Potential and limitations of plastic-eating microbes

Roger Pallàs Piqué, Lluc Rehues Garreta i Daniel Hernandez Carrasco
 
INTRODUCTION

Plastic is all over us. Probably you can touch some objects made of it just extending your hand from where you are. We use it in a daily basis and we couldn’t imagine a life without plastic since it can be found in a wide variety of products: synthetic textiles, packages, construction materials, toys, pipes, wires or cell phones, and those cases are just examples of an endless list.

This proliferation of plastic products, since its introduction on the market at the beginning of the XX century, is mainly due to three characteristics of the material: it has a low cost, it is easy to shape, and it is very resistant. So resistant that it can last for hundreds of years.  Paradoxically, the valuable quality of resistance has become a threat for ecosystems all over the world, mainly the marine ones, due to its accumulation (Vegter et al, 2014).

This major environmental issue has gradually gained global concern through the past decades and some solutions have been proposed in order to degrade the plastic compounds, such as thermal degradation (Misckolczi et al, 2004), catalytic degradation (Akpanudoh, N. 2005), and biodegradation (Masayuki, 2001). The development of biodegradation technics has been limited because of the lack of efficient polymer microbial biodegradation (Shah, A 2008). Lately, some crucial scientific events have changed the situation, mainly related to the research of more efficient microorganisms and the improvement of its enzymes.

The aim of this article is to introduce the lector to the current research situation on the plastic-eating microbes, and its potential applications on waste management.

IDENTIFICATION OF PLASTIC-EATING ORGANISMS

Research of organisms capable of plastic degradation comes out from the premise that they are composed by carbon polymers, and some organisms could have adapt to use this new carbon sources by developing enzymes capable of degrade this molecules (Whitney, 1991).

For doing so, some research groups had collected samples from garbage dumps and other places with high presence of plastic, where having the ability of feeding from the plastic could be an evolutionary advantage.

A variety of microorganisms have been described so far, including some fungi, but in general, they have very low degradation rates (Table 1). Nevertheless, Yoshida et. al. isolated a new microorganism (Ideonella sakaiensis) that mainly feeds from poly(ethylene terephthalate) also known as PET, and has renewed the interest in bioremediation of plastic waste.

Table 1: Representation of the different PET degrading microorganisms. 

Microorganism	Taxonomy	Metabolism	Degradation
Ideonella sakaiensis	Bacteria (Prokaryote)	Aerobic (Gram-negative)	.(Shosuke et al., 2016)
Nocardia sp.	Bacteria (Prokaryote)	Strict aerobic (Gram-positive).	The bacteria degrade 2% of the sample (1 cm2) in 200 days.(Chetna & Madhuri, 2013)
Bacillus subtilis	Bacteria (Prokaryote)	Strict aerobic	Very slow (slower than Nocardia).(Asmae, Noureddine, Moulay, Saâd Ibnsouda, & Fahim, 2015)
Penicillium funiculosum	Fungi (Eucariota)	Fermentator	It degrades a 40% PET composed and 60% Bionolle composed. (Nowak, Paja̧K, Labuzek, Rymarz, & Talik, 2011)

Nocardia sp. was cultivated on different cultivation media with PET as the only carbon source, and the best degradation rate was about 2% of the sample (1cm²)  in 200 days (Chetna & Madhuri, 2013), so Nocardia would spend more than 27 years to degrade the complete PET sample. This degradation rate is extremely low. Bacillus subtilis has also shown weak PET degradation activity using infrared spectroscopy (Asmae et al., 2015).

On the other hand, Penicillum funiculosum has been reported to have good PET biodegrading rates if it is combined with another aliphatic polyester called Bionolle®. Bionolle® is an easy biodegradable plastic, and combined with PET (40% PET 60% Bionolle®) the fungi can completely degrade it in 84 days (Nowak et al., 2011).

THE BIOCHEMICAL REACTION

As I. sakaiensis is the main focus microorganism on this research line we will put our attention on it. There are two facts revealed on the I. sakaiensis studies that set an inflexion point on the plastic biodegradability research:

• The conditions in which I. sakaiensis could consume PET were not difficult to reproduce at all. In fact, the bacteria culture where the reaction was identified for the first time was a warm bottle with plastic and some essential nutrients.

• The team that identified I. sakaiensis for the first time could identify the main enzyme that causes the PET degradation. Furthermore, they could also find the gene in the bacteria’s DNA that transcripts for the PET-digesting enzymes and manufacture them. The manufactured enzymes were made with the porpoise of demonstrating that the enzymes could digest PET alone, and they did.

Figure 1: Scheme of the enzymatic degradation of PET using the two enzymes that I. Sakaiensis present. Source: www.sciencemag.org 

The Figure 1 shows a simplification of the PET hydrolysis reaction process. Plastic biodegrading bacteria secrete PETase in order to break the polymeric structure into its monomers (MHET) using water to get an OH on each side. Then, MHET is absorbed by the bacteria and very efficiently transformed to Ethylene glycerol and terephthalate.

Teams from Korea, China, UK, US and Brazil have developed an informatics model of the PETase structure, showing the reaction mechanisms with a high accuracy level. Some of them have also detailed some small structural changes that improve its efficiency. The structure model shows that the protein has a tailor in order to bind PET surfaces (S. Joo et al, 2018, X. Hans et al, 2017, H. Austin et al, 2018).

However, using the PETase enzymes in a plastic digestion bioreactor is not efficient as semi-crystalline structures used in common plastic products are very difficult to break. On this way, PET semi-crystalline structure breaks into amorph structure at 70º C, what then lets the enzyme penetrate better into the material and increase its degradation efficiency.(Wei & Zimmermann, 2017).

