Abstract
Polyethylene (PE) is the second most abundant plastic worldwide due to its broad application in manufacturing disposable materials such as bags and bottles. Since it is highly resistant to natural biodegradation, it can accumulate in landfills, causing various ecological and toxicological consequences. Even though microbial degradation of PE has been extensively investigated, complete biodegradation of PE has yet to be achieved, and comparisons of PE biodegradation experiment findings are not practical. This may be due to the wide variety of PE substances used, and a wide variety of culture conditions, and many of the published findings still need key chemistry of PE materials, as well as basic biochemistry and microbiology. This review highlights the need for standardization in PE biodegradation studies and defining key biochemical terms. It aims to identify deficiencies and challenges in PE degradation experiments, enhancing future research for scientific and technological advancement. This review provides a biochemically based definition of PE biodegradation and summarizes microorganisms responsible for PE biodegradation. We also caution readers about the chemical makeup and structure of the PE, as well as the approaches utilized in microbes’ isolation. This review defines the fundamental elements required to conduct an effective PE biodegradation investigation.
Introduction
Plastic pollution occurs when plastic bodies and particles build up in the environment, posing a threat to humans, wildlife, and nature. Depending on size, plastic waste is categorized into micro, medium, and macro debris. While plastics are stronger than other materials and are suitable for a wide range of applications, they are difficult to degrade by natural processes due to their hydrophobic chemical nature. Combined with improper disposal, this causes plastics to accumulate and build up in the ecosystem. 1 Plastic pollution harms the water channels, land, and oceans, with coastal communities producing an estimated over eight million tons of plastic waste annually, much of which ends up in the ocean. 2 According to the National Academy of Sciences in the United States, the ocean’s gross load of plastic accumulating was predicted to reach approximately 8 million metric tons by 2022. In 2021, it was forecasted that rivers held over 2.6 million metric tons of plastic that eventually made their way into the ocean. Global plastics production reached an estimated 390.7 million metric tons in 2021, with most of this plastic being produced in Asia and China being the largest producer. Since the 1950s, an estimated 6.0 billion tons of plastic have been generated, with only nine percent being recycled and the remaining 12% being burned. 3 Overall, plastic pollution is a major issue that has far-reaching consequences for our planet.
PE, a synthetic material with untwisted saturated hydrocarbon and an elevated molecular structure represented as [CH2-CH2]n, accounted for about 30% of all plastic types in 2017. PE is divided into different grades based on the number of branches of the molecules. The most common grades are low-density PE (LDPE) and high-density PE (HDPE), medium-density PE (MDP), linear low-density PE (LLDPE), LDPE, HDPE, and very low-density PE (VLDPE). The global market volume of polypropylene in 2021 was approximately 75.6 million metric tons. 3 The hydrocarbon that makes PE hydrophobic and untwisted has a high molecular weight, resembling paraffin chemically. There are no covalent bonds between the individual macromolecules. PE polymers appear crystallized due to their harmonic molecular features, with increasing crystallinity leading to high stability and density. 4
The negative impact of PE pollution on organisms, especially marine organisms, includes physical consequences such as entanglement in plastic materials, health problems from ingesting plastic debris, and physiological interference due to exposure to chemicals in the plastics. The disruption of the hormonal mechanisms occurs due to direct or indirect consumption of plastics, which negatively impacts human health. 5 PE pollution remains a major threat to the biota, with significant negative impacts on biodiversity, especially in marine systems. The social and economic effects of PE polymer pollution have been determined. The consumption of tainted seafood and the building up of toxic materials in individuals due to PE would display a harmful effect on human health. Scuba divers who catch and tangle in abandoned fishing nets while diving face serious health risks. 6 Invertebrates ingest varying amounts, types, and shapes of plastic matter depending on their feeding habits and other factors. Microplastic, which includes PE, has a preference for metal compounds and may have ecotoxicological effects. 6
Published review articles on polyethylene biodegradation.
In this setting, we are seeking to express our views on the particular shortages of high-quality research and scientific methodology to strengthen present knowledge and comprehension across multiple particular factors, which would promote investigation, creativity, and growth on this widely announced topic despite the availability of many papers.10,13,15,20,27 There is a significant knowledge gap between the needs of society and scientific breakthroughs achieved in current circumstances confronting this essential challenge on the scientific understanding and oversight of PE regarding manufacturing to ecological hazards via the purchase cycle. The ability to degrade of PE remains an essential study subject that attracts public debate, and numerous novel studies and published findings have failed to tackle the basic and essential scientific problems involved, as previously stated.15,27 The key study and approach concerns related to this field are (1) the chemical makeup and structure of the PE, (2) the approaches utilized in microbes’ isolation, (3) the metabolic activity of the microbes as well as the biochemical mechanisms concerned, and (4) the tools used for assessing biodegradation levels. With this in thoughts, we are interested in expanding on each of them to demonstrate various seriously significant problems to promote the progress of basic research on this subject to better help society via the data provided in the most recent report as an illustration example. 20
As a result, in the present review, the PE characteristics as a substrate, microbes that trigger degradation, methodologies for evaluating, culture medium demands, standardization of isolation procedures, reactions and functions by microbes, and fresh viewpoints in this area of research are all extensively addressed to summarize the basic requirements for appropriate biodegradation examination as well as the analysis of the findings for an appropriate the presentation. Novel perspectives in this field of investigation are also offered to those engaged with this subject to improve the investigations.
Overview of PE biodegradation technology
Microorganisms can adhere to the hydrophilic polymer surface and begin to grow by utilizing the matrix as a carbon source. Pretreatment chemically or mechanically is typically necessary for PE, which has only CH2 groups, and since the water-resistant surfaces incorporate hydrophilic groups into the matrix polymer surface, increasing the hydrophilicity of the matrix.28,29 The major cleaves of the chain occur during the primary degradation, creating fragments of degrades called oligomers, which have low molecular weight and are subsequently re-degraded into dimers and even monomers. 30 Microbial extracellular enzymes are believed to cause degradation, and microbes use low-molecular-weight substances as energy sources.
