Plastic Degradation Using Pseudomonas Bacteria
Pseudomonas species are gram-negative, rod-shaped, chemoorganotrophic bacteria, many of which are able to metabolize plastics and are being researched for use in biorecycling. Those species or strains which are capable of degrading plastic are typically soil or sea-dwelling, solvent-tolerant extremophiles. They are metabolically diverse, which makes them ideal candidates for biotechnology as they present many possible solutions to bioengineering problems. Some, like Pseudomonas sp. TDA1, were discovered in soil samples from underneath a waste site containing abundant brittle plastic waste, a toxic environment due to the compounds produced by degrading plastic4.
P. sp. TDA1, the most recently discovered strain, is remarkable because of its ability to metabolize polyurethane and its subunits (including P. sp. TDA1’s namesake, 2,4-TDA), which make up polyurethane plastic. This could be of great utility for dealing with the massive amounts of plastic disposed of every year, which currently reside in landfills where they decay to produce toxic, sometimes carcinogenic chemicals8.
Other species and strains which have been researched for use in plastic recycling include Pseudomonas aeruginosa AKS9 and MZA-85, Pseudomonas sp. AKS2, Pseudomonas stutzeri JA1001, Pseudomonas sp. NCIM 2220, P. protegens Pf‐5, and P. mendocina.
History and Discovery
Pseudomonas species have been reported as plastic metabolizers since 1973 with the discovery of Pseudomonas sp. O-3 degrading polyvinyl alcohol (PVA). P. sp. AKS2 was shown to degrade low-density polyethylene (LDPE) and PES in 2013, marine Pseudomonas sp. were documented degrading high-density polyethylene (HDPE) in 2010, Pseudomonas stutzeri JA1001 demonstrated the ability to degrade PEG in 1991, Pseudomonas sp. NCIM 2220 was recorded degrading PS in 2002, Pseudomonas aeruginosa AKS9 was documented degrading polyurethane (PU) diol and a commercial form of polyester PU in 2011. Metabolization of the commercial form, Impranil DLN, by P. protegens Pf‐5 was recorded in 2016. Pseudomonas aeruginosa MZA-85 degraded polyester PU in 2013, and P. mendocina showed indications of the ability to metabolize PET.
The most recently discovered relevant strain, P. sp. TDA1, was first documented in a paper published March 27, 2020 in the Frontiers in Microbiology journal. A research team headed by Hermann J. Heipieper of the Helmholtz Centre for Environmental Research-UFZ in Leipzig, Germany isolated P. sp. TDA1 from a waste site containing many brittle, polyurethane plastics4. The team was searching for a microorganism capable of growing on and metabolizing subunits of polyurethane for use in biorecycling, and they were able to grow a culture of P. sp. TDA1 with only 2,4-TDA as a source of carbon and nitrogen. They used 16S rRNA gene sequencing and membrane fatty acid (MFA) profiling to identify the bacterium as a Pseudomonas sp.; alignment with the RDP database4. P. sp. TDA1 matches many of the characteristics of the Pseudomonas genus: rod-shaped, gram-negative, aerobic, chemoorganotrophic, and able to grow in the absence of organic material5. Pseudomonas species had been documented to attack polyurethane compounds previously, but sp. TDA1 was yet undocumented9.
P. aeruginosa is considered the type species of the Pseudomonas genus. Its chromosome is relatively large at 6,466,920 base pairs on average, with an average of 5,734 open reading frames. Only 17.5% of the genome is shared between different strains but is predominantly GC pairs throughout all of them. Genes encoding enzymes like dehydrogenases are common.
The P. sp. TDA1 genome was sequenced in the same study in which the bacterium was discovered in 2020. It is 5,910,795 base pairs long and as is typical of bacteria it is only one chromosome2. Since it was discovered and sequence so recently, information like guanosine-cytosine (GC) content is unavailable at this time. Sequencing was done using an Illumina MiSeq sequencer with a 250-bp paired-end protocol, demultiplexed by MiSeq reporter software, and assembled using the Velvet assembly program. Comparison to the UniprotKB genome database and the basic local alignment search tool (BLAST) database in NCBI was used to suggest enzymes and genes which might have been used in the digestion of polyurethane. These analyses identified many catabolic genes for aromatic compounds, including multiple oxygenases (which add oxygen from the air to compounds to degrade them)4.