APPLICATIONS AND LIMITATIONS OF PET DEGRADATION

These new achievements and this better acknowledge of the PET degradation shed a bit of light in the plastic waste issue, and although it’s just the beginning they are some relevant advances and new lines of research that are driving the research.

Currently the PET degradation process is very slow and it’s far from being applicable on a large scale, because of the low efficiency of the metabolic process. There are new studies centered in obtaining genetically modified strains that have a more efficient process of the metabolism, specifically enhancing the activity of the enzyme PETase. For instance, wild-type PETase has been reported to have a degradation rate of 8.2 mg per μmol·L−1 PETase per day, whereas three variants of the enzyme, R61A, L88F and I179F have been improved to reach rates of 13.5, 17.5 and 22.5 mg per μmol·L−1 PETase per day, respectively (Ma Y, 2018). The comparison of PET degradation rates between variants of the enzyme can be seen on the Figure 2.

 Figure 2: Weight-loss rate of  PET plastic with PETase and its variants. Source: Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering.

Another limitation of PET biodegradation is that its semi-crystalline structure makes all the molecules very compacted between them in a way that it is very difficult for the organisms to access and degrade these molecules (Sivan, A. 2011). To improve this, some researchers use the enzyme PETase alone, so it can penetrate more efficiently on the PET structure and in addition is no longer needed to ad nutrients and to wait for the bacteria to grow (Seo, H. 2018).

CONCLUSIONS  

Plastic biodegradation is still in developing stage and it is not ready to be applied as a bioremediation for environmental issues due to the evident limitations. The low degradation rate of the PETase shows that there are plenty of improvements to do in the protein engineering field. Another limitation for the use of this technology outside of a reactor is that the most efficient microorganisms are genetically modified, so they cannot be released to nature. The enzymes must be supplied every time as they get damaged or diluted. Finally, there is an increasing concern about the problems that plastic degradation microorganisms can cause to plastic structures.

BIBLIOGRAPHY

Akpanudoh, N. S., Gobin, K., & Manos, G. (2005). Catalytic degradation of plastic waste to liquid fuel over commercial cracking catalysts: effect of polymer to catalyst ratio/acidity content. Journal of Molecular Catalysis A: Chemical, 235(1-2), 67-73.

Asmae, N., Noureddine, E., Moulay, S., Saâd Ibnsouda, K., & Fahim, M. (2015). Biodegradation of poly (ethylene terephthalate) by bacillus subtilis. International Journal of Recent Advances in Multidisciplinary Research, 2(12), 1060–1062. https://doi.org/10.1002/da

Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., ... & Mykhaylyk, V. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), E4350-E4357.

Chetna, S., & Madhuri, S. (2013). Studies on Biodegradation of Polyethylene terephthalate: A synthetic polymer. Journal of Microbiology and Biotechnology Research, 2(2), 248–257. https://doi.org/10.4172/1522-4821.1000371.

Han, X., Liu, W., Huang, J. W., Ma, J., Zheng, Y., Ko, T. P., ... & Guo, R. T. (2017). Structural insight into catalytic mechanism of PET hydrolase. Nature communications, 8(1), 2106.

Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H. Y., Shin, T. J., ... & Kim, K. J. (2018). Structural insight into molecular mechanism of poly (ethylene terephthalate) degradation. Nature communications, 9(1), 382.

Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., ... & Yuan, Y. (2018). Enhanced Poly (ethylene terephthalate) Hydrolase Activity by Protein Engineering. Engineering.

Miskolczi, N., Bartha, L., Deák, G., Jover, B., & Kallo, D. (2004). Thermal and thermo-catalytic degradation of high-density polyethylene waste. Journal of Analytical and Applied Pyrolysis, 72(2), 235-242.

Nowak, B., Paja̧K, J., Labuzek, S., Rymarz, G., & Talik, E. (2011). Biodegradation of poly(ethylene terephthalate) modified with polyester “Bionolle®” by Penicillium funiculosum. Polimery/Polymers, 56(1), 35–44. https://doi.org/10.1007/s10973-009-0491-8.

Seo, H., Kim, S., Son, H. F., Sagong, H. Y., Joo, S., & Kim, K. J. (2018). Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli. Biochemical and biophysical research communications.

Shah, A. A., Hasan, F., Hameed, A., & Ahmed, S. (2008). Biological degradation of plastics: a comprehensive review. Biotechnology advances, 26(3), 246-265.

Shosuke, Y., Kasumi, H., Toshihiko, T., Ikuo, T., Hironao, Y., Yasuhito, M., … Kohei, O. (2016). A bacterium that degrades and assimilatespoly(ethyleneterephthalate). Research, 351(6278), 5. https://doi.org/10.1126/science.aad6359.

Sivan, A. (2011). New perspectives in plastic biodegradation. Current opinion in biotechnology, 22(3), 422-426.  

Vegter, A. C., Barletta, M., Beck, C., Borrero, J., Burton, H., Campbell, M. L., ... & Gilardi, K. V. (2014). Global research priorities to mitigate plastic pollution impacts on marine wildlife. Endangered Species Research, 25(3), 225-247.

Wei, R., & Zimmermann, W. (2017). Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate. Microbial Biotechnology, 10(6), 1302–1307. https://doi.org/10.1111/1751-7915.12714

Whitney, P. . (1991). Agricultural and synthetic polymers. Biodegradability and utilization. (G. Swift & J. E. Glass, Eds.), Endeavour (ACS Sympos, Vol. 15). Washignton, DC. https://doi.org/10.1021/bk-1990-0433.