Biodegradation of HDPE by Desert Bacteria: Insights from Isolates of the Lut-Desert, the Hottest Place on Earth

Biodegradation of HDPE by Desert Bacteria: Insights from Isolates of the Lut-Desert, the Hottest Place on Earth | 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 Biodegradation of HDPE by Desert Bacteria: Insights from Isolates of the Lut-Desert, the Hottest Place on Earth Setareh Hakakzadeh, Abbas Akhavan Sepahi, Parvaneh Maghami, Mohammad Mehdi Motaghi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7003856/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 environmental impact of plastic pollution, specifically from high-density polyethylene (HDPE), is significant due to its resistance to degradation. In this study, bacteria isolated from the Lut Desert, one of the hottest places on Earth, were investigated for their potential to degrade HDPE. Beta-hemolytic strains were prioritized due to their association with extracellular enzyme production and biosurfactant activity, which enhances surface adhesion and biodegradation. According to the BATH assay, 10 strains showed high hydrophobicity (34.44–37.38%), which improved bacterial attachment to polyethylene surfaces. HDPE degradation was evaluated through weight loss over 60 days, with values ranging from 5–14%. Strains 48, 44, 8 and 50 demonstrated the highest degradation efficiency, reducing HDPE the weight by 14.15%, 12.99%, and 12.01%, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed that polyethylene biodegrades by producing alkanes, carboxylic acids, and alcohols as byproducts. The identification of laccase (cotA), alkane monooxygenase (alkB), and phosphatase (phoD) genes was confirmed through PCR amplification, which revealed the enzymes that regulate HDPE degradation. The combination of hydrophobicity, biosurfactant production, and enzyme activity underscores the potential of extremophilic bacteria as effective tools for polyethylene bioremediation. This study's findings provide valuable insights into plastic degradation caused by microbial activity, which provides promising solutions for managing plastic waste in extreme environmental conditions. Biodegradation GC-MS High-density polyethylene Lut Desert. Phylogenetic tree SEM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Over the past decade, global plastic production and consumption have risen sharply, driven by widespread industrial applications in packaging, construction, textiles, and consumer goods [ 1 ]. Among fossil-derived plastics, HDPE, low-density polyethylene (LDPE), polyethylene terephthalate (PET), polystyrene (PS), and polypropylene (PP) are widely employed, particularly in packaging, due to their lightweight and flexible nature [ 2 ]. Polyethylene (PE) is the most widely produced synthetic plastic, with global annual production exceeding 140 million tons [ 3 ]. Projections estimate that plastic waste will reach 11 billion tons by 2025 and 34 billion tons by 2050 [ 4 ]. The COVID-19 pandemic and other global health crises have led to a surge in single-use plastic consumption, which has resulted in an exacerbation of plastic pollution worldwide [ 5 ]. The widespread use and environmental persistence of synthetic polymers have resulted in critical pollution challenges [ 6 ]. PE, in particular, resists biodegradation due to its chemically stable carbon–carbon (C–C) and carbon–hydrogen (C–H) covalent bonds, high molecular weight, and hydrophobic surface [ 3 ]. Consequently, plastic waste accumulates in landfills, aquatic systems, and ecosystems, where it pollutes coastlines, obstructs waterways, and puts wildlife at risk through entanglement, ingestion, and suffocation. Furthermore, degradation products from plastics can bioaccumulate through trophic levels, potentially posing significant risks to human health and ecosystem integrity [ 7 ]. The current waste management strategies fail to address the plastic crisis adequately. Only 9% is recycled, 12% is open-air burning, and the vast majority (79%) is either landfilled or enters natural environments [ 4 ]. Landfilling demands significant space and can result in groundwater contamination and methane emissions [ 8 , 9 ]. Incineration, though widely practiced, releases toxic compounds such as CO₂ and dioxins, which are associated with respiratory illnesses and cancers [ 8 ]. Recycling techniques such as mechanical recycling are energy-intensive and generate secondary pollutants. Although chemical recycling yields high-quality outputs, it remains expensive and technically complex [ 10 ]. As a sustainable alternative, microbial biodegradation represents an economically viable and environmentally benign strategy that exploits native metabolic pathways to completely mineralize synthetic polymers without yielding toxic intermediates [ 11 ]. Through this process, polymers are converted into smaller monomers, which microorganisms can further metabolize. The degradation rate varies depending on polymer type and environmental conditions [ 12 ]. Microorganisms possess diverse enzymes, genes, and proteins that modify plastic structures and initiate depolymerization [ 13 ]. Recent studies have identified over 100 bacteria and fungi involved in the biodegradation of synthetic polymers. [ 14 ]. The process of microbial degradation is enzymatically mediated through extracellular that include cutinases, lipases, esterases, carboxylesterases, and oxygenases like monooxygenases and dioxygenases. Polymer hydrophilicity is modified by oxidative enzymes, which facilitate microbial colonization and release of additional degradative enzymes. Laccase has also been implicated in the degradation of polyethylene [ 13 ]. However, enzyme activity is sensitive to environmental conditions and may be inhibited by extremes in pH, suboptimal ranges of thermal regimes, hydrostatic stress, or chemical composition. Extremophilic microorganisms, organisms that Flourish in extreme physicochemical conditions, are promising candidates for plastic biodegradation [ 15 ]. These microbes are naturally existing in environments such as salt-saturated lakes, deep-sea hot springs, xeric environments, volcanic zones, ocean depths, spacecraft, and cleanrooms. They produce robust and stable enzymes that retain activity under extreme stress. [ 16 – 18 ]. Desert ecosystems are increasingly recognized as reservoirs of extremophiles with novel metabolic traits. The Lut Desert, exhibiting the highest recorded land surface temperatures, provides a unique environment for isolating microorganisms with exceptional plastic-degrading capabilities [ 19 , 20 ]. These extremophiles exhibit adaptive features such as specialized protein structures and efficient DNA repair mechanisms that enable them to survive and degrade polymers in extreme heat and aridity [ 21 ]. Enzymes such as laccases, alkane monooxygenases, and phosphatases produced by these microbes enhance polymer hydrophilicity and promote biofilm formation and degradation [ 22 – 24 ]. Laccase-producing microbes, in particular, have demonstrated effectiveness in catalyzing oxidative reactions involved in polymer degradation [ 25 ]. The objective of this study was to isolate and identify bacteria that degrade high-density polyethylene (HDPE) from the Lut Desert, Iran, and characterize their enzyme and metabolic potential. The broader goal was to develop sustainable microbial strategies for plastic waste mitigation in extreme environments. 2. Materials and methods 2.1 Procurement of HDPE Granules Granules of HDPE (tracking number 5030EA ON LN14540 P.S:21/14253) were sourced from Tabriz Petrochemical Company (Tabriz, Iran). The granules were processed as the primary substrate to isolate polyethylene-degrading bacteria. Following recommendations from standard biodegradation protocols, these granules were utilized without prior chemical modification or sterilization. 2.2. Sample Collection Soil samples were collected from 31 locations across the Lut Desert in Iran during May and September 2022. GPS coordinates were recorded and converted into standard geographic coordinates using the dgmap.ir program. Samples were collected from the 5–10 cm depth layer to minimize contamination from transient microorganisms. Samples were stored in sterile tubes under cryopreservation (–20°C) for transit to preclude microbial community alterations. "Additional data are given in Online Resource 1". 2.3. Isolation of Bacteria One gram of each soil sample was suspended in 99 mL sterile distilled water (1:99 w/v) and homogenized by continuous shaking (5 h) to reduce microbial load and disperse soil particles. Serial dilutions (10⁻¹ to 10⁻⁷) were prepared, and aliquots from the dilutions were spread-plated on Plate Count Agar (PCA) medium. The plates were incubated at 30°C for 24–48 hours. The identification of emerging colonies was based on morphological characteristics such as size, shape, edge morphology, and pigmentation. The purified isolates were maintained at 4°C for further physiological and biochemical characterization. 2.4. Pretreatment of HDPE Pretreatment methods were applied to HDPE to improve its biodegradability, following established protocols [ 11 , 26 ]. 2.5 Hemolysis Study on Blood Agar Medium Hemolytic activity was assessed by cultured isolates on blood agar (5% v/v sheep erythrocytes) with incubation at 37°C for 24 hours. Hemolysis patterns (α, β, or γ) were recorded [ 27 ]. Colonies exhibiting beta-hemolysis were isolated and selected for further analysis due to their pronounced hemolytic activity. [ 28 ]. 2.6 Hydrophobicity Assay The hydrophobicity assay was conducted to assess the bacterial adhesion potential to polyethylene surfaces following the methodologies of Rosenberg et al. and Hadar & Sivan, with slight modifications. [ 29 , 30 ]. Beta-hemolytic strains were initially grown in nutrient broth at 30°C for 16 hours of incubation. Following incubation, the cultures were centrifuged at 8000 rpm for 20 minutes to pellet the cells. They were subsequently washed twice with phosphate urea magnesium (PUM) buffer (pH 7.1). After the washing, bacterial cells were resuspended in the same buffer (1.2 ml), followed by the introduction of 0.2 ml n-hexadecane, and the mixture was briefly vortexed to ensure homogeneous dispersion. The suspension was incubated at 30°C for 20 minutes in a shaker incubator, followed by a 2-minute settling period at room temperature. The aqueous phase's absorbance was determined by measuring it at 400 nm. The hydrophobicity percentage was determined using the equation below: $$\:H\%=\frac{{A}_{o}-A}{{A}_{0}}$$ 1 , where \(\:{A}_{o}\) ​ and A represent the absorbance of the aqueous phase at 400 nm before and after mixing with n-hexadecane, respectively [ 31 ]. A reduction in absorbance, compared to a cell-free control, indicated the extent of bacterial cell adherence to the hydrophobic phase. 2.7 Isolation of HDPE-degrading Bacteria HDPE-degrading bacteria were isolated following established methods [ 32 ] with modifications to suit local environmental samples. Beta-hemolytic bacterial strains exhibiting the highest levels of hydrophobicity were selected. The mineral salt medium (MSM) was prepared with the following composition (per liter): K₂HPO₄ (1 g), KH₂PO₄ (0.2 g), NaCl (2 g), NH₄NO₃ (1 g), MgSO₄·7H₂O (0.5 mg), CaCl₂·2H₂O (0.002 mg), KCl (0.015 g), and ZnSO₄·7H₂O (0.001 g), with the pH adjusted to 7.5 using sodium bicarbonate. To facilitate interaction between bacterial cells and the HDPE surface, Tween 80 was added at a concentration of 1 ml per 1000 ml of medium [ 33 ]. HDPE plates were pretreated, cut into 0.5 × 1 cm pieces, sterilized in 70% ethanol, dried, weighed, and aseptically transferred into 100 ml of MSM. Bacterial inoculum was prepared by culturing the isolated strains in nutrient broth until the logarithmic growth phase. 