Microbial Degradation of Environmental Microplastics

Hongqin Guo; Kai Yang; Li Cui

doi:10.7536/PC240706

Progress in Chemistry ›› 2025, Vol. 37 ›› Issue (1) : 112-123 DOI: 10.7536/PC240706

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Microbial Degradation of Environmental Microplastics

Hongqin Guo ¹^,²
, Kai Yang ¹
, Li Cui ¹^,^*

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Abstract

Due to the highly stable chemical properties of plastics, plastic wastes disposed into environments are difficult to degrade and can only be broken down into microplastics with smaller particle size and larger surface area through the weathering process. Microplastic pollution has become one of the most pressing environmental issues. There is an urgent need to reduce microplastic pollution in order to protect the ecological and human health. Biodegradation of microplastics can ultimately convert microplastics into environmentally friendly substances such as biomass, CO₂, CH₄ and H₂O or other valuable intermediates. It is thus an environmentally friendly technology to potentially make microplastics harmless and resourceful. This paper reviews the present understanding of microplastics biodegradation processes, the influencing factors, the microbial and enzymatic resources for microplastics degradation, and the up-to-date approaches for mining plastics-degrading microbial resources. It finally provides perspectives on the challenges of current research and the direction of future research on microplastic biodegradation.

Contents

1 Introduction

2 Microplastic biodegradation process

2.1 Degradation pathway

2.2 Influence factors

3 Microplastic biodegradation resources

3.1 Degrading bacteria

3.2 Catabolic enzymes

3.3 Synthetic community

4 Mining strategies for microplastics-degrading microorganisms

4.1 Culture-dependent methods

4.2 Culture-independent methods

5 Conclusion and outlook

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Keywords

microplastics / plastic biodegradation / biodegradation process / biodegradation resources / mining strategies

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Hongqin Guo, Kai Yang, Li Cui. Microbial Degradation of Environmental Microplastics. Progress in Chemistry, 2025, 37(1): 112-123 DOI:10.7536/PC240706

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1 Introduction

Plastics are high molecular weight compounds synthesized through polymerization reactions. Due to their durability, they are widely used in various aspects of modern society. With the extensive use of plastic products, plastic pollution has become one of the most pressing environmental issues facing humanity today, attracting significant international attention. According to statistics, by 2015, the total global production of plastics had reached 8.3 billion tons, with 4.9 billion tons of plastic being landfilled or released into the environment^[1]. Because of their stable physical and chemical structures, plastics are very difficult to naturally degrade in the environment, persisting for a long time with a half-life ranging from 0.19 to 5,000 years (Table 1)^{[Table 1][2-3]}. Discarded plastics continuously break down through weathering processes in the environment, forming microplastics (with diameters less than 5 mm) and even nanoplastics (with diameters less than 1,000 nm), which have smaller particle sizes and larger specific surface areas^[4⇓-6]. These are difficult to recover and process from the natural environment, posing a potential serious threat to ecosystems and human health. Microplastics can enter the human body through the respiratory and digestive tracts and have been detected in multiple tissues and organs, including the intestines, lungs, liver, kidneys, blood, and placenta, potentially harming the nervous, immune, and reproductive systems^[7]. The United Nations Environment Programme has listed plastics as a global emerging pollutant^[8]. In May 2022, China issued the "Action Plan for the Control of New Pollutants," further strengthening the management of plastic pollution at the national level. To protect the ecological environment and public health, it is imperative to reduce and control microplastic pollution.

The traditional methods of dealing with waste plastics are mainly landfill and incineration. However, these disposal methods are not environmentally friendly. The landfill process not only occupies a large amount of land resources but also easily causes secondary pollution, such as soil and groundwater contamination triggered by the leaching of harmful chemical additives in plastics (such as plasticizers, flame retardants, and antioxidants). Although incineration can reduce the volume of plastics, it emits greenhouse gases and volatile toxic organic compounds, which goes against the concept of "carbon neutrality." Environmental microorganisms are important drivers of material cycling in ecosystems. In the long-term evolutionary process, microorganisms have continuously adapted to various pollutants produced by human activities, and can even use specific biochemical reactions to degrade and transform them for obtaining matter and energy. Compared with traditional measures for reducing plastics, the microbial degradation of microplastics utilizes the widely present microorganisms in the environment to ultimately convert plastic waste into environmentally harmless substances like carbon dioxide and water, or other high-value intermediate products. This has significant potential for reducing environmental microplastic pollution. The research trends in the microbial degradation of microplastics mainly include: ① In scientific understanding, from the isolation and verification of degrading microorganisms, gradually deepening to the analysis of degradation pathways and functional mechanisms; ② In research methods, from traditional isolation and cultivation to the combined use of multi-omics technologies, single-cell technologies, and high-throughput screening technologies; ③ In engineering applications, from the in-situ colonization of efficient degrading bacteria to the design of synthetic functional bacterial communities and the genetic engineering modification of key degrading bacteria and enzymes. Key milestones include: ① The discovery of the biodegradation phenomenon of plastics: In 2010, Albertsson^[9] used radiolabeled isotopes to reveal the phenomenon of environmental microorganisms degrading plastics, proposing the possibility of plastic biodegradation; ② Screening of degrading bacteria and elucidation of degradation mechanisms: In 2016, Yoshida et al.^[10] first screened and identified an efficient PET-degrading bacterium (Ideonella sakaiensis 201-F6) and enzyme (PETase), elucidating the degradation mechanism. Since 2015, Yang et al. and Luo et al. have isolated plastic-degrading microorganisms from the intestines of plastic-degrading insects (such as mealworms, superworms, earthworms, and cockroaches)^[11⇓-13]; ③ Genetic engineering and synthetic biology: Since 2020, Zhu et al.^[14] and Tournier et al.^[15] have used genetic engineering and synthetic biology techniques to redesign the structure and function of specific enzymes involved in microplastic degradation, and combined engineered bacteria for the efficient expression and in-situ environmental remediation of key degrading enzymes. For example, the efficient expression of the PETase gene in E. coli achieved good PET degradation results^[14]; ④ Commercial application: In the past five years, multiple international research teams and companies have been dedicated to developing microbial degradation technologies that can be applied on a large scale, promoting the commercialization of plastic degradation technologies^[15]. These research trends and milestones mark the rapid development of the field of microplastic biodegradation, revealing the potential of microorganisms in plastic degradation. This article will review the degradation mechanisms, influencing factors, degradation resources, and exploration methods of microplastics, and will also look ahead to the challenges and future directions of current microplastic degradation research.

