Plastic-eating Bacteria

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Introduction

Starting from their introduction in the early 1900s, the use of plastics has grown exponentially, and currently, plastics have become widespread and essential in almost every society on earth to the point that life without plastics is unimaginable for most of us. None of the commonly used plastics are considered biodegradable (meaning that they cannot be dissolved or decomposed by natural agents). As a result, they accumulate, rather than decompose, in landfills or the natural environment. The continued widespread use of plastics, which does not seem to be decreasing anytime soon, has led to plastic pollution becoming an environmental hazard for which an efficient, easily employable and environmentally-friendly solution is yet to be found. The main plastic polymers that are produced worldwide are polyurethane, polyethylene, polyamide, polyethylene terephthalate, polystyrene, polyvinyl chloride, and polypropylene. These and other plastics have become ubiquitous throughout the world, even in the deepest parts of the world’s oceans such as the Mariana Trench. Plastic pollution in oceans continues to increase with plastic debris being found in all major ocean basins, with an estimated 4 to 12 million metric tons of plastic waste generated on land entering the marine environment in 2010 alone.

It has been conjectured that the great abundance of plastics has led to the evolution of plastic degrading enzymes such as PETase (PET-digesting enzyme) in bacteria. For a long time, much of the plastics that were being produced (basically, all of the plastics mentioned above) were thought to be impossible to degrade via natural agents as the polymers are designed to be durable and stable. Furthermore, no enzymes or organisms capable of breaking them down had been identified until 1977, when some lipases (extracellular enzymes that usually cleave esters in oils and fats) were reported to be able to attack ester bonds in some aliphatic polyesters and depolymerize such materials. In recent years, a lot of discoveries later, removal of plastics from the environment using microbes and their enzymes has become a focus of much research.

For the purposes of this page, plastic-eating bacteria are defined as bacteria that can break down and digest plastics such as polyethylene terephthalate (PET), polyethylene (PE) and polyurethane (PU). The recent discovery of Ideonella sakeasis, which can use the plastic PET as a carbon and energy source, and the following modifications to its genome which have enhanced its degrading capabilities have given new hope for the management of plastic pollution through the utilization of microbes and their enzymes. A number of other plastic-eating bacteria/microbes have also garnered much attention as the number of enzymes and microorganisms that are capable of breaking down several different types of plastics keeps growing. But, however exciting the applications of these organisms and their enzymes may seem, their capabilities are still limited and require further investigation and modification so that their potential can be fully exploited.

Cumulative plastic waste generation and disposal (in million metric tons). Solid lines show historical data from 1950 to 2015; dashed lines show projections of historical trends to 2050.

By Meheret Ourgessa

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Section 2

Plastics have mainly been introduced since the 1960s.(17) Given the relatively few decades since these polymers became available and widespread, nature has only had a very short time to evolve highly active enzymes. (17) In general, it is believed that the microbial degradation of human-made polymers is a very slow process. (17) The difficulty with degradation of plastics by microbes mainly stems from the high molecular weight of the polymers, strong C-C bonds and the extremely hydrophobic surface of plastics, which is very difficult to attack by enzymes.(17) Nonetheless, a number of bacteria and other microbes have been reported to be able to degrade a variety of plastics or contain enzymes that can. (17)(18)

As discussed earlier, researchers have discovered and characterized enzymes from bacteria other than I. sakeasis that are capable of breaking down PET. Currently, only a few bacteria (and fungi) have been described to be able to partially degrade PET to oligomers or monomers and all known PET hydrolases have relatively low turnover rates.(17) PETases represent the best-studied class of enzymes with respect to the hydrolysis of synthetic polymers. (17) Interestingly, the trait for PET degradation appears to be limited to a few bacterial phyla and is mostly found in bacterial isolates that are members of the Gram-positive phylum Actinobacteria.(17) Bacteria that have been found to contain PET hydrolases enzymes include Thermobifida fusca, Bacillus subtilis and species from the genera Thermomonospora.(17) However, none of them have been reported to contain MHETase, the enzyme that breaks down the monomers of PET after it is degraded by a PETase.(17) Overall, the I. sakaiensis PETase and MHETase enzymes are the best-studied models for PET degradation and it seems that they are the most potent as well. (17)(8)

