Safety

We aim to develop a GMO that can be used in an industrial process. Safety is therefore of utmost importance. We have payed attention to several aspects concerning safety in our design, during our laboratory work and in future applications of our product.

Lab safety

Safety is one of the most important things to take into account in a microbiological laboratory. We work with a number of different organisms, chemicals and other supplies, each with their own hazards and characteristics.
In our project, we work with the microorganisms Escherichia coli DH5α and BL21 DE3 Star, and Saccharomyces cerevisiae YPH499, BY4741 and BJ1991. The aforementioned E. coli strains are non-colonizing[1], and S. cerevisiae is ubiquitously present in nature and has a long history of safe usage[2]. Both are classified as risk group 1, which means that they don’t prove any danger to healthy people.
Therefore, all our lab work is performed in BSL-1 labs provided by the department of Biomolecular Sciences and Biotechnology of the University of Groningen. Furthermore, all team members performing lab work are in possession of the Safe Microbial Techniques certificate, issued by the university’s safety officer. Our team is working under the supervision of people with a lot of lab experience to offer more guidance.
The practical skills obtained during the safety course, along with the usage of a BSL-1 lab, will ensure that all organisms are contained in the lab and that the health of lab workers is secured.
Our desired product, styrene, is known to be toxic. Furthermore it might be carcinogenic, although available information is inconclusive[3,4,5]. Since styrene is also a volatile compound, it is essential to limit exposure. Therefore, styrene is handled in a fume hood as much as possible, and other personal protective equipment, like gloves, goggles, and lab coats are worn. These protective measures are used not only when working with styrene, but also whenever people are working with other hazardous chemicals, like strong acids, bases, or corrosive substances.

Beyond the lab

Undoubtedly, lab safety is very important, but it is important to keep a broader scope in mind. When we start upscaling our design towards industrial processes, novel safety challenges arise. The Dutch Governmental Institute for Public Health and Environment (RIVM) invited us to explore how our product could affect the world around us. This process resulted in more awareness of broader issues concerning our project. In order to adhere to the Safe-by-Design principle, which states that safety should be incorporated into every aspect of a project, from the early beginnings until the end, we had meetings with several stakeholders and experts in relevant fields. The main safety and environmental concerns which came up during this process, along with the outcome of the relevant discussions, are listed in the following. We have incorporated these insights into our design.

Biodegradability of plastics

A large portion of the styrene we produce with our product will most likely fuel the production of materials such as polystyrene and Acrylonitril-butadieen-styreen (ABS), which are not biodegradable. Afer use, a portion of the products made from these materials will be recycled. However a large part is stored in landfills or incinerated. In case the products are not collected and properly disposed of, they may also end up in the environment, either on land or in the world’s oceans. There, they may break down into micro- and nanoplastics, be taken up by organisms, and end up in the foodchain[6]. This can kill animals and likely poses a risk to human health[7].
Because we are aware of these environmental issues, we looked into other bioplastics that may have advantages in processing after usage, for example polylactic acid (PLA). However, during a meeting with Dr. Katja Loos we found out that this problem is not limited to non-degradable plastics. PLA, a so-called biodegradable plastic, in practice does not degrade when it enters the environment but behaves similarly to non-degradable plastics. The problem is therefore not that plastics are not degradable, but that disposal and recycling is not sufficient. This rings especially true for disposable plastic products like packaging and disposable bags. Thus, we decided to continue with our initial design to produce styrene, as the market cap and potential applications are much larger and on the short term more gains can be made regarding the reduction of CO2 emissions. It is easier to switch styrene suppliers than retooling a factory to use PLA, so switching to StyGreen should be a relatively easy step to go greener.

Styrene toxicity

As mentioned before, styrene is toxic and potentially carcinogenic. When upscaling our styrene production, the quantity of styrene in the reactor also increases. This could pose a hazard for public health in case of incidents. However, at the moment large facilities already exist which produce and process not only styrene, but a large selection of other hazardous petrochemical compounds. Using the knowledge that’s already present in the petrochemical field should help design safe facilities, to limit the risks in this aspect.