2 The Microbial Degradation Process Of Microplastics

The microbial degradation of environmental microplastics utilizes the microorganisms and enzymes present in the environment to ultimately convert microplastic waste into environmentally harmless substances such as biomass, CO₂, CH₄, and H₂O, or other high-value intermediate products. It is an environmentally friendly resource recovery and harmless control technology.

2.1 Degradation Process

The biodegradation process of microplastics can be divided into four stages, and the degradation of microplastics relies on microorganisms to collaboratively perform the four degradation steps^[16].

(1) Biodegradation: alters the chemical and physical properties of polymers. As a non-polar polymer, plastic has limitations in biodegradation due to its hydrophobic surface characteristics. The biodegradation process begins with the adhesion and colonization of microorganisms on the surface of the polymer. Through the formation of biofilms by microorganisms, the physicochemical properties of the polymer surface are modified, reducing the hydrophobicity and durability of the plastic material, thus promoting the microbial degradation of microplastics.

(2) Depolymerization: Microplastics are cleaved into oligomers, dimers, or monomers by enzymes released from microorganisms. Depolymerization mainly includes two primary reactions, namely, the reduction of polymer molecular weight and the oxidation of low-molecular-weight molecules^[17]. Microorganisms colonizing the surface of microplastics further secrete extracellular and intracellular enzymes, attacking the molecular chains of plastics, causing the breakage of hydrolyzable chemical bonds or the ends of molecular chains, producing monomers, dimers, and other oligomers, which leads to a decrease in the molecular weight of the microplastics. At the same time, under the action of oxygen and environmental ROS, further oxidation occurs, forming oxygen-containing functional groups such as hydroxyl, carbonyl, and carboxyl groups^[18-19], promoting the further degradation of microplastics. Hydrolases (such as lipases, esterases, proteases, etc.) and redox enzymes (such as laccases, monooxygenases, oxidases, peroxidases, alkane hydrogenases, etc.) are two major types of enzymes for degrading microplastics. Hydrolases are a class of enzymes with the ability to catalyze the hydrolysis of substrates, capable of degrading plastics like PET, PLA, PU, PVA, which contain easily hydrolyzable chemical bonds (such as C=O bonds, ester bonds). Redox enzymes are a class of enzymes with lignin-degrading capabilities, capable of degrading microplastics like PE, PS, PP, PVC, which contain less easily hydrolyzable C—C single bonds (Figure 1).

(3) Assimilation: The oligomers formed after chain cleavage are transported into the microbial cytoplasm during the assimilation stage. The oligomers released into the surrounding environment, with some of the lower molecular weight ones, enter the microbial cells through different membrane transport systems for further metabolism^[17].

(4) Mineralization: Oligomers that enter the microbial body undergo catabolism, forming CO₂, CH₄, and H₂O. Microplastic derivatives entering microbial cells are gradually stripped of carbon atoms from the polymer chains through a β-oxidation mechanism, generating acetyl-CoA which enters the tricarboxylic acid cycle (TCA cycle), oxidizing organic matter and producing ATP to provide energy for the cell^[20]. Ultimately, after a series of metabolic processes within the body, the oligomers undergo mineralization, converting into biomass within the cell while also producing CO₂, CH₄, and H₂O ^[21].