The exact process of PE biodegradation remains unidentified, but a number of both abiotic and biotic variables are considered important in PE biodegradation in the environment. 31 Pure cultures of bacteria or fungi that can degrade PE or microbial consortium from various outdoor and aquatic environments have been used in biodegradation studies.17,25,32 Bioaugmentation with specific microbial consortia may help with PE biodegradation. Waxworms have also been shown to accelerate the biodegradation of PE. 17
Under aerobic conditions, the final by-products of degradation are CO2 and H2O, besides biomass, while under anaerobic circumstances, microorganisms could biodegrade the PE, and the major byproducts are carbon CO2, H2O, methane, and biomass. However, the final by-products under sulfidogenic conditions are H2S, CO2, and H2O. 33 Environmental factors identify the group of microorganisms and the degradative mechanism. Recalcitrant synthetic polymers may take several hundred years to degrade completely, and commercial polymers may contain additives, antioxidants, and other stabilizers, but some of these could be harmful to microbes and may result in a decreased biodegradation rate. 33
Type of microorganisms used in PE biodegradation
Report on the biodegradation of PE using bacteria
Reports on the degradation of PE by bacteria.
The weight loss due to PE biodegradation ranged from 0.96 ± 0.02% and 1.72% in studies by Kumari et al. 37 and Khandare et al. 39 to 30% and 40% in studies by Hadad et al., 32 and Nadeem et al. 40 The incubation time with PE substrate was mostly concentrated at 90 days, although Nadeem et al. 40 extended the incubation period to 150 days, while Tribedi & Sil. 41 and Kavitha & Bhuvaneswari. 42 extended the incubation period of their isolates with PE substrate to 45 and 30 days, respectively. Pseudomonas species have been studied for their ability to decompose and digest various artificial substances, including plastics, polymers, and waste products. Pseudomonas species can degrade and metabolize these substances via extracellular oxidative and hydrolytic enzyme reactions, allowing them to take up and degrade polymer fragments and control interactions between biofilms and surfaces of polymers. 41 According to Tribedi & Sil. 41 research, Pseudomonas sp. AKS2’s degradation of LDPE induced the hydrophobic interaction necessary for creating biofilm on the surfaces of PE and resulted in a 5 ± 1% loss of the initial PE material weight after 45 days. A Pseudomonas strain that can break down LDPE with a weight decrease of 21% after 5 months of incubation was isolated in a recent study by Nadeem et al. 40
It has been reported that Stenotrophomonas sp. can biodegrade LDPE at rates of 8% in 60 days and 32% in 150 days, respectively, as stated in two separate investigations by Dey et al. 43 and Nadeem et al. 40 This bacterium can be used with a PE pre-treating additive for improved results. Two thermophilic bacteria, Brevibacillus borstelensis and Bacillus sp PE3, use carbons of LDPE as their sole energy source. As a result, 30% and 6.8% of the PE film weight were decreased during incubation periods of 90 and 30 days, respectively.32,42 According to Awasthi et al., 36 HDPE can be degraded by Klebsiella pneumoniae HDPE after thermal treatment, resulting in an 18.4% decrease in tensile strength and weight of the HDPE films within 2 months. SEM and atomic force microscopy (AFM) images have shown that bacteria can create surface roughness and cracks, indicating possible biodegradation of PE. Additionally, Serratia spp, a Gram-negative bacterium, has been shown to biodegrade LDPE, with weight loss reaching 40% after 150 days of incubation. 40 Genetic analysis of Exiguobacterium sp. LM-IK2 by Maroof et al. 44 revealed the existence of laccase and alkane hydroxylase genes related to LDPE degradation. The authors suggest Exiguobacterium sp. LM-IK2 could be further studied to increase its effectiveness in the bioremediation of LDPE. Khandare et al. 39 isolated four bacterial strains from the aquatic environment capable of biodegrading LDPE. Based on 16S rRNA gene sequencing, their bacterial isolates H-255, H-237, H-265, and H-256 were found to share significant similarities with Halomonas sp., Cobetia sp., Alcanivorax sp, and Exigobacterium sp., respectively. The authors said the bacterial isolate H-255 had the highest weight reduction of 1.72% LDPE.
Interestingly, a recent study by Khampratueng et al. 45 utilized SEM to examine surface changes in LDPE following a biodegradation experiment with Bacillus sp. AS3. The results showed that incubation with Bacillus sp. AS3 led to the formation of holes and rougher surfaces on the LDPE compared to the control sample. Moreover, biopolysaccharides, potentially from a biofilm, were observed on the LDPE surface, while the control sample remained consistently smooth. Additionally, Khampratueng et al., 45 used GC-MS to confirm the presence of depolymerized PE compounds and intermediate byproducts resulting from the incubation with the PE substrate. These findings highlight the importance of employing advanced techniques to analyze PE biodegradation.
The variability in results highlights the importance of environmental conditions, incubation time, and the specific bacterial strains used in the biodegradation process. Further research has focused on understanding and enhancing the biodegradation capabilities of these bacteria. 46 For instance, Pseudomonas species have shown varying degrees of success in breaking down polyethylene, with some strains achieving significant weight reductions. 46 Additionally, genetic studies have identified key enzymes, such as laccase and alkane hydroxylase, involved in PE degradation. These discoveries pave the way for optimizing bacterial strains and developing new biotechnological approaches to improve biodegradation efficiency. 22 As research progresses, biodegradation could become a viable complementary strategy to traditional PE waste management methods, helping mitigate plastic waste’s environmental impact.
Report on the biodegradation of PE using fungus
summarizes some fungi capable of degrading PE.
