Efficiency of Microorganisms and Effectiveness of Biodegradation Techniques on LDPE Plastics: A Systematic Review

Introduction

Plastics are synthetic polymeric molecules characterized by their versatility, lightness, low cost, and high durability.¹ Among the most common are polypropylene, polyethylene, nylon, and polycarbonate, which are considered highly persistent with a capacity for bioaccumulation; they also contaminate the soil, mainly cultivable areas, thus reducing the water filtration capacity and fertilization of plants.^2,3 Currently, they have become one of the most significant pollutants in marine ecosystems where most of these float and disintegrate into small fragments when exposed to the sun, taking the name of microplastics.⁴

Plastics are classified into¹: easily degradable, which includes biologically phase plastics such as compostable and biodegradable ones, and² difficultly degradable, among which thermoset and thermoplastic plastics such as polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polytetrafluoroethylene (PTFE), and low-density polyethylene (LDPE) can be mentioned.⁵ The latter, globally, are the most marketed, with a production of 390.7 million tons in 2021, of which 50% was produced in Asia and 22% in America.⁶ Polyethylene is the most commonly used plastic in everyday life, accounting for 96% of all plastics on the market.⁷

LDPE accounts for 64%, and is primarily used in the form of bags, wrappings, and containers, which are discarded after use.⁸ The mismanagement of plastic waste increases daily, mainly in Asian countries such as China, Indonesia, the Philippines, Vietnam, Malaysia, Thailand, and in Western countries like the United States. Of the plastics produced, considered to be 18 billion metric tons, 6% are incinerated, 23% are reused, 62% are disposed of in landfills, and 9% are considered recycled.¹

It has been demonstrated in-vitro that the ingestion of plastics by living beings produces a high impact on fauna. It is mentioned that they cause neurotoxic and degenerative damages in rodents, marine invertebrates, fish, and mammals, who are exposed to the presence of high levels of microplastics.⁹ Some studies in fish indicate that microplastic particles can cause oxidative damage to lipids in the gills and muscles, as well as neurotoxicity through the inhibition of acetylcholinesterase and alterations in neurotransmitter levels.¹⁰

Humans, as an important component of the ecosystem, are also affected by plastic waste. It has been estimated that a weekly intake of microplastics (MPs) with values ranging between 0.1 and 5 g can be found bound to food and drinking water, thereby generating adverse health effects.¹¹ In the city of Beijing, China, an analysis of feces conducted on young people between 18 to 25 years old who consumed water and food revealed the presence of microplastics such as polypropylene with a size of 20 – 800 nm.¹² Another study in Mexico found up to 30 microplastic particles in a series of foods such as energy drinks, tea, sodas, and beers¹³; another work conducted in Iran, in the analysis of bottled mineral water, found values of 8.5 ± 10.2 particles/L of PET, PS, PP.¹⁴

MPs are a globally recognized problem due to their prevalence in natural environments and the food chain, as well as their high impact on human health. Plastics directly affect living beings, either through ingestion or toxicity. It is noted that they could act as vehicles for invasive species and by adsorption on their surface of other synthetic chemical pollutants such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), or organochlorines, currently used by the chemical industry, thus potentiating or synergizing their toxic power due to components they contain such as plasticizers, heavy metal additives, etc.¹⁵ Studies on microbiota have allowed the assessment of the effect of MPs, especially PETs, on microbiota, demonstrating that they would act at the colon level, decreasing the values of Staphylococcus spp., Bifidobacterium spp., and Clostridium spp.¹⁶

An important aspect to consider is the degradation process of plastics, such as with LDPE, which can take up to 400 years to decompose.¹⁷ The process involves various microorganisms, mainly bacteria and fungi, that utilize mechanisms of photooxidation, thermal degradation, ozonation, mechano-chemical and catalytic degradation, and also biodegradation.⁵ The capability of hydroxylases, lipases, and laccases enzymes, secreted by LDPE-degrading microorganisms, which are responsible for breaking the polymer chain into low molecular weight fragments, must be mentioned.¹⁸ Extracellular enzymes play a very important role in biodegradation through the depolymerization of LDPE to form intermediate products that can be used as a carbon source by microorganisms,¹⁹ as they oxidize, reduce, hydrolyze, esterify, and cut the internal molecular structure of the polymer.²⁰

Microorganisms accelerate and increase the degradation process, making them an alternative to reduce the accumulation of petroplastics in the environment.²¹ There are reports of bacteria (Pseudomonas spp. and Bacillus spp.) and fungi (Aspergillus spp. and Fusarium spp.) that can degrade this plastic under laboratory conditions.^22,23 The use of more efficient microorganisms in the degradation of LDPE will allow the proper selection of bacteria or fungi with greater action and degradative efficiency of plastic.²⁴ At the industrial level, it will involve the handling of various effective methods of detection and quantification, such as gravimetry, scanning electron microscopy (SEM),^25–30 Fourier transform infrared spectroscopy (FTIR), and gas chromatography coupled with mass spectrometry (GC-MS), which complement the study of the polymer’s natural degradation.³¹

The purpose of this systematic review is to demonstrate the efficiency of LDPE-degrading microorganisms and the efficacy of the main biodegradation techniques on this type of plastics. The research question is: Which of the microorganisms is the most efficient and what techniques are the most effective in the biodegradation of LDPE-type plastics?