2.2 Influencing Factors

2.2.1 The Physicochemical Properties Of Microplastics

The types, structures, crystallinity, molecular weight, hydrophilicity and hydrophobicity, and additives of microplastics all affect the biodegradation efficiency of microplastics^[20]. The degradation of microplastics mainly depends on their chemical composition. Compared with other synthetic polymers, starch-based plastics, which are made primarily from starch and its derivatives, such as PLA, PVA, PHA, PBAT, etc., are more easily invaded by microorganisms that secrete enzymes to break down starch into smaller carbohydrates, thus exhibiting strong biodegradability^[22]. However, biodegradable plastics typically require controlled conditions for effective decomposition. For example, PLA and PBAT degrade at a rate of 90% within 180 days under anaerobic composting conditions at 70 ℃^[22]. In natural environments, such as seawater, soil, landfills, or through home composting, the degradation process is much slower, often requiring decades^[22]. Polyester microplastics, such as PET, PU, PCL, etc., contain hydrophilic groups like hydroxyl, carboxyl, and carbonyl, as well as hydrolyzable ester bonds, making them susceptible to microbial colonization and enzymatic degradation^[23]. On the other hand, polyolefin microplastics, which have a stable carbon chain backbone composed of high-energy C—C bonds and lack groups that are easily oxidized and hydrolyzed, are less prone to biodegradation^[22]. Additionally, the structure of microplastics also affects their biodegradability. For instance, different stereoisomers of PLA exhibit varying water absorption; meso-PLA, which has higher water absorption, degrades more efficiently under the action of proteinase K than racemic PLA^[24]. The crystallinity of microplastics also influences their degradation. Polymers with higher crystallinity have more stable chemical structures and properties, while semi-crystalline or amorphous polymers have looser molecular chain arrangements, providing more voids and active sites, making them more susceptible to attack by microorganisms and enzymes. Therefore, selecting plastics with lower crystallinity or reducing the crystallinity of microplastics can promote their biodegradation^[25]. Moreover, the molecular weight of microplastics is an important factor affecting their degradation. Lower molecular weights are more easily utilized and degraded by microorganisms and enzymes. For example, gut microbiota in yellow mealworms and barley worms can efficiently degrade low molecular weight (M_w<6.2 kDa) polyolefin microplastics like PP, PE, PS, but can only partially degrade high molecular weight microplastics^[26]. Microplastics with higher surface hydrophobicity limit the adhesion of microorganisms on the polymer surface, restricting biofilm formation and reducing the degradation of microplastics by microorganisms and enzymes within the biofilm. Furthermore, various additives in some microplastic products, such as preservatives, antioxidants, plasticizers, flame retardants, curing agents, biocides, and thermal stabilizers, also affect the biodegradation performance of microplastics. For instance, nucleophilic bromide ions produced by the decomposition of the flame retardant decabromodiphenyl ether at high temperatures can react with positively charged groups on the polycarbonate (PC) chain, accelerating the depolymerization of PC in subcritical water; whereas the nonpolar part of the plasticizer di-n-octyl phthalate embedded in the PC molecular chain inhibits further diffusion of solvent molecules into the polymer, suppressing the hydrolysis of PC^[27].

2.2.2 Environmental Factors

Environmental factors play a crucial role in enhancing the effectiveness of microbial growth on polymer surfaces. Environmental factors such as light, temperature, humidity, oxygen, and pH can alter the structure and properties of microplastics, thereby affecting their degradation. These factors also influence the diversity, relative abundance, and life activities and metabolic potential of microorganisms, which in turn affect the biodegradation of microplastics^[16,20]. For example, a low pH is conducive to the formation of hydrogen peroxide during photo-oxidation, thus promoting the degradation of microplastics^[28]. Environmental factors such as solar radiation, high temperatures, hydraulic impacts, abrasion by sand, and weathering can accelerate the aging of microplastics, making their surfaces rougher and less hydrophobic. Additionally, the oxygen-containing functional groups (such as carbonyl, carboxyl, and hydroxyl groups) formed during aging increase the likelihood of microbial contact and colonization, thereby enhancing the efficiency of microplastic degradation^[20].

2.2.3 Biological Factors

Biological factors include the diversity of species, differences in microplastic-degrading enzymes, and genetic variations. The differences in the ability of organisms such as bacteria and fungi to degrade microplastics are partly due to the types and active sites of enzymes produced by different microorganisms, which affect the contact efficiency between enzymes and substrates, thereby influencing the degradation of microplastics. On the other hand, extracellular polymers secreted by microorganisms can enhance their adhesion to the surface of microplastics, thus improving the degradation capability. In hotspots of microplastic pollution, long-term adaptation has led to the selection and evolution of intestinal, rhizosphere, and phyllosphere microorganisms in animals and plants, forming focal points for microplastic degradation. Animals such as cockroaches, earthworms, mealworms, and yellow mealworms can fragment microplastics through chewing and grinding, altering their physicochemical properties. Simultaneously, their gut microorganisms and secreted enzymes can oxidize microplastics, producing small molecular polymers, which promote the degradation of microplastics^{[13,29 -30]}. Some rhizosphere microorganisms, aquatic algae, and their secreted enzymes can cause physical changes in microplastics, promoting their degradation. For example, fungi like Alternaria alternata found in the pneumatophores of mangrove plants can colonize the surface of LDPE and secrete laccase and manganese peroxidase, disrupting the structure of microplastics and producing low-molecular-weight polymer fragments, thus facilitating the degradation of microplastics^[31]. Additionally, microbial consortia composed of multiple bacterial species often show more significant effects on microplastic degradation compared to single strains. For instance, a single degrading bacterium can reduce the weight of microplastics by 20%, whereas a combination of four strains can achieve a weight reduction of nearly 50% within 140 days^[32].

3 Microplastic-Degrading Bacteria and Degradation Enzymes

Severe environmental microplastic pollution urgently requires reduction and control. Mining for highly efficient and colonizing microplastic-degrading resources in environmental microorganisms, deeply analyzing their metabolic pathways, degradation mechanisms, and interaction relationships, designing and constructing synthetic microbial communities containing multiple specific functional microorganisms, optimizing the structure-function relationship, and elucidating its principles can help achieve enhanced targeted reduction and control of different environmental microplastics. This will improve the scientific and precise nature of microplastic pollution prevention and control, ensuring ecological environment and human health.