Fatima isolated Penicillium sp from the Red Sea in 2017, which showed a high percentage of reduction in PE weight (43.4%). 52 Harrat et al. 53 isolated Rhizopus spp with LDPE degradation capabilities in a minimal liquid medium, achieving a 20% weight decrease in Algeria. In 2019, Aisa and Omm-e discovered that Penicillium spp. Degraded PE 30% more effectively than Aspergillus niger (19%). 54 When we look at previous studies, it appears that HDPE is more difficult to degrade by fungal spp. For example, Mathur et al. 47 discovered that after 1 month of incubation with A. niger, there was a 3.44% decrease in the mass of PE. Singh et al. 55 demonstrated that after 30 days of incubation between isolated fungi and HDPE, A. terreus had the highest ability with 7.59% biodegradability, followed by A. versicolor with 3.79% biodegradability. Interestingly, Ameen et al. 56 isolated six fungal species that thrived on LDPE film without added carbon sources. Co-cultivation under aeration enhanced their LDPE degradation capacity, with microscopy showing significant fungal growth on the film’s surface. Compared to controls, these fungi exhibited increased biomass, higher ligninolytic enzyme production, and greater CO2 emissions, indicating their potential to degrade and utilize LDPE.
The efficiency of fungi in degrading PE is largely attributed to their unique ability to adhere to the hydrophobic surfaces of PE and secrete potent enzymes capable of breaking down the polymer’s complex structure.47,48 Unlike bacteria, fungi such as Penicillium and Aspergillus can thrive in nutrient-limited environments and under stress conditions, making them particularly effective in environments where other microorganisms might struggle.17,47 Combined with their enzymatic activity, this resilience allows fungi to penetrate and degrade PE more efficiently. Studies have shown that fungal genera like Rhizopus, Brown rot, White rot, and Cladosporium are also capable of degrading PE, highlighting the diverse fungal capabilities in this area. 53 These fungi degrade PE and contribute to the biofilm formation on the polymer surface, further enhancing the degradation process.
The extent of PE biodegradation by fungi can vary significantly depending on the species and environmental conditions. These variations underscore the importance of selecting the appropriate fungal species and optimizing incubation conditions to maximize the efficiency of PE degradation. Moreover, recent findings, such as those from Khruengsai et al. 49 and Saira et al. 51 demonstrate that certain fungal isolates can achieve significant degradation levels even with minimal nutrient supplementation, suggesting that these fungi could be deployed in natural environments with limited resources. Despite the promising results of LDPE, HDPE presents a greater challenge for fungal degradation. Studies have shown that HDPE is more resistant to fungal attack, with only modest reductions in weight observed even after extended incubation periods. 55 This resistance is likely due to HDPE’s higher crystallinity and density, making it less accessible to enzymatic attack.
Report on the biodegradation of PE using Algae and cyanobacteria
The biodegradation of LDPE by photosynthetic algae has been extensively studied. Microalgal biodegradation of PE is a practical and environmentally friendly alternative to traditional degradation methods. The colonization of PE bags in aquatic environments by various microalgal species has been documented, including Phormidium, Hydrocoleum, Lyngbya, Oscillatori, Nostoc, Chlorella, Stigeoclonium tenue, Pithophora, Anomoeoneis, Spirulina, and Nitzschia.57,58 Sanniyasi et al. 58 demonstrated the ability of algae to degrade PE by forming a round disc shape on the surface of the LDPE sheet. Kumar et al. 59 found that Anabaena spiroides treatment led to the highest percentage of PE degradation at 8.18%. However, studies showed that Cyanobacteria strongly stuck to the PE surface and could not be removed by a jet of water, indicating insufficient evidence that they can degrade PE. 58 Similar to these seven orders, nine families and 10 genera of microalgal species were counted from the various regions of Kota, Rajasthan. 60 The biodegradation of LDPE by photosynthetic algae presents a promising and eco-friendly alternative to conventional degradation methods. The above studies have documented the colonization of LDPE in aquatic environments by various microalgal species, which suggests their potential role in breaking down PE. Notably, Anabaena spiroides have achieved a degradation rate of 8.18%, highlighting their effectiveness in PE degradation. 58 However, the strong adhesion of certain algae, such as Cyanobacteria, to the PE surface without evidence of significant degradation points to the need for further research to better understand and optimize the biodegradation potential of these organisms.
A recent investigation conducted by Gowthami et al. 61 analyzed five marine microalgae strains, namely, Chloroidium saccharophilum, Picochlorum maculatum, Amphora sp., Hymenomonas globosa, and Limnospira indica, focusing on their efficacy in degrading LDPE over 45 days. The incubation of LDPE in microalgae culture led to the highest weight loss (20.16 ± 0.14%), a faster reduction rate (0.005/day), and a shorter half-life (138.4 days) in the LDPE treated with Picochlorum maculatum. The study employed advanced techniques such as SEM imaging to observe surface erosion in all treated LDPE samples, while ATR-FTIR spectra indicated the presence of new functional group peaks that crosspend to biodegraded PE byproducts. 61
Report on the biodegradation of PE using warm and larvae
Recent investigations have shown that mealworms can degrade PE. Brandon et al. 62 found that approximately 50% of the consumed PE particles were converted to carbon dioxide. Furthermore, Mealworms fed with PE had a weight loss of the ejected particles reduced by over 40.1 %. The second-generation sequencing technique used by the researchers to analyze the microorganisms in the worm’s gastric tract revealed spss such as Kosakonia sp. and Citrobacter sp., suggesting that these bacteria may contain enzymes crucial for PE biodegradation. Moreover, Galleria mellonella and Plodia interpunctella larvae can degrade LDPE without prior treatment.63,64 G. mellonella has a biochemical machinery for beeswax metabolism, which makes it useful for PE metabolism due to structural similarities between beeswax and PE. In just 12 h, 100 g of mellonella worms can result in over 90 mg weight reduction in PE. 26 Ren et al. 65 investigated the degradation of PE films by Enterobacter sp. D1 isolated from the gastric tract of the wax moth G. mellonella. After 2 weeks of incubation, they discovered that their isolates could build colonies surrounding the PE particles, resulting in particle disruption. Another study isolated two PE-degrading bacteria, Bacillus sp. YP1 and Enterobacter asburiae YT1 from the gut of P. interpunctella larvae. 66 These researchers found that gut microbes are essential in the breakdown of PE. More research is necessary to understand how these enzymes work and how they can break down other types of plastic.
