Overview

Polyethylene terephthalate (PET) is a widely used plastic in a variety of industries around the world. A direct demonstration of this is the amount of global PET bottle consumption, which surged from 1.5 million tons in 1950 to 27.64 million tons in 2018 [1]. Unfortunately, it is difficult to degrade PET naturally, taking around 450 years [2]. PET also easily fragments into microplastic particles, creating a wide spread of negative influence on the environment. These problems are exacerbated by environmentally unfriendly disposal methods, such as burying or burning. To address this, we explored an eco-friendly approach using PETase, an enzyme produced by bacteria, which can degrade PET microplastics. A suicidal switch was also applied to prevent leakage during the process.

Figure 1: diagram of microplastic impact [3]

The Effects of Microplastic Pollution

Nearly 10 million tons of plastic are dumped into the ocean every year [4], causing severe pollution. This not only harms marine ecosystems but also affects humans. "Conventional plastic materials are very resistant to degradation...The longevity of plastics is estimated to be hundreds or even thousands of years depending on properties of the plastics as well as the surrounding environmental conditions" [5]. These microplastics can enter marine organisms, and through consumption, they end up in our bodies. Even if we avoid seafood, microplastics are present in our water and food. Over time, accumulated microplastics and the harmful substances poses significant health risks.

Figure 2. Degrade of plastic to microplastic, the harm to the human body and ecosystem.

Current Challenges and Solutions

Although research on analyzing and addressing microplastic pollution has progressed, public awareness of the harmful effects of microplastic particles on ecosystems and human health remains low. Our team aims to contribute to attaining sustainable development and environmental protection, yet current technologies face limitations. Existing approaches, both chemical and biological, present distinct disadvantages, which hinder their overall effectiveness in combating microplastic pollution.

Chemical approaches, such as incineration and landfilling, often worsen the microplastic problem by releasing more particles. For instance, "In incineration sites, per metric ton of waste produced releases 360 to 102,000 microplastic particles after incineration" [6]. Additionally, microplastics in landfills can release toxic substances.

On the other hand, biological methods require specific, pH, temperature, and humidity levels that are often unachievable, conditions in natural environments or pose ecological risks. An example is the immune toxicity caused in white blood cells of Sparus aurata and Dicentrarchus labrax by Polyvinyl chloride (PVC) and polyethylene (PE) microplastics [7].

We began researching current solutions addressing these problems, however, many of these solutions also come with their own disadvantages. Current solutions, such as rectifying microplastic creation during laundry or the European Union's proposed sweeping ban on microplastics, often take a long time to implement, creating barriers for commerce and production or imposing additional costs on both consumers and companies.

We seek to address these gaps with innovative, more practical solutions. Therefore, we developed an alternative approach based on areas we identified for improvement.

Pictures from Pexels [8].

Our Solutions and Advantages

Recognizing the significant drawbacks of the existing solutions, our solution addresses the limitations of current approaches mentioned above, offering a more efficient and cost-effective method for reducing microplastic pollution. We utilize engineered cyanobacteria to degrade PET into TPA, followed by rhodobacteria that convert TPA into CO₂ and H₂O. This improves upon the inefficiency of existing EU strategies and the high costs associated with washing machine microplastic filters. Our approach is superior due to its continuous operation, increased efficiency, and reduced costs, making it a more practical and sustainable solution.

The aim of our project is to provide a sustainable solution to degrade microplastics without harming the environment. We genetically engineered E. coli to produce PETase, an enzyme that breaks down PET into monomers by cleaving ester bonds. To ensure efficient secretion, we attached E. coli's signal peptide to PETase. Using artificial intelligence, we identified mutations that led to two enhanced PETase variants: BhrPETase and IsPETase, which exhibit higher efficiency in PET degradation.

After engineering E. coli, we transferred the PETase sequence to cyanobacteria, which, due to photosynthesis, enables a self-sustaining bioreactor requiring minimal maintenance. When co-cultured with Rhodococcus, PET is degraded into H₂O and CO₂, which is further used by cyanobacteria for glucose production. This not only degrades microplastics but also prevents secondary pollution.

We designed bioreactors for both freshwater and saltwater environments:

  1. Freshwater bioreactor: A twin-membrane filter system traps microplastics for degradation by cyanobacteria, followed by Rhodococcus breaking down the resulting monomers into CO₂.
  2. Saltwater bioreactor: A freshwater pump initially cultivates cyanobacteria, which later releases enough PETase to continue degrading microplastics when the system transitions to saltwater. This ensures efficiency and adaptability with minimal modification between environments.

To prevent environmental contamination, we incorporated a suicide switch in cyanobacteria, triggered by the absence of iron ions, ensuring any leaked cells perish outside the bioreactor.

References

[1] Syama Sunil, et al. “Microplastics and Climate Change: The Global Impacts of a Tiny Driver.” Science of The Total Environment, Elsevier, 21 June 2024, www.sciencedirect.com/science/article/pii/S0048969724043080

[2] How long it takes for plastic to break down." PET Resin & PET Chips-Polyester Chip Supplier in China. (n.d.). https://wkaiglobal.com/blogs/how-long-it-takes-for-plastic-to-break-down

[3] Paul, I., Mondal, P., Haldar, D., & Halder, G. (2024). Beyond the cradle – Amidst microplastics and the ongoing peril during pregnancy and neonatal stages: A holistic review. Journal of Hazardous Materials, 469, 133963. https://www.sciencedirect.com/science/article/abs/pii/S0304389424005429?via%3Dihub

[4] Plastic Oceans International. (2018). Plastic Pollution Facts. Plastic Oceans International. https://plasticoceans.org/the-facts/

[5] Adelhafidi, A., Aidene, S., Andrady, A. L., Aragaw, T. A., Arhant, M., Beltrán-Sanahuja, A., Bläsing, M., Cadée, G. C., Cai, L., Cesa, F. S., Chen, X., Cheremisinoff, N. P., Chubarenko, I., Crawford, C. B., Debroas, D., Dris, R., Eerkes-Medrano, D., Eriksen, M., Fairbrother, A., … Pal, P. (2021, January 23). Understanding plastic degradation and microplastic formation in the environment: A Review. Environmental Pollution. https://www.sciencedirect.com/science/article/abs/pii/S0269749121001329

[6] Yang Z, Lu F, Zhang H, Wang W, Shao L, Ye J, He P, Is Incineration the Terminator of Plastics and Microplastics?, Journal of Hazardous Materials (2020), https://www.sciencedirect.com/science/article/abs/pii/S0304389420314187?via%3Dihub

[7] You Li et al 2021 IOP Conf. Ser.: Earth Environ. Sci. 631 012006 https://iopscience.iop.org/article/10.1088/1755-1315/631/1/012006

[8] (N.d.). Tenerifeweekly.com. Retrieved October 2, 2024, https://tenerifeweekly.com/2023/09/08/the-beaches-of-macaronesia-are-sinks-for-microplastics-according-to-a-study-by-the-ull-and-the-university-of-the-azores/