic film inoculated with isolated bacteria resulted in significant weight loss (16%-26%). Both the SEM and Optical Microscope images at 2000x, and 1000x magnifications respectively, revealed surface topology modifications in forms of erosion, cracks, grooves, and minor holes. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the transformation of recalcitrant LDPE film.16S rRNA gene sequencing identified the potential bacteria as \u003cem\u003eBacillus pumilus\u003c/em\u003e strain FMA1, \u003cem\u003eBacillus aerius\u003c/em\u003e strain FMA2, \u003cem\u003eBacillus\u003c/em\u003e sp. (in: firmicutes) strain FMA3, \u003cem\u003eBacillus cereus\u003c/em\u003e strain FMA4, and \u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain FMA5. A significant finding was the detection of \u003cem\u003eBacillus aerius\u003c/em\u003e strain FMA2 and \u003cem\u003eBacillus atrophaeus\u003c/em\u003e strain FMA5, which have no previous LDPE degradation records. More importantly the \u003cem\u003eBacillus aerius\u003c/em\u003e strain FMA2 showed the best degradation potential among the five isolates. A Phylogenetic Tree constructed using the DNA sequence data signified the evolutionary relationship among the bacterial species. This research reveals the prevalence of \u003cem\u003eBacillus\u003c/em\u003e species as soil bacterium in Dhaka city along with their inherent biodegradation capabilities, emphasizing an eco-friendly management of emerging plastic pollution.\u003c/p\u003e","manuscriptTitle":"Rapid Biodegradation of Low-Density Polyethylene (LDPE) Without Pre-Treatment by Bacillus Strains Isolated from Garbage Dumpsites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:08:35","doi":"10.21203/rs.3.rs-9075950/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e52807b3-beb1-477e-a782-cc2653f80696","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T21:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:08:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9075950","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9075950","identity":"rs-9075950","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}