A recent investigation delved into the potential of LDPE-degrading bacteria found in the gut symbionts of lesser waxworm (Achroia grisella) larvae for the efficient biodegradation of LDPE. 67 Among all isolates in the worm gut, two bacterial strains displayed the most significant reduction in tensile strength, with reductions of 51.3% and 58.3%, respectively. These bacterial strains correspond to the molecularly identified species Citrobacter freundii and Bacillus sp. 67 Another recent study isolated an LDPE-degrading bacterial strain from the gut of earthworms collected from a plastic waste dump site. 68 The isolate was Bacillus gaemokensis strain SSR01, which successfully degraded LDPE film after 14 days of incubation, showcasing a maximum weight loss of 4.98%. 68 Both studies examined the deteriorated film using FTIR to identify degraded products and conducted SEM analysis to observe surface alterations. Energy dispersive X-ray spectroscopy testing also confirmed decreased basic carbon content in the broken-down LDPE film.67,68
In another investigation, Zaman et al. 69 conducted a study on the biodegradation of LDPE and LLDPE using Zophobas atratus larvae. The study involved the isolation and genome sequencing of gut bacteria collected from larvae frass. The results showed that the larvae consumed 24.04% of LDPE and 15.12% of LLDPE over 36 days, with 85% and 87% survival rates, respectively. In a similar study, Ding et al. 70 investigated the PE degradation capability of Tenebrio obscurus and Tenebrio molitor larvae under high-purity HDPE, LLDPE, and LDPE powders diets for 21 days at 65 ± 5% humidity and 25 ± 0.5°C. The results indicated that the larvae digested nearly 40% of the ingested three PE types and showed similar survival rates and weight changes, but their fat content decreased by 30%–50% over the 21 days. These studies highlighted the essential role of insects and worms’ gut microbes in the breakdown of PE, emphasizing the need for further research to understand the mechanisms of these enzymes and their potential application in the degradation of PE.
Mechanism of polyethylene biodegradation
Biodeterioration
The biodegradation process can be divided into three stages: biodeterioration, bio fragmentation, and assimilation. Figure 1 provides a diagrammatic representation of this process. Biodeterioration is referred to occasionally as a superficial decomposition that alters the substance’s mechanical, physical, and chemical characteristics. When a PE is subjected to abiotic variables in its natural setting, its framework weakens, allowing for additional deterioration.
71
The compression (mechanical), temperature, light, and environmental chemicals are all abiotic variables that affect these original changes. While biodeterioration tends to be the initial phase of biodegradation, it can sometimes occur concurrently with bio fragmentation.
71
Mechanism of enzymatic activity. The enzyme’s active site breaks long chains of PE polymer into smaller monomers or dimers. In the second stage of the reaction, the microorganism can build up and utilize PE as the sole energy source.
The biodeterioration mechanism starts with the oxidation of the PE exterior by oxidative enzymes or oxidizing agents like U-V light. Hydrolases represent the main enzymes incorporated in the decomposition of environmental PE. 72 These enzymes are essential for breaking chemical chains in aquatic media, leading to the cleavage of larger molecules into smaller ones. They split long carbon chains in half and decreased the number of carbonyl groups, converting them into carboxylic acids. This speeds up the oxidation of PE and cleaves the polymer carbon chains into fragments, releasing intermediate products such as long-chain aliphatic compounds like alkenes and alkanes. 72
Bio fragmentation
Bio fragmentation of a PE is the destructive procedure whereby bonds inside a PE are split, forming oligomers and monomers. PE exists in a hydrophobic form in nature. Extracellular enzymes produced by various microorganisms initially adhere to the surface of PE using hydrophobic interactions in the initial process of the polymer-enzyme interaction. 71 The enzyme’s active site breaks down the long chains of PE polymer into monomers or dimers, which are smaller. In the next phase of the reaction, the microorganisms can build up and utilize PE as the sole energy source. 72 The steps needed to break down these substances vary depending on the framework’s oxygen level. Aerobic digestion is the degradation of substances by microbes when oxygen exists, and anaerobic digestion, on the other hand, is the decomposition of substances when oxygen doesn’t exist. 73 The primary distinction between those procedures is that anaerobic procedures generate gases like methane while aerobic processes lack it (both processes produce CO2, H2O, some debris, and intriguing biomass). Furthermore, aerobic breakdown usually happens faster than anaerobic breakdown, whereas anaerobic digestion reduces the size and weight of the substance more effectively. 71
Assimilation
In the assimilation phase of polyethylene biodegradation, fragmented polyethylene particles are absorbed by microorganisms through various mechanisms. 71 Membrane transporters facilitate the direct uptake of some fragmented molecules across the microbial cell membrane, allowing them to enter the cell in their fragmented form. 30 However, other fragments are less readily transported and require preliminary biological transformations. These transformations convert the polyethylene fragments into simpler compounds that can be more easily transported across the cell membrane. 30 Once inside the cell, these compounds undergo catabolic processes, crucial for the microorganism’s metabolic activities. Polyethylene-derived fragments are further broken down into smaller molecules through enzymatic reactions. 46 This catabolic activity generates adenosine triphosphate (ATP), which provides the energy necessary for the microorganism’s cellular functions. 71 Additionally, some of the breakdown products are utilized as building blocks for synthesizing cellular structure components, contributing to the growth and maintenance of the microorganism. 71 This dual role of the catabolic processes—energy production and biosynthesis—highlights the integral role of assimilation in enabling microorganisms to utilize polyethylene fragments effectively. 30
Enzymes involved in PE biodegradation
PE biodegradation requires enzymes that can break down the polymer. Most PE-degrading enzymes are oxidoreductases, including peroxidase, hydroxylase, laccase, and soybean peroxidase. Azotobacter beijerinckii HM121 contains hydroquinone peroxidase, which can degrade PE. However, most PE-decomposing enzymes can oxidize the lateral and adjacent carbon. The alkaline hydrolysis enzymes family can degrade n-alkanes, which are the essential component of PE, through hydroxylation reactions. Peroxidase and laccase can result in terminal oxidation. 74 Laccases is a glycoprotein enzyme found in fungi, higher plants, and bacteria. The blue-copper family of laccases and oxidases can provoke the oxidation of various arylamines and phenol. Copper significantly affects laccase induction and activity, leading to PE degradation. Heat-treated PE can degrade due to lipase activity secreted from Streptomyces species. 75 Similarly, after about 2 weeks of incubation with P. chrysosporium MTCC-787, about 70 percent of a pre-oxidized HDPE sample was degraded. The degradation of PE by such organisms depends heavily on the extracellular peroxidases. 76
An LDPE-degrading laccase was purified from R. rubber C208. Copper significantly affects laccase induction and activity because they have four copper ion bonding sites, which leads to PE degradation. 73 Johnnie et al. 77 used a laccase extracted from Trichoderma viride to break down LDPE. The weight reduction of LDPE after 10 days of enzyme treatment was about 2.3%. It has been investigated whether a depolymerase can degrade PE succinate. 78 This enzyme was purified and cloned after being extracted from Aspergillus fumigatus. 79 According to reports, this enzyme works best at slightly acidic media and a temperature of about 55 ± 1.0°C when acting on PE.
