During the course of our project, we proactively reached out to experts to help shape our experimental design and understand how to best implement our model in the real world. We chose experts in science, law, and engineering, both in the academic and industrial fields to create a well-rounded collection of advice and information. With all the following experts, we offered each team we collaborated with the chance to send in questions during our interviews, which you will find linked in the below sections, alongside key quotes, recommendations, important details and records of our interviews.
Integrated Human Practice
During the course of our project, we proactively reached out to experts to help shape our experimental design and understand how to best implement our model in the real world. We chose experts in science, law, and engineering, both in the academic and industrial fields to create a well-rounded collection of advice and information. With all the following experts, we offered each team we collaborated with the chance to send in questions during our interviews, which you will find linked in the below sections, alongside key quotes, recommendations, important details and records of our interviews.
Experimental Design Experts
In this section you will find experts who helped us improve the design of our experiments through advice and information.Dr. Dietmar Schlosser
Group Leader Environmental Mycology at the Department of Environmental Microbiology, Helmholtz Center for Environmental Research - UFZ Expertise: LaccasesFind them here:https://www.ufz.de/index.php?de=39080Helped us with: Advice on different algae laccases, optimal growth conditions and lab advice.
Dr. Dietmar Schlosser
How this impacted our project: Using his lab recommendations, our Chlamy team added copper to growth media and identified several possibilities for the low protein yield, ranging from potential glycosylation issues to problems related to growth conditions (see Chlamy result page for more details). Also, he gave some advice with regard to screening methods, which we could implement if time and resources would not be limiting.
Key recommendations:Copper in medium, which is known to regulate laccase gene expression at the transcriptional level and is a part of the catalytic center of laccases, may increase laccase production. Medium concentration recommendation is 10-20 µM (not exceeding 200 µM). Glycosylation issues could be a reason for lower production due to original constructs coming from fungus, where laccase glycosylation commonly takes place. One native function of laccase may relate to detoxification of plant-derived organic compounds.Usually, when using ABTS as a reagent for laccase screening during fungal growth on agar plates, color reactions (formation of a blue-green semi-stable cation radical from ABTS) may occur immediately after inoculation with mycelia-containing agar plugs derived from active fungi with strong laccase production. Less active fungi may need some days up to 2 weeks to produce a laccase-indicating color reaction. Recommended ABTS concentration is 1-2 mM. ’
Dr. Sabine Sané Lecturer and researcher at the Institute for Microsystem Technology - IMTEK at Albert-Ludwigs-Universität Freiburg Expertise:Bio-electrochemical enzymatic systems to produce electricity and to degrade micropollutants in wastewater, project focused on Tametes versicolorFind them here:https://www.biooekonomie-bw.de/fachbeitrag/aktuell/pilzsuppe-als-klaeranlage-und-biobrennstoffzelle Helped us with:Advice on laccase experimental conditions and laccase-based system placement within a wastewater treatment plant, discussed her work with laccase that led to her winning the international Huber Technology Prize 2014: Future Water.
How this impacted our project: We were very concerned about the effects of the wastewater pH on the laccase reactivity, which is one of the reasons we chose laccase constructs which could still perform in more neutral pH ranges. This helped to inform us in which sections of the wastewater treatment plant we could effectively implement our project into. She also made recommendations for laboratory use of T. Versiocolor laccase, our positive control purchased laccase, and what problems might arise during the course of experiments.
Key recommendations: Because purifying laccase is very time-consuming and costly, bypass purification and add medium directly to the water. Let the model organism grow in the culture medium and use the supernatant directly, thus avoiding the purification, then use an ABTS test to see how much laccase is through the reaction of the substrate. When designing a bioreactor to hold microorganisms, Dr. Sané’s previous project used a dialysis hose attached to the fuel cell with filters put in front so that fungus did not grow in fuel cells, however the filters were often clogged. Filters contained cellulose acetate filters and PET, however after a short time, the filter became blocked and the laccase no longer passed through. Important design information for the implementation of a bioreactor installed in an active plant. Placement of bioreactor should be in a pH range appropriate for the laccase to react with the substrate. Fungal laccase is normally optimally active at pH5, while wastewater is generally neutral or alkaline. An important point to consider is whether there are steps to adjust the pH range of the wastewater for the reaction if utilizing a laccase that only functions at a certain pH range. Only after the biological stage is it possible to acidify something.Prof. Dr. Antonio Pierik Professor of Biochemistry at Technical University of Kaiserslautern Expertise: Biochemistry and metallic proteins (such as laccase)Find them here:https://www.chemie.uni-kl.de/en/pierik/members-of-the-group/prof-dr-antonio-pierik/ Helped us with:Proteins with metallic atoms such as laccase, gave recommendations for production in algae and how to avoid potential difficulties that could arise.