Metabolizing different plastics is achieved by enzymes which are both secreted extracellularly and intracellularly. Oxidations can increase the ability of bacteria to attach to and degrade plastics; depolymerases and hydrolases break down large polymers into more manageable units which can be moved into the cells and degraded by dehydrogenases, hydroxylases, and oxidases. Esterases, lipases, and cutinases are particularly essential hydrolases which break ester bonds by attacking carbonyl carbon atoms. Transporters for plastic oligomers are also an essential component of plastic metabolism. Metabolic processes for the degradation of plastics like PEG, PE, PS, polyester PUR (but not PUR monomers and oligomers), PET, PES, PEG, and PVA have all been investigated.
The ability to metabolize polyurethane (PUR) monomers and oligomers (byproducts and subunits rather than raw plastic) is the hallmark characteristic of P. sp. TDA1 and the reason it was discovered. Polyurethane polymers and components were able to replace both carbon and nitrogen in the P. sp. TDA1 environment without depriving the bacteria of the nutrients they needed for survival4. The primary polyurethane component focused on is 2,4-TDA, a toxic element which had never been biodegraded before.
P. sp. TDA1 was grown on an agar plate in a mineral medium with 2,4-TDA (a polyurethane subunit) as the only source of carbon. It did not grow on plates with no carbon source, showing that the bacterium was not autotrophic and was indeed using the 2,4-TDA to survive. Strain TDA1 grew in a nitrogen-deficient media as well, in which 2,4-TDA was both the only carbon and nitrogen source. In both instances, the growth of Pseudomonas sp. TDA1 corresponded to a decrease in 2,4-TDA; high performance liquid chromatography (HPLC) was used to show that the decrease of 2,4-TDA was significantly greater on plates containing P. sp. TDA1 than on control plates with no bacteria4. This demonstrated that P. sp. TDA1 was metabolizing and living off of 2,4-TDA.
Researchers proposed a metabolic, oxidative pathway through which the bacterium breaks down the 2,4-TDA. This pathway begins with an electron transferring group hydroxylating the methyl group of 2,4-TDA to a primary alcohol. Next, an alcohol dehydrogenase and an aldehyde dehydrogenase catalyze the production of 2,4-diaminobenzoate (4-aminoanthranilate). This anthranilate is proposed to be converted to energy by benzoate 1,2-dioxygenase, with 4-aminocatechol as an intermediate. The ring of 4-aminocatechol could be cleaved through an extradiol and homoprotocatechuate meta-pathway4. As of yet, however, this mechanism still needs to be confirmed by additional biochemical analysis.
Environmental and Industrial Implications
Plastic is one of the most ubiquitous materials used by humans, and therefore in the world. One of the reasons for this is its resilience; unfortunately, this makes it difficult to recycle and when placed in a landfill, plastics (like many petroleum products) can release toxic compounds which seep into the ground, damaging ecosystems. For this reason, developing cheaper more efficient methods of recycling is essential to slowing the destruction of the Earth and its inhabitants.
Polyurethane is one of the most universally used plastics, predominantly found in furniture, construction materials, and appliances. Demand for the material is expected to exceed 56 billion U.S. dollars by 2021, with 16 million tons estimated demand in 20166. Part of the reason polyurethane is so popular is its durability, particularly to heat. It can take up to 1000 years to degrade and is expensive to recycle3. When left in landfills, as it often is, polyurethane introduces toxic chemicals as it slowly breaks down, harming the environment and potentially exposing humans to carcinogens.
It is clear that there is an urgent need for a more effective way to deal with polyurethane waste (in addition to reducing plastic consumption). Researchers are looking for this solution in microbes, leading to the discovery of P. sp. TDA1. While this bacterium could conceivably be recruited to digest plastic waste, researchers have a more ambitious (and industrially applicable) solution in mind: figuring out the mechanism by which this bacterium degrades polyurethane8. This would enable industrial recycling centers to chemically digest polyurethane to its key components en masse. Even more ambitiously, there is the potential to modify these systems to convert harmful, difficult to digest plastic waste into more ecofriendly, biodegradable plastic or simply create novel bioplastics for human use9. These are all future prospects, however, for which the groundwork is only beginning to be laid.
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