How did it all start?
In the middle of May we had already decided to do our project about diclofenac and use laccase enzymes in the project. This decision had been made strongly because we wanted to aid the Baltic Sea but we had also been influenced by a partnership project between three German teams from 2020 (Darmstadt, Kaiserslautern, and Stuttgart). These teams had already worked with diclofenac and laccases so it was natural for us to continue from where they had left off. We just approached the topic from a slightly different point of view. In order to gain more information about their projects, we contacted these three German teams. After we did so, we got invited to an already scheduled meeting with the Kaiserslautern team from 2020 and some other teams planning to work with laccases. The teams attending this laccase meeting were UChicago 2021, TU Kaiserslautern 2021, DUT China 2021, CPU China 2021 and TU Kaiserslautern 2020.
The meeting was held in Zoom and it came to be a really big turning point in our project because after it, our Student team leader, Malin, contacted another member, Ethan, from the UChicago team. They texted for a while and then organized a meeting with our teams. We met each other for the first time on May 27th and slowly started to get to know each other. In the first meeting, we shared our project topics and after hearing of the other team’s plans, we agreed to start a partnership together. It was nice to have another team using laccases to plan the project further with. You can access UChicago’s wiki page from here.
Our partnership formed to happen every week in an one hour Zoom meeting first on Wednesdays but later on Thursdays. We usually started our meetings with updating each other on what had happened during the past week and then we moved onto things we needed to discuss. We ended every meeting by taking a picture of us all. Here is the document where you can see all of the pictures taken since May and also look more carefully into what happened each week:
The formation of the partnership
Even though we were super excited to have another team to work with us, the formation of a both ways beneficial partnership took a while. However, after some research and discussion we came up with a plan. We were both working on polluting agents among wastewater and trying to find a way to degrade a specific pharmaceutical compound that was harmful for our local bodies of water. We decided it would be a good idea to base the partnership on us both producing some additional information to give to the other team to improve their project. This was an especially suitable plan for us because we were able to access the lab differently. We here in Finland, had an opportunity to access a wet lab during the summer whereas the team in Chicago was only going to work in a dry lab.
However, we had one problem to start with because we weren’t going to study the same drug. Aboa’s drug of choice was diclofenac but UChicago was planning to study triclosan. We couldn’t just easily switch the drug to triclosan because it’s not a widely used drug here in Finland and therefore it doesn’t pose much damage to the marine environment and vice versa. We also came across another problem since the laccase that would’ve been the most suitable to degrade our drug wasn’t necessarily the most suitable for degrading their drug.
We overcame these challenges by deciding that they would study the behaviour of our laccases in addition to what they had already decided to study. They would’ve produced some theoretical data and our input would’ve been to replicate their simulation conditions in a wet lab environment and thus provide experimental data for comparison. This way we would both receive additional results for our projects that our teams would otherwise not have been able to produce with the available resources. Data comparison from dry lab and wet lab could also increase the credibility of used methodologies if both teams would obtain results in agreement with each other.
Prior to the decision, team UChicago also helped us to decide which laccases we would use in our study. They had proposed to us two laccases, CotA and CueO. They were used by team Darmstadt to degrade diclofenac the year before. The team had never made it to the wet lab experiments so it was a good idea for us to continue from where they had left. We still wanted to add something of our own to the plan. We did some literature research and found a laccase called Yak (from Yersinia enterocolitica) which could work in pH 7 with diclofenac. It was also a novelty factor for us because it hadn’t been used before in iGEM. We discussed with UChicago if they’d be able to do simulations on all these three laccases. They had agreed to study the CotA and CueO because there were molecular structures easily available online for them. However, the situation for Yak wasn’t the same because there was no published molecular structure available. We decided to continue with our plan anyway.
Practical plans for the partnership
One practical plan we had in mind for the partnership was that UChicago would’ve done molecular dynamics simulations on our laccases in GROMACS and then give us propositions of mutations we could’ve tested out in the wet lab during summer. They would’ve studied the structures of our laccases, especially the active site binding activity, changing some amino acids and seeing what happens theoretically. This way we could’ve gained information on how to better for example the stability or specificity of our chosen laccases. Thus we could’ve also taken this into account when designing our constructs to degrade diclofenac more efficiently in order to reach the ultimate goal, bettering the situation of the Baltic Sea. Unfortunately, we never made it to this point due to a lack of resources but we were excited to plan our partnership to include something like this.