2 ml of the bacterial suspension, adjusted to match a 0.5 McFarland standard (equivalent to approximately 1.5 × 10⁸ CFU/ml), was introduced into each flask [ 34 ]. The culture flasks underwent 60 days of continuous shaking (180 rpm) at 30°C in a temperature-controlled orbital shaker incubator. 2.8 Estimation of HDPE Degradation After the 60-day incubation period, HDPE films were aseptically retrieved from the mineral salt medium and thoroughly cleansed to remove microbial biomass and extracellular polymers. The films were first treated with 2% (v/v) aqueous sodium dodecyl sulfate (SDS) solution for 15 minutes at room temperature (25 ± 2°C) with gentle agitation, followed by three sequential 10-minute washes with sterile distilled water to ensure complete removal of detergent residues. The purified films were oven-dried at 45°C for 24 hours until they reached constant weight. Pre- and post-incubation weights were precisely measured using an analytical microbalance (± 0.01 mg precision) for subsequent weight loss was calculated using the following equation: $$\:Percentage\:of\:weight\:loss\:=\frac{Initial\:weight\:-\:Final\:weight\:}{Initial\:weight\:}\times\:100$$ 2 This method follows the weight loss calculation approach outlined in previous studies evaluating the biodegradation of HDPE [ 32 ]. 2.9 Scanning Electron Microscopy (SEM) of Polyethylene Surfaces To characterize bacterial-induced morphological alterations on the PE surface, PE films exposed to bacteria for 60 days were freeze-dried to maintain surface integrity. Following that, SEM was used to analyze the samples. 2.10 Molecular Characterization of Bacterial Strains Based on the analysis of bacterial growth, hemolytic activity, hydrophobicity, and weight loss, genomic DNA was isolated from 10 selected bacterial strains out of the 50 initially obtained. Genomic DNA was extracted using the saturated phenol method. [ 35 ]. Specific primers were designed and synthesized to target genes involved in polyethylene degradation, namely laccase, alkaline monooxygenase, and phosphatase. Target gene amplification, including that of the 16S rRNA gene, was performed using Polymerase Chain Reaction (PCR). Universal primers 27F (5′-AGAGTTTGATCCGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were employed for 16S rRNA amplification. PCR reactions were conducted in a thermocycler under conditions optimized for each gene. [ 33 ]. Amplification success was confirmed by 2% agarose gel electrophoresis, followed by Sanger sequencing of representative amplicons for precise gene identification [ 36 ]. The sequencing data was analyzed using Chromas Lite software for quality control and editing. Subsequently, the sequences were compared to the NCBI database using BLAST to assess their similarity to known bacterial strains. Phylogenetic analysis was performed using the neighbor-joining method implemented in MEGA7 [ 37 ]. 2.11 GC-MS Analysis GC-MS analysis revealed HDPE degradation products after after 60 days of microbial treatment. Culture supernatants were filtered to remove bacterial cells and particulate matter and extracted with toluene. The organic phase containing the degradation products was concentrated by a rotary evaporator under reduced pressure. The concentrated extracts were subjected to analysis using GC-MS. Standard operating conditions for the GC-MS analysis were employed, with mass spectra obtained and compared against the National Institute of Standards and Technology (NIST) mass spectral library [ 38 ]. 3. Results 3.1 Bacterial Isolation and Characterization The Lut Desert is renowned for its extreme environmental conditions, such as high surface temperatures, as recorded by NASA. Despite this, the region supports a diverse array of heat- and drought-resistant bacteria [ 19 ]. Fifty bacterial strains were isolated and purified by analyzing samples from 31 geographically distinct locations across the desert. All isolates were characterized through comprehensive morphological and biochemical profiling, including Gram staining, catalase/oxidase/urease/hemolytic activity, motility tests, and starch hydrolysis, among other tests, revealing distinct phenotypic patterns among strains. These assays provide insights into the metabolic potential of the strains, particularly their role in polyethylene degradation. "Additional data are given in Online Resource 2" 3.2 Culture in Blood Agar Medium Among the 50 bacterial strains isolated and cultured on blood agar medium, 18 isolates demonstrated alpha hemolysis, 22 exhibited beta hemolysis, and 12 showed gamma hemolysis. Strains exhibiting beta hemolysis progressed to hydrophobicity testing due to their potential for enhanced biofilm formation and biodegradation activity. 3.3 Bacterial Cell Surface Hydrophobicity The hydrophobic nature of the bacterial cell surface significantly contributes to its ability to adhere to hydrophobic polymers like HDPE. This characteristic was measured by performing the Bacterial Adhesion to Hydrocarbons (BATH) assay on beta-hemolytic strains (Fig. 1 ). The percentages of hydrophobicity among the strains ranged from 16.33% (strain 21) to 37.38% (strain 50). Strains with higher hydrophobicity, such as strain 50 (37.38%), strain 44 (34.44%), strain 8 (32.26%), and strain 48 (31.67%), demonstrated enhanced capacity to adhere to hydrophobic surfaces, such as HDPE. Ten strains that had the highest hydrophobicity values were chosen for subsequent analyses. These findings are consistent with previous studies, which reported that bacteria with hydrophobic cell surfaces exhibit better efficiency in degrading hydrophobic polymers such as HDPE [ 39 ] . 3.4 HDPE Weight Loss The degradability of the 10 bacterial strains with the highest hydrophobicity was quantitatively evaluated through gravimetric analysis of HDPE films following 60 days of incubation in the mineral salt medium under optimized conditions. The percentage of HDPE weight loss ranged from 5–14%, with strains 48, 44, 8, and 50 demonstrating the highest degradation activities, achieving weight losses of 14.15%, 12.99%, 12.99%, and 12.01%, respectively (Fig. 2 ). These findings highlight the capability of these bacterial strains to degrade HDPE under the specified experimental conditions. 3.5 Surface Morphology Analysis by SEM To investigate the structural changes on HDPE surfaces, SEM was performed on HDPE films exposed to bacterial treatments. SEM images showed bacterial adhesion and colonization on treated HDPE films compared to untreated films (Fig. 3). 3.6 Molecular Characterization and 16S rRNA Gene Sequencing Ten beta-hemolytic strains were selected for molecular characterization, including genomic DNA extraction and 16S rRNA gene sequencing. This recognized method of bacterial taxonomy elucidates the genetic determinants of the strains' polyethylene-degrading capacity, facilitating their application in bioremediation and plastic waste management. Agarose gel electrophoresis was employed to evaluate the integrity and purity of the extracted DNA, which revealed distinct and high-quality bands. PCR amplification was conducted to detect genes associated with polyethylene degradation, including laccase ( cotA ), alkane monooxygenase ( alkB ), and phosphatase ( phoD ), based on methods described by Smith et al. [ 40 ] and Xiao et al. [ 41 ]. The PCR products were visualized through electrophoresis, yielding distinct bands at 1200 bp for the phosphatase gene, 600 bp for the laccase gene, and 700 bp for the alkane monooxygenase gene. The positive control was Bacillus subtilis, and the negative control consisted of a PCR without template (primers only). (Fig. 4). “The original uncropped gel image corresponding to Fig. 4 is provided in Online Resource 3”. The presence of all three genes in eight strains highlighted their strong potential for polyethylene degradation. Among these, the five most promising strains, as determined by gene amplification results, underwent 16S rRNA sequencing for species identification. BLAST analysis indicated that there is significant homology between established bacterial species. The generated sequences were submitted to GenBank under accession numbers PV425917, PV425446, and PV606519 (Figs. 5 a- 5 b). These results underscore the significant biodegradation potential of these strains and their genetic capacity for contributing to environmental plastic degradation efforts. 3.7 GC-MS Profiling of Plastic Degradation Compounds GC-MS analysis revealed metabolic by-products of HDPE degradation. The major degradation products discovered were alkanes and carboxylic acids, as depicted in Table 1 . The corresponding chromatograms are presented in Fig. 6. Detected compounds such as hexanoic acid, oleic acid, dodecane, and octadecane indicate the microbial breakdown of HDPE into simpler organic molecules. Carboxylic acid, including hexadecanoic acid, suggests the oxidative cleavage of polyethylene, while alkanes such as hexadecane, heptadecane, and octadecane are consistent with the hydrocarbon backbone of HDPE are metabolized by bacterial enzymes. The findings are consistent with the well-known biodegradation pathways for plastic polymers [ 22 , 42 ]. These findings are consistent with established pathways for plastic biodegradation, underscoring the capability of the isolated strains to transform polyethylene into simpler compounds. Table 1 Major degradation products identified by GC-MS during HDPE biodegradation by selected bacterial strains. A checkmark (✓) indicates the presence of the compound in GC-MS profiles from the respective strain. S.no Compound name Strains 44 Strain 50 Strain 8 Strain 48 1 Dodecane ✓ ✓ 2 Hexacosane ✓ 3 Eicosane ✓ ✓ 4 Decane ✓ ✓ ✓ ✓ 5 Hexadecane ✓ ✓ 6 Phthalic acid ✓ ✓ 7 Heptadecane ✓ 8 Benzaldehyde ✓ ✓ 9 Tetradecane ✓ ✓ 10 Octadecane ✓ ✓ ✓ 11 Undecane ✓ 12 Nonahexacontanoic acid ✓ 13 Methoxyacetic acid ✓ 14 Hexadecadiene ✓ 15 Heptadecanone ✓ 16 Cyclotetradecane ✓ 17 Pentadecane ✓ 4. Discussion and Research Perspectives The global prevalence of plastics, particularly HDPE, is due to their durability, versatility, and cost-effectiveness. These beneficial material properties promote environmental persistence and accumulation. This study aimed to characterize polyethylene-degrading bacteria isolated from Iran's Lut Desert, one of Earth's most extreme hyper-arid ecosystems. The extremophilic nature of these bacteria suggests that they produce heat-stable enzymes that can catalyze the breakdown of HDPE [ 43 ]. Our findings confirm that these desert bacteria can degrade HDPE, as evidenced by enhanced biofilm formation, bacterial adhesion, and the production of degradation by-products identified by GC-MS. Identification of terminal oxidation products (carboxylic acids, alcohols) and intermediate metabolites (ketones, esters) demonstrates progressive HDPE depolymerization. The degradation potential of Bacillus, Staphylococcus, and Pseudomonas species is similar to that shown in previous research [ 44 ]. Among studied polyethylene-degrading actinomycetes, Rhodococcus strains consistently demonstrate superior performance with pre-treated substrates [ 45 ]. Most existing studies on PE degradation are limited to mesophilic soil bacteria, overlooking other microbial sources [ 46 , 47 ], composting agricultural [ 48 ], hot springs [ 49 ] and landfills [ 50 ]. This study is unique in showing that desert isolates can degrade HDPE in laboratory conditions. Our research provides new insights into the degradation of HDPE by extremophiles from arid desert environments, addressing a critical gap in bioremediation studies. 