Why did we talk to him?

• Patrick Schröder is a research assistant at the German Federal Environmental Agency. He is specified on biodegradation of pharmaceuticals and the development of antimicrobial resistances in the environment.

• He brought common mistakes in disposal of pharmaceuticals to our attention. In most regions it is sufficient to dispose them in household waste, but in others they must be handed in at special collection points or pharmacies.
• He also told us about particularly pharmaceuticals that cause an immense burden on the environment. These include ibuprofen and hormones like estrogen, since they pose dangers to aquatic flora and fauna and are still used by most of us on a regular basis.
• Furthermore, he pointed out that diclofenac, a substance that we focus our attention on, has never received an environmental risk assessment as of today and is nevertheless used by the general public without hesitation.

• In order to bring the information about proper disposal of pharmaceuticals to the public, we have translated the flyer from the German Federal Environmental Agency into English, in order to sensitize people internationally.
• We also decided to take a closer look at the degradation of estrogen and ibuprofen by laccases. Since laccases also show dismantling of both substances, we try to make our contribution here.
• Our goal is to educate the average citizen. If illness or symptoms exist, it is unevitable to use drugs in the process of recovery, but excessive consumption leads to environmental pollution and species extinction.[5]

text über Prof. Dr. Jörg Oehlmann

Why did we talk to him?

• Jörg Stülke is the Head of the Department of General Microbiology at the Institute of Microbiology and Genetics at the University of Göttingen. He is an expert for B. subtilis and designed a strain where the genes sinR and tasA are knocked out (GP1622).^[1] This strain would be useful for testing, if we could bring our enzymes as fusion proteins with TasA into the biofilm matrix. (Verlinkung Text TasA sinR knockout)

What information did we gather?

• Besides providing us the needed strain GP1622, he agreed with our assumption, that a knockout of sinR would not have any bad impacts on our biofilm.

How did we adjust our project?

• Getting the GP1622 B. subtilis strain enabled us to design our experiments and would have shortened the needed time in the lab.

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Why did we talk to him?

• PhD Yunrong Chai is an assistant professor at the College of Science at the Northeastern University in Boston and an expert for microbial genomics and biofilm formation. We talked to him about our ideas to engineer and improve our biofilm.

Which information did we gather?

• Concerning our plan to knockout the sinR gene of our B. subtilis to simplify the induction of the biofilm formation, PhD Chai explained that this knockout will make our biofilm more robust. The effects on other properties of our B. subtilis and our biofilm should be minor.
• Endospores are not able to produce our degradation enzymes, which is why preventing sporulation of our B. subtilis would be useful. As the sigma factor F (σ^F) regulates genes associated with sporulation, he suggested a σ^F knockout to avoid sporulation. PhD Chai predicted no impact on gene expression outside of sporulation.
• The combination of a σ^F and sinR knockout would lead to a B. subtilis that can not sporulate and forms a more robust biofilm.
• A fusion protein of the matrix protein TasA with our enzymes would be a perfect way to display the degradation enzymes in the biofilm matrix. Because TasA is very abundant, many enzymes would be located in the matrix. Furthermore, he successfully generated fusion proteins of TasA with fluorescence proteins in his lab.
• A mixed biofilm of different microorganisms could increase the robustness of our biofilm but would also complicate our assays and would be an advanced step in your project.

How did we adjust our project?

• We continued planning our fusion proteins with TasA and the knockout of sinR. Additionally, we integrated the knockout of σ^F into our project to avoid sporulation of our B. subtilis.

Why did we talk to her?

• Professor Dr. Lackner is the head of the department for sewage management at the TU Darmstadt since 2016. She is known for her work with microbial communities regarding wastewater purification in Germany, but also abroad, which is why an interview with her was of great interest for us. More so compelling was the fact that she has significant experience with the degradation of harmful pharmaceutical residues.

What information did we gather?

• Prof. Lackner told us about possible complications regarding GMOs in wastewater treatment plants and the difficulties surrounding this topic. She could confirm the danger pharmaceuticals pose in wastewater, among them diclofenac, as she has been working on its degradation herself.
• Hence, she was able to assure us that an oxidation of diclofenac was more than possible – as this has already been done. For this, it was even more helpful that she sent us continuative literature regarding diclofenac, as one of her Ph.D. students has worked on degrading the substance.
• When asked about other harmful substances, Prof. Lackner mentioned ibuprofen and carbamazepine as well as others.
• Due to her vast experience, she also gave us immense input regarding a carrier material for our biofilm and told us about the most efficient environmental conditions.

How did we adjust our project?

• She did not only confirm the alarming toxicity of diclofenac and that there is a need of a project idea like ours, but also pointed us in the direction of searching for other substances like carbamazepine.
• Her willingness to be at our disposal regarding literature or information about diclofenac and wastewater purification made her a great asset for our project. Thanks to her, and her research group, we were able to get crucial information on how and to which extent diclofenac can be oxidized by laccases.
• Since we were undecided on which carrier material we should apply for our biofilm, her recommendation of the ones she uses and the offer to provide us with them has meant a great deal for the thrive of our project.

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Visit of a WWTP

• Since our project is to be integrated into wastewater treatment plants in the future, we wanted to have a first-hand look at such a plant. For that, we visited the wastewater treatment plant from ENTEGA in Darmstadt. This visit gave us the opportunity to see by ourselves how wastewater treatment can work and which steps are necessary to achieve a clean outcome. We learned how different waste water treatment plants can be and how differently they have to deal with new guidelines concerning the quality of the treated wastewater. The wastewater treatment plant in Darmstadt is built of a primary treatment clarifier, followed by a biological wastewater treatment and a final clarifier.
  For the implementation of our biofilm, a new clarifier for an additional purification stage would be necessary in the mentioned wastewater treatment plant. Space for another tank in this plant is available but we have to consider that especially wastewater treatment plants in bigger cities wouldn’t have enough space for this. Considering this issue, we thought about different options to implement our project (link Biofilm in chemischer Stufe, Filmis).

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