3.1 Microplastic-Degrading Microorganisms

More than 416 types of microorganisms with the potential to degrade microplastics have been reported^[33], mainly distributed in the phyla Pseudomonadota, Actinobacteriota, Firmicutes, and Bacteroidota^[16,22] (Figure 2). Bacteria from families such as Actinomycetaceae, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacillaceae, and Flavobacteriaceae are the primary functional microorganisms for plastic degradation. Among them, Pseudomonas aeruginosa, Rhodococcus sp., Amycolatopsis sp., Aspergillus sp., Bacillus sp., and Cladosporium sp. are the main bacterial genera and species proven to be associated with microplastic degradation^[34]. For example, Pseudomonas aeruginosa can degrade microplastics such as PS, PET, PE, and PVC, while Bacillus sp. can degrade microplastics like PS, PET, PE, and PP^[17,33,35]. In recent years, research on the degradation of plastics by insects has provided new directions and strategies for the efficient biodegradation and resource recovery of waste plastics. Over the past decade, the gut microorganisms and enzymes of lepidopteran and coleopteran larvae, such as mealworms, cotton bollworms, superworms, lesser mealworms, and waxworms, have been found to degrade various microplastics, including PS, PE, PP, PVC, PLA, and PET^[20,36]. The main potential microplastic-degrading bacteria in insect guts include genera such as Pseudomonas, Spirochaeta, Enterococcus, and Acinetobacter^[21,37].

In addition to bacteria, fungi also possess the ability to degrade microplastics. The filamentous branched structure of fungi has a powerful capacity for extension and penetration, with hyphae releasing enzymes through exocytosis to degrade microplastics. Fungi have a robust enzymatic system, adsorption capabilities, and the ability to produce natural biosurfactants, allowing them to utilize microplastic substrates as sources of carbon and electrons, providing materials and energy for cells^[38]. Currently, more than 256 species of fungi capable of degrading microplastics have been reported, among which Ascomycota, Basidiomycota, and Mucoromycota are the main fungal phyla involved in microplastic degradation^[33] (Figure 3). For example, fungi such as Rhizopus, Mucor, and Aspergillus can degrade PLA, while Mucor, Aspergillus flavus, and Streptomyces strains are also involved in the degradation of starch-based PE^[23]. Bacteria of the genus Penicillium can degrade various types of microplastics, including PLA, PU, PE, PCL, PHA, and PP^[33].

It is worth noting that although a large number of microplastic-degrading bacteria have been screened from environmental microbial communities, the degradation capabilities of some microorganisms have not been fully verified due to differences in characterization methods and experimental operations. Even when the degradation capability has been confirmed, the degradation effects of these microplastic-degrading bacteria in practical environmental applications are not ideal, still facing challenges such as weak colonization ability, poor stability of key degrading enzymes, and low degradation efficiency^[39]. Additionally, the vast majority (> 90%) of environmental microorganisms cannot be isolated and cultured in laboratory environments^[40], with major limiting factors including unknown metabolic requirements, unclear physiological states, and low species abundance. However, many uncultured microorganisms may play significant roles in the in situ environmental degradation of microplastics. Moreover, in situ degradation may involve the division of labor and cooperation among multiple microorganisms^[41], making it difficult for a single strain to effectively degrade and reduce microplastic pollution in the environment^[42]. Finally, microplastics themselves can easily adsorb toxic pollutants from the surrounding environment, which may be toxic to microplastic-degrading microorganisms^[43], thereby inhibiting their degradation activity. Therefore, there is an urgent need to use culture-independent techniques to find strains or microbial consortia that have efficient in situ microplastic degradation capabilities and can colonize in polluted environments.

3.2 Enzymes For Microplastic Degradation

Enzymes are highly efficient biological catalysts that offer numerous advantages over chemical processes, including: (1) high catalytic efficiency, which can accelerate each fundamental reaction step by up to 10²⁰times^[44]; (2) low environmental impact, reducing energy consumption and waste generation^[44]. Enzymes are considered as renewable and biodegradable catalysts, which can be achieved through less expensive and environmentally friendly fermentation processes. Currently, the commonly used databases for plastic-degrading genes and enzymes include the Kyoto Encyclopedia of Genes and Genomes (KEGG)^[45], Carbohydrate-Active Enzymes Database (CAZy)^[46], Metabolic Pathways and Enzyme Database (MetaCyc)^[47], Microbial Metabolism and Genome Database (BioCyc)^[48], Plastic Microbial Degradation Database (PMDB)^[49], Plastic-Degrading Microbes and Proteins Database (PlasticDB)^[33], and Bioremediation Enzyme Prediction Database (RemeDB)^[50], among others. These databases provide information on microbial metabolic pathways, microbial enzymes, and functional genes for microplastic degradation, thus facilitating the assessment of biodegradation processes. More than 20 types of enzymes have been reported to be involved in the biodegradation of various microplastics, including laccases, esterases, hydrolases, hydroxylases, dehydrogenases, monooxygenases, oxidoreductases, peroxidases, chitinases, cutinases, lipases, etc.^[33](Figure 4). Enzyme-mediated depolymerization is one of the initial and critical steps in the biodegradation process of microplastics^[51]. Depolymerizing enzymes such as PLA depolymerase, PCL depolymerase, PHA depolymerase, etc., can catalyze the hydrolysis of polymer backbones, leading to the cleavage of polymers into oligomers and monomers, making microplastics more accessible to other enzymes^[44]. Oxidases, such as laccases, oxygenases, monooxygenases, peroxidases, etc., promote the oxidation and breakdown of complex microplastic molecules into smaller molecules or soluble compounds, thereby facilitating further degradation of microplastics^[21,52]. For example, laccases can oxidize the polymer chains of PS, PE, PVA, and other microplastics, while monooxygenases can cleave PS aromatic rings through main-chain and side-chain cleavage. Lignin peroxidase and manganese peroxidase play significant roles in the degradation of PE. Hydroxylases can catalyze hydroxylation reactions, increasing the hydrophilicity of microplastics, making substrates more water-soluble and easier for further microbial degradation and metabolism^[44,53-54]. Hydrolases can further accelerate the breakdown of polymer chains into smaller, more easily metabolized components, playing a crucial role in the biodegradation of microplastics. Hydrolases have been reported to have the ability to degrade plastics such as PCL, PET, PLA, PA, and PHA^[55]. For instance, two hydrolases, PETase and MHETase, produced by Ideonella sakaiensis, can hydrolyze PET and its intermediate product, mono(2-hydroxyethyl) terephthalate, respectively, generating terephthalic acid and ethylene glycol^[10]. Dehydrogenases are also essential for the microbial metabolism of the decomposition products generated from microplastic degradation. When microplastics are broken down into smaller organic molecules by other enzymes, dehydrogenases are responsible for catalyzing the oxidation of these molecules, producing reduced coenzymes (NADH or NADPH), which are subsequently used in the electron transport chain to generate ATP. This process allows microbes to obtain energy from the decomposition products of microplastics, promoting their growth and the degradation process^[56].