4.1 Challenges and Future Research Directions Despite the promising results, the rate of polyethylene degradation is still slow, reporting a rate of only 4% after 90 days under optimal conditions (Gupta et al., 2023). These findings underscore the critical need to systematically explore underexplored extremophilic ecosystems for novel biocatalysts with enhanced plastic degradation efficiency under industrially relevant conditions. Polyethylene degradation occurs in three stages: bio-deterioration, bio-fragmentation, and mineralization [ 51 ]. Our study supports this understanding by demonstrating biofilm formation and the presence of intermediate degradation products through GC-MS analysis. 4.2 Enzymatic Activity and Genetic Potential of Desert Isolates Extremophilic bacteria, such as those isolated in our study, produce stable enzymes under extreme environmental conditions [ 52 , 53 ]. We identified genes encoding laccase, alkane monooxygenase, and phosphatase in the desert bacterial isolates. Laccase's four-copper active site enables the oxidation of recalcitrant pollutants through radical-mediated mechanisms, offering unique advantages for bioremediation applications [ 25 ]. Oxygenases, such as alkane monooxygenase, can enhance the hydrophilicity of polyethylene polymers, promoting bacterial adhesion and facilitating biodegradation. The discovery of phosphatase genes in these bacteria complements the oxidative capabilities of this strain and indicates dual bioremediation potential for contaminated sites containing plastic and heavy metals. Our study focused mainly on oxidative enzymes, but phosphatases are also a significant bioremediation tool, specifically for heavy metal detoxification through phosphate-mediated precipitation and mineralization. [ 54 ]. Future work must characterize whether the observed phosphatase activity synergizes with oxidative degradation pathways, potentially enabling concurrent HDPE depolymerization and metal ion precipitation (as metal-phosphate complexes). 4.3 Future Prospects The presence of genes encoding laccase, alkane monooxygenase, and phosphatase in the isolates highlights their potential for future research in microbial plastic degradation and environmental bioremediation. However, one major challenge lies in cultivating extremophilic bacteria in laboratory settings, as many of these organisms remain unculturable. Novel biocatalysts with significant potential for polyethylene degradation may be present in these unculturable species. Microbial biodegradation represents a sustainable pathway toward circular plastic economies by valorizing waste polymers into reusable metabolites while decreasing petrochemical dependence and ecological footprints [ 55 , 56 ]. However, a critical aspect of future research must focus on the environmental impact of degradation products, such as alkanes and carboxylic acids. While these intermediates naturally occur during biodegradation, their accumulation requires stringent monitoring to prevent ecotoxicological effects. 5. Conclusion The biodegradation potential of extremophilic bacteria isolated from the Lut Desert is highlighted in this study to combat HDPE pollution. By identifying bacterial strains equipped with genes encoding laccase, alkane monooxygenase, and phosphatase enzymes, we demonstrated their ability to degrade HDPE. The GC-MS analysis confirmed that these bacteria were responsible for the metabolic breakdown of HDPE, with the detection of essential degradation intermediates like alkanes and carboxylic acids. The discovery of phosphatases has paved the way for their use in other areas besides plastic degradation, such as bioremediation of heavy metals and pesticides. Exploring desert extremophiles increases our understanding of plastic degradation under harsh conditions, highlighting their importance in addressing plastic waste globally. A critical research priority involves systematically optimizing culture parameters (aeration, C: N ratio, trace elements) to enhance biomass yield and enzyme titers from these extremophilic isolates. In addition, studying microbial communities from other extreme environments could lead to more effective degraders that could accelerate the breakdown of polyethylene and other persistent pollutants. The use of extremophilic bacteria in environmental management strategies can offer long-term and innovative solutions to global issues, such as managing waste and restoring ecosystems. In this work, the value of extremophiles as potential candidates for future biotechnology applications in pollution mitigation and environmental recovery is underlined, with the potential to deal with plastic pollution and other environmental pollutant. Declarations Author Contribution A. A. conceptualized the study, helped with material preparation, and supervised and assisted with drafting the manuscript. S. H. prepared the materials, provided the results, wrote the manuscript, and prepared figures and tables. P. M. and M. M. provided manuscript review and writing assistance. A. A., P. M., and M. M. supervised the project and provided overall guidance. All authors reviewed and approved the final version of the manuscript. References Newrick, B.A., et al., Enhanced biodegradation of high-density polyethylene microplastics: Study of bacterial efficiency and process parameters. Journal of Hazardous Materials, 2025. 485 : p. 136822. Salwa, H., et al., Green bio composites for food packaging. Int. J. Recent Technol. Eng, 2019. 8 (2): p. 450-459. Baldera-Moreno, Y., et al., Biotechnological aspects and mathematical modeling of the biodegradation of plastics under controlled conditions. Polymers, 2022. 14 (3): p. 375. Shilpa, N. Basak, and S.S. Meena, Microbial biodegradation of plastics: Challenges, opportunities, and a critical perspective. Frontiers of environmental science & engineering, 2022. 16 (12): p. 161. Parashar, N. and S. Hait, Plastics in the time of COVID-19 pandemic: protector or polluter? Science of the Total Environment, 2021. 759 : p. 144274. Cowger, W., et al., Global producer responsibility for plastic pollution. Science Advances, 2024. 10 (17): p. eadj8275. Adekanmbi, A.O., et al., Assessing the environmental and health impacts of plastic production and recycling. World Journal of Biology Pharmacy and Health Sciences, 2024. 17 (2): p. 232-241. Asiandu, A.P., A. Wahyudi, and S.W. Sari, A review: plastics waste biodegradation using plastics-degrading bacteria. Journal of Environmental Treatment Techniques, 2021. 9 (1): p. 148-157. Ong, G.H., et al., Screening of Native Fungi For Biodegradation of High-Density Polyethylene (HDPE) Plastic in Mangroves Ecosystem. Malaysian Applied Biology, 2024. 53 (6): p. 97-103. Knickmeyer, D., Social factors influencing household waste separation: A literature review on good practices to improve the recycling performance of urban areas. Journal of cleaner production, 2020. 245 : p. 118605. Crystal Thew, X.E., et al., Enhancing plastic biodegradation process: strategies and opportunities. Critical reviews in biotechnology, 2024. 44 (3): p. 477-494. Hussain, A., et al., Low and high-density polyethylene and expanded polystyrene biodegradation by the greater wax moth Galleria mellonella L reveals a key role of the gut microbiome. Ecotoxicology and Environmental Safety, 2025. 294 : p. 118074. Zhou, Y., et al., Challenges and opportunities in bioremediation of micro-nano plastics: A review. Science of the Total Environment, 2022. 802 : p. 149823. Zhang, Y., et al., Biodegradation of polyethylene and polystyrene: From microbial deterioration to enzyme discovery. Biotechnology Advances, 2022. 60 : p. 107991. Mutlu-Ingok, A., et al., Applications of extremozymes in the food industry. Microbial Extremozymes, 2022: p. 197-206. Schröder, C., C. Burkhardt, and G. Antranikian, What we learn from extremophiles. ChemTexts, 2020. 6 : p. 1-6. Chia, X.K., et al., Role of extremophiles in biodegradation of emerging pollutants. Topics in Catalysis, 2024: p. 1-18. Margesin, R. and F. Schinner, Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles, 2001. 5 (2): p. 73-83. Shirsalimian, M.S., et al., Isolation of a mesophilic and halotolerant strain of Kocuriapolaris from Gandom Beryan area in the Lut Desert of Iran, moderately resistant to gamma radiation and desiccation. Biosciences Biotechnology Research Asia, 2016. 13 (4): p. 2343-2350. Shahsavari, N., F. Kafilzadeh, and M. Kargar, Isolation and identification of thermophiles bacteria from one of the hottest places on the planet (Lut Desert, Iran) and measuring their enzyme activities. Geomicrobiology Journal, 2021. 38 (10): p. 850-858. Atanasova, N., et al., Plastic degradation by extremophilic bacteria. International Journal of Molecular Sciences, 2021. 22 (11): p. 5610. Mohanan, N., et al., Microbial and enzymatic degradation of synthetic plastics. Frontiers in Microbiology, 2020. 11 : p. 580709. Chu, Y.-H., et al., Purification and characterization of alkaline phosphatase from lactic acid bacteria. RSC advances, 2019. 9 (1): p. 354-360. Hou, L. and E.L.-W. Majumder, Potential for and distribution of enzymatic biodegradation of polystyrene by environmental microorganisms. Materials, 2021. 14 (3): p. 503. Shome, R., Role of microbial enzymes in Bioremediation. ELifePress, 2020. 1 (1): p. 15-20. Balasubramanian, V., et al., Enhancement of in vitro high-density polyethylene (HDPE) degradation by physical, chemical, and biological treatments. Environmental Science and Pollution Research, 2014. 21 : p. 12549-12562. Buxton, R., Blood agar plates and hemolysis protocols. American Society for Microbiology, 2005. 15 : p. 1-9. Bamba, T., et al., High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar. Biology Methods and Protocols, 2024. 9 (1): p. bpae044. Rosenberg, M., D. Gutnick, and E. Rosenberg, Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol lett, 1980. 9 (1): p. 29-33. Hadar, Y. and A. Sivan, Colonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber. Applied microbiology and biotechnology, 2004. 65 : p. 97-104. Vinderola, C., M. Medici, and G. Perdigon, Relationship between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell wall protein profiles in indigenous and exogenous bacteria. Journal of Applied Microbiology, 2004. 96 (2): p. 230-243. Gupta, K.K., et al., Degradation of high density polyethylene (HDPE) through bacterial strain from Cow faeces. Biocatalysis and Agricultural Biotechnology, 2023. 48 : p. 102646. Montazer, Z., M.B. Habibi Najafi, and D.B. Levin, In vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella. Canadian Journal of Microbiology, 2021. 67 (3): p. 249-258. Andrews, J.M., Determination of minimum inhibitory concentrations. Journal of antimicrobial Chemotherapy, 2001. 48 (suppl_1): p. 5-16. Sambrook, J. and D.W. Russell, Purification of nucleic acids by extraction with phenol: chloroform. Cold Spring Harbor Protocols, 2006. 