Introduction
Since the discovery of plastics degrading microbes such as Ideonella sakaiensis 201-F6 (Yoahida et al., 202) in 2016, there has been an increase of interest in the potential of engineered microbes for the purpose of plastic waste management and valorization. As shown in Table 1, this is reflected within the iGEM community as we see an increasing trend in the number of projects working with plastics degradation, for purposes such as upcycling of plastic waste or the bioremediation of microplastic pollution. Given that the microbial degradation of plastic has emerged as a promising alternative to the traditional methods of plastic degradation via chemical and thermal processes, the team found it concerning that there was a lack of safety analysis surrounding the study and engineering of plastic-degrading microbes. The team thought it necessary to conduct a deep analysis on the risks associated with this technology not only to reflect on the use of PETase in our system, but also to serve the community by providing future teams with necessary information to act cautiously and prevent unnecessary consequences. The team chose to approach this issue through two ways: a biosecurity and a biosafety analysis
Defining Key Terms
When dealing with these two terms, it is important to highlight their differences. Biosecurity is the measure taken to stop the intentional spread of harmful organisms in the environment. The purpose of biosecurity is to protect agriculture and human health, preventing, controlling and managing the biological risks (Mandal, 2010). In comparison, biosafety is the practice of working safely when handling biological materials to prevent the accidental or unintentional release of these materials. Technologies, specific designed principles, and containment principles should be used to prevent unintentional exposure and accidental release of infectious agents and pathogens into the environment. Responsible laboratory practices are essential to biosafety, effectively preventing unauthorized access, loss, theft, misuse, diversion, and intentional release (World Health Organization, n.d.).
Dual use assessment is a part of biosecurity that refers to life sciences research, knowledge, information, or products that have beneficial purposes but could be potentially misused to cause harm or to be used maliciously (“Dual-Use Research,” n.d.). It encompasses activities that might negatively impact public health and safety, agriculture, the environment, and national security (WHO, 2020). For our project, the biosecurity aspect entailed a dual use assessment on microbes containing plastic-degrading enzymes, specifically PETase. The biosafety aspect was an environmental analysis covering the potential risks of accidental release.
Biosecurity and Dual Use Assessment
| Year | Number of iGEM teams with Projects Relating to Plastics Degradation Via Plastic-degrading Enzymes |
|---|---|
| 2020 | 7 |
| 2021 | 11 |
| 2022 | 7 |
| 2023 | 18 |
| 2024 | 19 |
When the team first started researching the use of engineered microbes in plastic degradation in the scientific community, we started to realize the prevalence of plastic, especially in the medical field. We came upon the article “Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation” which mentioned the discovery of Pap1, a functional plastic degrading enzyme from RPM52859.1, in a clinical setting. The enzyme was found to be capable of degrading PCL, a plastic with medical relevance. The same article mentioned the risk of plastic degrading pathogens using plastic medical implants—such as PET vascular implants—as a food source to facilitate further growth. This led to the question of the potential dual use of plastic degrading enzymes: How easily can these engineered microbes be used for harm in a medical setting? With PET being one of the most commonly used plastics—present in packaging, textiles, automotive components, and electronic components (Tomy Muringayil Joseph et al., 2024) —that is also widely applied to the medical field, we decided to narrow down our dual use assessment to the impact of PET-degrading microbes in the medical field.
Based on our preliminary research, the team quickly saw a few gaps in our knowledge. Not many studies on the specific implementations of PET plastic in the medical field existed. Based purely on existing papers, we had no way of strongly verifying that PET was as widespread within the medical industry as it is in daily life. To rectify this issue, the team secured an interview with Dr. Robert Lai, a plastic surgeon from Elite Aesthetic Plastic Clinic. As a professional within the medical industry, he would be able to provide insider information on the presence of PET. In our interview, he stated that materials made of PET—such as PET mesh used for support and structural reconstruction within the body—are used in plastic surgery and are expected to rise in demand because of their structural integrity in comparison with other plastics. “In plastic surgery, you need to put plastic objects in the right place in the body. You need to find a safe space to reduce plastic poisoning. PET solves that issue.” He clarified that traditional plastic was hard to work with because it needed to be placed inside the body in places that are in good health and have good circulation so that the toxins released by the plastic would not build up and cause plastic poisoning. With PET, there is no longer that worry because it is more stable in the human body and releases less toxins. PET is more suited to innate use than other plastics because it does not cause strong reactions with body tissue. This means that PET use in plastic surgery is likely to experience an upwards trend, resulting in an increase in PET plastic in the medical industry. This further justifies our dual use assessment by highlighting an area in the medical industry that could have severe implications in the case of malevolent use of PET degrading microorganisms.
Additionally, the team also had correspondence with Jiahui International Hospital (Shanghai), inquiring about the use of PET within the medical industry and existing precautions against the degradation of PET within a medical setting. The hospital’s response further supported the initial finding that PET was very widespread within the medical industry. “As a type of aseptic packaging, PET is very important in maintaining an aseptic environment. Within the hospital, PET is commonly present as sterile barrier systems, suture packaging, bottles, and CT scans.” Jiahui stated that they were aware of the existence of plastic-degrading microorganisms, but that naturally occurring strains should pose no significant threat to existing plastic infrastructure because of their slow degradation rates and specific growth requirements such as optimal pH and temperature. However, this means that engineered microorganisms with genetic constructs designed specifically for plastic degradation and remediation that may have less requirements, such as our PETase-based construct, may pose a threat to plastic infrastructure within medical settings. Though our cells were experimentally unable to degrade PET that was not dissolved in solution due to their inability to break the bonds existing in PET, the same may not hold true for microorganisms designed by other teams.