Enzymes that degrade microplastics are closely related to those that degrade natural polymers, and it has been reported that the pathways for degrading PS and lignin are highly similar^[21]. The ability of enzymes to degrade microplastics may stem from their long-term selection, adaptation, evolution, and specialization in degrading natural polymers, which helps microorganisms and their hosts utilize specific nutrients (e.g., microplastics feeding insect larvae) and adapt to environmental changes (e.g., microplastic pollution)^[52]. Therefore, more enzymes capable of degrading natural polymers need to be further explored for their potential in degrading microplastics.

3.3 Synthetic Microbiota

Synthetic microbial communities are artificially constructed consortia of multiple species co-cultured for specific functions. Due to the metabolic interactions and functional complementarity among the microorganisms, synthetic microbial communities exhibit stronger colonization ability, better functionality, and higher environmental adaptability compared to single-species cultures. In the field of microplastic degradation, synthetic microbial communities can be designed to carry specific enzymes or to have synergistic effects, working together to effectively degrade specific types of microplastics, thus helping to address the issue of microplastic pollution. For example, a consortium consisting of four strains, including Aneurinilyticillus aneurinilyticus, Brevibacillus agri, Brevibacillus sp., and Brevibacillus brevis, isolated from activated sludge in wastewater treatment plants and landfills, can cause mass losses of 58.2%, 46.6%, and 56.3% in LDPE, HDPE, and PP, respectively, within 140 days, whereas individual strains only caused mass losses of 22.2% to 28.2% in microplastics^[32]. Additionally, studies have shown that marine microbial consortia have better degradation effects on polymers when they work synergistically^[57]. Similarly, synthetic microbial communities constructed using microplastic-degrading strains isolated from plastic waste processing plants show higher PE degradation efficiency than single strains^[58]. Moreover, the rational configuration of synthetic microbial communities can utilize microplastics to produce other high-value-added products. For instance, Qi Qingsheng et al. designed a co-culture system of PET-degrading yeast (Yarrowia lipolytica Polf) and PHB-producing bacteria (Pseudomonas stutzeri), achieving the simultaneous degradation of PET and synthesis of the economically valuable biodegradable plastic PHB^[59].

Quantitative and targeted design based on synthetic microbial communities, and the customization of functional microbial consortia for degrading and synthesizing microplastics, can provide new strategies for the enhanced and targeted reduction of microplastics, achieving the prevention and control of environmental microplastic pollution. At the same time, it is necessary to pay attention to the biosafety and environmental risks during the use of synthetic microbial communities, ensuring the safety and sustainability of their applications.

4 Methods For Mining Microbial Resources For Microplastic Degradation

Microplastic-degrading microorganisms can convert environmental microplastics into harmless substances such as carbon dioxide and water, or other high-value intermediate products, achieving the harmless treatment and resource utilization of environmental microplastics. Developing efficient and highly sensitive methods for mining and characterizing microplastic-degrading microbial resources helps to further explore these microbial resources and screen for in-situ highly effective degrading microorganisms, which is of great significance for the management of environmental microplastic pollution. The main methods are divided into culture-dependent and culture-independent categories (Figure 5).

4.1 Culture-Based Methods

The isolation and cultivation of microorganisms is a classic method for exploring microbial resources and is also the most critical step in understanding and utilizing microorganisms. Microorganisms, having evolved over long periods in various ecological niches within natural environments, have formed a rich and diverse genetic resource, which holds significant potential for environmental remediation of pollutants. Microplastic-degrading microorganisms are typically isolated and cultivated from areas heavily contaminated with microplastics, such as agricultural film soil, marine sediments, waste recycling stations, and insect intestines. The traditional process of isolation and cultivation involves inoculating environmental samples, enriched with microplastic-degrading microorganisms, into selective media where microplastics serve as the sole carbon source. This is followed by enrichment and subculturing under suitable conditions. Potential plastic-degrading microorganisms are then isolated and purified using gradient dilution or streak plate methods. Subsequently, the plastic degradation capabilities of the purified degrading microorganisms are verified, and their phylogenetic species are analyzed based on 16S rRNA gene sequences^[10,60-61]. Currently, many researchers use this method to isolate strains that can utilize microplastics as their sole carbon and energy sources, and study their degradation pathways and molecular mechanisms. Yoshida et al.^[10] isolated and cultivated a PET-degrading bacterium, Ideonella sakaiensis 201-F6, from a waste recycling station, which could use PET as its primary energy and carbon source. They identified a key PET hydrolase responsible for degradation, providing an in-depth insight into the degradation mechanism. Ju Feng et al.^[52] isolated a PVC-degrading bacterium, Klebsiella sp. EMBL-1, from the intestines of insect larvae, capable of depolymerizing and utilizing PVC as its sole energy source.