2006 (1): p. pdb. prot4455. Xie, M., et al., Comparative impacts of polyethylene and biodegradable film residues on soil microbial communities and rapeseed performance under field conditions. Frontiers in Microbiology, 2025. 16 : p. 1553807. Kumar, S., G. Stecher, and K. Tamura, MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 2016. 33 (7): p. 1870-1874. Farzi, A., et al., Biodegradation of high density polyethylene using Streptomyces species. J. Coast. Life Med, 2017. 5 (11): p. 474-479. Harshvardhan, K. and B. Jha, Biodegradation of low-density polyethylene by marine bacteria from pelagic waters, Arabian Sea, India. Marine pollution bulletin, 2013. 77 (1-2): p. 100-106. Smith, C.B., et al., Alkane hydroxylase gene (alkB) phylotype composition and diversity in northern Gulf of Mexico bacterioplankton. Frontiers in Microbiology, 2013. 4 : p. 370. Xiao, Y., et al., An alkaline thermostable laccase from termite gut associated strain of Bacillus stratosphericus. International Journal of Biological Macromolecules, 2021. 179 : p. 270-278. Shah, A.A., et al., Biological degradation of plastics: a comprehensive review. Biotechnology advances, 2008. 26 (3): p. 246-265. Atalah, J. and J.M. Blamey, Isolation and characterization of a novel laccase from an Antarctic thermophilic Geobacillus. Antarctic Science, 2022. 34 (4): p. 289-297. Kumar, S., A. Hatha, and K. Christi, Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags. Revista de biología Tropical, 2007. 55 (3-4): p. 777-786. Fontanella, S., et al., Comparison of the biodegradability of various polyethylene films containing pro-oxidant additives. Polymer degradation and stability, 2010. 95 (6): p. 1011-1021. Koutny, M., et al., Soil bacterial strains able to grow on the surface of oxidized polyethylene film containing prooxidant additives. International Biodeterioration & Biodegradation, 2009. 63 (3): p. 354-357. Jeon, H.J. and M.N. Kim, Degradation of linear low density polyethylene (LLDPE) exposed to UV-irradiation. European polymer journal, 2014. 52 : p. 146-153. Dang, T.C.H., et al., Plastic degradation by thermophilic Bacillus sp. BCBT21 isolated from composting agricultural residual in Vietnam. Advances in Natural Sciences: Nanoscience and Nanotechnology, 2018. 9 (1): p. 015014. Baltaci, M.O., et al., Isolation and characterization of thermophilic bacteria from geothermal areas in Turkey and preliminary research on biotechnologically important enzyme production. Geomicrobiology journal, 2017. 34 (1): p. 53-62. Gumbi, A., et al., Isolation and characterization of pseudomonas aeruginosa and brevibacillus species and their potential to biodegrade polyethylene material. Science World Journal, 2019. 14 (4): p. 57-61. Gu, J.-D., Biodegradability of plastics: the issues, recent advances, and future perspectives. Environmental Science and Pollution Research, 2021. 28 (2): p. 1278-1282. Gunjal Aparna, B., S. WaghmodeMeghmala, and N. Patil Neha, Role of extremozymes in bioremediation. Research Journal of Biotechnology, 2021. 16 : p. 3. Mesbah, N.M., Industrial biotechnology based on enzymes from extreme environments. Frontiers in Bioengineering and Biotechnology, 2022. 10 : p. 870083. Yu, X., et al., Heavy metals remediation through bio-solidification: Potential application in environmental geotechnics. Ecotoxicology and Environmental Safety, 2023. 263 : p. 115305. Garcia-Garcia, G., et al., Environmental impact of different scenarios for the pyrolysis of contaminated mixed plastic waste. Green Chemistry, 2024. 26 (7): p. 3853-3862. Tan, A.F., et al., Reimagining plastics waste as energy solutions: challenges and opportunities. npj Materials Sustainability, 2024. 2 (1): p. 2. Additional Declarations No competing interests reported. Supplementary Files OnlineResource1.docx OnlineResource2.docx OnlineResource3UncroppedGelpdf.pdf 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. 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1","display":"","copyAsset":false,"role":"figure","size":50404,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of hydrophobicity of 22 selected strains.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/ec7449cf7e3cd2468af664b0.png"},{"id":92501655,"identity":"ddd74be9-fcde-4ea4-b5e0-fe8bcbeab227","added_by":"auto","created_at":"2025-09-30 11:33:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36755,"visible":true,"origin":"","legend":"\u003cp\u003eGravimetric analysis of HDPE film degradation by selected bacterial isolates after 60 days of incubation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/954d71899023c24f22c94545.png"},{"id":92501707,"identity":"411f8392-9087-4740-9b73-932a1c3a37b4","added_by":"auto","created_at":"2025-09-30 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(700 bp) genes. Lanes 1–3 (from the right) correspond to phosphatase; lanes 5–7 correspond to laccase; lanes 4-8 contain the molecular weight marker; and lanes 9–11 correspond to alkane monooxygenase. For each gene, the first lane contains the bacterial sample, the second is the negative control (no DNA template), and the third is the positive control (Bacillus subtilis).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/2afe3ce99a82e30bce25e59f.png"},{"id":92501674,"identity":"24e48646-9188-4bb2-bc54-c9527e77d726","added_by":"auto","created_at":"2025-09-30 11:33:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":226061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-b)\u003c/strong\u003e Maximum-likelihood phylogenetic trees of HDPE-degrading isolates based on 16S rRNA gene sequences. (a) Bacillus spp. (b) Streptomyces sp.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/ecf6df7346c2b1b3c79ae8df.png"},{"id":92501671,"identity":"43ee2c12-395a-4af8-b35d-b1328d5f1a69","added_by":"auto","created_at":"2025-09-30 11:33:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":192449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-e) \u003c/strong\u003eGC-MS chromatograms of HDPE degradation by bacterial strains isolated from the Lut Desert. \u003cstrong\u003e(a)\u003c/strong\u003estrain 44, \u003cstrong\u003e(b)\u003c/strong\u003e strain 50, \u003cstrong\u003e(c)\u003c/strong\u003e strain 48, (\u003cstrong\u003ed\u003c/strong\u003e) strain 8, and (\u003cstrong\u003ee\u003c/strong\u003e) the abiotic control (no bacterial treatment).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/37eb694af397fa66de681a9e.png"},{"id":96245266,"identity":"3f934669-20ac-444e-9b30-a5b0d2c24e38","added_by":"auto","created_at":"2025-11-19 07:20:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1765469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/debea25b-1220-4d5b-b969-181409cc34c0.pdf"},{"id":92501680,"identity":"cefa43ed-49d0-48e8-a472-4ff342a6b4db","added_by":"auto","created_at":"2025-09-30 11:33:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17247,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/4a38569b7408817e86b12305.docx"},{"id":92501722,"identity":"3a116be1-a60f-4329-8d42-5dc5160f2220","added_by":"auto","created_at":"2025-09-30 11:33:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31613,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/42040ab9bffd5a052881c3e0.docx"},{"id":92501685,"identity":"ba01b31a-4358-4671-93c9-bc259e46cbf7","added_by":"auto","created_at":"2025-09-30 11:33:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":181403,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource3UncroppedGelpdf.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7003856/v1/17b03a91e041cbe53a233e8d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodegradation of HDPE by Desert Bacteria: Insights from Isolates of the Lut-Desert, the Hottest Place on Earth","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the past decade, global plastic production and consumption have risen sharply, driven by widespread industrial applications in packaging, construction, textiles, and consumer goods [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among fossil-derived plastics, HDPE, low-density polyethylene (LDPE), polyethylene terephthalate (PET), polystyrene (PS), and polypropylene (PP) are widely employed, particularly in packaging, due to their lightweight and flexible nature [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePolyethylene (PE) is the most widely produced synthetic plastic, with global annual production exceeding 140\u0026nbsp;million tons [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Projections estimate that plastic waste will reach 11\u0026nbsp;billion tons by 2025 and 34\u0026nbsp;billion tons by 2050 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The COVID-19 pandemic and other global health crises have led to a surge in single-use plastic consumption, which has resulted in an exacerbation of plastic pollution worldwide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe widespread use and environmental persistence of synthetic polymers have resulted in critical pollution challenges [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. PE, in particular, resists biodegradation due to its chemically stable carbon\u0026ndash;carbon (C\u0026ndash;C) and carbon\u0026ndash;hydrogen (C\u0026ndash;H) covalent bonds, high molecular weight, and hydrophobic surface [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, plastic waste accumulates in landfills, aquatic systems, and ecosystems, where it pollutes coastlines, obstructs waterways, and puts wildlife at risk through entanglement, ingestion, and suffocation. Furthermore, degradation products from plastics can bioaccumulate through trophic levels, potentially posing significant risks to human health and ecosystem integrity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe current waste management strategies fail to address the plastic crisis adequately. Only 9% is recycled, 12% is open-air burning, and the vast majority (79%) is either landfilled or enters natural environments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Landfilling demands significant space and can result in groundwater contamination and methane emissions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Incineration, though widely practiced, releases toxic compounds such as CO₂ and dioxins, which are associated with respiratory illnesses and cancers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recycling techniques such as mechanical recycling are energy-intensive and generate secondary pollutants. Although chemical recycling yields high-quality outputs, it remains expensive and technically complex [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a sustainable alternative, microbial biodegradation represents an economically viable and environmentally benign strategy that exploits native metabolic pathways to completely mineralize synthetic polymers without yielding toxic intermediates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Through this process, polymers are converted into smaller monomers, which microorganisms can further metabolize. The degradation rate varies depending on polymer type and environmental conditions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Microorganisms possess diverse enzymes, genes, and proteins that modify plastic structures and initiate depolymerization [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies have identified over 100 bacteria and fungi involved in the biodegradation of synthetic polymers. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe process of microbial degradation is enzymatically mediated through extracellular that include cutinases, lipases, esterases, carboxylesterases, and oxygenases like monooxygenases and dioxygenases. Polymer hydrophilicity is modified by oxidative enzymes, which facilitate microbial colonization and release of additional degradative enzymes. Laccase has also been implicated in the degradation of polyethylene [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, enzyme activity is sensitive to environmental conditions and may be inhibited by extremes in pH, suboptimal ranges of thermal regimes, hydrostatic stress, or chemical composition. Extremophilic microorganisms, organisms that Flourish in extreme physicochemical conditions, are promising candidates for plastic biodegradation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These microbes are naturally existing in environments such as salt-saturated lakes, deep-sea hot springs, xeric environments, volcanic zones, ocean depths, spacecraft, and cleanrooms. They produce robust and stable enzymes that retain activity under extreme stress. [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDesert ecosystems are increasingly recognized as reservoirs of extremophiles with novel metabolic traits. The Lut Desert, exhibiting the highest recorded land surface temperatures, provides a unique environment for isolating microorganisms with exceptional plastic-degrading capabilities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These extremophiles exhibit adaptive features such as specialized protein structures and efficient DNA repair mechanisms that enable them to survive and degrade polymers in extreme heat and aridity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Enzymes such as laccases, alkane monooxygenases, and phosphatases produced by these microbes enhance polymer hydrophilicity and promote biofilm formation and degradation [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Laccase-producing microbes, in particular, have demonstrated effectiveness in catalyzing oxidative reactions involved in polymer degradation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe objective of this study was to isolate and identify bacteria that degrade high-density polyethylene (HDPE) from the Lut Desert, Iran, and characterize their enzyme and metabolic potential. The broader goal was to develop sustainable microbial strategies for plastic waste mitigation in extreme environments.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Procurement of HDPE Granules\u003c/h2\u003e\u003cp\u003eGranules of HDPE (tracking number 5030EA ON LN14540 P.S:21/14253) were sourced from Tabriz Petrochemical Company (Tabriz, Iran). The granules were processed as the primary substrate to isolate polyethylene-degrading bacteria. Following recommendations from standard biodegradation protocols, these granules were utilized without prior chemical modification or sterilization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Sample Collection\u003c/h2\u003e\u003cp\u003eSoil samples were collected from 31 locations across the Lut Desert in Iran during May and September 2022. GPS coordinates were recorded and converted into standard geographic coordinates using the dgmap.ir program. Samples were collected from the 5\u0026ndash;10 cm depth layer to minimize contamination from transient microorganisms. Samples were stored in sterile tubes under cryopreservation (\u0026ndash;20\u0026deg;C) for transit to preclude microbial community alterations. \"Additional data are given in Online Resource 1\".\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Isolation of Bacteria\u003c/h2\u003e\u003cp\u003eOne gram of each soil sample was suspended in 99 mL sterile distilled water (1:99 w/v) and homogenized by continuous shaking (5 h) to reduce microbial load and disperse soil particles. Serial dilutions (10⁻\u0026sup1; to 10⁻⁷) were prepared, and aliquots from the dilutions were spread-plated on Plate Count Agar (PCA) medium. The plates were incubated at 30\u0026deg;C for 24\u0026ndash;48 hours. The identification of emerging colonies was based on morphological characteristics such as size, shape, edge morphology, and pigmentation. The purified isolates were maintained at 4\u0026deg;C for further physiological and biochemical characterization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Pretreatment of HDPE\u003c/h2\u003e\u003cp\u003ePretreatment methods were applied to HDPE to improve its biodegradability, following established protocols [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Hemolysis Study on Blood Agar Medium\u003c/h2\u003e\u003cp\u003eHemolytic activity was assessed by cultured isolates on blood agar (5% v/v sheep erythrocytes) with incubation at 37\u0026deg;C for 24 hours. Hemolysis patterns (α, β, or γ) were recorded [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Colonies exhibiting beta-hemolysis were isolated and selected for further analysis due to their pronounced hemolytic activity. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Hydrophobicity Assay\u003c/h2\u003e\u003cp\u003eThe hydrophobicity assay was conducted to assess the bacterial adhesion potential to polyethylene surfaces following the methodologies of Rosenberg et al. and Hadar \u0026amp; Sivan, with slight modifications. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Beta-hemolytic strains were initially grown in nutrient broth at 30\u0026deg;C for 16 hours of incubation. Following incubation, the cultures were centrifuged at 8000 rpm for 20 minutes to pellet the cells. They were subsequently washed twice with phosphate urea magnesium (PUM) buffer (pH 7.1). After the washing, bacterial cells were resuspended in the same buffer (1.2 ml), followed by the introduction of 0.2 ml n-hexadecane, and the mixture was briefly vortexed to ensure homogeneous dispersion. The suspension was incubated at 30\u0026deg;C for 20 minutes in a shaker incubator, followed by a 2-minute settling period at room temperature. The aqueous phase's absorbance was determined by measuring it at 400 nm. The hydrophobicity percentage was determined using the equation below:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:H\\%=\\frac{{A}_{o}-A}{{A}_{0}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{o}\\)\u003c/span\u003e\u003c/span\u003e​ and \u003cem\u003eA\u003c/em\u003e represent the absorbance of the aqueous phase at 400 nm before and after mixing with n-hexadecane, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A reduction in absorbance, compared to a cell-free control, indicated the extent of bacterial cell adherence to the hydrophobic phase.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Isolation of HDPE-degrading Bacteria\u003c/h2\u003e\u003cp\u003eHDPE-degrading bacteria were isolated following established methods [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with modifications to suit local environmental samples. Beta-hemolytic bacterial strains exhibiting the highest levels of hydrophobicity were selected. The mineral salt medium (MSM) was prepared with the following composition (per liter): K₂HPO₄ (1 g), KH₂PO₄ (0.2 g), NaCl (2 g), NH₄NO₃ (1 g), MgSO₄\u0026middot;7H₂O (0.5 mg), CaCl₂\u0026middot;2H₂O (0.002 mg),\u003c/p\u003e\u003cp\u003eKCl (0.015 g), and ZnSO₄\u0026middot;7H₂O (0.001 g), with the pH adjusted to 7.5 using sodium bicarbonate. To facilitate interaction between bacterial cells and the HDPE surface, Tween 80 was added at a concentration of 1 ml per 1000 ml of medium [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHDPE plates were pretreated, cut into 0.5 \u0026times; 1 cm pieces, sterilized in 70% ethanol, dried, weighed, and aseptically transferred into 100 ml of MSM. Bacterial inoculum was prepared by culturing the isolated strains in nutrient broth until the logarithmic growth phase. 2 ml of the bacterial suspension, adjusted to match a 0.5 McFarland standard (equivalent to approximately 1.5 \u0026times; 10⁸ CFU/ml), was introduced into each flask [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The culture flasks underwent 60 days of continuous shaking (180 rpm) at 30\u0026deg;C in a temperature-controlled orbital shaker incubator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Estimation of HDPE Degradation\u003c/h2\u003e\u003cp\u003eAfter the 60-day incubation period, HDPE films were aseptically retrieved from the mineral salt medium and thoroughly cleansed to remove microbial biomass and extracellular polymers. The films were first treated with 2% (v/v) aqueous sodium dodecyl sulfate (SDS) solution for 15 minutes at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) with gentle agitation, followed by three sequential 10-minute washes with sterile distilled water to ensure complete removal of detergent residues. The purified films were oven-dried at 45\u0026deg;C for 24 hours until they reached constant weight. Pre- and post-incubation weights were precisely measured using an analytical microbalance (\u0026plusmn;\u0026thinsp;0.01 mg precision) for subsequent weight loss was calculated using the following equation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Percentage\\:of\\:weight\\:loss\\:=\\frac{Initial\\:weight\\:-\\:Final\\:weight\\:}{Initial\\:weight\\:}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis method follows the weight loss calculation approach outlined in previous studies evaluating the biodegradation of HDPE [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Scanning Electron Microscopy (SEM) of Polyethylene Surfaces\u003c/h2\u003e\u003cp\u003eTo characterize bacterial-induced morphological alterations on the PE surface, PE films exposed to bacteria for 60 days were freeze-dried to maintain surface integrity. Following that, SEM was used to analyze the samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Molecular Characterization of Bacterial Strains\u003c/h2\u003e\u003cp\u003eBased on the analysis of bacterial growth, hemolytic activity, hydrophobicity, and weight loss, genomic DNA was isolated from 10 selected bacterial strains out of the 50 initially obtained. Genomic DNA was extracted using the saturated phenol method. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Specific primers were designed and synthesized to target genes involved in polyethylene degradation, namely laccase, alkaline monooxygenase, and phosphatase. Target gene amplification, including that of the 16S rRNA gene, was performed using Polymerase Chain Reaction (PCR). Universal primers 27F (5\u0026prime;-AGAGTTTGATCCGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-GGTTACCTTGTTACGACTT-3\u0026prime;) were employed for 16S rRNA amplification. PCR reactions were conducted in a thermocycler under conditions optimized for each gene. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Amplification success was confirmed by 2% agarose gel electrophoresis, followed by Sanger sequencing of representative amplicons for precise gene identification [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The sequencing data was analyzed using Chromas Lite software for quality control and editing. Subsequently, the sequences were compared to the NCBI database using BLAST to assess their similarity to known bacterial strains. Phylogenetic analysis was performed using the neighbor-joining method implemented in MEGA7 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 GC-MS Analysis\u003c/h2\u003e\u003cp\u003eGC-MS analysis revealed HDPE degradation products after after 60 days of microbial treatment. Culture supernatants were filtered to remove bacterial cells and particulate matter and extracted with toluene. The organic phase containing the degradation products was concentrated by a rotary evaporator under reduced pressure. The concentrated extracts were subjected to analysis using GC-MS. Standard operating conditions for the GC-MS analysis were employed, with mass spectra obtained and compared against the National Institute of Standards and Technology (NIST) mass spectral library [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Bacterial Isolation and Characterization\u003c/h2\u003e\n \u003cp\u003eThe Lut Desert is renowned for its extreme environmental conditions, such as high surface temperatures, as recorded by NASA. Despite this, the region supports a diverse array of heat- and drought-resistant bacteria [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Fifty bacterial strains were isolated and purified by analyzing samples from 31 geographically distinct locations across the desert. All isolates were characterized through comprehensive morphological and biochemical profiling, including Gram staining, catalase/oxidase/urease/hemolytic activity, motility tests, and starch hydrolysis, among other tests, revealing distinct phenotypic patterns among strains. These assays provide insights into the metabolic potential of the strains, particularly their role in polyethylene degradation. \u0026quot;Additional data are given in Online Resource 2\u0026quot;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Culture in Blood Agar Medium\u003c/h2\u003e\n \u003cp\u003eAmong the 50 bacterial strains isolated and cultured on blood agar medium, 18 isolates demonstrated alpha hemolysis, 22 exhibited beta hemolysis, and 12 showed gamma hemolysis. Strains exhibiting beta hemolysis progressed to hydrophobicity testing due to their potential for enhanced biofilm formation and biodegradation activity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Bacterial Cell Surface Hydrophobicity\u003c/h2\u003e\n \u003cp\u003eThe hydrophobic nature of the bacterial cell surface significantly contributes to its ability to adhere to hydrophobic polymers like HDPE. This characteristic was measured by performing the Bacterial Adhesion to Hydrocarbons (BATH) assay on beta-hemolytic strains (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The percentages of hydrophobicity among the strains ranged from 16.33% (strain 21) to 37.38% (strain 50). Strains with higher hydrophobicity, such as strain 50 (37.38%), strain 44 (34.44%), strain 8 (32.26%), and strain 48 (31.67%), demonstrated enhanced capacity to adhere to hydrophobic surfaces, such as HDPE. Ten strains that had the highest hydrophobicity values were chosen for subsequent analyses. These findings are consistent with previous studies, which reported that bacteria with hydrophobic cell surfaces exhibit better efficiency in degrading hydrophobic polymers such as HDPE [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e] .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 HDPE Weight Loss\u003c/h2\u003e\n \u003cp\u003eThe degradability of the 10 bacterial strains with the highest hydrophobicity was quantitatively evaluated through gravimetric analysis of HDPE films following 60 days of incubation in the mineral salt medium under optimized conditions. The percentage of HDPE weight loss ranged from 5\u0026ndash;14%, with strains 48, 44, 8, and 50 demonstrating the highest degradation activities, achieving weight losses of 14.15%, 12.99%, 12.99%, and 12.01%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings highlight the capability of these bacterial strains to degrade HDPE under the specified experimental conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Surface Morphology Analysis by SEM\u003c/h2\u003e\n \u003cp\u003eTo investigate the structural changes on HDPE surfaces, SEM was performed on HDPE films exposed to bacterial treatments. SEM images showed bacterial adhesion and colonization on treated HDPE films compared to untreated films (Fig. 3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Molecular Characterization and 16S rRNA Gene Sequencing\u003c/h2\u003e\n \u003cp\u003eTen beta-hemolytic strains were selected for molecular characterization, including genomic DNA extraction and 16S rRNA gene sequencing. This recognized method of bacterial taxonomy elucidates the genetic determinants of the strains\u0026apos; polyethylene-degrading capacity, facilitating their application in bioremediation and plastic waste management. Agarose gel electrophoresis was employed to evaluate the integrity and purity of the extracted DNA, which revealed distinct and high-quality bands. PCR amplification was conducted to detect genes associated with polyethylene degradation, including \u003cem\u003elaccase\u003c/em\u003e (\u003cem\u003ecotA\u003c/em\u003e), \u003cem\u003ealkane monooxygenase\u003c/em\u003e (\u003cem\u003ealkB\u003c/em\u003e), and \u003cem\u003ephosphatase\u003c/em\u003e (\u003cem\u003ephoD\u003c/em\u003e), based on methods described by Smith et al. [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] and Xiao et al. [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. The PCR products were visualized through electrophoresis, yielding distinct bands at 1200 bp for the \u003cem\u003ephosphatase\u003c/em\u003e gene, 600 bp for the \u003cem\u003elaccase\u003c/em\u003e gene, and 700 bp for the \u003cem\u003ealkane monooxygenase\u003c/em\u003e gene. The positive control was Bacillus subtilis, and the negative control consisted of a PCR without template (primers only). (Fig. 4). \u0026ldquo;The original uncropped gel image corresponding to Fig. 4 is provided in Online Resource 3\u0026rdquo;.\u003c/p\u003e\n \u003cp\u003eThe presence of all three genes in eight strains highlighted their strong potential for polyethylene degradation. Among these, the five most promising strains, as determined by gene amplification results, underwent 16S rRNA sequencing for species identification. BLAST analysis indicated that there is significant homology between established bacterial species. The generated sequences were submitted to GenBank under accession numbers PV425917, PV425446, and PV606519 (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eThese results underscore the significant biodegradation potential of these strains and their genetic capacity for contributing to environmental plastic degradation efforts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 GC-MS Profiling of Plastic Degradation Compounds\u003c/h2\u003e\n \u003cp\u003eGC-MS analysis revealed metabolic by-products of HDPE degradation. The major degradation products discovered were alkanes and carboxylic acids, as depicted in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The corresponding chromatograms are presented in Fig.\u0026nbsp;6. Detected compounds such as hexanoic acid, oleic acid, dodecane, and octadecane indicate the microbial breakdown of HDPE into simpler organic molecules. Carboxylic acid, including hexadecanoic acid, suggests the oxidative cleavage of polyethylene, while alkanes such as hexadecane, heptadecane, and octadecane are consistent with the hydrocarbon backbone of HDPE are metabolized by bacterial enzymes. The findings are consistent with the well-known biodegradation pathways for plastic polymers [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. These findings are consistent with established pathways for plastic biodegradation, underscoring the capability of the isolated strains to transform polyethylene into simpler compounds.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMajor degradation products identified by GC-MS during HDPE biodegradation by selected bacterial strains. A checkmark (✓) indicates the presence of the compound in GC-MS profiles from the respective strain.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS.no\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrains 44\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain 50\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain 8\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain 48\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDodecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexacosane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEicosane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexadecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhthalic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeptadecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBenzaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTetradecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOctadecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUndecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNonahexacontanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethoxyacetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexadecadiene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeptadecanone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCyclotetradecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePentadecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e✓\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion and Research Perspectives","content":"\u003cp\u003eThe global prevalence of plastics, particularly HDPE, is due to their durability, versatility, and cost-effectiveness. These beneficial material properties promote environmental persistence and accumulation.\u003c/p\u003e\u003cp\u003eThis study aimed to characterize polyethylene-degrading bacteria isolated from Iran's Lut Desert, one of Earth's most extreme hyper-arid ecosystems. The extremophilic nature of these bacteria suggests that they produce heat-stable enzymes that can catalyze the breakdown of HDPE [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our findings confirm that these desert bacteria can degrade HDPE, as evidenced by enhanced biofilm formation, bacterial adhesion, and the production of degradation by-products identified by GC-MS. Identification of terminal oxidation products (carboxylic acids, alcohols) and intermediate metabolites (ketones, esters) demonstrates progressive HDPE depolymerization. The degradation potential of Bacillus, Staphylococcus, and Pseudomonas species is similar to that shown in previous research [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Among studied polyethylene-degrading actinomycetes, Rhodococcus strains consistently demonstrate superior performance with pre-treated substrates [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Most existing studies on PE degradation are limited to mesophilic soil bacteria, overlooking other microbial sources [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], composting agricultural [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], hot springs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and landfills [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This study is unique in showing that desert isolates can degrade HDPE in laboratory conditions. Our research provides new insights into the degradation of HDPE by extremophiles from arid desert environments, addressing a critical gap in bioremediation studies.