The climate and CO2 emissions

It is an established fact that the rising level of CO2 emissions by human activity causes temperatures to rise and climate to change. This change has a detrimental effect on the earth, and should be limited as much as possible, for example by stopping the use of fossil fuels[8]. Our method of styrene production could contribute to the reduction of global CO2 emissions and simultaneously secure production of styrene-based plastics that are in common use around the world. In order to compare our CO2 emissions to fossil fuel-based methods, we performed a Carbon Footprint Analysis (CFA).
The CFA shows that the amount of CO2 emitted to produce StyGreen are up to 70% lower than the fossil fuel-based production methods in current use. If worldwide styrene production would switch to our new method, this would result in a large reduction of CO2 emissions.

Containment

A potential risk of any GMO-related project would be the escape of an engineered organism, either from the lab or from a facility later on in the production process, into the environment.
In nature, cellulose is abundant in plants and trees. One could imagine that our modified yeast strain could utilize the cellulose of living trees or plants. During the development of our modified S. cerevisiae strain, we however discovered that this strain can only degrade cellulose under very specific conditions. Even under these conditions, the rate of cellulose degradation is extremely slow. Furthermore, trees and plants are not pure cellulose, but also contain other materials, like hemicellulose and lignin, and all these fibers are densely packed, limiting accessibility. We argue that this slow growth, combined with the metabolic burden of protein overexpression and inaccessibility of cellulose in plants and trees. will lead to a substantial evolutionary disadvantage over other microorganisms already inhabiting the environment, thus preventing eventual colonisation. Since there are almost no places where cellulose is the only carbon source, there is no niche where only the cellulose-degrading strain can live. This means that at almost all places it has to compete, where it will likely lose out.
While it is good to reduce the effects of a potential escape, the best way to limit this risk is to prevent an escape in the first place. Therefore proper containment protocols should be put in place. While the cells will be lysed in order to extract the styrene as efficiently as possible, some might survive. Therefore all contaminated waste streams should be thoroughly sterilized before leaving the facility. To prevent escape before lysis, the reactor should be sealed off, and all vents should have filters able to catch particles the size of yeast cells. Clothing worn by employees of the facilities should be autoclaved before disposal, and should not leave the sterile zone. Cleaning and sanitation procedures should be in place to keep the facility as clean as possible.

Besides the strive towards a more bio-based economy, another present societal challenge is the rise of antibiotic resistance. In case we would have an organism with several antibiotic resistance cassettes, this would increase the potential dangers associated with escape into the environment. In our project, we switched from genetic modification techniques using antibiotic selection to CRISPR-Cas9 to integrate the construct into the host organism. This removes the reliance on antibiotics, and the related antibiotic resistance.

References

[1] University of Edinburgh. (unknown). Risk Assessment Example. E. Coli K-12 Derivate Expressing Human Growth Hormone. http://www.chem.ed.ac.uk/sites/default/files/safety/documents/GMOform_example.PDF

[2] United States Environmental Protection Agency. (1997). Attachment I -- Final Risk Assessment Of Saccharomyces Cerevisiae. https://www.epa.gov/sites/production/files/2015-09/documents/fra002.pdf

[3] Sciencelab (2005). Material Safety Data Sheet. Styrene (Monomer) MSDS. http://www.sciencelab.com/msds.php?msdsId=9925112

[4] National Toxicology Program. (2011). Styrene. https://web.archive.org/web/20110612085554/http://www.niehs.nih.gov/about/materials/styrenefs.pdf

[5] Danish Environmental Protection Agency. (2011). CLH report. Proposal for Harmonised Classification and Labelling. Substance Name: Styrene. https://www.compositesworld.com/cdn/cms/uploadedFiles/danish_epa_styrene_review(2).pdf

[6] C.B. Crawford, B. Quinn. (2016) Microplastic Pollutants. 1st edition, Elsevier Science. ISBN: 9780128094068

[7] M. Revel, A. Châtel, C. Mouneyrac. (2018) Micro(nano)plastics: A threat to human health? Current Opinion in Environmental Science & Health. Volume 1, February 2018, Pages 17-23

[8] NASA. (2018) Causes. NASA’s Jet Propulsion Laboratory. Accessed 13-10-2018. https://climate.nasa.gov/causes/