Methods

The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) methodology was used, which is established for systematic reviews and meta-analysis statements.³² The information was extracted from articles obtained from various databases such as: Scopus, Web of Science (WOS), Embase, and Google Scholar. The identification, screening, and eligibility of scientific articles were organized through the Zotero Bibliographic Manager.³³ The protocol of the systematic review was registered in PROSPERO (International prospective register of systematic reviews) under the number CRD42024506168.

The search strategy in all databases consisted of managing Boolean operators (AND, OR, and NOT), keywords (biodegradation, low-density polyethylene), years of publication (2010 – 2022), type of document (original article), language (Spanish, English), and open access publications. The DeCS/Mesh search terms were: Low-Density Polyethylene, LDPE, efficiency, biodegradation, microbial consortia, fungi, and bacteria.

The auxiliary search strategy included:

((TITLE-ABS-KEY (BIODEGRADATION) AND TITLE-ABS-KEY (LOW DENSITY POLYETHYLENE)) AND (LIMIT-TO (LANGUAGE,“English”) OR LIMIT-TO (LANGUAGE,“Spanish”)) AND (LIMIT-TO (PUBYEAR,2010) OR LIMIT-TO (PUBYEAR,2011) OR LIMIT-TO (PUBYEAR,2012) OR LIMIT-TO (PUBYEAR,2013) OR LIMIT-TO (PUBYEAR,2014) OR LIMIT-TO (PUBYEAR,2015) OR LIMIT-TO (PUBYEAR,2016) OR LIMIT-TO (PUBYEAR,2017) OR LIMIT-TO (PUBYEAR,2018) OR LIMIT-TO (PUBYEAR,2019) OR LIMIT-TO (PUBYEAR,2020) OR LIMIT-TO (PUBYEAR,2021) ORLIMIT-TO (PUBYEAR,2022)) AND (LIMIT-TO (EXACTKEYWORD,“Article”)) AND (LIMIT-TO (DOCTYPE,“ar”)) AND (LIMIT-TO (SRCTYPE,“j”)))

(ALL=(biodegradation)) AND ALL=(low density polyethylene) and 2022 or 2021 or 2020 or 2019 or 2018 or 2017 or 2016 or 2015 or 2014 or 2013 or 2012 or 2011 (Publication Years) and 28 Article (Document Types) and Article (Document Types) and All Open Access (Open Access) and English (Languages)

“biodegradation AND polyethylene AND low AND density AND [2010-2022]/py AND [article]/lim AND ([english]/lim OR [spanish]/lim”

The advanced search in this database included the exact phrase “LDPE biodegradation”; at least one of the following terms: Fungi, bacteria, or microbial consortia; terms mentioned in all scientific articles. It was also possible to delimit the years and languages of publication for each study.

Results

Figure 1 shows the PRISMA methodology, whose search protocol identified 483 primary articles, of which 133 duplicates, 279 by title, 5 by abstract, 11 by access, and 20 for not meeting the inclusion terms were excluded. A total of 35 full-text articles were obtained, and 15 were included from previous review.

Figure 1. Flowchart of identification, screening, eligibility, and included articles.

Table 1 indicates the number of articles found, totaling 50, with the highest quantity produced between the years 2018 to 2022. Also, it is observed that 100% of the articles correspond to experimental research works. Likewise, it points out the study techniques, with the most frequent being: SEM, gravimetry, and FTIR. Regarding the microorganisms identified in each of the studies, the most frequent in the phylum bacteria belonged to the genera Pseudomonas and Bacillus; as for the phylum of fungi, Aspergillus spp. predominated. As for the isolation site, the most used and mentioned in the reviewed articles were landfill soil and sanitary landfill.³²

Table 2 allows for the descriptive observation of microorganisms classified into bacteria, fungi, and microbial consortia, in quantities of 23, 17, and 9 respectively. Also, it specifies the species and the efficiency of LDPE-degrading microorganisms expressed in weight loss from highest to lowest and the days used for complete degradation, highlighting among bacteria Enterobacter spp., Pantoea spp., P. spp., Escherichia coli, and B. spp. which obtained an ED range of 9.00-70.00%, 24.00-64.00%, 1.15 – 61.00%, 45.00%, and 1.50-40.00% with TD of 4-150, 120, 4-150, 30, and 30-120 days, respectively; in the case of fungi, the main microorganisms are Neopestalotiopsis phangngaensis, Colletotrichum fructicola, and Thyrostroma jaczewskii with ED of 54.34, 48.78, and 46.34%, respectively, and TD of 90 days; and, the most efficient microbial consortia were from E. spp. and Pantoea sp. with ED of 38.00 – 81.00%, and TD of 120 days; and, P. protegens, Stenotrophomonas sp., B. vallismortis, Paenibacillus sp. with ED of 55.00 – 75.00% and TD of 120 days.