However, producing and testing potential mutations wasn’t the main plan we had in mind for our partnership. We were both studying laccases and also working with the wastewater treatment processes of our hometowns so it was natural for us to combine these elements together. In July we both got to visit our local WWTPs, Aboa physically and UChicago virtually. We discussed these visits in our meeting and came to a conclusion that the conditions of the wastewater were actually quite similar in the processes of our two cities. We had agreed that studying the functionality of our laccases in conditions simulating our WWTPs would give us valuable information especially regarding our proposed implementations. As these were found to be similar, neither team would need to perform additional experiments to accommodate for the other team’s local wastewater conditions.
We had previously contacted our local WWTP experts and found out that the best place to add our photobioreactor would be right after the aeration phase. (To find out why, see our Proposed Implementation). We shared this information with UChicago and concluded that together we wanted to produce theoretical and practical data on the functionality of our laccases in realistic pH and temperature conditions. The conditions would thus mimic the realistic conditions of the particular part of the process in the actual wastewater treatment plant. In the partnership, our part would’ve been to do kinetic measurements in a few preselected pH and T conditions to produce quantitative data of our enzymes’ activities. We were supposed to use ABTS assay to find out whether the designed and produced laccases were active and then study their degradation efficacy with diclofenac using LC-MS. UChicago would’ve, on the other hand, done molecular dynamics simulations of our enzymes reacting with diclofenac in similar conditions to produce comparative data for our wet lab results. If there had been differences with our results, we could’ve discussed together what went wrong and why. There was also another benefit for UChicago in this plan. If we were to find out our practical results were in agreement with their theoretical results, we could’ve interpreted it as their simulations working. This means they could better trust their simulation results and thus tentatively conclude that successful practical results with their laccase could be achieved.
The preselection of realistic pH and T levels was also a common task. We read some literature and found out the optimal pH and T levels for the chosen laccases. Then we adjusted this with the information we’d got from the WWTP experts. Finally we decided to do the measurements on temperatures 11 and 22 degrees Celsius because we wanted to study the functionality in winter and summer conditions. We also decided on pH levels as we were going to study pH 5 and 7. The latter one was because the pH level of the wastewater is 7. The former one was because pH 5 was the optimal pH level for UChicago’s laccase. The optimals for our laccases were hard to find from literature. One article said however, that the optimal pH for CueO was 6,5 and pH 7 was close enough to that. By studying pH 5 we could’ve also seen at the same time if the efficiency of our laccases would’ve been higher in pH 5 than 7.
This was however the original plan and unfortunately, we had to narrow down the alternatives as the time went on. We decided to continue with the summer temperature because if the enzymes would work at a sufficient efficiency in that temperature, we would at least know that the system could be utilized a part of the year and we could continue to study the lower temperatures. If, on the other hand, the enzymes wouldn’t work well enough at 22 degrees, we would know they would most probably not work in the lower temperatures either. In this case, for example, protein engineering options should be explored.
How did we adjust to challenges?
It was such a pity but we actually never made it to the final part of our partnership plan. You can read more about what we actually managed to do with these insufficient resources from the Results page. You can also read there more thoroughly why we didn’t manage to do all the measurements we had planned to do. On UChicago's side, they ran out of computational power by the end of the competition so they also couldn’t completely execute the plans we had made together. In a nutshell, we had a lot of good plans and ideas but we were far too optimistic in the beginning. When reality started hitting us in the head during summer, we realized that we’re not actually able to do all that we planned to do. At that point we created a plan B as our backup plan.
Our plan B was about changing the mutual benefit of the partnership outside of the lab. We decided that we would implement the partnership in the Human Practices side. We had both visited at that point, our local WWTP’s and made some connections to experts in those plants. We thought we could use them to our mutual benefit. Our plan was that we would’ve first finalized our proposed implementation with our local WWTP experts to find out whether they’re actually doable in a real life environment. Then we would’ve shared the finalized plans of our proposed implementations with each other. Lastly, we would’ve discussed the other team’s proposed implementation with our local WWTP experts to find out whether it would actually be doable on the other side of the world regarding the technical side, laws and legislations.