How this impacted our project: Made us aware of the problem that the secreted laccase maybe didn’t get the copper-ions it needed to fold properly, and he proposed that we determine the copper. He also gave us tips regarding the purification of the laccases with a Ni2+ column. But due to time constraints we could not implement his suggestions.
Implementation Experts
In the following section you will find the experts who helped us understand the organization of the wastewater treatment system, explained the hurtles of real-world implementation for our project and advised us how to overcome these limitations through future work and data collection. Find our plan for the implementation here. Dr. Ulrich Ehlers
Helped us with: Understanding wastewater treatment plant restrictions for GMO in Germany and bioreactor specifications.
How this impacted our project: We understood that with implementation, we would need to reach out to the appropriate local state authority to be approved as a genetic engineering facility and that with our safeguards to prevent our genetically modified organisms in place (containing them within a bioreactor to prevent distribution throughout the entire plant system or into water sources), we would be considered a contained use system in the eyes of the BVL. We also learned that the BVL is primarily concerned with food, feed, or genetically modified organisms (GMOs) in industrial applications and that because our project would retain our organisms and only allow the release of the enzymatic products of our GMOs we wouldn’t be required to obtain a permit under the Genetic Engineering Act through the BVL. We were advised to contact our state authority to learn more specific information to our project’s required regulations, which we did in a later interview.
Direct quotes: ‘So, what you have described now, where you just ensure by that the genetically modified [organisms] do not leave the sewage treatment plant, then one could even regard this as a ‘contained use,’ as a genetic engineering plant for example. However, this would then have to be decided by the competent state authority. […] The approval procedures for genetically technical installations are much shorter. So, they may take a few months, and the requirements, specifically for how the data is to be presented, are much lower than when it comes to placing genetically modified microorganisms on the market.’‘If you only use the enzymes, these enzymes are neither food nor feed, then you do not need a permit under genetic engineering law for the use of these enzymes. Apart from GMOs (genetically modified organisms), only food and feed obtained from genetically modified organisms must have a permit under genetic engineering law. I cannot tell you now whether you need a permit in any other legal area, so to speak, for the use of these enzymes, but in terms of genetic engineering, with us it runs under industrial applications, i.e. neither food, feed, nor GMOs. You do not need a permit under the Genetic Engineering Act.’ ‘General tip: If you plan to do [a project involving GMOs outside a laboratory setting], contact the BVL in good time so we give advice and answer questions. As I said, if it is an , that is to say, time limited, in the case of microorganisms we would also be the licensing authority, [however] when it comes to placing them on the market, we would no longer be the licensing authority, but we would at least be able to answer questions when it comes to the authorization procedure. So as I said, within the scope of our possibilities, as far as we have the time and capacity, we are always available for advice.’
Dr.-Ing. Henning Knerr
Helped us with: Understanding major micropollutants in Germany (Diclofenac specific) and Rheinland-Pfalz wastewater treatment plant design and smaller treatment plant methods of cleaning micropollutants.How this impacted our project: This enabled us to focus our implementation efforts by understanding what the needs and requirements were for smaller plants currently in operation in Germany.
Regional State Authority of Rhineland-Palatinate Marc Wollenschläger: Industrial Wastewater TreatmentMichael Körner: Communal Wastewater Treatment Dr. Thomas Kaplan: Genetic EngineeringExpertise: Legal requirements of implementing a genetic engineering project into Rheinland-Palatinate wastewater treatment systems.Find them here:https://sgdsued.rlp.de/de/ueber-uns/behoerdenleitung/Helped us with: Advising over state regulations for implementation and Rheinland-Pfalz wastewater treatment plant design, information on smaller treatment plant methods of cleaning micropollutants.
How this impacted our project: After speaking with the BVL, we chose to reach out to the Regional State Authority to better understand what would be needed for implementation of our project and what data would be required to get approval. Because we were so fortunate to have the expertise of three very qualified individuals during the course of our presentation and interview, we were able to gather a lot of foundational knowledge and recommendations from the team over how to effectively implement our bioreactor in a real local wastewater plant. We were also highly encouraged to update the office on our progress as they were interested in the outcome and what it could mean for the future of treatment options in smaller facilities, and even offered to advise us going forward.