LDPE: Low-density polyethylene. IL: Isolation Location. MI: Identified Microorganism. TE: Study Technique. FTIR: Fourier Transform Infrared Spectroscopy. FTIR-ATR: Fourier Transform Infrared Spectroscopy - Attenuated Total Reflectance. SEM: Scanning Electron Microscopy. GC-MS: Gas Chromatography–Mass Spectrometry. EDS: Energy Dispersive X-ray Spectroscopy. FE-SEM: Field Emission Scanning Electron Microscopy. HT-GPC: High Temperature Gel Permeation Chromatography. TGA: Thermogravimetric Analysis. AFM: Atomic Force Microscopy. XRD: X-ray Diffraction. BATH: Bacterial Adhesion to a Hydrocarbon. GC-FID: Gas Chromatography-Flame Ionization Detector. DSC: Differential Scanning Calorimetry. NMR: Nuclear Magnetic Resonance.

Discussion

According to the data analyzed (Table 1), in recent years there has been an increase in studies on LDPE-degrading microorganisms with the aim of minimizing environmental impacts through bioremediation.⁷⁸ The requirement for special technologies allows understanding the degree of polymer disintegration and the nature of its resulting products. In the SR, up to 20 study techniques used in the biodegradation of LDPE have been detected. From our point of view, we consider highlighting in this article those methods that, due to frequency and especially efficacy, stand out among others. These methods include the following: SEM,^67,69–71 gravimetry,^{25,26,27,28,29,30,35,36,39,40} and FTIR.^20,28,60,66

The SEM study technique,^{19,42–44,46,49–54,56,62,63,70,72} is used to detect the biodegradation of LDPE and is employed to monitor changes on the surface of the LDPE film.^72,74–76 The adhesion of microorganisms to the surface is essential for biodegradation.⁶⁵ After incubating LDPE with selected degrading microorganisms on the surface, some characteristics such as erosion, holes, and cavities are observed, which are attributed jointly to the formation of bacterial film and the penetration of fungal hyphae.^60,68 Erosion is considered the primary cause of the mass reduction of the surface due to the secretion of enzymes and microbial extracellular metabolites.²⁰

Another frequently used technique in the reviewed articles is gravimetry,^{20,42–46,48–55,68–71} which is a simple and highly precise test to determine the polymer weight reduction, originating as a consequence of being used as a source of carbon and energy by microorganisms.^{19,62–65,72–74,76} This weight loss is considered proportional to the surface area, as biodegradation starts on the polymer’s surface.³⁸ With Gram-positive bacteria, the degradation efficiency (DE) in terms of LDPE weight loss has been reported: Streptomyces sp. DE 5.2% and degradation time (DT) of 90 days⁴⁰ and Bacillus amyloliquefaciens DE 11% and DT 60 days.³⁸

It was found in the review that various articles mentioned the FTIR technique,^{19,25,26,27,28,29,30,35,37,40,44,46,50,51,64,67–70,74–76} as the third most frequent of those applied to determine biodegradation; in addition, the cited studies consider it analytical and efficient, useful for identifying the chemical configuration of organic, polymeric, and inorganic material, and the morphological changes, which are supported by the chemical structural changes at the level of the carbon chains, observing new functional groups (alkoxy, acyl, carboxyls, and nitro) or absence of them, and modifications in the chains such as breaks, stretches, and formation of double bonds; moreover, this technique determines the carbonyl index (CI), which measures the degree of degradation of the LDPE and in which its value depends on the degraded carbonyl bonds.²⁸ In reality, it involves measuring the concentration of carbonyl groups (CG) corresponding to acids, aldehydes, and ketones.³⁵ In the process of LDPE biodegradation, the initial weight corresponds to the oxidation of the chain that leads to the formation of CG, and subsequently, these form carboxylic groups that are degraded by β-oxidation and then through the citric acid cycle to CO₂ and H₂O.⁷⁷

