INSA Safety department

For the experimentation part of the project we were hosted by the TBI, a laboratory in the biotechnology field. Nathalie Doubrovine is the head of the safety department and the person in charge of risk prevention. Her role is to ensure that the various safety rules are respected so the employees can work safely. Before starting wet lab activities, we attended two training courses: one with Nathalie Doubrovine herself on a zoom meeting, and the other one on the lab intranet, as a training program through the software NEO. We learned about the multiple risks that can be found in a laboratory and took a test to validate our knowledge.

Legislation and French Labor Law

We were working in a French Laboratory, thus we had to respect the French national regulations about working conditions and GMO handling. As we were working with microorganisms, we were affected by the regulation on workers protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 from May 4th 1994). It also includes human endoparasites which may cause infections, allergies or be toxic. This Decree is the French transposition of Directive 90/679/EEC and is also transcribed in the Labor Code (Articles L4421-1 R4421-1 to R4427-5). In addition, the Decree of July 16th 2007 (NOR: MTST0756429A, revised on February 16th 2018) describes the technical preventive measures that must be followed in research laboratories, where workers are likely to be exposed to biological pathogens. We had to follow the rules of health, safety, and preventive medicine applied in public services in France (Decree No. 82-453). This decree refers to the Labor Code, Public Health Code and Environmental Code.

Training

Two members of our team attended an autoclave training to allow us to sterilize our equipment by ourselves. This training taught us the dangers of explosions and implosions of an autoclave, and the safety measures to take to ensure the protection of the operator. A lab coat, heat resistant gloves and glasses are required for the manipulation of the autoclave.

We also had training in the use of Ethidium Bromide and UV light for revealing our gel electrophoresis. There is a specific lockable room to handle this solution, where nothing can exit the room once it comes into contact with a contaminated object. Lab coats, gloves and glasses are mandatory in this particular room. The room is equipped with specific bins for the different types of waste, such as electrophoresis gels or consumables, to avoid all contamination of the environment.

Safety in the lab

Basic rules

In all laboratories, some basic safety principles must be applied:

It is forbidden to smoke in all rooms.
It is forbidden to drink and eat in the laboratory rooms.
It is compulsory to wear appropriate individual personal equipment.
Long hair must be tied back.
Mouth pipetting of any substance is prohibited.

There are also other precautions when working with biological organisms:

We must wash our hands regularly.
It is compulsory to wear gloves except for the use of an electric or gas burner.
In some cases (UV light, projection risk), it is compulsory to wear protection glasses.
The basic clothing: A conventional lab coat, pants and closed shoes.

Materials and Machines

We must always be vigilant when using certain equipment. There are many risks related to electricity, as well as the risk of injuries through improper use of various equipment, such as:

Centrifuge
Risk of injury if incorrectly handled. We must check that the lid is in place and that the centrifuge is well balanced before launching.
Electrophoresis tank
Risk of electrocution. We must keep the Safety Lid on the cell during electrophoresis.
Syringe and Blade
Risk of pricking or cutting. We must discard them into a specific bin.
Water-baths
Electrical or burn risk. We must check that the material is in good condition before using it.
Ethidium Bromide confinement
Risk of contamination and exposure of personnel to a CMR. All manipulations are performed in a specific room with appropriate equipment.
Chemical hood
Risk of inhalation of toxic compounds.
Electrolyzer
Risk of explosion if accumulation of H₂. The electrolyzer allows us to better control the gas flow.
CO₂ gas bottle
Risk associated with the use of compressed gas tanks. Only to be handled by a trained person.
Risk of anoxia. Gas bottles must be manipulated in ventilated rooms.

Storage

Three different cupboards are used to separate the different kinds of chemicals: flammable, acids and bases.

Waste management

Different trash containers are available in the lab:

Biological waste (which is autoclaved before being thrown out)
Common waste
Special container for chemicals
Special container for waste in contact with ethidium bromide
Biological safety cabinets

To work in a sterile area and thus avoid external contaminations by unwanted microorganisms, we used biological safety cabinets (FASTER Ultrasafe). These BSCs were cleaned with ethanol before and after each manipulation, and were sterilized by UV light every night. A maintenance control is done each year. The last control was executed on december the 5th of 2019.

