Microbial degradation of polyethylene terephthalate: a systematic review

Plastic pollution levels have increased rapidly in recent years, due to the accumulation of plastic waste, including polyethylene terephthalate (PET). Both high production and the lack of efficient methods for disposal and recycling affect diverse aquatic and terrestrial ecosystems owing to the high accumulation rates of plastics. Traditional chemical and physical degradation techniques have caused adverse effects on the environment; hence, the use of microorganisms for plastic degradation has gained importance recently. This systematic review was conducted for evaluating the reported findings about PET degradation by wild and genetically modified microorganisms to make them available for future work and to contribute to the eventual implementation of an alternative, an effective, and environmentally friendly method for the management of plastic waste such as PET. Both wild and genetically modified microorganisms with the metabolic potential to degrade this polymer were identified, in addition to the enzymes and genes used for genetic modification. The most prevalent wild-type PET-degrading microorganisms were bacteria (56.3%, 36 genera), followed by fungi (32.4%, 30 genera), microalgae (1.4%; 1 genus, namely Spirulina sp.), and invertebrate associated microbiota (2.8%). Among fungi and bacteria, the most prevalent genera were Aspergillus sp. and Bacillus sp., respectively. About genetically modified microorganisms, 50 strains of Escherichia coli, most of them expressing PETase enzyme, have been used. We emphasize the pressing need for implementing biological techniques for PET waste management on a commercial scale, using consortia of microorganisms. We present this work in five sections: an Introduction that highlights the importance of PET biodegradation as an effective and sustainable alternative, a section on Materials and methods that summarizes how the search for articles and manuscripts in different databases was done, and another Results section where we present the works found on the subject, a final part of Discussion and analysis of the literature found and finally we present a Conclusion and prospects.

• PET-degrading fungi and bacteria possess enzymes such as PETase, which are hydrolases that cleave ester bonds. • The biodegradation of PET is a slow process that leads to the use of pretreatments to increase its efficiency

Introduction
Excessive use of plastic has had devastating effects on the environment, causing major damage to ecosystems [1,2]. The annual environmental report on plastic pollution issued by the United Nations in 2019 has revealed extremely high amounts of this polymer on the planet, with approximately 13 million tons of plastic entering the oceans annually [3]. If this trend continues, it is expected that 26 billion tons of plastic waste will be generated and 12 billion tons will be accumulated in diverse ecosystems by 2050 [3,4]. Of the total plastic waste, around 9% is recycled, 12% is incinerated, and 79% is disposed of in the environment [4,5]. Polyethylene terephthalate (PET) is one of the seven major types of plastics included in the Plastics Identification System [6]. It is a long-chain polymer molecule with aromatic polyester composed of comonomers and aliphatic monomers that are susceptible to hydrolysis [5,7,8]. This polymer is widely used to manufacture singleuse disposable products, such as bottles, food containers, jars, and pillow stuffing; therefore, its production has been higher than that of the other plastic types [4,5,8].
Colombia, Mexico, Bolivia, Brazil, India, and the United States have implemented a series of physical and chemical methods to process PET, which have significantly reduced PET levels [9]. However, recycling, landfilling, and incineration have led to new problems due to the generation of toxic waste, which in turn affects living beings and causes a loss of ecosystems [10,11]. These problems and the limitations of the mentioned techniques, increase the use of biological methods based on microorganisms capable of transforming this polymer into simpler forms [2,[12][13][14].
Biological methods harness the metabolic capabilities of some microorganisms that naturally use polymers, such as plastic, as a source of carbon [2,9]. These methods also use genetically modified microorganisms, whose genome has been modified by genetic engineering to introduce new traits [5,[15][16][17]. Microbial enzymes, such as PET hydrolase (PETase), are important players in these biological techniques since they degrade PET through the hydrolysis of ester bonds, yielding simpler forms of the polymer [18,19].
Considering the detrimental effects on the environment caused by the use of physical and chemical methods for plastic waste management, biological methods have emerged as the best option to mitigate plastic pollution. The alarming accumulation of plastic in ecosystems, as reported by major organizations around the world, is expected to increase unless decisions are made to reduce the use and production of plastics, especially PET, which is present in many single-use products. Therefore, this review aims to present the existing publications on wild and genetically modified microorganisms with the potential of degrading PET to make them available for future work and to contribute to the eventual implementation of an alternative, an effective, and environmentally friendly method for the management of plastic waste such as PET.