The depolymerase isolated from bacteria poses a clear hallo on agar plates with PE as the sole source of energy. 78 The strain was used to extract the depolymerase enzyme, and the clear hallo demonstrated its ability to degrade various PE types. 75 Cytochrome P450 (CYP, P450) is a multifunctional enzyme that can recognize a variety of substrates and provoke a wide range of reactions. 79 Using the P450 enzyme that can degrade PE into short chains is a promising approach, as other degrading enzymes can only oxidize PE. 79 PE’s structural makeup is similar to alkane, which consists of C-C and C-H bonds. Alkane mono-oxygenase may be a candidate for PE degradation, with AlkB being attributed to PE degradation.79,80
Challenges associated with PE biodegradation experiments
The investigation into PE biodegradation reveals both progress and challenges in understanding and optimizing this complex process. The review highlights the diverse methods and findings in the field by analyzing 40 studies across various research approaches, including those focused on bacteria, fungi, worms, algae, and cyanobacteria (Figure 2). Bacteria and fungi have emerged as the primary focus, with a substantial body of research dedicated to their roles in degrading PE. In contrast, research on algae and cyanobacteria remains limited, suggesting that these organisms may offer untapped potential for PE degradation but still need to be more extensively studied. Summary of studies involved in this review. A. A total of 41 studies were considered in this study, of which 16 addressed bacteria genera, 14 involved fungal genera, six examined worms and larvae, and only five covered algae and cyanobacteria. B. Most of the previous studies have used LDPE as a substrate. C. The majority of the bacteria belonging to the phyla Pseudomonadota and Bacillota, while Penicillium and Aspergillus species are among the most common fungal genera capable of degrading PE. D. weight loss was the comments method used for assessment of PE biodegradation. E. application of basic concepts by previous studies. F. source of samples for previous studies.
The positive trend in research interest over the past 5 years indicates a growing recognition of the potential for biological methods to address PE pollution. This interest is driven by evidence showing that while PE degradation in nature is slow, it is achievable through various microbial communities. However, the review also identifies several critical challenges that hinder the practical application of these findings. Variability in the effectiveness of different microorganisms, slow degradation rates, and the need for specific environmental conditions are significant hurdles that must be addressed. To improve the understanding and efficiency of PE biodegradation, there is a need for more standardized experimental protocols and a deeper exploration of the mechanisms involved. The challenges summarized in Figure 3 highlight the importance of developing more effective microbial and enzymatic degradation strategies. Addressing these challenges will be essential for advancing biological methods as viable solutions for managing PE waste. Shows the key concepts and approach concerns related to polyethylene biodegradation experiments: the chemical makeup and structure of the PE, the approaches utilized in microbes’ isolation, the metabolic activity of the microbes, the biochemical mechanisms concerned, and the tools used for assessing biodegradation levels.
The chemistry and structure of the PE substrate
The chemical makeup of what is being studied must be properly understood and described in every scientific inquiry, and that criterion likewise relates to PE. 20 Even though PE polymers are polymerization objects, and the corresponding monomers and molecular mass are crucial data for identifying a substance class, It is rare to come across data regarding the PE chemical structure in previous studies. 15 Among the most significant and basic challenges, that is likely deliberately dismissed by several studies, is the lack of basic polymer chemical properties of the PE selected for any examinations as there’s barely any minimal description information on the pureness, molecular mass, and molecular weight distribution of the PE before the start of any study. 15
Characteristics of common types of PE.
In PE biodegradation trials, two significant variables should be stated: weight-averaged molar mass (Mw) and number-averaged molar mass (Mn). Mw is the entire weight of the polymers divided by the total quantity of particles in the sample, whereas Mn is determined from the mole fraction distribution of different-sized molecules. 18 The Mw of the main polymer chain is typically greater than 30,000 g/mol, indicating the needed enzymatic strength to decompose the longest polymeric chain. 81 A few notifications in the scientific literature specify the Mw of the polymers examined. Other research has stated the Mw, Mn, and polydispersity (PD) of the initial and surged photo-degraded PE, in addition to thermally split LDPE film specimens that included degradable PE additives pro-oxidants.3,19 PE characteristics such as Mw or point of melting typically are noticed in reported findings, but only before any additional processes such as UV treatment, nitrogen gas thawing, extrusion, or solvent dissolution. 3 Because the dimension of the PE chain may be altered before degradation by microbes, changes in Mw or Mn can be interpreted incorrectly as merely caused by microorganism enzyme activity. The characteristics of PE instantly before it is subjected to microbial decomposition have to be stated to determine the true PE-degradation capacity of microbes.
