Biofilms are communities of microorganisms living surrounded by an extracellular matrix^[1]. The microorganisms are either attached to each other or to a surface. Since they have cell-to-cell contact all the time, they are in a non-motile stadium, distinguishing them from planktonic cells. This also leads to a higher biomass per unit of volume than usually found with free living microorganisms^[2].

Most of the biomass of the biofilm is made up by the matrix. It consists of extracellular polymeric substances (EPS), produced by the microorganisms interacting with each other. It stabilizes and protects the bacteria from detachment, harsh environmental conditions and toxic substances^[3]. They also determine microcolonies within the biofilm, which are separated by pores and channels, supplying the cells with all the nutrients needed by passively resorbing them into the biofilm^[4]. A big part of the EPS are the proteins which are secreted into the biofilm matrix. These can be proteolytic enzymes as well as hydrophobic proteins that form a skin to protect the biofilm from dehydration^[5]. Proteolytic enzymes degrade the nutrients gained through sorption or dissolve the surface on which the cells are growing.

Since biofilms can have different gradients, e.g. an oxygen gradient, biofilm forming bacteria are often able to differentiate into e.g. motile cells, matrix-producing cells, sporulating cells or competent cells^[6]. Some bacteria even become cannibals, if nutrients are not available and digest their surrounding cells. Furthermore, biofilms in nature are usually very biodiverse, consisting of mixed species, living in the habitat most suitable for them and cooperating with each other by exchanging necessary molecules^[7].

Intriguingly, microorganisms can interact with another using use electrical communication via nanowires or they can communicate by using quorum sensing^[8,9]. When quorum sensing takes place, chemicals are sent from cell to cell, leading to a reaction cascade.

When a biofilm is getting too crowded, single cells are set free and can disperse in the environment making space for the remaining bacteria. The dispersed cells reattach somewhere else, forming a new community and thus leading to the spread of the bacterial biofilm^[10].

In nature, biofilms can be found for example in the soil, on plant roots or on rocks ^[11]. However, in medical applications biofilms are often seen as a problem as they can be pathogenic and attach onto prosthetics, gums or catheters^[12].

Bacterial biofilms are communities that produce an extracellular matrix^[2]. During the development of Bacillus subtilis biofilms, motile cells differentiate into non-motile “matrix producers” organized in cell chains^[13]. During biofilm maturation, some cells induce sporulation; others differentiate to generate a heterogeneous population with different physiologies and gene expression^[13]. Gene expression during biofilm formation in B. subtilis is regulated by a complex network which we want to adjust to the needs of our project.
A biofilm matrix consists of several extracellular components produced by the bacteria, including exopolysaccharides, proteins, lipids, and extracellular DNA^[3]. When a B. subtilis population continues to grow, production of extracellular signaling molecules is amplified by a positive feedback loop. Genes associated with fiber production (tapA-sipW-tasA operon) and EPS production (epsA-O operon) are repressed by SinR and AbrB in motile cells and are expressed during biofilm formation^[14]. This project focusses on a few key proteins which help us use B. subtilis biofilms in wastewater treatment; one of which is the major biofilm matrix component (TasA).

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.

B. 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^[15]. 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^[16]. 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 a 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^[17].

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 fungi^[16]. Since B. subtilis is a natural biofilm former, it has been used as model organism for studying biofilms in the last years^[13]. 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), unlike other bacteria which are often pathogenic like E. coli or Staphylococcus aureus^[18,19]. 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^[20]. Spores are highly resistant towards extreme temperature, dehydration, radiation and chemicals^[21]. 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 metabolically inactive but can germinate once nutrients are sufficiently available or when they experience high pressure^[22].

When confronted with the word micropollutants, the majority would come to think of water pollutants like microplastics that accumulate in the environment and our bodies. But besides this undoubtably problematic pollution factor, there are many more water pollutants most of us might not be aware of. One of these is the anti-inflammatory painkiller called diclofenac. Chemical compounds, like diclofenac itself, pose no danger to the human organism and most people tend to use painkillers frequently without ever wondering of the consequences of its disposal into wastewater^[23]. So, what exactly happens to the mentioned compounds?

Not only diclofenac, but other substances like azithromycin and carbamazepine are used in many different forms like pills or gels. As they are excreted via urine for instance, often not completely dismantled, they enter the wastewater treatment plants (WWTPs)^[24]. Although WWTPs contain mostly three but also up to four wastewater purification stages, the German Environment Agency found out that substances like diclofenac are still not sufficiently degraded and reside in open waters in comparably high concentrations, as shown on the figures below (Figures 1 and 2) (published data by the German Environment Agency)^[24,25].

Considering the increasing age of the German population, medical consumption constantly intensifies and as a result leads to more excessive contamination and environmental impacts^[24].

