Why did we talk to him?

• Patrick Schröder is a scientific administrator at the German Environment Agency. He is specialized in biodegradation of pharmaceuticals and the development and spread of antimicrobial resistances in the environment.

• He brought common mistakes in disposal of pharmaceuticals to our attention. In many regions in Germany it is sufficient for them to be disposed of via household waste, but in others they must be returned to designated collection facilities or pharmacies. In Germany, consumers can find out the correct disposal method on arzenitmittelentsorgung.de
• He also told us about pharmaceuticals that cause an immense burden on the environment due to their high consumption such as ibuprofen and hormones like estrogen. These pose risk to aquatic flora and fauna.
• Furthermore, he pointed out that diclofenac, a substance that we focus our attention on, has never undergone an environmental risk assessment as of today since it was authorized before 2005, like many other pharmaceuticals.

• In order to bring the information about proper disposal of pharmaceuticals to the broader public, we translated the flyer from the German Environment Agency into English, in order to increase the awareness of the general public internationally.
• We also decided to take a closer look at the degradation of estrogen and ibuprofen by laccases. Since laccases also are able to transform both substances, we try to make our contribution here.
• Our goal is to educate the citizens. If illness or symptoms exist, it is necessary to use pharmaceuticals in the process of recovery, but excessive consumption leads to environmental pollution and species extinction^[1].

Why did we talk to him?

• We wanted to know more in detail how micropollutants effect the aquatic world in our waters. Therefore, we contacted Prof. Dr. Jörg Oehlmann. He has been Professor for aquatic ecotoxicology at the Goethe University in Frankfurt am Main since 2001.

Which information did we gather?

• Prof. Oehlmann informed us about the evaluation of a substance toxicity and why harmful effects are difficult to prove.
• Once again, he told us how harmful Diclofenac (PNEC 50 ng/L) is and that in a lot of waters in Germany the recommended threshold values are exceeded.
• Additionally, he mentioned the negative effects of especially Carbamazepine, 17-alpha-ethynyl estradiol as well as endocrine disruptors such as Bisphenol A.
• With regard of our implementation, he drew attention to our plastic based biofilm carriers. Through them, we would introduce plasticizers as well as microplastics into the water. In addition, he made us aware of the spatial limitation municipal wastewater treatment plants (WWTP) have, which could make the inclusion of an additional clarifier for our biofilm impossible.

How did we adjust our project?

• We did a lot of research on the aforementioned substances and found that the laccases we use are already able to degrade all of them. As a future outlook when we could possibly go in to the laboratory, we plan to test laccase activity in assays with those compounds.
• Furthermore, we thought about new strategies for implementation of our project besides that of an additional clarifier. We want to avoid using plastic carriers and developed a system which fits to the conditions in municipal WWTPs as well.

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).^[2] 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.

Why did we talk to her?

• Dr. Sabine Sané did her subsequent PhD studies at the department of Microsystems Engineering at the University Freiburg and investigated biotechnological approaches to regenerative energy systems. Among other things, her research also concentrated on the degradation of micropollutants in wastewater. Therefore, her work and experience with laccases made her a great asset for our project. Sané was interviewed by our friends from iGEM Kaiserslautern.
  Sané mainly worked with the laccase of the fungus Trametes versicolor.

What information did we gather?

• The purification of laccases is very laborious and expensive, which is why we would benefit from avoiding it. An ABTS assay can be used to detect the most efficient laccase concentration.
• The degradation of estrogen via laccases is theoretically possible and was proved successful with the laccase of Trametes versicolor.


• The pH optimum of the laccases needs to be considered and the pH of the wastewater must eventually be adjusted accordingly.

How did we adjust our project?

• We were able to gather information in the conversation with Dr. Sané, thanks to the framework of our partnership with iGEM Kaiserslautern and iGEM Stuttgart (als Link). As a result, all her input has helped us to acquire more knowledge about the functionality of laccases, like the degradation of estrogen, which is also a problem in wastewater (Verlinkung liste pharmis). For our experimental approach (verlinkung zu experiments) we can resort to her contribution.

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 sigF knockout to avoid sporulation. PhD Chai predicted no impact on gene expression outside of sporulation.
• The combination of a sigF 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 sigF into our project to avoid sporulation of our B. subtilis.

• Dr. Dietmar Schlosser is group leader for Environmental Mycology in the Department of Environmental Microbiology at the Helmholtz Centre for Environmental Research in Leipzig. Although he is more of an expert for fungal laccases, we still considered his experience with laccases in general as valuable. All given answers and input from below were gathered from the interview protocol provided by iGEM Kaiserslautern, since they spoke directly with Dr. Schlosser and offered to ask our question in collaboration

Which information did we gather?