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Challenges and Future Research Directions\u003c/h2\u003e\u003cp\u003eDespite the promising results, the rate of polyethylene degradation is still slow, reporting a rate of only 4% after 90 days under optimal conditions (Gupta et al., 2023). These findings underscore the critical need to systematically explore underexplored extremophilic ecosystems for novel biocatalysts with enhanced plastic degradation efficiency under industrially relevant conditions. Polyethylene degradation occurs in three stages: bio-deterioration, bio-fragmentation, and mineralization [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our study supports this understanding by demonstrating biofilm formation and the presence of intermediate degradation products through GC-MS analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Enzymatic Activity and Genetic Potential of Desert Isolates\u003c/h2\u003e\u003cp\u003eExtremophilic bacteria, such as those isolated in our study, produce stable enzymes under extreme environmental conditions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We identified genes encoding laccase, alkane monooxygenase, and phosphatase in the desert bacterial isolates. Laccase's four-copper active site enables the oxidation of recalcitrant pollutants through radical-mediated mechanisms, offering unique advantages for bioremediation applications [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Oxygenases, such as alkane monooxygenase, can enhance the hydrophilicity of polyethylene polymers, promoting bacterial adhesion and facilitating biodegradation. The discovery of phosphatase genes in these bacteria complements the oxidative capabilities of this strain and indicates dual bioremediation potential for contaminated sites containing plastic and heavy metals. Our study focused mainly on oxidative enzymes, but phosphatases are also a significant bioremediation tool, specifically for heavy metal detoxification through phosphate-mediated precipitation and mineralization. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Future work must characterize whether the observed phosphatase activity synergizes with oxidative degradation pathways, potentially enabling concurrent HDPE depolymerization and metal ion precipitation (as metal-phosphate complexes).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Future Prospects\u003c/h2\u003e\u003cp\u003eThe presence of genes encoding laccase, alkane monooxygenase, and phosphatase in the isolates highlights their potential for future research in microbial plastic degradation and environmental bioremediation. However, one major challenge lies in cultivating extremophilic bacteria in laboratory settings, as many of these organisms remain unculturable. Novel biocatalysts with significant potential for polyethylene degradation may be present in these unculturable species. Microbial biodegradation represents a sustainable pathway toward circular plastic economies by valorizing waste polymers into reusable metabolites while decreasing petrochemical dependence and ecological footprints [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, a critical aspect of future research must focus on the environmental impact of degradation products, such as alkanes and carboxylic acids. While these intermediates naturally occur during biodegradation, their accumulation requires stringent monitoring to prevent ecotoxicological effects.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe biodegradation potential of extremophilic bacteria isolated from the Lut Desert is highlighted in this study to combat HDPE pollution. By identifying bacterial strains equipped with genes encoding laccase, alkane monooxygenase, and phosphatase enzymes, we demonstrated their ability to degrade HDPE. The GC-MS analysis confirmed that these bacteria were responsible for the metabolic breakdown of HDPE, with the detection of essential degradation intermediates like alkanes and carboxylic acids. The discovery of phosphatases has paved the way for their use in other areas besides plastic degradation, such as bioremediation of heavy metals and pesticides. Exploring desert extremophiles increases our understanding of plastic degradation under harsh conditions, highlighting their importance in addressing plastic waste globally. A critical research priority involves systematically optimizing culture parameters (aeration, C: N ratio, trace elements) to enhance biomass yield and enzyme titers from these extremophilic isolates. In addition, studying microbial communities from other extreme environments could lead to more effective degraders that could accelerate the breakdown of polyethylene and other persistent pollutants. The use of extremophilic bacteria in environmental management strategies can offer long-term and innovative solutions to global issues, such as managing waste and restoring ecosystems. In this work, the value of extremophiles as potential candidates for future biotechnology applications in pollution mitigation and environmental recovery is underlined, with the potential to deal with plastic pollution and other environmental pollutant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA. A. conceptualized the study, helped with material preparation, and supervised and assisted with drafting the manuscript. S. H. prepared the materials, provided the results, wrote the manuscript, and prepared figures and tables. P. M. and M. M. provided manuscript review and writing assistance. A. A., P. M., and M. M. supervised the project and provided overall guidance. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNewrick, B.A., et al., \u003cem\u003eEnhanced biodegradation of high-density polyethylene microplastics: Study of bacterial efficiency and process parameters.\u003c/em\u003e Journal of Hazardous Materials, 2025. \u003cstrong\u003e485\u003c/strong\u003e: p. 136822.\u003c/li\u003e\n\u003cli\u003eSalwa, H., et al., \u003cem\u003eGreen bio composites for food packaging.\u003c/em\u003e Int. J. Recent Technol. Eng, 2019. \u003cstrong\u003e8\u003c/strong\u003e(2): p. 450-459.\u003c/li\u003e\n\u003cli\u003eBaldera-Moreno, Y., et al., \u003cem\u003eBiotechnological aspects and mathematical modeling of the biodegradation of plastics under controlled conditions.\u003c/em\u003e Polymers, 2022. \u003cstrong\u003e14\u003c/strong\u003e(3): p. 375.\u003c/li\u003e\n\u003cli\u003eShilpa, N. Basak, and S.S. Meena, \u003cem\u003eMicrobial biodegradation of plastics: Challenges, opportunities, and a critical perspective.\u003c/em\u003e Frontiers of environmental science \u0026amp; engineering, 2022. \u003cstrong\u003e16\u003c/strong\u003e(12): p. 161.\u003c/li\u003e\n\u003cli\u003eParashar, N. and S. Hait, \u003cem\u003ePlastics in the time of COVID-19 pandemic: protector or polluter?\u003c/em\u003e Science of the Total Environment, 2021. \u003cstrong\u003e759\u003c/strong\u003e: p. 144274.\u003c/li\u003e\n\u003cli\u003eCowger, W., et al., \u003cem\u003eGlobal producer responsibility for plastic pollution.\u003c/em\u003e Science Advances, 2024. \u003cstrong\u003e10\u003c/strong\u003e(17): p. eadj8275.\u003c/li\u003e\n\u003cli\u003eAdekanmbi, A.O., et al., \u003cem\u003eAssessing the environmental and health impacts of plastic production and recycling.\u003c/em\u003e World Journal of Biology Pharmacy and Health Sciences, 2024. \u003cstrong\u003e17\u003c/strong\u003e(2): p. 232-241.\u003c/li\u003e\n\u003cli\u003eAsiandu, A.P., A. Wahyudi, and S.W. Sari, \u003cem\u003eA review: plastics waste biodegradation using plastics-degrading bacteria.\u003c/em\u003e Journal of Environmental Treatment Techniques, 2021. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 148-157.\u003c/li\u003e\n\u003cli\u003eOng, G.H., et al., \u003cem\u003eScreening of Native Fungi For Biodegradation of High-Density Polyethylene (HDPE) Plastic in Mangroves Ecosystem.\u003c/em\u003e Malaysian Applied Biology, 2024. \u003cstrong\u003e53\u003c/strong\u003e(6): p. 97-103.\u003c/li\u003e\n\u003cli\u003eKnickmeyer, D., \u003cem\u003eSocial factors influencing household waste separation: A literature review on good practices to improve the recycling performance of urban areas.\u003c/em\u003e Journal of cleaner production, 2020. \u003cstrong\u003e245\u003c/strong\u003e: p. 118605.\u003c/li\u003e\n\u003cli\u003eCrystal Thew, X.E., et al., \u003cem\u003eEnhancing plastic biodegradation process: strategies and opportunities.\u003c/em\u003e Critical reviews in biotechnology, 2024. \u003cstrong\u003e44\u003c/strong\u003e(3): p. 477-494.\u003c/li\u003e\n\u003cli\u003eHussain, A., et al., \u003cem\u003eLow and high-density polyethylene and expanded polystyrene biodegradation by the greater wax moth Galleria mellonella L reveals a key role of the gut microbiome.\u003c/em\u003e Ecotoxicology and Environmental Safety, 2025. \u003cstrong\u003e294\u003c/strong\u003e: p. 118074.\u003c/li\u003e\n\u003cli\u003eZhou, Y., et al., \u003cem\u003eChallenges and opportunities in bioremediation of micro-nano plastics: A review.\u003c/em\u003e Science of the Total Environment, 2022. \u003cstrong\u003e802\u003c/strong\u003e: p. 149823.\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al., \u003cem\u003eBiodegradation of polyethylene and polystyrene: From microbial deterioration to enzyme discovery.\u003c/em\u003e Biotechnology Advances, 2022. \u003cstrong\u003e60\u003c/strong\u003e: p. 107991.\u003c/li\u003e\n\u003cli\u003eMutlu-Ingok, A., et al., \u003cem\u003eApplications of extremozymes in the food industry.\u003c/em\u003e Microbial Extremozymes, 2022: p. 197-206.\u003c/li\u003e\n\u003cli\u003eSchr\u0026ouml;der, C., C. Burkhardt, and G. Antranikian, \u003cem\u003eWhat we learn from extremophiles.\u003c/em\u003e ChemTexts, 2020. \u003cstrong\u003e6\u003c/strong\u003e: p. 1-6.\u003c/li\u003e\n\u003cli\u003eChia, X.K., et al., \u003cem\u003eRole of extremophiles in biodegradation of emerging pollutants.\u003c/em\u003e Topics in Catalysis, 2024: p. 1-18.\u003c/li\u003e\n\u003cli\u003eMargesin, R. and F. Schinner, \u003cem\u003ePotential of halotolerant and halophilic microorganisms for biotechnology.\u003c/em\u003e Extremophiles, 2001. \u003cstrong\u003e5\u003c/strong\u003e(2): p. 73-83.\u003c/li\u003e\n\u003cli\u003eShirsalimian, M.S., et al., \u003cem\u003eIsolation of a mesophilic and halotolerant strain of Kocuriapolaris from Gandom Beryan area in the Lut Desert of Iran, moderately resistant to gamma radiation and desiccation.\u003c/em\u003e Biosciences Biotechnology Research Asia, 2016. \u003cstrong\u003e13\u003c/strong\u003e(4): p. 2343-2350.\u003c/li\u003e\n\u003cli\u003eShahsavari, N., F. Kafilzadeh, and M. Kargar, \u003cem\u003eIsolation and identification of thermophiles bacteria from one of the hottest places on the planet (Lut Desert, Iran) and measuring their enzyme activities.\u003c/em\u003e Geomicrobiology Journal, 2021. \u003cstrong\u003e38\u003c/strong\u003e(10): p. 850-858.\u003c/li\u003e\n\u003cli\u003eAtanasova, N., et al., \u003cem\u003ePlastic degradation by extremophilic bacteria.\u003c/em\u003e International Journal of Molecular Sciences, 2021. \u003cstrong\u003e22\u003c/strong\u003e(11): p. 5610.\u003c/li\u003e\n\u003cli\u003eMohanan, N., et al., \u003cem\u003eMicrobial and enzymatic degradation of synthetic plastics.\u003c/em\u003e Frontiers in Microbiology, 2020. \u003cstrong\u003e11\u003c/strong\u003e: p. 580709.\u003c/li\u003e\n\u003cli\u003eChu, Y.-H., et al., \u003cem\u003ePurification and characterization of alkaline phosphatase from lactic acid bacteria.