We aim to convert micropollutants in wastewater treatment plants using an enzyme-producing biofilm and came to the decision that Bacillus subtilis is the best choice as our biofilm forming organism.

Bacillus subtilis is a gram-positive bacterium which is usually found in soil. Gram-positive bacteria only have one cell membrane and are thus often used for protein secretion^[1]. On the one hand, secreting proteins is beneficial in protein production because no cell disruption is necessary to collect the protein and the formation of inclusion bodies can be prevented^[2]. On the other hand, and in our case specifically, we avoid the issue of bringing enzyme substrates across the membrane in the cell as well as potential cell toxicity of the substrate and degradation product. We want to leverage the ability of B. subtilis to secret proteins to immobilize our target enzymes in the biofilm matrix by fusing them to a matrix protein. This is easier to realize with B. subtilis as gram-positive model organism than with the gram-negative bacteria, e.g. Escherichia coli. In addition, B. subtilis is a facultative anaerobic organism which means it can grow in oxygenic, as well as anaerobic conditions, which can be of advantage when using the biofilm underwater in the clarifier^[3].

In nature B. subtilis forms biofilms on plant roots as it needs a nutrient source. The advantage for the plants is that B. subtilis protects it from pathogenic infections by funghi^[4]. Since B. subtilis is a natural biofilm former, it has been used as model organism for studying biofilms in the last years^[4]. By using this natural biofilm former, we do not have to worry about how our organism will form a biofilm. Furthermore, B. subtilis is classified as generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA)^[5], unlike other bacteria which are often pathogenic like E. coli or Staphylococcus aureus^[6]. It is very important for us to use an organism which is generally safe given the fact that we want to bring it into a wastewater treatment plant.

Another characteristic of B. subtilis is its ability to form endospores when exposed to extreme environmental conditions^[7]. Spores are highly resistant towards extreme temperature, desiccation, radiation and chemicals. This resistance makes spores interesting for industrial processes, e.g. by displaying enzymes on the surface of spores which can then be used in harsh reaction conditions. Furthermore, they are metabolic inactive but can germinate once nutrients are sufficiently available or when they experience high pressure^[8].