Part 1

Before we started working practically in the laboratory, we obtained a safety instruction from our biology safety officer of the department (Prof. Dr. Heiko Liesegang). We were informed about the following topics:

• Behavior in a laboratory (e.g. long clothes, hair tied back, no running, no eating, no drinking, no smoking)
• Emergency plan of the institute
• Wearing of appropriate personal protection equipment (e.g. lab coat, googles, gloves)
• How to prevent and respond to accidents (e.g. wearing PPE, fire extinguishers, whole-body and eye shower, elicit fire alarm, gas warning, first aid)
• How to meat safety measures of S1 organisms (e.g. disinfection, collecting and autoclaving waste)
• How to work with chemicals and learning about chemical hazards (e.g. transportation, use of chemical fume hood)
• Proper Documentation of experimental work

Our labwork was always done under supervision and it was strictly controlled that we follow the rules we learned about. When we had any safety concerns, we could evaluate them with our biology safety officer of the department as well as with the occupational safety officer of the department and the employees of the university's staff office for safety and environmental protection.

Part 2

For our project we were just working with S1 organisms, classified by the German DSMZ. They are not known to be harmful to humans or the environment.

• Escherichia coli strains: DH5-alpha, XL-1 blue MRF, TOP10, CopyCutter EPI 400
• Solventogenic clostridia: Clostridium saccharoperbutylacetonicum ATCC 27021, Clostridium beijerinckii ATCC 51743, Clostridium beijerinckii NRRL B-593
• Acetogenic bacteria: Acetobacterium woodii ATCC 29683, Clostridium ljungdahlii ATCC 55383

We did not release our organisms, their products (such as butanol) or our engineered Clostridia outside the lab. For experiments that had to be conducted sterile, we worked under a laminar flow hood. Chemicals that could generate toxic or hazardous gases, vapors or fumes were stored and only open in a chemical fume hood to avoid inhalation.
All waste (solid and liquid) that might have been contaminated with biological S1 material was collected and autoclaved before disposal.

Part 3

When anaerobic flasks were autoclaved, the autoclave was not opened before the liquid in the flasks had reached a temperature of max. 60 °C to avoid bursting of the glass when it rapidly comes into contact with colder air. Additionally, we collected and sealed all needles that we needed to transfer anaerobic media in a special container to prevent sticking and infection when handling the waste. One risk that we specifically had to deal with was the usage of highly explosive hydrogen gas. We used a mixture of H2 and CO2 to grow our solventogenic bacteria and our engineered clostridia without added sugars in the medium. Therefore, we opened the hydrogen gas cylinder only for the time of usage and worked under a fume hood. Moreover, the department was equipped with a gas warning system that set off an emergency alarm when free hydrogen gas was detected in the laboratories. Moreover, hydrogen gas was also a component of the atmosphere in our anaerobic tent. However, the level of hydrogen gas in the atmosphere was kept so low that there was no danger of explosion even during electroporation in the tent.