4.2 Culture-Independent Methods

4.2.1 Omics Technologies And Big Data Algorithms

In recent years, with the advancement and cost reduction of sequencing and detection technologies, the widespread application of omics technologies has provided a cultivation-independent approach for studying and understanding the composition and functions of microorganisms in complex environments. Metagenomic technology, through high-throughput sequencing of environmental sample DNA, followed by gene sequence assembly, annotation, and analysis, offers a wealth of information on the composition of in situ environmental microbial communities and the functional genes involved in microplastic degradation^[62⇓-64]. For example, Zhou et al.^[63] used metagenomic technology to study microplastic degradation genes in a subtropical estuarine environment in China, finding that human activities significantly impact the abundance of plastic degradation genes and using metagenome binning techniques to reveal the potential for microplastic degradation in various microbial groups. Metatranscriptomic technology, through sequencing of transcripts from environmental samples, can be used to study active microorganisms and the functional activity of microplastic degradation in the environment, as well as to compare differentially expressed genes and degradation pathways under varying conditions. Mamtimin et al.^[21] conducted metatranscriptomic sequencing of gut microbes in mealworms, discovering that the pathways for degrading polystyrene (PS) and lignin are highly similar, suggesting that the plastic degradation ability of insects may originate from their long-term selective adaptation to natural lignin degradation.

The integration of multiple omics data, such as genomics, transcriptomics, proteomics, and metabolomics, can provide a more comprehensive understanding of the degradation process and mechanisms of microplastics^[65]. Wright et al.^[61] used multi-omics techniques to study the succession process of marine microbial communities in the degradation of PET, isolating two strains of PET-degrading bacteria and further elucidating their degradation mechanisms. Meyer-Cifuentes et al.^[41] utilized integrated multi-omics analysis to investigate the degradation process of poly(butylene adipate-co-terephthalate) (PBAT) by marine microbial enrichments, finding that the degradation was completed through the division of labor among various microorganisms. It revealed that the depolymerization process was mainly carried out by attached biofilm communities, while all microbial communities participated in the degradation process of monomers. Although multi-omics technologies reflect the degradation potential of microplastics, they also provide new insights into cultivating microplastic-degrading microorganisms in situ environments. Most microorganisms in natural environments are still difficult to isolate and culture. Metagenomic binning tools can assemble draft genomes of environmental microplastic-degrading microorganisms based on sequencing results, thereby allowing for the analysis and estimation of their growth conditions, nutritional requirements, and metabolic physiology^[66⇓-68]. This provides a basis for cultivating key strains of microplastic degraders in complex communities and brings hope for utilizing the rich genetic resources of uncultured microorganisms.

The challenge of omics technology lies in bioinformatics analysis and heavily relies on the information already available in databases. The microbial diversity in natural environments is extremely high, while the known genomic information in databases is relatively limited. Moreover, the development speed of algorithms and software for genome analysis lags far behind the progress of sequencing technologies and the massive amount of sequencing data generated^[42]. Additionally, the information in databases helps to understand microbial metabolic pathways, thereby facilitating their isolation and cultivation by altering culture conditions^[67-68]. The combination of big data with artificial intelligence and machine learning algorithms has opened up new prospects for exploring the functional potential and mechanisms of microplastic-degrading microorganisms. Based on the existing information in databases regarding genes or enzymes related to microplastic degradation, algorithms can be used to predict potential degradative gene resources in microbial sequencing data. For example, Sankara Subramanian et al.^[50] developed the RemeDB tool using a Hidden Markov Model (HMM)-based search algorithm, simplifying the identification of microplastic-degrading microorganisms. Danso et al.^[69] identified 504 potential PET hydrolase genes from the database using an HMM algorithm and functionally verified four of them. Using a similar approach, Tan et al.^[70] identified a dioxygenase, HIS1, capable of degrading polypropylene, which was heterologously expressed and its degradation function was verified. Furthermore, using global genomic databases to obtain information on degradative genes and potential degrading microorganisms can promote understanding of core degrading communities and their degradation patterns. Qian Haifeng et al.^[71] used a machine learning model to predict and map the global oceanic microplastic degradation potential, finding that regions severely polluted by microplastics have a high potential for microbial degradation, with Proteobacteria being the main group of microplastic-degrading bacteria.