Materials and methods
A literature search about PET degradation worldwide was carried out. Articles published between 2004 and 2022 were retrieved from different databases. The exclusion criteria were reviews on the subject, articles lacking evidence of methods of degradation by microorganisms, and articles where the molecular structure of PET had been modified. The keywords "microorganism", "biodegradation", "PET," and "PET hydrolase" and their combinations were used in English and Spanish. In addition, Boolean operators such as "AND, " "OR, " and "NOT" and quotation marks were used.

Results
A total of 137 publications focusing on PET degradation by wild or genetically modified microorganisms were used for the analysis.
The reviewed studies provided data to differentiate between microorganisms with a natural capability of degrading PET and those that were genetically modified. Among the publications analyzed, 51.8% (71 articles) used wild microorganisms (Table 1), 32.8% (45 articles) used genetically modified microorganisms (Table 3), 0.7% (1 article) used both types of microorganisms [20] and the remaining 14.6% (20 articles) corresponded to reviews (Fig. 1A). Furthermore, the articles that did not specify the genus or species but broadly refer to the group of bacteria [9,21], actinomycetes [22], bacterial consortia [23], and/or fungal consortia [24] were also included.
Wild-type microorganisms capable of degrading PET were isolated mostly from soil (22.6%, forest soil) [5, 7, 36-38, 42, 45], followed by aquatic habitats (12.4%, salt and fresh water) [12,[46][47][48][49], from landfills (8.0%) [27,33,34,40,50] and 56.9% of the publications didn't specify (Fig. 1C). The first report on this topic was published in 2004 [21,51], so the publications in this review cover the period from 2004 to 2022. The largest number of articles on the subject were published between 2015 and 2019, therefore, only the latter are shown in Table 1. On the other hand, some of the most recent publications are shown in Table 2, highlighting that 36 papers were published in 2021 of which most were reviews.

Microorganisms genetically modified for PET degradation
From the analysis, 32.8% of the publications (45/137 reviewed articles) used microorganisms that were genetically modified to enhance their PET degradation potential. As shown in Table 3, 50 strains of Escherichia coli were modified in studies from different countries, such as Germany, Austria, and Japan. In addition, the yeast species Pichia pastoris [54] and Saccharomyces cerevisiae [20], microalgae Chlamydomonas reinhardtii [16] and Phaeodactylum tricornutum [55], bacterial species Bacillus subtilis [53,56] and Clostridium thermocellum [52] were also used as genetically modified microorganisms instead of E. coli.

Enzymes used to generate genetically modified microorganisms
The most widely used enzymes were PETase from Ideonella sakaiensis and other enzymes derived from it. The genes of these enzymes were introduced in the genome of others microorganisms [5,16,17,53,[55][56][57]65]. Furthermore, there were cutinase Thc_Cut1 from Thermobifida cellulosilytica and other recombinant enzymes derived from it, which were used to genetically modify microorganisms [61,64]. In addition, cutinase TfCut2, whose tfcut2 gene from Thermobifida fusca was introduced in seven microorganisms [28,30,52,59,29]. Figure 2 shows the frequency of use of all mentioned enzymes in the reviewed publications, classified according to their biological activity, i.e., esterases, cutinases, nonspecific hydrolases, and fungal hydrophobins.

Description of the literature
Few studies have been performed on PET degradation by microorganisms in comparison to those conducted on the biodegradation of other polymers, such as polyurethane, low-density polyethylene (LDPE), polyhydroxyalkanoates, or biodegradable plastics [66,67]. The same is true when compared to the existing studies on plastic waste management in general [4,68,69]. Most studies on PET degradation have focused on proving the capability of some microorganisms to initiate polymer degradation by forming biofilms [70,71]. Other published studies have focused on microbial consortia with the potential to degrade PET [25,38,42,71], detecting modifications in functional groups of polymers during biodegradation for assessing the process efficiency [12,38], identifying enzymes involved in PET degradation [25,42,72], intermediate hydrolysis products of PET degradation and its inhibition [28,72], or interaction between marine microorganisms and PET microplastics [12]. In addition to research on wild microorganisms, a significant number of studies have focused on producing genetically modified microorganisms with increased potential for PET degradation. Genes encoding enzymes involved in the degradation process have been introduced and expressed mainly in E.coli [5,17,53,57,58,60,61,63,65,73]. Several studies are conducted with these modified microorganisms to investigate the products or intermediate metabolites of PET degradation using recombinant enzymes [5,8,28,74], and assess structural changes in enzymes to reduce their activation temperature or increase their degrading efficiency [15,18,56,57,59,62,75], search for amino acid sequence homology among different cutinases and compare their activity [20,29], elucidate the thermodynamics and kinetics of the conformational and colloidal stability of a hydrolase [54], and express and measure the catalytic activity of a PETase in microalgae [16,55].