The pureness of substances in manufacturing processes is an inevitable problem and crucial data, but numerous researchers used readily accessible PE gadgets instead of 100% pure glue to form or mold samples for their studies, lacking recognition of the reality that the items comprise an array of chemicals besides to their foundation polymers. 20 In this setting, the proper conversion of polymers into goods necessitates the inclusion of plasticizing agents, filler, and molding chemicals. Because pure resins made from polymers can’t be transformed into high-quality products without them, this method incorporates chemicals into the end items. 82 Any deterioration detected on items cannot be attributed solely to PE. 15 More research is concerned about this problem, which impacts the stated degradability of using these as only one carbon source. Before performing studies about the selection of PE, researchers, and ecological scientists must pay special attention to the experimental substances.
The majority of the studies that were reviewed did not take into account some significant factors that might have an impact on their findings. Among these factors, solvents were used to make it easier for PE substrates to dissolve. Solvents used to increase the solubility of PE substrates can impact their chemical characteristics and molecular architectures. 83 Another factor is the amount of surface interaction across the microbes, and the kind of PE substrate (particle, powder, or film) used in the investigations can affect how quickly PE degrades. 83 The biodegradation process cannot start unless the microorganisms or extracellularly produced enzymes can interact directly with the PE interface. 83 Interestingly, retail items, particularly for packaging applications, incorporate oxidants that improve the degradation of the PE. As a result, the noticed deterioration is probably largely due to the formulation’s oxidant during manufacture. 30 Another element that is sometimes disregarded is UV light from sunlight after plastic disposal, which adds greatly to the deconstruction of PE outdoors due to their accessibility and occurring naturally.
Microbes’ isolation and microbial consortium demands
The second aspect of a successful PE biodegradation experiment is the microorganisms. Any assertion of PE biodegradation needs to be facilitated by a living microbe and its metabolic capacity to affect the PE chemical structure, namely the level of polymerization and molecular mass dissemination. 84 Sadly, many of the articles that have been published lack crucial details before asserting deterioration.
To isolate probable microbes, first enrichment and transmission with pure PE as the sole carbon source for microbes picking is needed, and parallel transforms are additionally needed to eliminate non-degrading microbes while enriching the community of capable members. 85 Previously, this well-established approach to PE degradation had not been substantially applied to biodegradation research. 19 The microbiological and biochemical reactions implicated must be further explained now that an efficient microorganism will be produced. Several published research findings on PE degradation are now based on a culture medium that contains a rich source of energy that promotes the growth of several microbes, such as sugar glucose, extract of yeast, and malt liquor extracts. 19 The reported development of numerous microbes on PE or in cultures containing this mixture content is not attributable to using the PE carbon atoms, certainly rather than the backbone carbon, as a supply of carbon and energy for propagation. 15 Simultaneous assessments, whether on the growth of microbes in the media or the analysis of recovered polymers throughout incubation, are similarly deceptive. The purified and identified microbes were not specifically enriched for their capacity to degrade PE of high innocence and known chemistry, and they thus lack the needed biochemical enzymatic processes in breaking down the PE to survive in this cultivation media since no breeding tension is placed on the microbial community. 15
Another important aspect of PE biodegradation research is enriching a consortium colony or isolating a single type of strain so that degradation studies can be conducted using recognized microbes or an established community in consortia. 86 Enriching ought to be used to do this, and this step is frequently missed across numerous research. 20 The origin of the inoculum is also critical in the enriching procedure, as is a basic understanding of the site’s chemical and physical features. 87 Numerous investigations employ this stage as a secure safety for achieving the data check rather than putting any attention to detail into maximizing the benefits of this method.15,19 The energy and time invested in the inoculum could eventually pay off if additional effort is put into it effectively and persistently.
The fluctuation between the microbial community and the percentage of substrate ought to be tracked and demonstrated by the findings. 87 In many studies, no microbial community details are supplied; rather, substrate level decreases are demonstrated as the only proof of deterioration, ignoring the control data sans inoculation. 15 Variations in the microbe and substrate/PE levels ought to be presented concurrently in this situation to enable assessment of potential PE breakdown by the involved microbes throughout the culture. 20 Furthermore, to validate microbe PE degradation, an abiotic control needs to be set up in tandem with the metabolically active processes so that chemical reliability may be assessed despite biases from chemical changes, such as hydrolysis.19,20 To demonstrate any impacts from damaged proteins in the inoculum cells, a further control that ought to be provided but is seldom used is dead or sterilized inoculum. 85 Considering all of these factors, the methodology can give good data on the degradability of contaminants by pure cultures or consortiums of microbes. Inevitably, a great majority of published research lacks an adequate grasp of enrichment and transmission techniques and the focus on what is required for the rules to be implemented in reality, resulting in brash and hasty claims or conclusions. 20
The metabolic activity of the microbes and the biochemical mechanisms concerned
Identifying biodegradation biochemical reaction pathways of any molecules of organic matter or polymers cannot be demonstrated unless extensive evidence on chemistry and biology to back it up. 88 Suppose the chemical makeup of PE substances and the microbial medium of cultivation content are in doubt. In that case, it is hard to solidly pinpoint the biochemical process pathways and the relevant enzymes needed as crucial outcomes. 20 Microbes are unlikely to play a function in the alteration of PE. Because non-selective enzymes are not particular to any substrate, particularly polymeric substances, biological or artificial, no biochemical pathways for changing the flexible covalent nature—C-C bond, the substance structure’s backbone can be created. 20 Lipase generation, for instance, can be triggered by many inorganics as well as organic substances, but the enzymatic response is instead a unique splitting of the trash foundation C-C bonds that provides the significant splitting of the polymer framework, leading to a reduction in molecular mass and an alteration in the dissemination to smaller molecular weights as the dominant portion in the entire plastic materials. 74 When the underlying chemistry of PE degradation is examined carefully, more inquiries than solutions appear in the existing literature. Alkanes have the most structural similarities to PE, and the recognized breakdown mechanism for long-chain alkanes is the oxidation underneath aerobic conditions catalyzed by monooxygenase. 88 Furthermore, despite the large number of microbes identified and purified for “degradation” of PE, no novel biochemical reaction type has been revealed, and no disruption of the PE CC bond has been proven.15,19