However, we were hoping to get at least some information from Aboa’s late lab experiments in September and therefore, we didn’t start the plan B process earlier. After the last experiments in the lab, we got affirmed that we didn’t have enough information for the original partnership plan to be fulfilled so we had to start plan B. Unfortunately, we didn’t have as much time left as we would’ve hoped for the whole process of plan B, so we decided to implement it in a slightly different way. We decided to finalize the proposed implementations by ourselves and share the documents with each other. Then we sent emails with the other team’s document to our local WWTP experts and hoped to receive answers early enough to share them with each other. Thus, we were hoping to gain practical information of our own proposed implementations’ distributive possibilities.
Here are the questions we created for this survey:
- FIN: Onko meidän suunnitelma oikeasti toteutettavissa paikallisessa jätevedenpuhdistamossamme? Jos ei, niin mitä pitäisi ottaa huomioon?
ENG: Could our plan actually be implemented at our local wastewater treatment plant? If not, what should be considered?
- FIN: Mitä lakeja ja säädöksiä pitää ottaa huomioon suunniteltaessa GMO-materiaa sisältävää lisäosaa osaksi vedenpuhdistusprosessia?
ENG: What laws and regulations need to be considered when planning a system containing GMOs that would be integrated as a part of the wastewater purification process?
- FIN: Olisiko yhteistyöjoukkueemme suunnittelema lisäys toteutettavissa meidän paikallisessa jätevedenpuhdistusprosessissa? Jos ei, niin miksi?
ENG: Could the plans of our partnership team be implemented at our local WWTP. If not, why?
- FIN: Miten jätevedenpuhdistuslaitosten eri vaiheet eroavat kaupunkiemme välillä? Mihin projekteissamme suunnittellut prosessit sopisivat?
ENG: How are the stages of wastewater treatment plants different between our two cities, and where would each project’s implementation fit into that process?
As we were waiting for the answers from the experts we realized that we were actually able to answer the last question ourselves since we had shared relevant videos, documents and links to our WWTP’s homepages earlier with each other. Thus we decided to make a thorough comparison of the two WWTP’s located on different sides of the world in order to find out what was actually similar between them. We also wanted to know if there were any remarkable differences we should’ve taken into account still at this point. By making a comparison between the processes and techniques of at least these two WWTP’s, we would be able to consider with more expertise the transferability of our proposed implementations to other types of wastewater treatment plants in the world.
Comparison of the two WWTPs
Turun seudun puhdistamo Oy is responsible for treating wastewater in Turku. They provide services to 14 municipalities which includes almost 300,000 residents in the Turku region (Kakolanmäen jätevedenpuhdistamon toiminta). They also process the industrial wastewater of the region. This company cleans 90 000 m³ of wastewater per day in the Kakolanmäki wastewater treatment plant (Turun Seudun Puhdistamo). On the contrary, The Stickney Water Reclamation Plant (WRP) is responsible for the wastewater treatment and stormwater management of the City of Chicago and of 125 Cook County communities. It is a part of the Metropolitan Water Reclamation District of Greater Chicago (MWRD) and provides services for over 2.3 million people. An average of 700 million gallons of wastewater per day is cleaned there.
Already at this point we can notice that when designing our proposed implementation for only Kakolanmäki wastewater treatment plant, we would have a problem when trying to scale it up to the size of Chicago WWTP. However, in both plants, the cleaning process is trying to be quite natural and thus they’re rather similar to each other. The process in Turku is based on mechanical, chemical and biological treatments. In Chicago there’s also physical and biological treatments in the beginning but the chemical treatment is only used if necessary in the end. You can see the processes depicted in figures 1 and 2.