• Dr. Schlosser has confirmed the native function of laccases: their ability to detoxify. In fungal laccases that reaction leads to the formation of alkaline compounds.
• The laccase has a poor water solubility.
• The reaction time of an ABTS assay to determine the most efficient laccase concentration can range from a few days up to two weeks. As optimal concentration of ABTS he recommended 2 mM.
• To monitor the degradation of the starting materials, Dr. Schlosser uses the method of UHPLC coupled with MS/MS in his research.
• The public opinion on GMOs reveals a dread of unprecedented affects but an acceptance when used for medicinal applications.
• He investigates the toxicity of products by means of bacterial inhibition tests, which provide an EC₅₀ value.

How did we adjust our project?

• The information we gained from the conversation with Dietmar Schlosser within the framework of our partnership with iGEM Kaiserslautern and iGEM Stuttgart has helped us to develop further understanding for working with laccases. In our experimental planning we can resort to the bundled knowledge we have collected, as Dr. Schlosser’s approach on the tracing of product degradation is similar to our approach with HPLC. In correspondence to his toxicity assessments, we are confirmed in our experimental approach.

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.

Which information did we gather?

• He told us about the reoccurring problems associated with commonly used procedures like ozonolysis and the use of active carbon.
• Udo Bäuerle assured us that a project like ours would be really helpful and advised us to implement our system in an additional clarifier as fourth purification step. Our engineered Bacillus subtilis probably will not survive in the activated sludge clarifier which is the second purification step.
• Udo Bäuerle was the first person to mention rotating disc filters on which our biofilm could grow on and could be implemented into the WWTP.

How did we adjust our project?

• On this basis we were able to discuss our project with plant managers of our local WWTPs. We did research about the diving bodies and after discussing this idea with other experts we finally came to the biofilm carriers we want to use.

Why did we talk to him?

• In order to implement our biofilm into wastewater treatment plants (WWTP) we have to deal with legal requirements in Germany. Therefore, the team from Kaiserslautern had the idea to contact Dr. Ulrich Ehlers from the Federal Office for Consumer Protection and Food Safety (BVL) and since we are working close together, we were able to ask him a few questions. He has been the head of the "release and placing on the market" section in the BVL’s "Genetic Engineering" department.

Which information did we gather?

• A distinction is made between content use and work outside a closed system. The latter is divided into “release” and “placing on the market”. Both of them are no applicable options for our project. (See more -->here[link zu langen Versionen])
• For our project, he recommended content use with measures to ensure the biofilm is not leaving the WWTP. The treatment plant in which we would implement our project would then have to be classified as a “genetic engineering plant” by the local state authorities.

How did we adjust our project?

• Now we do know whom we have to contact, if we plan to implement our system into the first WWTP. We have also put our focus more on creating a system that enables working in content use and getting a WWTP classified as genetic engineering plant. Besides various efforts we designed a kill switch.

Visit of a WWTP in Darmstadt, Germany

• 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).

Visit of a WWTP in Archbold, Ohio

• Wastewater treatment is a global problem. In our project we not only aim for implementation in wastewater treatment plants&nsbp;(WWTPs) in Germany but also in other countries. Therefore, it is important to look into the structure of WWTPs around the globe. Our partnership with the iGEM team from Kaiserslautern enabled us to get into contact with Mike Short and Dan Avers from the WWTP in Archbold, Ohio. Dan Avers is a water distribution chief and Mike Short is a wastewater superintendent.
• We found out that there are five different classes of plants in the United States, ranging from one to five with five being the highest. The higher the class, the bigger the plant and the amount of water purified daily. In addition, more and more complex purification stages are installed with increasing class^[X]. The WWTP in Archbold belongs to class three with a 2.5 MGD (million gallons per day) design flow. It contains bacterial sewage sludge as secondary treatment in seven aeration tanks with two settling tanks installed behind. The effluents from those tanks are treated with chlorine to kill pathogenic organisms. Sodium chloride is used to remove any remaining chlorine before the effluent flows into the Brush Creek^[X]. Mr. Short told us that they do not measure the concentration of micropollutants in the incoming wastewater as it is done in the WWTP we visited in Darmstadt, Germany. Instead, there is mandatory annual testing by the state. He told us that as far as he knows there are no federal laws or regulations prohibiting the use of GMOs in WWTPs. But the decision whether GMOs are allowed is still up to the Environmental Protection Agency (EPA).
• Another key factor is funding. As WWTPs are always being redesigned and new components are added there is a need for financial security. Therefore, costs for the installation and operation of our project have been estimated and it has been shown that the use of B-TOX is cheaper than currently available methods for removing micropollutants from wastewater. Besides, the fact that the WWTPs are constantly redesigned benefits us. The implementation if B-TOX can be combined with normal refurbishments or other new installations. Making it easily implementable and also easy to remove from the system.
• By this WWTP tour we learned more about the differences that exist between WWTPs around the world. Understanding the different types and classes of WWTPs helped us to think about an implementation strategy that fits for many different plants and reminded us that it would be good to think about alternative approaches.

