Introduction

With our project, we want to address the plastic pollution problem by improving the production of PHA with synthetic biology. This involves different safety aspects. Firstly, we addressed the issues microplastics cause for the environment and for human health, and how PHA would be a solution. Secondly, our project itself needed to be executed safely. We addressed the rules and regulations relating to the genetic engineering aspects of our project. The extraction of PHA from the biomass in particular requires safety considerations, since standard protocols make use of dangerous solvents. To address this, we used a tool to decide the best alternative and safest approach.

Project Safety

Dangers of Microplastics

Microplastics present a major risk to both human health and the environment. In terms of human health, research has found microplastics in essentially all tissues of the body1. The non-degradability of microplastics means that they accumulate and cause both short-term and long-term adverse effects, negatively affecting organs such as the heart and brain2. During our interview with developmental biology expert Michael Richardson , he also highlighted how nanoplastics could possibly traverse barriers like the placenta and impact developing embryos3. This demonstrates the pressing need for removing microplastics from our food production-cycle to prevent accumulation in the human body and the generational negative effects associated with this.

Microplastics also present a major problem for the environment, as they endure and accumulate in various ecosystems where they disrupt habitats, affect reproductive patterns, and accumulate toxic chemicals4. In soil, microplastics can contaminate crops and hinder their growth, impacting agriculture and food safety5. They can also alter microbial communities in the environment, causing disruption to healthy communities and allowing the growth of harmful microorganisms6. Overall, microplastics have far-reaching and detrimental effects on the environment, underlining the urgent need to reduce their release and mitigating their adverse impact on ecosystems, wildlife, and human well-being.

Safety of PHA

The degradability of PHA is a promising characteristic for a multitude of applications, offering the potential to mitigate environmental harm associated with traditional plastics. However, before advocating for large-scale use of PHA, the safety and impact on environmental settings and on human health need to be considered.

In soil environments, it has been shown that PHA degrades without causing adverse effects on microbial communities7. This suggests that PHA would be a promising alternative material for reducing microplastic soil pollution. The effect of PHA on aquatic ecosystems has also been examined, indicating that PHA breaks down in seawater environments without posing significant toxicity concerns for the ecosystem8. Furthermore, the safety of PHA for pharmaceutical and therapeutic applications has also been assessed9. PHA has been found to be biocompatible with human systems, and no toxic effects have been found10. Together, this underscores the potential for PHAs to be utilized safely in agriculture and food production contexts.

However, while the existing research shows promising results concerning PHAs' safety in soil, water, and for human applications, more studies are needed for mapping the exact migration patterns and long-term consequences of PHA in the environment. Continued research is essential to ensure a complete understanding of their implications.

Use of GMO-based bioplastics in food production

During our lecture at the NGL , an association for people interested in natural science and medicine, we asked our audience how they felt about the safety of GMO-based bioplastics in agriculture, where they scored the safety a 3.5 out of 5. This is a small sample size for measuring the full general opinion, but gives an indication of why it is important to be transparent and open to the public about the production process and the precautions taken to ensure the safety of GMO-based products. We also discussed our project with experts , Petra Hogervorst and Mirjam Schuijff from the Dutch National Institute for Public Health and the Environment (RIVM), who highlighted that we may encounter difficulties with public opinion regarding the use of GMO derived products in agriculture. Taken together, we have a good understanding of the importance of having a solid plan to ensure GMO-safety to mitigate the concerns raised by the public.

Our containment strategies

    In our project, we will take three key steps during the production process to guarantee that the final product is GMO-free. Firstly, we would take measures to ensure bio-containment of the bacteria. In our project, we knocked-out the carotenoid pathway, which causes the organism to become more UV sensitive and reduces bacterial viability outside of the lab or production environment11. Additionally, it can be considered to engineer the bacteria to exclusively feed on methanol, which would prevent escape as the substrate is then not readily available outside of the bioreactor. Secondly, human and environmental health could be ensured during the extraction of PHA from the bacteria, which is either executed by suspending the cells in a heated solvent over a period of time or by introducing large volumes of lysing agents to the cells12. To this step we could consider adding chlorine, which denatures DNA and ensures that no functional modified genetic material is released into the environment13. Finally, the extracted PHA will also be heated to temperatures that are lethal to the bacteria during the molding of PHA into a product, as described by the creators of Happy Cups . Together, these steps ensure that there are no live organisms and no modified genetic material left in the product or released into the environment. We would make the safety measures around our project transparent to help decrease the stigma surrounding GMO-based products.

Lab Safety Measures

Risk assessment GMO

Working with genetically modified organisms (GMO) includes several environmental regulations and safety risks for the employee. Firstly, all our team members received instructions on how to work in a laboratory with GMO. Then, before starting our work with GMOs, we conducted a risk assessment. This firstly addressed environmental risks, where we explored whether the organism could harm the environment if it is accidentally released. Secondly, the assessment addressed the risks to the people in the laboratory, for example, if the GMO is pathogenic or produces toxins. Prior to starting work in the lab, we carried out an evaluation with the help of the bio-safety officers (BSO) of our university of the health, safety and environment (HSE) department of the Faculty of Science. Following conventional GMO risk assessment guidelines, the host, the vectors, and the inserts were evaluated separately. Herein, the component with the highest risk was considered as the determining factor for choosing the necessary laboratory level.