In conclusion, potential for dual use of PET-degrading microbes within the medical industry does exist because of the prevalence of PET. The release of these microorganisms in a medical setting can cause great damage to aseptic environments and packaging, as well as threatening the structural integrity of medical devices and implants. This will decrease the safety of medical procedures and increase risk of accidents occurring and designing microorganisms with the aim of plastics degradation is strongly advised.
Biological Safety
In the case of accidental release, parts of our project could cause damage to the environment and pose a threat to human safety. Our project uses plasmids with broad host ranges (such as from the pSEVA series identified as suitable for P. putida), meaning that accidental release would result in the risk of horizontal gene transfer of PET degradation genes, which can result in the unintentional degradation of plastics beyond the intended waste streams, or give antibiotic resistance to native microbes. The transfer of antibiotic resistance could threaten modern medicine and give rise to pathogens that are much harder to treat (Martínez & Baquero, 2014). Our project also engineers Escherichia coli BL21 DE3 and Pseudomonas putida KT2440 strains. The E. coli strain BL21 is nonpathogenic and unlikely to survive in host tissues and cause disease (Chart et al., 2000). In the case of accidental release, it would not survive easily in the outside environment or cause much harm. While P. putida is soil dwelling and more likely to survive outside of the lab, the KT2440 strain is non-pathogenic (Belda et al., 2016).
Though the two types of bacteria are unlikely to cause environmental damage on their own, their genetically engineered PET degradation capabilities could potentially disrupt natural ecosystems if released.
Environmental Analysis
However, the degradation of plastic by our engineered bacteria is unlikely to cause environmental harm because the intermediate and resulting products of PET degradation are unlikely to reach the concentrations needed to pose an environmental threat due to their fast degradation rates.
Terephthalic acid (TPA) is not considered toxic to aquatic organisms at low concentrations. Its short-term effects on the environment and humans are minimal, as studies on rats show no severe impact unless they are exposed to large amounts of TPA (Hernandez, 2001).
Ethylene glycol (EG) has a range of half-lives, spanning from 2 to 24 days in surface water, groundwater, and soil (Staples, Williams, Craig, & Roberts, 2001). In the atmosphere, it may take about 10 days to break down, while in soil and water environments degradation generally occurs within several days to a week (Agency for Toxic Substances and Disease Registry Division of Toxicology and Environmental Medicine, n.d.). At low concentrations, EG has minimal effects. However, at higher doses, it can deplete dissolved oxygen in bodies of water, harm aquatic and terrestrial animals, and contaminate groundwater (Environment Canada Health Canada, 2000). As oxygen levels decline, aquatic organisms may experience adverse effects (Hydratech, n.d.). EG exposure can also affect growth, mortality, and muscular coordination in various species (Environment Canada Health Canada, 2000).
MHET is obtained through PET hydrolysis, being one of the main products (Erika et al., 2022). The degradation rate of MHET ranges from various pH conditions (Meyer-Cifuentes & Öztürk, 2021).
Overall, terephthalic acid and ethylene glycol pose minimal environmental and toxicity risks when present at low concentrations, but high levels can negatively affect aquatic and terrestrial organisms. In contrast, the environmental impact of MHET remains less understood, as current research is limited. Taken together, the byproducts of PET degradation generally do not present significant ecological concerns under low-concentration conditions, though continued study is necessary to fully assess long-term risks.
Based on the dual-use assessment and environmental analysis, a kill-switch is strongly advised when designing cells with PETase or PET-degrading abilities in order to prevent misuse or accidental harm when exposed to the environment.
Chemical Safety
Three types of hazardous chemicals were used: chloroform, methanol, and ethanol. These three chemicals were used in our analytical technique, where chloroform and methanol were used for Gravimetric analysis, and ethanol was used to disinfect lab benches.
Chloroform can pose human health and environmental hazards. If chloroform is inhaled at high concentrations it has the potential to cause drowsiness, dizziness, nausea, and excitement following along with ingestion problems such as abdominal pain (UK, 2024). It could also cause skin and eye irritation, is suspected of causing cancer and fertility damage, and may harm the liver, kidneys, and respiratory tract through long exposure. In addition, it may be harmful to aquatic organisms (ILO & WHO, 2024). Throughout the use of chloroform, protocols such as operating in certified fume hoods, wearing appropriate personal protective equipment, and using containers appropriate for chloroform were adapted to guarantee safe usage of the chemical (LABORATORY SAFETY GUIDELINE, 2019).
Methanol, while a common laboratory solvent, poses significant health risks including high toxicity through ingestion, inhalation, or skin absorption as the body metabolises it into formic acid, which can lead to metabolic acidosis, neurological damage, and blindness, alongside being highly flammable (National Institute for Occupational Safety and Health [NIOSH], 2019). In the context of gravimetric analysis, where it was used as a solvent to wash precipitates, these hazards were mitigated through strict safety protocols. These protocols mandated the use of a fume hood, personal protective equipment including chemical-resistant gloves and safety goggles, and the elimination of all ignition sources to ensure its safe handling and prevent exposure (Furman, 1979).
Ethanol is a highly effective disinfectant, but its main risk include high flammability, which can create explosive vapor-air mixtures, and its toxicity, which can cause central nervous system depression, respiratory irritation, and with chronic exposure, organ damage (World Health Organization [WHO], 2010). To mitigate these hazards when disinfecting lab surfaces, safety protocols mandate applying ethanol in well-ventilated spaces away from ignition sources, using it in small volumes to minimize vapor accumulation, and allowing surfaces to fully air dry before operating electrical equipment to prevent fires (U.S. Centers for Disease Control and Prevention [CDC], 2024).