Another plastic polymer bacteria have been shown to degrade is polyurethane (PUR/PU).(17)(4) There are a number of different types of polyurethane polymers, but microbes act on only those that have ester links.(17) Although ether linked polyurethane plastics exist, it seems that no enzymes capable of acting on polyurethane ethers have been described.(17) With respect to bacteria capable of degrading PUR, Gram-negative Betaproteobacteria from the genus Pseudomonas have been most frequently linked with PUR activities.(17) One Pseudomonas species that has been studied for its PUR-degrading activity is able to grow on a PU-diol solution, a polyurethane oligomer, as the sole source of carbon, energy and nitrogen. (4) This bacterial strain was obtained from soil samples and is capable of degrading both, an oligomeric PU and a PU building block.(4) While the list of PU-active bacteria is steadily increasing, various fungal species have also been identified as PU degraders. (17)

Another plastic polymer that has been associated with bacterial degradation is polyethylene (PE). Research performed in polyethylene biodegradation, both with pure strains and complex microbial communities has proved that biodegradation of this material although slow is actually happening in nature.(19) Possible PE degradation has been affiliated with a surprisingly large number of bacterial genera among which are the Gram-negative species with the genera Pseudomonas, Ralstonia, and Stenotrophomonas but also many Gram-positive taxa. (17) A few studies have also linked PE-degrading microbes with the complex gut microbiomes of invertebrates.(17)(5) One such study indicated that during short-term exposure, the intestinal microbiome of the larvae of G. mellonella (a greater wax moth) is intricately associated with polyethylene biodegradation in vivo.(5) The study identified microorganisms in the genus Acinetobacter that appeared to be involved in this biodegradation process.(5) A number of fungal genera have also been affiliated with assumed PE degradation. (17)

Another plastic that has been targeted for bacterial degradation is polyamide.(17) Polyamide (PA) is a polymer of repeating units of aliphatic, semiaromatic, or aromatic molecules linked via amide bonds.(17) Because the monomers for making this polymer can be very versatile, there are many different types of synthetic polyamides, with the most popular being nylon and Kevlar.(17) Although no microorganisms capable of fully degrading the intact high-molecular-weight polymer have been reported, several studies are available on bacteria acting on either linear or cyclic nylon oligomers with short chain lengths.(17) Examples include .(17) The only enzyme that has been reported to act on high-molecular-weight nylon fibers so far originated from a white rot fungus.(17) Some Pseudomonas species have been reported to utilize 6-aminohexanoate-dimers, oligomers of polyamide, as a sole carbon and nitrogen source.

Polystyrene(PS), a synthetic polymer widely used for packaging industries and many daily use articles, has also been a target of possible bacterial degradation.(17) Unfortunately, much like polyamide, there is no known enzyme that can degrade the high molecular-weight polystyrene polymer.(17) Styrene itself is able to be used as a carbon source for growth by some microorganisms.(17)Styrene degradation in bacteria is well studied in Pseudomonas, Xanthobacter, Rhodococcus, Corynebacterium, and others.(17) It appears to be a widespread metabolism.(17) Rhodococcus ruber has been shown to form biofilms on PS and partially degrade it.(20)(17) A biofilter consisting of Brevibacillus sp. has been shown to remove 3 kg of styrene in a day.(20) In addition, Several bacteria have been reported to form either alone or as members of consortium biofilms on polystyrene films and particles, thereby degrading the polymer.

(17)

In sharp contrast to the huge global production rates of polypropylene and polyvinyl chloride(PVC), hardly any information is available on microbial degradation of either of these important polymers.(17) Though there are a very few reports that describe the degradation of these polymers based on weight loss and using mixed species microbial communities have been published(21)(22), it is likely that these reports were in part misled by the degradation of the chemical additives rather than the polymer.(17) As a result, no defined enzymes or pathways that are responsible for the degradation of either of these two high molecular-weight polymers are known.(17)

Arctic microorganisms may have unique potential for biodegradation of plastics due to the environmental conditions of polar oceans, which differ from other marine ecosystems.(23) Bacteria from these regions respond quickly to changing environmental patterns. (23) Although there isn’t much research conducted on the interactions between plastic and marine microbiota, the growing amount of plastic waste might force the microorganisms in these environments to adapt to new substrates.(23) Thus, these organisms are prime area for future research on biodegradation of plastics.(23) The table below lists some microorganisms isolated from cold environments and able to degrade bacteria.(23)

Mechanisms of Degradation

Understanding the mechanisms by which bacteria and other microbes degrade plastic polymers or oligomers can lead to identification of enzymes involved in the processes which can be targets for biotechnological engineering to lead to more efficient microbial degradation of plastics.