Why did we talk to him?

• Prof. Dr. Ralf Möller is the head of the Space Microbiology group at the German Aerospace Center (DLR) in Cologne.
• He is also Professor of Space Microbiology at the Bonn-Rhein-Sieg University of Applied Sciences.
• Prof. Dr. Möller has more than 20 years of experience with biofilms and especially with B. Subtilis.

• Overexpressing a tasA enzyme fusion protein in B. subtilis could not only be used for wastewater treatment on earth but also for biomining so-called rare earth elements, especially for the later application in the field of asteroid mining.
• In order to be able to purify wastewater with the B-TOX system e.g. on a space station, the stability of our biofilm would have to be increased. This is necessary because biofilms behave differently in space than they do on earth due to the equal distribution of liquids in space.
• Our kill switch is a good method to ensure biocontainment.

• Prof. Dr. Möller inspired us to broaden our horizon and to look into the implementation of our biofilm in space. (Biofilm in space as link)
• Furthermore, he encouraged us in the development of our kill switch, as he thinks it is of great interest to be able to make the growth rate of the biofilm dependent on certain situations or desired environments.

• Prof. Dr. Sibylle Gaisser is a professor for industrial biotechnology and her field of teaching includes bioethics among other subjects at the University of Applied Sciences Ansbach. Whereas her field of research consists of Synthethic Biology and antibiotics-biosynthesis, her research about socio-economic analyses and technology assessment in life sciences was of great interest to us.

Which information did we gather?

• Prof. Dr. Gaisser advised us to guarantee the compliance with ethical standards, such as that there would come no harm to the public or non-beneficiaries, that we would provide accessibility to all, preserve the environment and protect all wildlife with our project.
• In the case of an implementation of our project idea into WWTPs, we would have to prove that private households pose to be the primary source of wastewater pollution in order to justify possible tax increases. This way, we would employ the polluter-pays principle.
• She advised us to always ensure a closed system, when it comes to the application of GMOs. In addition, we should provide a physical containment as well in order to prevent a break out of bacteria. Those can be achieved in form of cartridges and immobilization steps.
• No WWTP personnel should get in contact with GMOs without prior safety instructions.
• The public opinion on GMOs reveals a dread of unprecedented affects but an acceptance when used for medicinal applications.
• She suggested to prevent misuse (dual use issue) by adding a safety form.

How did we adjust our project?

• To comply with ethical standards, we kept all standards in mind during the development of our project. To justify possible tax increases because of our project, we had to prove private households as the primary source of wastewater pollution – which we were also told by Prof. Dr. Oehlmann (click here). To provide proper safety, we strictly complied to safety regulations for laboratories and developed a kill switch system. Furthermore, we incorporated the concept of enzyme immobilization. To assure personnel safety, we created a “How to use B-TOX” instruction to educate WWTP employees beforehand. To minimalize the risk of misuse we decided to add a safety form for our project. For a more detailed review of our interview with Prof. Dr. Gaisser, click here.

Why did we talk to him?

• The ethical evaluation of our project is a major part in our research. Andreas Jürgens is professor at the Department of Biology at the Technische Universität Darmstadt and is part of the ethics committee at this university. The ethics committee evaluates if research projects are in agreement with ethical standards. Therefore, he is most suited to help us find flaws and strengths of our considerations. Together with Prof. Dr. Jürgens we talked about how to evaluate our project and further considerations.

Which information did we gather?

• To evaluate the properties of a project, one part is to make risk evaluations, considering not just biological aspects like horizontal gene transfer but also the implementation. Therefore, thinking about the worst-case scenario is really important. Prof. Dr. Jürgens told us, that the use of the endogenous B. subtilis laccase helps to stay genetically closer to the wild type. Thereby, we reduce the risk of unexpected effects if cells do somehow escape into the environment. To evaluate the concrete risks of our project he suggested to talk to experts of B. subtilis or the staff of wastewater treatment plants.
• We have to consider a cost-benefit evaluation not only in a financial way but also concerning potential risks and benefits for humans and the environment.
• He confirmed our assumption, that we need to train the people getting in contact with our GMO.

How did we adjust our project?

• We wrote a "safety instruction form” for the staff of WWTPs on how to handle our biofilm, regarding the safety of the user.
• Our next steps in realization of our project would be to talk to B. subtilis experts about the risk of horizontal gene transfer and to WWTP experts about the physical containment possibilities.
• We are continuing on making our project safer and reduce potential risks.