Most common study techniques
All studies evaluated PET degradation through changes in the appearance or topology of the polymer surface, such as the occurrence of pores, cracks, and change in the color or crystallinity, using analytical techniques, such as electron microscopy and X-ray diffraction [14,28,63], through variations in polymer mass and weight [5,69,76], changes in PET atomic absorption spectra [50] detection of the appearance and disappearance of functional groups by Fourier-transform infrared spectroscopy (FTIR) [12,38,47,63], colonization of the polymer surface by Scanning electron microscopy (SEM) [14,16,51,63], or quantification of intermediate products of PET degradation, such as mono(2-hydroxyethyl) terephthalate (MHET), bis(2hydroxyethyl) terephthalate (BHET), terephthalic acid (TPA), and ethylene glycol by high-performance liquid chromatography [16,20,61,65]. SEM and FTIR are the most commonly used techniques in the determination of PET degradation. SEM images allow the study of cell division stages, individual colonization of microorganisms, and biofilm formation on a PET surface [25,31,33,61,62], a process that seemed to be a prerequisite for biodegradation and which indicates that microorganisms are able to utilize PET as a carbon source for their growth [25,71,77]. This result in several articles is confirmed by FTIR results showing the absorption spectra of the appearance or disappearance of functional oxidized groups during the biodegradation process of the polymers [58,63]. SEM and FTIR are complementary techniques used in several papers that have shown a good approximation of the degree of degradation in synthetic polymers if are used in combination to confirm the data [12,27,63]. These techniques are non-invasive and allow the study of a process as slow as PET biodegradation [78].

Percentage of PET degradation
PET degradation is a slow and difficult process that depends on the structure and quantity of the polymer as well as on the interactions between the environment and the degrading microorganisms [1,79]. Studies measuring the degradation through polymer weight loss over 30 days, 6 weeks, 6 months, and up to 1 year have observed degradation values that did not exceed 45% [2,14,70,80]. Based on the present literature review, one of the highest PET degradation yields was reported by Dang et al. [81], who observed a 43.05% decrease in the weight of particulate pretreated PET by the action of Bacillus sp. BCBT21. The only study that used PET microplastics and a protozoan, Spirulina sp., reported degradation of 48.61% [12], whereas another work on microorganisms isolated from a marine environment determined degradation of 22% and 35% by Aspergillus sp. and Vibrio sp., respectively [14]. Taniguchi et al. [25] observed a decrease of 75% in carbon, which was catabolized to CO 2 , using a microbial consortium composed of bacteria, yeasts, and protozoa. These two characteristics, i.e., the low degradation rates and the long time involved, are the limiting factors that have led to increased use of genetically modified microorganisms or use pretreatments. The presence of several amorphous regions in its structure makes PET one of the polymers that are the most susceptible to attack by microorganisms [29,82]. Different pretreatments have been performed to favor the hydrolysis of ester bonds. Among them is the addition of functional groups, such as carbonyls, alcohols, phenols, and hydroxyls [69,76], genetic engineering of microorganisms to increase the production of surfactants, favoring cell adhesion to the surface of materials [67,77,80,83], or UV irradiation of PET to favor its colonization [27,70,82].