The first part of the treatment process in both of these plants is mechanical purification. Large debris is filtered out when wastewater goes through a coarse screen. In Turku, sand separation is used, too. Some solids and organic matter are allowed to settle in the bottom of the tanks and from there, they’re collected for further use. In Chicago they are used as fertilizers and in Turku as biogas. Air is also added among the wastewater in this step to balance the oxygen concentration. Fat, oil and grease are allowed to rise to the top of the water and scraped out of there at least in Chicago. This wasn’t mentioned separately in Kakolanmäki’s operation description but we recall it was also done there as we visited the plant in July. In Turku, they also add a chemical already at this point to remove phosphorus which doesn’t happen in WRP as chemical treatment isn’t a necessary step there. (Turun Seudun Puhdistamo in English; MWRD, Water Reclamation Plants; Stickney Water Reclamation Plant.)
After these steps there’s an aeration phase where microbes are used to remove organic matter from the wastewater in both processes. The aerobic microbes need oxygen to thrive, so air is pumped to the water. Also anaerobic microbes are used in Kakolanmäki but a similar method isn’t mentioned in the operation description of WRP. Both aerobic and anaerobic microbes multiply and eat the remaining organic materials and nutrients in the wastewater. In the tanks where aerobic microbes live, nitrogen is also being removed in Kakolanmäki. After this treatment, the microbes clump together and settle to the bottom of the tank where they become part of the organic residual, the sludge. This sludge is then removed and most of it is conducted and reused back in the aeration phase in both processes. The rest is disposed of.
The last part of the process is tertiary treatment which is mandatory in Kakolanmäki but obligatory in WRP. In Kakolanmäki they use sand filtration to clean the water up to 99% purity level from organic compounds, phosphorus and solids (Kakolanmäen jätevedenpuhdistamon toiminta). The purified water is then discharged into the Linnanaukko harbour basin in Turku. In WRP they may use sand filtration if it’s required to have over 95% of the contaminants removed. They also might remove ammonia in this part or use other disinfection methods such as chlorination and dechlorination or UV light. Afterwards the water is released to the Chicago Sanitary and Ship Canal.
What is left after the partnership?
Already before comparing the two WWTPs, we were aware of placing our proposed implementation right after the aeration phase in Kakolanmäki WWTP. After the comparison we found out that the Secondary clarification step in Turku is the same as the function of the Final settling tank in Chicago. Before this step the main idea in both WWTP’s is the same so the best place to add our photobioreactor would be before the previously stated steps. However, we might have to think more about the possible effects of using chlorine and UV light to our laccases.
On the other hand, the timeline for the execution of our plan B was quite short and therefore, we were also prepared for not receiving answers from the experts. Ultimately, we didn’t wait for them to answer anymore and we actually commented on UChicago's proposed implementation ourselves. We sent them a document with propositions of improvement because we felt like we were able to give them some good feedback. We wanted to help them make their proposed implementation actually more doable in real life. We offered some feedback on the realistic expenses of e.g. GMO waste, materials, equipment and wages for personnel they’d included in their original plan.
But all in all, we are extremely happy to have had this partnership even though we weren’t actually able to make much concrete happen. We met each other weekly and were there to troubleshoot together from time to time. We made progress and discussed many problems together as one big team. In addition to gaining mutual benefit for the scientific side of our project, we also benefited in other ways of the partnership. As the jamboree was this year organized remotely, we felt like there was a big part missing of the competition. Our partnership relieved the feeling and gave us a sense of the international community. Our partnership with UChicago also gave us some much needed peer pressure as it created deadlines for finishing each step on the way on time. It started from communication in the Zoom chat and we’re thinking the interaction will continue even after wiki freeze. This has been an awesome journey.
- Kakolanmäen jätevedenpuhdistamon toiminta. Retrieved Oct 14th, 2021 from https://www.turunseudunpuhdistamo.fi/toiminta.
- MWRD, Water Reclamation Plants. Retrieved Oct 14th, 2021 from https://mwrd.org/water-reclamation-plants.
- Stickney Water Reclamation Plant. Retrieved Oct 14th, 2021 from https://mwrd.org/sites/default/files/documents/Fact_Sheet_Stickney.pdf.
- Turun Seudun Puhdistamo in English. Retrieved Oct 14th, 2021 from https://www.turunseudunpuhdistamo.fi/in-english.
- Turun Seudun Puhdistamo. Retrieved Oct 14th, 2021 from https://www.turunseudunpuhdistamo.fi/.