4.2.2 Nano Secondary Ion Mass Spectrometry

Studying environmental microorganisms at the single-cell level can overcome the limitations of pure cultures and enable in situ functional studies under complex environmental conditions. Due to methodological constraints, current methods for determining the degradation of microplastics by microorganisms, such as measuring the weight loss of microplastics, using infrared spectroscopy to determine surface oxidation, and observing surface morphology with electron microscopy, are all indirect and cannot conclusively determine whether the degradation is caused by microbial or abiotic processes. Additionally, these methods lack sensitivity and directness, requiring time-consuming experimental procedures. Moreover, existing methods struggle to track the transformation of carbon from microplastics into degradation products or its conversion into microbial biomass. Nanoscale secondary ion mass spectrometry (NanoSIMS) is currently the most spatially resolved and sensitive analytical technique in the field of environmental microbiology. It works by bombarding the sample surface with a primary ion beam, generating a series of secondary ions for detection, thereby enabling high-resolution imaging of the elemental composition and isotope distribution of the sample. Vaksmaa et al.^[72] used ¹³C-labeled, difficult-to-degrade plastic PE co-incubated with Rhodotorula mucilaginosa yeast isolated from the ocean, demonstrating that UV-aged ¹³C-PE could be assimilated into their own biomass within 5 days, with an estimated degradation rate of approximately 3.8% of its total mass per year. Furthermore, Sander et al.^[73] used ¹³C stable isotope-labeled biodegradable plastic PBAT co-incubated with soil, and imaged the incubated samples using NanoSIMS, finding that PBAT could be assimilated by filamentous fungi in the soil to form their own biomass, and measured the amount of ¹³CO₂ produced during the 42-day incubation process. However, since NanoSIMS is a destructive technique, it is not possible to conduct important downstream studies on the detected microplastic-degrading microorganisms, such as sorting, sequencing, or subsequent cultivation, making it difficult to elucidate the metabolic pathways of degrading microorganisms and limiting a deeper understanding and utilization of these functional microorganisms.

4.2.3 Fluorescence Labeling Technology

In recent years, fluorescence analysis methods have been increasingly used for the screening of microplastic-degrading microorganisms or enzymes due to their high sensitivity and high-throughput detection. The key issue in applying fluorescence screening is how to convert the function of microplastic degradation into a fluorescence signal. Currently, the fluorescence detection of microplastic degradation mainly focuses on polyester plastics, utilizing esterases that can specifically hydrolyze carboxylic ester bonds, leading to the release of a fluorescent probe, thereby achieving the effect of screening esterases. The principle involves modifying small molecular esters of microplastic monomers with fluorescent groups^[74-75]. Under normal covalent bonding, the fluorescence is in a quenched state; only after enzymatic hydrolysis does the fluorescent molecule get released, thus generating a fluorescence signal. Zumstein et al.^[76] embedded fluorescein dilaurate into a polyester matrix, characterizing the hydrolysis process of polyesterase by detecting the production of fluorescein. In addition, combining this principle with a microfluidic platform allows for the screening of microorganisms or hydrolases with degrading functions at the single-cell level, providing new technical means for exploring microplastic degradation resources. Du Wenbin et al.^[75] developed a fluorescence-activated droplet sorting platform for high-throughput screening of PET-degrading microorganisms or enzymes (PETase). By dispersing individual microorganisms in single droplets and incubating them with a PET analog substrate, fluorescein dibenzoate, the substrate can be hydrolyzed by PETase, emitting a fluorescence signal for high-throughput sorting. Using this method, nine different genera of PET-degrading bacteria were obtained from wastewater samples from a PET textile factory, and two potential new PET-degrading enzymes were identified from highly active strains, which were heterologously expressed to verify their degrading functions^[75]. Xu et al.^[74] proposed a polyurethane analog fluorescent probe, modifying naphthalimide derivative molecules on both sides of the polyester monomer. When degraded by polyesterase, two probe molecules are released, emitting fluorescence. Furthermore, the feasibility of using this probe for screening polyester-degrading microorganisms or enzymes was verified in combination with droplet microfluidics. Although these approaches provide new high-throughput technical means for exploring microplastic-degrading microorganisms and enzymes, current strategies rely on the design and synthesis of fluorescent probes and plastic monomers.

4.2.4 Single-Cell Raman Technology

Raman spectroscopy is a molecular vibrational spectroscopy that can provide phenotypic fingerprint information of bacteria at the single-cell level, such as proteins, nucleic acids, lipids, carbohydrates, and pigment molecules. When combined with stable isotope labeling (such as ¹³C, ²D, ¹⁵N), because the heavy isotopes in newly synthesized biomolecules replace the light isotopes in the original biomolecules after microorganisms assimilate the isotope-labeled substrates, causing a red shift in the corresponding Raman peaks, it can effectively analyze the functional activity of individual microorganisms in situ^{[77⇓⇓⇓-81]}. Single-cell Raman combined with stable isotope labeling has been used to study in situ degrading bacteria in complex environments. For example, Huang Wei et al. used single-cell Raman combined with ¹³C-labeled polycyclic aromatic hydrocarbons to analyze in situ degrading bacteria in groundwater samples at the single-cell level, and simultaneously linked different species and in situ degradation activities through fluorescence in situ hybridization^[82], finding that uncultivated bacteria play a key role in the in situ environmental degradation of polycyclic aromatic hydrocarbons^[83]. Additionally, for systems without suitable stable isotope-labeled substrates, single-cell Raman can be combined with heavy water labeling to study metabolically active bacteria. For instance, in soil with only fixed-state phosphorus, after incubation with heavy water, the activity of phosphorus-solubilizing microorganisms was significantly higher than that of non-phosphorus-solubilizing microorganisms, producing characteristic C-D Raman peaks, while low or inactive non-phosphorus-solubilizing microorganisms had weak or no C-D peaks. The C-D Raman marker peak can be used to identify in situ phosphorus-solubilizing bacteria in the soil environment^[84]. Using a similar principle, this method can also be applied to the study of cellulose-degrading bacteria. In cases where cellulose is the sole carbon source, it can reveal the heterogeneity of cellulose degradation activity at the single-cell level^[85]. Furthermore, by using single-cell Raman combined with heavy water labeling, in situ identification of antibiotic-resistant bacteria (under antibiotic pressure) and metabolically active bacteria in the biofilm on microplastics can be achieved, revealing the metabolic activity and physiological state of microplastic-associated microorganisms^[86-87].