In Table 2, as observed, the microorganisms frequently reported in the articles analyzed in Table 1 include bacteria from the genera Bacillus, Brevibacillus, Cellulosimicrobium, Comamonas, Delftia, Enterobacter, Escherichia, Idonella, Kocuria, Lysinibacillus, Paenibacillus, Pantoea, Pseudomonas, Rhodococcus, Rhodotorula, Stenotrophomonas, and Streptomyces,^{25,28,30,42,46,58,59} with the genera Pseudomonas, Bacillus, and Streptomyces predominating. Other microorganisms are fungi, with Aspergillus sp. most frequently cited.^{39,48,50,52,63} Among the less frequently cited species include Rhizopus oryzae, Paenibacillus sp., Streptomyces coelicoflavus, Thiobacillus, Clostridium, Achromobacter denitrificans, Penicillium oxalicum, P. chrysogenum, Pycnoporus sanguineus, Enterobacter cloacae.^{29,37,44,45,54,55,61,74}

In the same table, it is analyzed that among the most efficient bacteria in the degradation of LDPE according to the weight loss of the polymer include several species such as Enterobacter spp. with an ED of 9.00 – 70.00%, and TD of 4 -150, Pantoea spp. with an ED of 24.00 – 64.00% and TD of 120, Pseudomonas spp. with an ED of 1.15 – 61.00% and TD of 4-150, Escherichia coli with an ED of 45.00% and TD of 30, and finally, Bacillus spp. with an ED of 1.50 – 40.00% and TD of 30-120. Bacillus sp. is also considered as another important species in the biodegradation process, having a consumption rate of 0.0019 g of the polymer per day⁴⁵; or, participating in consortia such as the one constituted by Bacillus vallismortis, Pseudomonas protegens, Stenotrophomonas sp., and Paenibacillus sp.⁴²

It has been determined that P. aeruginosa cultured on LDPE as the only carbon source has an ED of 0.0015 g of LDPE per day and a TD of 462 days to reduce a polyethylene film from 1g to 0.5g²⁰; Enterobacter cloacae AKS7 and Escherichia coli possess another type of degradative action, and it is due to the secretion of extracellular polymeric substances and the high hydrophobicity of the microorganism’s cell wall, which allows a greater formation and adherence of the bacterial biofilm.^36,61 In the case of Pantoea sp., its efficiency can be measured either individually or in consortium with Enterobacter.⁴¹

Fungi (Table 2), like bacteria, are considered LDPE-degrading microorganisms. The most efficient are: Neopestalotiopsis phangngaensis, Colletotrichum fructicola, and Thyrostroma jaczewskii with EDs of 54.34, 48.78, and 46.34%, respectively, and a TD of 90 days. The mycotic activity is considered to be due to their great capacity for adherence. In the polymer biodegraded for 40 days, the biofilm formed by the strongly adhered fungi is observed; at 80 days, surface deformation is evident and microcracks are differentiated.²⁸ Other efficient species are also reported, such as A. clavatus with an ED of 35.00% and a TD of 90 days²⁷ and A. versicolor with 40.60% and a TD of 90 days.⁴³

The degradative efficiency of microbial consortia has also been reported in different studies,^35,41,63,70 showing the most efficient to be the one formed by Enterobacter spp. and Pantoea sp., and the one of Pseudomonas protegens, Stenotrophomonas sp., B. vallismortis, and Paenibacillus sp. The cooperation of different microorganisms allows the use of different and complementary metabolic capacities for their growth, forming pure or mixed biofilms (fungi and bacteria), more resistant and metabolically more active.⁷⁹

LDPE-degrading microorganisms form a biofilm on the polymer and use it as a carbon source for consumption, an event that is reflected in weight loss. The biodegradation by microorganisms is a process of high metabolic activity, in which the count of viable cells, the concentration of surface proteins, and the efficiency in degradation expressed as polymer weight loss must be taken into account.²⁰ The most frequent place of isolation of microorganisms with LDPE-degrading capacity came from landfill soil and sanitary landfills with the presence of plastics. Various studies have indicated that bacteria and fungi adapt under different environmental conditions, a process mediated by complex cellular changes at the enzymatic level,^19,56 maintain physiology and metabolism, thus ensuring the survival of microorganisms. It has been demonstrated that bacteria such as Pseudomonas putida is a resistant and efficient xenobiotic decomposer because it presents an effective efflux pump; similarly, Streptomyces atacamensis shows xerotolerant latency and spore response to desiccation, and upregulation of proteins that are functional during xeric stress,⁸⁰ which probably explains why certain microorganisms are more efficient at degrading LDPE compared to others.

Considering that degradation is a slow process (this activity occurs before 60 days of incubation), and that degradation methods are heterogeneous, some authors conclude that there is no standard methodology in relation to analytical methods.¹⁵ However, in this article, we present the various biodegradation techniques, so a more precise vision could be had to assess which of them is the most consistent and effective according to their ED and TD.

Finally, based on the detection and quantification tests of polymer degradation, the exposed microorganisms constitute a sustainable alternative, useful for bioremediation and minimization of environmental impacts, with the aim of reducing environmental pollution by LDPE.

Conclusions