Bacillus sp. major PET degrader
The affinity of certain species of microorganisms for different types of polymers is evident. This review shows that species belonging to the genus Bacillus are able to degrade PET more efficiently than other microorganisms. Bacillus cereus and Bacillus gottheilii have been shown to adapt to other polymers, such as polyethylene (PE), polypropylene (PP), and polystyrene (PS) [76]. Pseudomonas sp. degraded LDPE [83,84], Aspergillus sp. degraded LDPE and high-density polyethylene [85,86], and Acinetobacter sp. was reported as a good polyurethane-degrading microorganism [76,87]. Altogether, the results indicate that these microorganisms possess specific enzymatic mechanisms for the transformation of polymers into simpler forms [69,76,83]. The ability of Bacillus sp. to utilize these substrates as a source of carbon and energy is evinced in their adaptation to PET-contaminated environments [76,88,89], and it could have been favored by evolutionary strategies specific to this genus, the so-called "Bacillus lifestyle". Zeigler and Nicholson [90] have stated that "organisms with the Bacillus lifestyle have not only survived but also thrived on the Earth-and perhaps beyond" [90]. Among the Bacillusspecific characteristics are their ability to form endospores [90,91], persistence and ability to colonize surfaces through the formation of multicellular communities, such as biofilms and swarming [92,93], and ability to produce surfactants such as surfactin [79,80], and peculiar cell wall structure [70,94,95]. Endospore and biofilm formation are evolutionarily ancient strategies found in several species of bacteria and archaea that allow them to survive in lownutrient environments such as the surface of polymers like PET [89][90][91]. The mechanism of endospore formation, production of exopolysaccharides (EPS) for biofilm formation, and production of biosurfactants are finely regulated in Bacillus sp. by two-component signal transduction systems (TCSs). TCSs consist of 36 histidine kinase (HK) sensor proteins and 35 response regulators (HK/RR) [88,89] are activated during the stationary phase of growth and sense various physical and chemical stimuli from the environment [96,97]. B. subtilis harbors at least three coupled HK/ RR systems that control the expression of genes involved in the competition, synthesis of degrading enzymes, and endospore formation called ComP/ComA, DegS/DegU. and Spo0F/Spo0B/Spo0A, respectively [89,98]. During the stationary phase of a Bacillus sp. culture, cell subpopulations able to import and export DNA from and into the medium are also generated [90,99], leading to extensive taxonomic differentiation at the species (sp.) level in this bacterial genus. This could explain why several species of isolated microorganisms with the potential to degrade PET are not determined and reported in some publications, appearing instead as Bacillus sp. BCBT21, Bacillus sp. AIIW2, or simply Bacillus sp. [42,49,81]. As previously mentioned, another evolutionary strategy of Bacillus sp. to adapt to hostile environments or to grow upon a limited carbon source is its relatively hydrophobic cell wall [70,95], which is one of the key factors in the degradation of plastics in general [79,83]. The cell wall composition of microorganisms is known to be directly involved in bioremediation processes by enabling cell adhesion to the surface of the material [67,100]. Species of the genus Bacillus, in particular Bacillus sp. C.I.P. 76-1 11 and B. subtilis, have a distinctive structure that differs from that of other gram-positive bacteria. Their cell wall contains a high percentage of very long polysaccharides, other than peptidoglycan, which favors polymer hydrolysis [94,95].

Enzymes that transform PET
Some authors state that polymer biodegradation is determined by the extracellular enzymes secreted by microorganisms and the amount of these secreted enzymes, rather than by the cell wall and the production of biosurfactants [77,82]. Such enzymes act according to the surface properties of the polymer and lead to hydrolytic cleavage, breaking down the material into monomers, dimers, and oligomers that can then be used by the microorganisms as an energy source [69,79,86,81]. In this review, numerous thermophilic and mesophilic microorganisms were found and some of them were isolated from compost, such as I. sakaiensis, Bacillus sp. BCBT21, T. fusca, and Saccharomonospora viridis AHK190 [25,68,81]. These microorganisms produced hydrolase-type enzymes, such as esterases, lipases, and cutinases, which oxidize PET ester bonds [1,79,83] at temperatures near 55 °C as Bacillus sp. BCBT21 167 or at 30 °C as the PETase from I. sakaiensis [2]. Other enzymes that also act at high temperatures are p-nitrobenzylesterase (BsEstB) from B. subtilis whose optimal temperature is 40 °C [58] and TfH from T. fusca whose activation temperature is 60 °C [73]. Therefore, in parallel with this work, cutinases were found to be the enzymes most widely used to modify microorganisms for enhancing their potential for PET degradation [5,15,19,29,73]. According to Taniguchi et al. [25], the mechanism of PET degradation into monomers involves the activity of PETase and a mono(2-hydroxyethyl) terephthalate hydrolase (MHETase), which has only been studied in I. sakaiensis to date [2,72,74].