Most contaminants in our surroundings are hydrophobic, which means they have very poor solubility in water. Consequently, they fail to disintegrate effectively within water and media for growth, restricting bacteria’ utilization of them as energetic sources. 19 Much of the information provided includes such compounds at levels much over their physical dissociation limit, and deterioration of these compounds has been shown. 13 Even if a solvent was employed to assist in dissolving the substances, these findings should be interpreted with care since the hydrophobic nature of the PE will not render them suitable for absorption and then metabolism. 18 Because the soluble quantities utilized are too inadequate, the measured deterioration might be biased or solely due to loss through combustion, adsorption of the chemical substances to culture bottles, and chemical binding onto cell walls biomass. 18
PE biodegradation assessment tools
Reviewing the available data reveals that numerous studies have examined PE-degrading microbes using readily available polymers, which may contain various chemical ingredients and additives that could impact the technique and degree of biodegradation. 19 In most earlier investigations, the weight loss and functional group modifications on the polymer’s surface were calculated, and structure change evaluation was performed using the EM. However, it needs to be clarified whether the degradation of additives, which frequently make up a substantial percentage of the polymer, causes weight loss and surface structure changes. 89 PE Weight loss and colonization of microorganisms on PE-containing agar plates is insufficient evidence for total PE biodegradation. 20 Comprehensive biodegradation needs to be validated, which has been one of the key issues with biodegradation investigations. Furthermore, these techniques are unsuitable for studying PE breakdown in open or aquatic conditions, where oxygen and nutritional levels, temperature, and microbial communities vary greatly. 90 Hence, more significant adjustments are required to discern the true degradation of PE while minimizing the possibility of artifacts caused by additive degradation. 91 When using PE polymers as their sole carbon source, microorganisms produce carbon dioxide as one of the principal metabolites by oxidizing the carbon source under aerobic environments, whereas carbon dioxide and methane are generated as final byproducts under anaerobic settings. 90 As a result, the amount of these products or the quantity of oxygen utilized during decomposition can be used as analytical criteria to determine the ultimate biodegradable characteristics.
In this context, Rose et al. proposed a reliable method in 2020 to assess the degradation of PE by measuring carbon dioxide via gas chromatography as a consequence of bacterial respiration and degradation. 83 Their method is reliable and repeatable and allows for monitoring microbial activity without interfering with the culture content or attempting to eliminate adherence cells, lowering the likelihood of impurities or disrupting biofilms. 83 Because it was modified to account for the various nutrient requirements of each species, their approach could be modified to replicate a specific aquatic environment. 90 Because land-based, aquatic, and marine biological environments are fundamentally different, future studies using this method may replace or supplement other methods to produce definitive results. 90
There were no real positive attempts to perform a truly academic contrast across the inoculated and the appropriate controls (ignoring any inoculation and inoculated with sterilized inoculum or only exopolymers (EPS) of the microbes that inhabit the growing medium) by the basic tenets of scientific techniques since persuasive research verification needs to be developed on interventions with only one factor as a particular parameter to conclude the effectiveness of the procedures. 20 The control lacking inoculation of microbes of the growing medium having supplemental carbon from an organic origin is a bogus control that cannot be compared effectively to the infected ones. 91 Furthermore, when the substance shifts, the EPS and microbial byproducts stick to PE samples, producing conflicting signals to the laboratory findings for a false positive. 20 Bacterial EPS attaches fast to polymers, producing spectra that vary from those obtained without inoculation as a control. Such control data must be gathered to provide a credible explanation of the PE deterioration findings. Similarly, extra organic substances in the media might have similar outcomes, leading to an incorrect understanding of deterioration. 91 Unfortunately, only a few significant detailed studies have been conducted to assess this basic and essential problem in biodegradation evaluation through measurement of neat PE, those under abiotic conditions, with interaction with microbes, and various forms of clearing to record the reliability of techniques and applications. Science advances via novel methods of experimentation, and the findings gained have to be reliable and repeatable.
PE biodegradation technique, future innovation
The future of PE biodegradation is increasingly intertwined with advances in biology and biotechnology, which offer promising avenues for addressing the persistent challenge of plastic pollution. These fields are poised to drive significant innovation by leveraging microorganisms’ natural capabilities and enhancing them through genetic and biochemical modifications.
One major biological approach involves discovering and characterizing new microbial species and enzymes capable of degrading PE. 91 Researchers have recently identified several bacteria and fungi with natural abilities to break down PE, often through the secretion of specific enzymes like oxidases and hydrolases. By isolating these organisms from diverse environments, such as soil, marine ecosystems, and landfills, scientists are expanding the catalog of potential biodegraders. Advances in metagenomics and metatranscriptomics allow the identification of previously unknown microbial species and their associated genes directly from environmental samples without culturing. 92 This speeds up the discovery process and provides a more comprehensive understanding of the microbial communities involved in PE degradation.
Microbial synergy and recombination technology, two exciting future breakthroughs for PE biodegradation technology that were overlooked in many prior studies, should be considered in the forthcoming studies. 85 Complex microbial communities and combinations of chosen bacteria have also been studied to ascertain whether there are synergistic effects on biodegradation by certain bacteria. 84 The justification for adopting the microbial consortium is that different bacteria use various metabolic processes and express various oxidizing enzymes while cultivated with various kinds of PE, and this mixture could facilitate improved microbial-induced PE biodegradation. 92 Ensembles of bacteria are more effective, resilient, and manageable than single strains. This is due to several factors, including the fact that multiple enzymes and metabolic processes work in concert to degrade PE polymers in a complex manner. 92 According to Zhang et al., 24 microbial complexes also produce fresh environmental conditions for species, which may stimulate some inactive metabolic processes under a single-species culture environment.