Key recommendations: The concept for a bioreactor system using laccase we presented has not been implemented in Rheinland-Palatinate at this time and would be an interesting tool for smaller plants to use. Each treatment plant and each state have different regulations and any system would need to abide by these to be approved for implementation. To verify our bioreactor can properly contain and filter the algae, we were recommended to test the filtration system by plating fluid to ensure no growth occurs post-filtration. It’s important to also verify this at different temperatures to confirm the filter works effectively in both summer and winter conditions (4C vs Room Temperature comparison).
Thorsten Jung and the rest of the crew at Kaiserslautern Water Treatment Plant Head of the Central Wastewater Treatment Plant in Kaiserslautern Expertise: German Wastewater Treatment PlantFind them here:www.stadtentwaesserung-kaiserslautern.deHelped us with: Comparisons between US water treatment and German water treatment methods.
How this impacted our project: Understanding the parameters needed for implementing our project. We were able to take photos of the generators, pools, and sand trap, learned about the order of wastewater treatment stages and use these references to understand the best placement for our bioreactor in a small treatment plant.
Mike Short
Village of Archbold Wastewater Superintendent Expertise: American Wastewater Treatment PlantFind them here:http://archbold.com/wastewater/
Mike Short
Helped us with: Comparisons between US wastewater treatment and German wastewater treatment methodHow this impacted our project: This enabled us to focus our implementation efforts by understanding what the needs and requirements were for smaller plants currently in operation in Germany.
Human Practice Experts
In the following section you will find the experts who helped us with human practice and educating the masses. With interviews, tours, and panel discussions to encourage discussion, we used these resources to help inform both our local community in Kaiserslautern and those around the world. Find more details on education page and the Livestream below. Dan Avers
Village of Archbold Water Distribution Chief Expertise:American Water Treatment PlantFind them here:http://archbold.com/water/
Dan Avers
Helped us with: Comparisons between US water treatment and German water treatment methods.How this impacted our project: By focusing on both water treatment (which cleans water from a local source and provides it to the city) and wastewater treatment (which cleans wastewater and releases it back into the environment), we could understand the important circular feedback involved in city water management, especially at an international level. This helped us grow our project design to be more flexible to both systems through the implementation of a pH neutral laccase and bioreactor setup and enable more treatment plants to better serve their environments and communities using our methods. We were also very fortunate enough to share this experience with a class of US students, and were able to use this tour to teach them about micropollution and wastewater treatment.
Dr. Gerd Maack
Helped us with: Understanding major micropollutants in Germany (Diclofenac specific) and worldwide environmental effects.How this impacted our project: By understanding the concerns micropollutants raise to the environment and community health on the scale of Germany, we were able to better implement our human practice and educational initiatives and better inform the public.
Christian Kaiser Member of the Initiative Progressive Agrarwende, author of ‘Initiative for a Progressive Turnaround in Agricultural Science’ (‘Initiative für progressive Agrarwende’) Expertise: Biology student at the University of Bonn, focus in pharmaceutical biology on bacterial natural substances and their biosynthesisFind them here:https://progressive-agrarwende.org/ueber-uns/Helped us with: Our project’s design and realistic implementation, discussion on GMO regulations and micropollutants impact on the genral populous.
Panel Discussion: Sebastian Brockmann from the iGEM-team Stuttgart and Linda Müller from the iGEM-team Kaiserslautern spoke over many topics over the course of the podcast, with a focus on the themes ‘Influence of man-made water pollution on aquatic ecosystems’ and ‘GMOs to save the environment.’ With this discussion, we succeeded in raising public awareness over the concerns water pollution and fostered better community understanding of GMOs.
How this impacted our project: We chose this expert to help reinforce our community message of understanding GMO’s and reverse some popular misconceptions. We had previously performed a survey with our community to better understand misunderstandings held about GMOs. Below are some key questions answered by our expert and compared with our survey answers from the community. Watch our Podcast with Stuttgart: here
During the course of our project, we proactively reached out to experts to help shape our experimental design and understand how to best implement our model in the real world. We chose experts in science, law, and engineering, both in the academic and industrial fields to create a well-rounded collection of advice and information. With all the following experts, we offered each team we collaborated with the chance to send in questions during our interviews, which you will find linked in the below sections, alongside key quotes, recommendations, important details and records of our interviews.