Raman spectroscopy is a non-destructive testing method. When combined with single-cell sorting, it provides a new strategy of "separation before cultivation," enabling the targeted sorting, sequencing, and even cultivation of Raman-identified target functional bacteria, thereby achieving a joint analysis of phenotype and genotype^[88]. Currently, based on the single-cell Raman sorting platform, it has been possible to perform single-cell sorting and targeted metagenomic studies on Raman-identified soil antibiotic-resistant bacteria^[89], as well as the Raman identification^[84] and sorting cultivation^[90] of environmental phosphorus-solubilizing bacteria. Although there are no reports yet on using single-cell Raman identification for microplastic-degrading bacteria, these examples indicate that this technology has great potential in exploring microbial resources for microplastic degradation.

4.2.5 Synthetic Biology Technology

Due to the fact that some microplastic-degrading bacteria themselves possess antibiotic resistance genes or virulence factors, their direct application in the environment may pose potential ecological risks. Constructing safer engineered bacteria through genetic engineering for the large-scale production of key degrading enzymes and in situ environmental remediation of microplastics is an important direction for development. With the advancement of molecular biology and genetic engineering technology, significant progress has been made in developing new highly efficient microplastic-degrading microorganisms through genetic manipulation^[91]. By transferring the genes synthesizing microplastic-degrading enzymes into engineered bacteria for heterologous expression, it is possible to artificially construct highly efficient microplastic-degrading engineered bacteria for plastic degradation. For example, Zhu et al.^[14] used genetic engineering methods to introduce the PETase enzyme gene into E. coli for high-efficiency expression, achieving good results in degrading PET in a simulated wastewater treatment plant effluent environment. Hu et al.^[92] further constructed a co-expression system of adhesion proteins and PETase degrading enzymes in E. coli, increasing the adhesion force between the engineered E. coli and PET, thus enhancing its efficiency in PET degradation. Tournier et al.^[15] proposed another solution to improve degradation efficiency by modifying the degrading enzyme itself to be more heat-resistant. At low temperatures, PET is in a crystalline state, making it difficult for degrading enzymes to effectively bind and "digest" it. Under high-temperature conditions (65°C), PET can soften and form an amorphous state, allowing the degrading enzyme to interact with PET more easily. However, under high-temperature conditions, degrading enzymes are prone to inactivation. By redesigning genetically modified enzymes to have higher thermal stability, it is possible to achieve efficient PET degradation at high temperatures. Additionally, Alper et al.^[93] utilized a machine learning system to predict the impact of mutations on the performance of the PETase enzyme. Through engineering and testing of mutants, they identified an enzyme with significantly improved thermal stability and degradation activity, proposing a potential pathway for industrial-scale use of degrading enzymes for microplastic recycling. In summary, the development of genetic engineering and artificial intelligence technologies enables the redesign of the structure and function of specific enzymes involved in microplastic degradation, combined with the high-efficiency expression of key degrading enzymes in engineered bacteria. The integration of interdisciplinary technologies such as bioinformatics, artificial intelligence, and synthetic biology in the future will help find green, efficient, and sustainable solutions for the biodegradation of microplastics.

5 Conclusion and Prospects

Environmental microorganisms are a crucial driving force in the material cycles of ecosystems and have significant potential for addressing the increasingly severe problem of environmental microplastic pollution. Although some important progress has been made in the microbial degradation of microplastics, many challenges still remain.

(1) Understanding the degradation patterns of microplastic-associated microorganisms: Although the biofilm on environmental microplastics contains potential degrading bacterial communities, the degradation efficiency is relatively low, making it difficult to utilize directly. By deciphering the assembly mechanisms and degradation patterns of microplastic-associated degrading bacterial communities, we can identify core degrading microorganisms at spatiotemporal scales, which will promote the efficient reduction and control of environmental microplastics. Additionally, future research should focus on the impact of environmental factors such as pH, humidity, and temperature on the structural characteristics and spatial distribution of microplastic-degrading bacterial communities, thereby providing a theoretical basis for optimizing degradation efficiency.

(2) Develop in-situ analysis methods for microplastic degradation: Due to the widespread presence of additives in microplastics, many existing indirect research methods for plastic degradation are not rigorous. Therefore, it is necessary to further standardize the characterization and analytical research of the degradation process, and continuously improve the field of microbial degradation research on microplastics. Developing highly sensitive in-situ analysis methods and characterization techniques will provide important technical support for a deeper understanding of the in-situ degradation processes of microplastics, their molecular mechanisms, and the exploration of efficient in-situ microbial degradation resources.

(3) Mining in-situ efficient degradation microbial resources: The currently discovered microplastic-degrading microbial resources, whether in terms of the number of strains or degradation efficiency, are still far from sufficient. At present, the rapid development of omics technologies, single-cell technologies, artificial intelligence algorithms, and synthetic biology provides a series of powerful tools for discovering, designing, and acquiring microplastic-degrading genes and enzymes, which will greatly promote the mining of various efficient in-situ environmental degradation resources. In addition, through the artificial construction of synthetic microbial communities, utilizing metabolic interactions and functional complementarity among the microbial populations, it is possible to form stable species compositions with high degradation capabilities and strong environmental adaptability. By using these communities for the synthesis of high-value products, there is hope to truly achieve green reduction and waste-to-resource transformation of microplastic pollution.

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