Molecular methodologies for manipulating genes implicated are also a standard lab practice, allowing PCR amplification of a specific genetic material, quantifying the gene quantity, and monitoring gene expression. 93 Biologists and non-biologists easily employ High-throughput genome sequencing technologies to disclose microbial population data for considerably more thorough information on microbe population makeup. 93 Transcriptomics and proteomics are utilized to identify active metabolic reactions and expressed proteins. 94 Furthermore, steady isotope probing allows for an immediate connection across the substrate being studied and the microbes that use it in pure culture or under environmental circumstances, greatly improving the investigation’s capacity for better comprehending the in-situ population and metabolically active microbes instead of removing them from their natural niche. 94 Moreover, Recombinant DNA technology, on the other hand, might be utilized to recombinant enzyme-generating genes to boost the effectiveness of enzymes in decomposing PE. 95 The MHETase gene was successfully cloned in plasmids using an in-silico technique. Recombinant plasmids with MHETase gene sequences were created, and MHETase enzymes can be manufactured for the plastic decomposition analyzing process. Regarding PE biodegradation, there is a dearth of technology that can produce pure types of PE-degrading enzymes without the need for purification. 95
In addition to these approaches, applying omics technologies, such as genomics, proteomics, and metabolomics, provides deeper insights into the molecular mechanisms underlying PE biodegradation. 92 These technologies allow researchers to track the expression of genes, the production of proteins, and the changes in metabolic pathways in real time as microbes degrade PE. 96 This information is crucial for identifying key regulatory genes and metabolic bottlenecks that can be targeted for genetic or chemical modifications to enhance degradation efficiency. For instance, transcriptomic analyses have revealed that certain stress-response genes are upregulated during PE degradation, suggesting potential targets for improving microbial resilience and performance. 97 In sum, biology and biotechnology are at the forefront of advancing PE biodegradation, offering innovative solutions that harness and enhance the natural capabilities of microorganisms. By integrating approaches such as metagenomics, genetic engineering, synthetic biology, directed evolution, and omics technologies, researchers are paving the way for more efficient and sustainable strategies to address plastic pollution. As these fields continue to evolve, they hold the potential to revolutionize how we manage and mitigate the environmental impact of polyethylene and other persistent plastics.
Based on the above comment on the biodegradation report, we recommended the following strategies for optimizing the future PE biodegradation experiments prospects: 1. When purchasing PE, it is advised to identify the chemical composition and physical characteristics of the product and any additives (the existence of additives may alter the technique utilized for estimating biodegradation and the degree of PE biodegradation). 2. PE preparation: PE sheets rather than powder should be utilized when preparing PE for microbial biodegradation investigations because PE sheets provide surfaces for microbial colonization and biofilm development, which may speed up the biodegradation process. Additionally, it’s advised to avoid using strong solvents when preparing PE because they could affect its structure and properties, leading to a bias in the result. 3. Experimental conditions: For best results, the biodegradation experiment should be conducted in conditions ideal for microbes, including temperature, pHH, and humidity. 4. Biodegradation level estimation: To estimate the biodegradation level, avoid methods that are affected by the presence of PE additives, such as measuring PE weight loss, and instead use a more advanced technique, such as quantifying CO2 using gas chromatography as a result of microbial degradation and respiration and monitoring the microbial growth curve, to obtain more reliable and meaningful results. 5. Prospects: Microbial synergy techniques and recombination technologies are recommended, alongside methodologies such as metagenomics, genetic engineering, synthetic biology, directed evolution, and omics technologies. These approaches aim to facilitate the development of more efficient and sustainable strategies for tackling pollution from PE.
Conclusion and further research directions
Research is an exhaustive procedure that involves a sound theory combined with trustworthy techniques to improve and enhance knowledge. Given all of those above, it is obvious that the foundations of PE biodegradation are still not being addressed adequately to progress the basic investigations on biodegradability, comprising previous publications. The initial and most significant bit of fundamental data is the PE chemistry, which should be given with not only the monomer designation but also the molecular mass, level of the polymerization, and the arrangement of molecular mass as an adequate and fundamental characterization of the substances applied, corresponding to the quality of other (inorganic and organic) chemicals utilized in studies. Furthermore, microbiology must be refined for a mechanical comprehension of enrichment, parallel transporting, and medium content than isolation and characterization of isolates from non-meaningful media used with no deciding on stress, and confirmation of PE degradation is going to be conducted with the more appropriate techniques for accurate identification and proof than a single method. Furthermore, PE should be created deliberately from purified polymer rather than manufacturing machines to ensure the highest level of the findings and prevent interference with chemical compounds. Simultaneously, appropriate control reference materials must provide excellent study outcomes. Finally, upcoming creative PE biodegradation research should concentrate on microbial synergy, recombinant DNA technology, and methodologies such as metagenomics, genetic engineering, synthetic biology, directed evolution, and omics technologies. Without comprehensive prior information, organization, and methodology, the findings gained cannot validate the aims of an investigation, and such information has sadly permeated the academic literature. As a result, younger researchers must be prepared with fundamental, vital data to make crucial and selected decisions to add to scientific knowledge via a novel paradigm based on reliable scientific facts. Through study built on a strong basis, technological advances may be promoted to address real-world challenges with improved expertise and profound understanding.
Footnotes
Credit authorship contribution statement
Babbiker Gorish Conceptualization, Data curation, Formal analysis, Investigation, Writing — original draft; and Writing — review & editing. Waha Abdelmula: Conceptualization, Data curation, Formal analysis, Investigation, Writing — original draft; and Writing — review & editing, Hisham N. Altayeb: review & editing. Daochen Zhu: Supervision review & Funding acquisition.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Key R&D Program of China (Grant No. 2023YFC3403600) and the Key Research and Development Program of Jiangsu Province (Grant No. BE2021691).
Data availability statement
No data was used for the research described in the article.