Risk Assessment GMO of our project

    The entirety of our lab work was done with either E. coli TOP10 or Methylobacterium extorquens AM1. According to Dutch GMO law, both these species are listed under Appendix 2 section A1, meaning they are regarded as safe for a Risk-Level 1 laboratory. For all of the vectors we used, the sequences were known and the backbones were derived from plasmids which are on List A2 of Appendix 2 of the Dutch GMO law, meaning it is regarded as safe for a Risk-Level 1 laboratory. As for the inserts, we either expressed mCherry, PhaC, GroEL/ES or bacteriophage holins and endolysins. Since phaC and groEL/ES are native gene constructs, and mCherry is regarded as safe, these GMOs would be considered Risk-Level 1.

    For the bacteriophage genes, however, there was a lack of knowledge. It had not been tested whether concentrated exposure to bacteriophage holins or endolysins would lead to toxicity in humans. After discussing with the BSO and reviewing literature we found that the T4 bacteriophage itself is safe to work with, indicating that synthesizing a part from the phage would be safe14. Additionally, the holin/endolysins are designed to attack a bacterial cell wall, and not a eukaryotic one which would suggest that accidental exposure to the gene products will not be harmful15. Therefore, all our research could take place in a Risk-Level 1 lab.

Work with dangerous chemicals

In the lab, the risks of the chemicals used need to be assessed before you can start conducting experiments, to prevent possible health damage. A helpful system is the STOP strategy (Fig. 1, Substitution, Technical measures, Organizational measures, Personal protection). Article 55 of the REACH regulation in EU law states that carcinogenic, mutagenic, or reprotoxic (CMR) substances have to be substituted for less harmful substances where possible. Alternatively, measures need to be taken to limit the personal exposure. For our project, we used the tool for Risk Assessment of Hazardous Substances, provided by the VGM (Safety, Health and Environment) department of Leiden University to evaluate and improve our experimental setup.

REACH:

A system for the registration, evaluation and the admission of chemicals that are produced or imported in the European Union. The name 'REACH' means Registration, Evaluation, Authorization and restriction of CHemicals.

STOP strategy
Fig. 1 | STOP-strategy: When working with hazardous substances, it should first be considered to Substitute the chemical for a safer alternative. If that is not possible, Technical measures can be taken where a person is shielded from the substance by, for example, a closed system. Organizational measures include using smaller amounts, or reducing time and frequency of the exposure. Finally Personal protection measures can be taken, such as gloves or safety goggles. However, these measures are least effective and should come as a last resort.
Excel tool

The Risk Assessment of Hazardous Substances excel tool is freely available upon request by the AMD (Occupational Health, Safety and Environmental Service) of Leiden University. They are willing to instruct you on how to apply the tool correctly, since the tool is rather complicated. The tool is constantly being improved, so it is encouraged to check with the AMD whether or not a new version is available. You can contact the AMD through: amd@science.leidenuniv.nl

Risk Assessment of Hazardous Substances for PHA extraction

    Since our project used chemical solvents for the PHA extraction and purification, we carried out a chemical safety assessment. For example, chloroform is often used for PHA extraction, but is highly dangerous and can cause acute toxicity when swallowed, and is carcinogenic and a reproductive toxin17. It also penetrates latex and nitrile gloves within minutes17. The tool for Risk Assessment of Hazardous Substances takes into consideration the evaporation kinetics, volume, concentrations and hazardous properties of chemicals to determine a risk exposure score. Next to that, a fume hood containment calculation is done, to check if the hazardous fumes of the substance cannot ‘escape’ the containment of the fume hood, thus making sure that the user is protected.

    Using this, we learned that it would be too dangerous to use in an open system, which is why we should use a system such as the one shown in the dropdown menu below. However, referring back to the STOP strategy, we also explored less harmful alternatives to chloroform. We found that dimethyl carbonate (DMC) can also be used for PHA extraction. DMC extraction produces a slightly lower PHA yield than chloroform extraction, however, DMC is a much safer substance, with the only danger being its flammability18. Based on this research and the results of the tool, our setup was discussed with the university’s chemical safety officer and with experimental chemists to determine safety and feasibility. Following these discussions, feedback was implemented and cross-checked with the Excel tool. This process was carried out in an iterative manner until all parties agreed on the safety of the setup and planned experimental procedure.

    Ultimately, we decided to focus on DMC-based extraction. In addition to this, we also worked on developing an autolysis system for chemical-free extraction, as this would allow for complete removal of the risk factors related to hazardous chemicals. In the context of autolysis based extraction, DNAse could be used for denaturing the modified genetic material instead of chlorine, as this may be better suited to this process and more environmentally sustainable. However, more research is needed in order to use DNAse for this purpose.

How to set up a PHA extraction with hazardous solvents

To perform a safe solvent-based PHA extraction we used a reflux setup (Fig. 2). In this process the solvent undergoes boiling and subsequent condensation, with the condensed liquid cooling down and returning to the initial flask. This procedure was performed in a fume hood to ensure gasses will not escape into the lab. Appropriate gloves were used when handling hazardous chemicals. Appropriate gloves can be determined by, for example, using the Gloves Chemical Resistance Guide from ShieldSkin . This guide describes the permeation times of chemicals, so that it can be checked if a certain glove is resistant enough for certain chemicals and thus provides enough protection.

Setup Extraction
Fig. 2 | PHA extraction reflux set-up.
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