Major habitat for PET degrading species
In terms of habitats, the soil was the most cited in the reviewed articles [5,7,36,37,42,45]. The wide variety of microorganisms in the soil makes it an environment of high biodiversity; one gram of soil is estimated to harbor millions of culturable bacteria [7,101]. These microorganisms participate in different processes such as nutrient cycling and production of metabolites that promote plant growth and soil mineralization [102], and soil fertility [101,103]. Microorganisms are responsible for the high rates of metabolic activity observed in soil [104]. Among soil microorganisms, bacteria stand out, being one of the largest groups including a large number of species, followed by actinomycetes, fungi, soil algae, and protozoa [101]. Hug et al. [105], in their approach to a new tree of life, showed that the domain Bacteria includes the largest number of described lineages (92 phyla), followed by the domain Archaea (23 phyla), and are a part of terrestrial habitats such as soil. The high diversity of bacterial species increases the probability of finding bacterial genera with the potential to degrade PET as evinced in this study [106]. Some researchers have reported that fungi possess a higher enzymatic potential to degrade plastics compared to that bacteria [39,68]. Nevertheless, bacteria grow faster and have lower nutritional and environmental requirements than fungi and are therefore preferable for research.
Several microorganisms that compose the soil microbiome remain unknown [107]. Soils are exposed to numerous contaminants, mainly heavy metals, hydrocarbons and their derivatives, and plastic waste [108,109]. Some microorganisms have adapted to these polluted environments and use those contaminants as a source of carbon and nitrogen for their survival [2,9]. Bacterias have largely developed the ability to adapt to these habitats and diverse environmental conditions, showing, in some cases, changes in their structure as a survival strategy [110,111].
In environments such as soil, PET degradation is favored by aerobic conditions that accelerate the oxidation of the polymeric molecule to form a polar complex of carbonyl groups and generate water and CO 2 as final products [66]. Plastic biodegradation in general can occur under both aerobic and anaerobic conditions, depending on the microorganism involved and the environmental conditions [11,66].

Conclusion and perspectives
The present systematic review revealed that among wild microorganisms, bacterial and fungal genera such as Bacillus sp. and Aspergillus sp., showed the greatest enzymatic potential for degrading PET. Further studies using these two microorganisms together are warranted to obtain increased polymer degradation rates. An alternative could be the use of compost where these two microorganisms, in association with others, might show enhanced degradation. Janczak et al. [7] showed the biodegradation capacity of rhizosphere microorganisms in the compost where a fungal strain (Laccaria laccata) and a bacterial strain (Serratia plymuthica) were able to degrade biodegradable polymers such as polylactic acid and conventional polymers such as PET.
Our review reveals that the world is working at an accelerated pace to address this issue by taking fundamental actions, banning single-use products at the national level, and creating public-private partnerships for waste management. Additionally, this work demonstrates the exceptional potential of microorganisms in substantially reducing PET emissions to the environment; therefore, we propose their use for the management of this type of waste. For example, composting plants enriched with microbial consortia having a high PET-degrading potential could be implemented through partnerships between government and private companies. In particular, different microalgae species should be included to exploit their high diversity in different countries. Furthermore, it is worth studying whether enzymes such as PETase or similar ones are present in other PET-degrading microorganisms, such as Bacillus sp. and Aspergillus sp., to confirm their involvement in the degradation process and to thoroughly elucidate the mechanism of PET degradation by microorganisms.
We believe that the benefits of using biological methods for the degradation of polymers such as PET should be further explored to replace the physical and chemical methods causing harmful effects on the environment.
Lastly, our goal with this literature review is to encourage scientific-based initiatives that promote the use of biological techniques on a commercial scale for the mitigation of the high rates of plastic pollution. Funding This research was funded by the Direccion General de Investigaciones of the Universidad Santiago de Cali. The role played by the latter was only to finance the research Project.
Data availability Data sharing is not applicable to this article, as no data sets were generated or analyzed during the present study. We used articles published in different databases (ScienceDirect, Springer Link, Scopus, Pubmed, Taylor and Francis Online, Wiley Online Library, and Scielo), that are referenced throughout the paper and appear in the bibliography of the paper.

Declarations
Conflict of interest The authors declare that they have no competing interests.
Ethical approval Not applicable.

Consent for publication Not applicable.
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