External risks

TBI is subject to many different types of hazards due to its wide spectrum of scientific activities. One important hazard category includes risks that are not directly linked to the TBI research activity such as working conditions (noise, thermal atmosphere, heatwave during summer, etc.), electrical hazards, working on computer screens, falling, and so on. The main hazard category covers the substances handled for research purposes such as class 1 microorganisms (GMO or not), urban wastewaters, chemical products including CMRs, or cryogenic fluids to name a few. A secondary main hazard category relates to equipment and pilot plants with specific risks such as equipment using pressurized liquids, gases or steam (autoclaves), instruments generating non-ionizing radiations (Laser, UV lights) and electromagnetic radiation (RMN).

Safety for our project

We chose our project with the goal of sending the system to space inside a spacecraft. This means that safety is at the very center of our project, since we cannot afford to send an object that could endanger the lives of the crew aboard a spacecraft. We have studied the important safety points related to our reactor during its design on earth. In addition, we also took into account the safety problems that our system could generate when it is launched into space and when it is used by astronauts. For instance, we consider microgravity, scarce resources, lack of room, takeoff, etc. We have received the support of many experts in the space field (CNES expert, astronaut, doctor…), in order to be as close as possible to the safety standards of the systems already embarked on spaceships. Additionally, the part concerning GMOs from a nutritional standpoint has been dealt with the help of various specialists.

The safety aspect of our project is present at all stages of the life cycle of our system. We invite you to take a look at the implementation page for more information on the safety aspects of our project in its application in space or on earth.

Microorganisms used in our project and associated instructions

Our microorganisms

Throughout our project, we had to use many different microorganisms.
In our coculture, we manipulated the bacterium Clostridium ljungdahlii and several different strains of Saccharomyces cerevisiae.
We also modified the bacterium Escherichia coli and inserted the modifications in our yeast Saccharomyces cerevisiae.

Classification of our modified and non-modified microorganisms

Organisms are classified into four distinct groups based on their pathogenicity and the risk they pose to public health, or the environment.

We only handled group I organisms.

Like natural biological agents, GMOs are classified into 4 groups, according to the risks they present for public health or the environment. Each group defines the class of containment in which the GMO is to be handled.

The 4 classes of containment:

Containment class 1 corresponds to operations using GMOs from Group I, and for which the risk to human health and the environment is limited or negligible.
Containment class 2 corresponds to operations using GMOs from Group II and of low risk to human health and the environment.
Containment class 3 corresponds to operations using GMOs from Group III and of moderate risk to human health and the environment.
Containment class 4 corresponds to operations using GMOs from Group IV and which pose a high risk to human health and the environment.

We only handled containment class 1 organisms.

Working practices for class C1 (group 1) organisms for GMOs

Work surfaces are cleaned and disinfected daily and immediately after any incident that might lead to the release of organisms containing recombinant DNA molecules.
All biological waste and fluids are sterilized (or otherwise inactivated by validated procedures) before destruction or disposal. Materials contaminated with genetically modified microorganisms such as glassware and laboratory equipment must be decontaminated prior to washing, reuse or destruction.
Mechanical pipetting equipment must be used, mouth pipetting is prohibited (see Safety in the lab).
Handling must be done in such a way as to minimize the creation of aerosols.
Eating, drinking, storing food, and any activities not related to the experiment are not permitted in the work area.
Wearing a lab coat or specific laboratory clothing is required. Laboratory clothing is not worn off-site (see Safety in the lab).
Personnel should wash their hands after contact with organisms containing recombinant DNA molecules and also when leaving the laboratory. Special containment equipment is not required at this level (see Safety in the lab).

Safety rules concerning the use of our device :

Use of CO₂ as a supply for our system

Carbon dioxide is a colorless, almost odorless and tasteless gas. It is therefore practically undetectable by humans. Carbon dioxide is not considered toxic. However, carbon dioxide can have an influence on health when its volume concentration is higher than the natural 0.04% in the atmosphere. This impact depends on the CO₂ content. In order to handle carbon dioxide safely, it is essential to know the properties of this gas and to take appropriate safety measures. To manipulate it in the safest possible way, we were careful to use it in a highly ventilated room and to vented the gas coming out of the reactor outside the building.