Most WWTP reveal a lack of an adequate removal of micropollutants, especially chemical compounds which may bioaccumulate at some point in water bodies^[25]. Approximately 70 % of pharmaceuticals in wastewater originate from private households. Diclofenac, an analgesic, was found in wastewater effluent and surface water at concentrations of 65 ng/l for the former and 647 ng/l for the latter in 2013^[26]. In 2016, the environmental quality standard of 0.1 µg/L was exceeded at 21 out of 24 German measuring points. Hence, even by surpassing up to a tertiary purification stage in WWTPs, pharmaceuticals still end up in water bodies and lead to detrimental effects in aquatic fauna - such as liver or kidney damage in fish. But not only does it endanger aquatic fauna, other kinds of animals are also affected via the food chain. In worst cases, this can even lead to species extinction^[23].

Therefore, it is our responsibility to step in. Complying to ethical standards, it is our duty to adhere to certain principles like the polluter-pays-principle and the precautionary principle, as we were told by Prof. Dr. Sibylle Gaisser. That means that we have the responsibility to provide a suitable and optimized system in order to achieve a significant deterioration in wastewater pollution, caused by humans to begin with. We as society, the primary cause for wastewater pollution through pharmaceuticals in wastewater effluent, are the only accountable parties with the obligation to preserve our environment. To do so, we strive to provide a way to minimize detrimental effects micropollutants cause. This is where B-TOX comes into play. By fusing degradation enzymes to a modular B. subtilis biofilm, we want to reduce the ratio of pharmaceuticals in wastewater. With our project, we can make an impact and come one step closer to the conservation of the environment.

For save implementation, the organism should be contained inside the biofilm environment. How does the individual cell know that it is in the biofilm? Is there a mechanism that can be modified towards our desired application? Can survival of one cell be linked to its presence in the biofilm?

As discussed in our text Evolution of the kill switch design, we examined and discarded many different approaches. Instead of killing the microorganisms with a toxin as soon as the corresponding antidote is no longer produced (Kill switch variants and precursors), we can also take essential components away from the microorganisms. Individual bacterial cells should then only receive this substance when remaining under the predetermined conditions. A cell leaving our biofilm would then be faced with drastically impaired fitness, ultimately leading to cell death. In the sections quorum sensing and comQXPA system we give further background to these concepts.

Quorum sensing is generally defined as a process of cell-cell communication that allows individuals in bacterial communities to gather information about their surroundings and enable interaction ^[27]. In this regard, interaction most often involves changing the gene expression of individual cells ^[28]. The production and concentration of autoinducers increases with higher cell density ^[7].

Which processes are regulated by quorum sensing?

Quorum sensing regulates a variety of physiological processes including symbiosis, competence, sporulation, and biofilm formation. Individuals able to participate in this multicellular and multispecies interaction pattern can be found throughout the domain of bacteria including gram-positive and gram-negative bacteria ^{[27, 29]}.

Is there a difference between gram-negative and gram-positive quorum sensing?

Gram-negative bacteria use acylated homoserine lactones as autoinducers (HSL inducers). LuxI-like proteins are responsible for the biosynthesis of these substances and LuxR-like proteins bind the inducers in a concentration dependent manner after the threshold concentration in the surrounding environment is surpassed ^[29].

In comparison, gram-positive bacteria like B. subtilis secrete processed oligopeptides as autoinducers to communicate between individual cells. Secretion takes place via an ATP-binding cassette (ABC). In contrast to the LuxR-type, two-component adaptive response proteins are used for detection of autoinducers. Ligand binding of these inducers activates a phosphorylation cascade that ends in the phosphorylation of a response regulator protein. The last transmission of a phosphorylation signal enables the response regulator protein to bind to a specific DNA sequence, leading to an altered transcription of the respective genes ^[29]. LuxS homologues have been identified in gram-positive and gram-negative species, but it remains to be studied if and how quorum sensing works between the two classes^[29].

Why is it important for us?

As mentioned above B. subtilis is a gram-positive bacterium that is able to form biofilms and uses quorum sensing (figure 3) as a communication mechanism ^{[16, 30]}. This provides a perfect intrinsic system and target we can exploit for our kill switch.

As described, B. subtilis is a natural biofilm former and already uses quorum sensing ^{[16, 31, 32, 33]}. One of these systems is the Com-system, which is naturally used by the organism to regulate its natural competence as well as the process of sporulation. B. subtilis carries two peptides that act as autoinducers, namely the competence pheromone (ComX) and competence and the sporulation factor (CSF).

This is the mechanism of the comQXPA system (figure 4) ^{[16, 33]}:

First, pre-ComX is produced. Depending on the strain pre-ComX is a 53-58 amino acid long peptide. Cleavage of pre-ComX into one larger amino acid peptide and the 6-10 amino acid long ComX oligopeptide results in isoprenylation of tryptophane at position W53 (conserved) by ComQ prior to excretion^[33]. ComX binds to ComP (receptor protein kinase) on the extracellular side of the membrane. ComP phosphorylates itself after ligand binding and is then able to phosphorylate ComA to ComA-P. By direct binding to the target DNA sequence, ComA-P regulates the gene expression of many genes including genes under the control of the degQ promoter.

In this process ComA-P accumulates via a positive feedback loop: ComA-P induces the production of CSF which then inhibits the aspartate phosphatase rapC. As rapC acts as a negative regulator for ComA-P, the concentration of ComA-P steadily increases. The increased concentration levels of ComA-P lead to stronger and more frequent induction of the degQ promoter.