\u003c/em\u003e RSC advances, 2019. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 354-360.\u003c/li\u003e\n\u003cli\u003eHou, L. and E.L.-W. Majumder, \u003cem\u003ePotential for and distribution of enzymatic biodegradation of polystyrene by environmental microorganisms.\u003c/em\u003e Materials, 2021. \u003cstrong\u003e14\u003c/strong\u003e(3): p. 503.\u003c/li\u003e\n\u003cli\u003eShome, R., \u003cem\u003eRole of microbial enzymes in Bioremediation.\u003c/em\u003e ELifePress, 2020. \u003cstrong\u003e1\u003c/strong\u003e(1): p. 15-20.\u003c/li\u003e\n\u003cli\u003eBalasubramanian, V., et al., \u003cem\u003eEnhancement of in vitro high-density polyethylene (HDPE) degradation by physical, chemical, and biological treatments.\u003c/em\u003e Environmental Science and Pollution Research, 2014. \u003cstrong\u003e21\u003c/strong\u003e: p. 12549-12562.\u003c/li\u003e\n\u003cli\u003eBuxton, R., \u003cem\u003eBlood agar plates and hemolysis protocols.\u003c/em\u003e American Society for Microbiology, 2005. \u003cstrong\u003e15\u003c/strong\u003e: p. 1-9.\u003c/li\u003e\n\u003cli\u003eBamba, T., et al., \u003cem\u003eHigh-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.\u003c/em\u003e Biology Methods and Protocols, 2024. \u003cstrong\u003e9\u003c/strong\u003e(1): p. bpae044.\u003c/li\u003e\n\u003cli\u003eRosenberg, M., D. Gutnick, and E. Rosenberg, \u003cem\u003eAdherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity.\u003c/em\u003e FEMS Microbiol lett, 1980. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 29-33.\u003c/li\u003e\n\u003cli\u003eHadar, Y. and A. Sivan, \u003cem\u003eColonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber.\u003c/em\u003e Applied microbiology and biotechnology, 2004. \u003cstrong\u003e65\u003c/strong\u003e: p. 97-104.\u003c/li\u003e\n\u003cli\u003eVinderola, C., M. Medici, and G. Perdigon, \u003cem\u003eRelationship between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell wall protein profiles in indigenous and exogenous bacteria.\u003c/em\u003e Journal of Applied Microbiology, 2004. \u003cstrong\u003e96\u003c/strong\u003e(2): p. 230-243.\u003c/li\u003e\n\u003cli\u003eGupta, K.K., et al., \u003cem\u003eDegradation of high density polyethylene (HDPE) through bacterial strain from Cow faeces.\u003c/em\u003e Biocatalysis and Agricultural Biotechnology, 2023. \u003cstrong\u003e48\u003c/strong\u003e: p. 102646.\u003c/li\u003e\n\u003cli\u003eMontazer, Z., M.B. Habibi Najafi, and D.B. Levin, \u003cem\u003eIn vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella.\u003c/em\u003e Canadian Journal of Microbiology, 2021. \u003cstrong\u003e67\u003c/strong\u003e(3): p. 249-258.\u003c/li\u003e\n\u003cli\u003eAndrews, J.M., \u003cem\u003eDetermination of minimum inhibitory concentrations.\u003c/em\u003e Journal of antimicrobial Chemotherapy, 2001. \u003cstrong\u003e48\u003c/strong\u003e(suppl_1): p. 5-16.\u003c/li\u003e\n\u003cli\u003eSambrook, J. and D.W. Russell, \u003cem\u003ePurification of nucleic acids by extraction with phenol: chloroform.\u003c/em\u003e Cold Spring Harbor Protocols, 2006. \u003cstrong\u003e2006\u003c/strong\u003e(1): p. pdb. prot4455.\u003c/li\u003e\n\u003cli\u003eXie, M., et al., \u003cem\u003eComparative impacts of polyethylene and biodegradable film residues on soil microbial communities and rapeseed performance under field conditions.\u003c/em\u003e Frontiers in Microbiology, 2025. \u003cstrong\u003e16\u003c/strong\u003e: p. 1553807.\u003c/li\u003e\n\u003cli\u003eKumar, S., G. Stecher, and K. Tamura, \u003cem\u003eMEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets.\u003c/em\u003e Molecular biology and evolution, 2016. \u003cstrong\u003e33\u003c/strong\u003e(7): p. 1870-1874.\u003c/li\u003e\n\u003cli\u003eFarzi, A., et al., \u003cem\u003eBiodegradation of high density polyethylene using Streptomyces species.\u003c/em\u003e J. Coast. Life Med, 2017. \u003cstrong\u003e5\u003c/strong\u003e(11): p. 474-479.\u003c/li\u003e\n\u003cli\u003eHarshvardhan, K. and B. Jha, \u003cem\u003eBiodegradation of low-density polyethylene by marine bacteria from pelagic waters, Arabian Sea, India.\u003c/em\u003e Marine pollution bulletin, 2013. \u003cstrong\u003e77\u003c/strong\u003e(1-2): p. 100-106.\u003c/li\u003e\n\u003cli\u003eSmith, C.B., et al., \u003cem\u003eAlkane hydroxylase gene (alkB) phylotype composition and diversity in northern Gulf of Mexico bacterioplankton.\u003c/em\u003e Frontiers in Microbiology, 2013. \u003cstrong\u003e4\u003c/strong\u003e: p. 370.\u003c/li\u003e\n\u003cli\u003eXiao, Y., et al., \u003cem\u003eAn alkaline thermostable laccase from termite gut associated strain of Bacillus stratosphericus.\u003c/em\u003e International Journal of Biological Macromolecules, 2021. \u003cstrong\u003e179\u003c/strong\u003e: p. 270-278.\u003c/li\u003e\n\u003cli\u003eShah, A.A., et al., \u003cem\u003eBiological degradation of plastics: a comprehensive review.\u003c/em\u003e Biotechnology advances, 2008. \u003cstrong\u003e26\u003c/strong\u003e(3): p. 246-265.\u003c/li\u003e\n\u003cli\u003eAtalah, J. and J.M. Blamey, \u003cem\u003eIsolation and characterization of a novel laccase from an Antarctic thermophilic Geobacillus.\u003c/em\u003e Antarctic Science, 2022. \u003cstrong\u003e34\u003c/strong\u003e(4): p. 289-297.\u003c/li\u003e\n\u003cli\u003eKumar, S., A. Hatha, and K. Christi, \u003cem\u003eDiversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags.\u003c/em\u003e Revista de biolog\u0026iacute;a Tropical, 2007. \u003cstrong\u003e55\u003c/strong\u003e(3-4): p. 777-786.\u003c/li\u003e\n\u003cli\u003eFontanella, S., et al., \u003cem\u003eComparison of the biodegradability of various polyethylene films containing pro-oxidant additives.\u003c/em\u003e Polymer degradation and stability, 2010. \u003cstrong\u003e95\u003c/strong\u003e(6): p. 1011-1021.\u003c/li\u003e\n\u003cli\u003eKoutny, M., et al., \u003cem\u003eSoil bacterial strains able to grow on the surface of oxidized polyethylene film containing prooxidant additives.\u003c/em\u003e International Biodeterioration \u0026amp; Biodegradation, 2009. \u003cstrong\u003e63\u003c/strong\u003e(3): p. 354-357.\u003c/li\u003e\n\u003cli\u003eJeon, H.J. and M.N. Kim, \u003cem\u003eDegradation of linear low density polyethylene (LLDPE) exposed to UV-irradiation.\u003c/em\u003e European polymer journal, 2014. \u003cstrong\u003e52\u003c/strong\u003e: p. 146-153.\u003c/li\u003e\n\u003cli\u003eDang, T.C.H., et al., \u003cem\u003ePlastic degradation by thermophilic Bacillus sp. BCBT21 isolated from composting agricultural residual in Vietnam.\u003c/em\u003e Advances in Natural Sciences: Nanoscience and Nanotechnology, 2018. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 015014.\u003c/li\u003e\n\u003cli\u003eBaltaci, M.O., et al., \u003cem\u003eIsolation and characterization of thermophilic bacteria from geothermal areas in Turkey and preliminary research on biotechnologically important enzyme production.\u003c/em\u003e Geomicrobiology journal, 2017. \u003cstrong\u003e34\u003c/strong\u003e(1): p. 53-62.\u003c/li\u003e\n\u003cli\u003eGumbi, A., et al., \u003cem\u003eIsolation and characterization of pseudomonas aeruginosa and brevibacillus species and their potential to biodegrade polyethylene material.\u003c/em\u003e Science World Journal, 2019. \u003cstrong\u003e14\u003c/strong\u003e(4): p. 57-61.\u003c/li\u003e\n\u003cli\u003eGu, J.-D., \u003cem\u003eBiodegradability of plastics: the issues, recent advances, and future perspectives.\u003c/em\u003e Environmental Science and Pollution Research, 2021. \u003cstrong\u003e28\u003c/strong\u003e(2): p. 1278-1282.\u003c/li\u003e\n\u003cli\u003eGunjal Aparna, B., S. WaghmodeMeghmala, and N. Patil Neha, \u003cem\u003eRole of extremozymes in bioremediation.\u003c/em\u003e Research Journal of Biotechnology, 2021. \u003cstrong\u003e16\u003c/strong\u003e: p. 3.\u003c/li\u003e\n\u003cli\u003eMesbah, N.M., \u003cem\u003eIndustrial biotechnology based on enzymes from extreme environments.\u003c/em\u003e Frontiers in Bioengineering and Biotechnology, 2022. \u003cstrong\u003e10\u003c/strong\u003e: p. 870083.\u003c/li\u003e\n\u003cli\u003eYu, X., et al., \u003cem\u003eHeavy metals remediation through bio-solidification: Potential application in environmental geotechnics.\u003c/em\u003e Ecotoxicology and Environmental Safety, 2023. \u003cstrong\u003e263\u003c/strong\u003e: p. 115305.\u003c/li\u003e\n\u003cli\u003eGarcia-Garcia, G., et al., \u003cem\u003eEnvironmental impact of different scenarios for the pyrolysis of contaminated mixed plastic waste.\u003c/em\u003e Green Chemistry, 2024. \u003cstrong\u003e26\u003c/strong\u003e(7): p. 3853-3862.\u003c/li\u003e\n\u003cli\u003eTan, A.F., et al., \u003cem\u003eReimagining plastics waste as energy solutions: challenges and opportunities.\u003c/em\u003e npj Materials Sustainability, 2024. \u003cstrong\u003e2\u003c/strong\u003e(1): p. 2.\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, GC-MS, High-density polyethylene, Lut Desert. Phylogenetic tree, SEM","lastPublishedDoi":"10.21203/rs.3.rs-7003856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7003856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe environmental impact of plastic pollution, specifically from high-density polyethylene (HDPE), is significant due to its resistance to degradation. In this study, bacteria isolated from the Lut Desert, one of the hottest places on Earth, were investigated for their potential to degrade HDPE. Beta-hemolytic strains were prioritized due to their association with extracellular enzyme production and biosurfactant activity, which enhances surface adhesion and biodegradation. According to the BATH assay, 10 strains showed high hydrophobicity (34.44\u0026ndash;37.38%), which improved bacterial attachment to polyethylene surfaces. HDPE degradation was evaluated through weight loss over 60 days, with values ranging from 5\u0026ndash;14%. Strains 48, 44, 8 and 50 demonstrated the highest degradation efficiency, reducing HDPE the weight by 14.15%, 12.99%, and 12.01%, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed that polyethylene biodegrades by producing alkanes, carboxylic acids, and alcohols as byproducts. The identification of laccase (cotA), alkane monooxygenase (alkB), and phosphatase (phoD) genes was confirmed through PCR amplification, which revealed the enzymes that regulate HDPE degradation. The combination of hydrophobicity, biosurfactant production, and enzyme activity underscores the potential of extremophilic bacteria as effective tools for polyethylene bioremediation. This study's findings provide valuable insights into plastic degradation caused by microbial activity, which provides promising solutions for managing plastic waste in extreme environmental conditions.\u003c/p\u003e","manuscriptTitle":"Biodegradation of HDPE by Desert Bacteria: Insights from Isolates of the Lut-Desert, the Hottest Place on Earth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 11:32:23","doi":"10.21203/rs.3.rs-7003856/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":"81f9d7e6-bbbc-44e0-b1c1-813d6c61f0a3","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T21:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 11:32:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7003856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7003856","identity":"rs-7003856","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}