Use of H₂ as a supply for our system

H₂ can cause asphyxiation at high concentrations. Symptoms may include loss of consciousness or motor skills. It is also an explosive gas that requires precautions for use. In order to gradually produce H₂, in small quantities, we used an electrolyser and evacuated the gas outside the building. Thus, throughout our handling, we have taken care to ventilate our work environment.

Safety during Covid-19

This year with the particular context of the Covid-19 pandemic, we have been authorized to manipulate in the laboratory provided that we follow some additional safety rules. Wearing a mask was mandatory, as well as washing and/or applying hydroalcoholic gel on our hands when entering or leaving the building or any room. Each member of the team was given their own equipment (pipette, lab coat…) and we had to be careful not to exchange our equipment. During tasks such as meetings, benchling designs or more generally any task that does not need to be in the laboratory, working from home was encouraged.

These rules have evolved over time alongside the evolution of the pandemic in France. For instance, we had to wear a mask in all situations at the beginning of the project. After a few weeks, we were allowed to take it off when working alone in a well-ventilated room (open windows), provided that we always clean our working space afterwards with a virucide.

The pandemic has affected our project, particularly in terms of the handling areas, which were restricted. Some rooms and equipment were inaccessible or required a certain person to perform the task. Additionally, our supervisors could not be present in the laboratory all day long. They had to plan their visits and respect the rules of social distancing.

Other side effects were the delivery delay of the electrolyzer. Indeed, our supplier was from the USA and had been very impacted by the pandemic. As a result, our experiments on C. ljungdahlii were significantly delayed.
On the other hand, if someone had any symptoms of the COVID-19, they had to stay at home for 7 to 14 days and get tested if possible. Among our members, some of them endured health issues. Cécilia had a fever because of a dental infection, so she had to stay at home for a week. Pierre also bacame sick and had several symptoms of the COVID-19. So he had to stay at home and do a PCR test. The same happened to Solène and Cécilia a few days later. We are only eight members and the presence of everyone is paramount for the experiments. Even though the tests were negative, these three confinements in a row have unfortunately delayed us as well.

References

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[2] “CNRS - Informations aux laboratoires.” http://www.cnrs.fr/infoslabos/reglementation/CGGrecommand.htm (accessed Oct. 20, 2020).

[3] “Evaluation des risques et contraintes règlementaires lors de manipulation d’OGM/MGM - Biotechnologies-Biochimie Génie biologique.” https://genie-bio.ac-versailles.fr/spip.php?article231 (accessed Oct. 20, 2020).

[4] L. Bodineau et al., “Comité de pilotage et rédacteurs,” p. 52.

[5] “La sécurité des véhicules à hydrogène.” https://www.h2-mobile.fr/dossiers/securite-vehicules-hydrogene/ (accessed Oct. 26, 2020).

[6] “Snapshot.” Accessed: Oct. 26, 2020. [Online]. Available: https://www.h2-mobile.fr/dossiers/securite-vehicules-hydrogene/.

[7] “FR.FDS_.15-Fiche-de-donnees-de-securite-Hydrogene.pdf.” Accessed: Oct. 26, 2020. [Online]. Available: http://www.airflow.fr/wp-content/uploads/2014/02/FR.FDS_.15-Fiche-de-donnees-de-securite-Hydrogene.pdf.

[8] “pangas-consignes-de-securite-manipulation-du-dioxyde-de-carbone-co2_tcm557-114545.pdf.” Accessed: Oct. 26, 2020. [Online]. Available: https://www.pangas.ch/fr/images/pangas-consignes-de-securite-manipulation-du-dioxyde-de-carbone-co2_tcm557-114545.pdf.

[9] ClemCed, “règles de sécurité, usage, stockage.,” CarboGlacedom. https://www.carboglacedom.com/regles-de-securite/ (accessed Oct. 26, 2020).

[10] “MSA - Risque CO₂.” https://loire-atlantique-vendee.msa.fr/lfy/risque-co2 (accessed Oct. 26, 2020).

[11] M. de l’Intérieur, “Monoxyde de carbone : attention, danger !,” https://www.interieur.gouv.fr/A-votre-service/Ma-securite/Conseils-pratiques/A-votre-domicile/Monoxyde-de-carbone-attention-danger. https://www.interieur.gouv.fr/A-votre-service/Ma-securite/Conseils-pratiques/A-votre-domicile/Monoxyde-de-carbone-attention-danger (accessed Oct. 26, 2020).

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