One of the most well-studied mechanisms of biodegradation of plastics is the pathway for PET catabolism in I. sakeasis. It is thought that I. sakeasis first excretes PETase enzymes (classified as esterase) from the cells onto PET surfaces. (24)(8) Appendages that grow extracellularly might be involved in the delivery of PETase onto PET surfaces.(8) Extracellular PETase hydrolyzes PET to produce MHET as the major product and TPA(terephthalic acid).(24) The PET hydrolysis products are then transported into the periplasmic space through an outer membrane protein such as porin.(24) MHETase, predicted to be an outer membrane anchored lipoprotein, hydrolyzes MHET into TPA and EG(ethylene glycol).(24) TPA is taken up into the cytoplasm through the TPA transporter coupled with TPA-binding protein and then integrated via protocatechuic acid (PCA) to the tricarboxylic acid (TCA) cycle.(24)(8) Ethylene glycol is metabolized via glyoxylic acid to the TCA cycle.(24)

Interestingly, the expression of the PETase gene was dramatically upregulated in the presence of PET (but not TPA) in a culture of I. sakaiensis, raising the question of which molecule(s) induces its expression.(24) However, TPA does upregulate the expression of a gene cluster that highly identical with two TPA degradation gene clusters identified in Comamonas sp. strain E6.(24) In other bacteria acting on PET,

Researchers have also proposed a degradation pathway for the oligomers of polyurethane by a Pseudomonas strain.(4) This proposed pathway is summarized in the figure to the right.(4) There a number of enzymes involved in this pathway. One of the first enzymes identified to act on PUR was the PueB lipase from Pseudomonas chlororaphis, a different Pseudomonas species.(17) This organism codes for at least one additional enzyme active on PU, which was designated PueA.(17) Both enzymes are lipases (which catalyze the ester-linked lipids) and similar to PET degradation with microbes, PU is degraded by the secreted hydrolases, and the degradation is tightly regulated.(17) As mentioned previously, enzymes and microbes described as capable of degrading organisms were all acting on ester-linked PU.(17) There have yet to be enzymes described to act on polyurethane ethers in scientific literature.(17)

To date, no microorganisms capable of degrading the intact high-molecular-weight polyamide polymer have been reported, but there are many bacteria that act on the oligomers of polyamide.(17) Three main enzymes have been reported as essential for the initial hydrolysis of cyclic and linear 6-aminohexanoate oligomers.(17) The first one is a cyclic-dimer hydrolase (NylA), the second a dimer hydrolase (NylB), and the third an endo-type oligomer hydrolase (NylC).(17) Once the oligomers are hydrolyzed, the monomers are metabolized by different aminotransferases.(17)

As with polyamides, there is no known microorganisms capable of degrading the high molecular-weight polystyrene polymer and neither are there any enzymes, but there are many microbes that degrade the oligomers of the plastic.(17) Under aerobic conditions, styrene is oxidized by two different pathways. The first one involves attacking the vinyl side chain and the second one is through attacking a rather nonspecific aromatic ring, thereby forming primarily the intermediates 3-vinylcatechol, phenylacetic acid, and 2-phenylethanol. These intermediates are channeled into the Krebs cycle after ring cleavage. The degradation of the vinyl side chain involves the action of three key enzymes, a styrene monooxygenase(styA and styB), a styrene oxide isomerase(styC), and a phenylacetaldehyde dehydrogenase(styD). The respective genes for side-chain oxygenation are frequently located in a single conserved gene cluster, often designated styABC(D).(17) The styrene monooxygenase attacks the vinyl side chain to release epoxystyrene, which is then subjected to isomerization to form phenylacetaldehyde. Phenylacetaldehyde is then oxidized to phenylacetic acid through the involvement of a dehydrogenase. In P. putida, the phenylacetic acid is activated to phenylacetyl-coenzyme A (CoA) and then subjected to β-oxidation to yield acetyl-CoA, which is directly fed into the Krebs cycle(26). (17)The expression of the conserved cluster is regulated through either a two-component regulatory system or LysR-type regulators.

Research performed on PE degradation by microbes so far is mainly of descriptive nature, with a few studies devoted to polyethylene degradation mechanisms or the isolation of enzymes belonging related to this process.(19) However these findings are limited and further evidence is required to identify the complete mechanisms for polyethylene degradation. (19) Similarly, the few studies that describe degradation of polyvinyl chloride and polypropylene by microbial communities do not detail the enzymes and pathways involved in the process.(17)(25)

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2018, Kenyon College.