On the 28th of July 2010 the United Nations General Assembly recognized access to clean drinking water and sanitation as a human right [1]. Therefore, it is important that we as a society continue to pursue improvements in our own local waters as well as to work on new ways to improve the water quality in places where access to clean water is still highly limited to this day. However, to achieve clean water quality is now more difficult than ever. Through the industrial revolution the demand for oil skyrocketed and large factories simply drained their wastewater into nearby rivers. This wastewater often contained residuals of crude and with that polycyclic aromatic hydrocarbons (PAHs). Still today these PAHs can be found in rivers across the world. In addition to this continuous pollution, the globalization of the world has resulted in huge amounts of oil being transported by sea every day and is still increasing. Along with this come oil disasters which spill large quantities of this dangerous material into our world’s rivers and oceans, with devastating effects on the local flora and fauna. And even though much oil can be retrieved from the water after such a disaster, large amounts of PAHs are still spread into the environment. That’s why it is so important that this dangerous and often overlooked PAH pollution in our environment is finally being addressed through our project.

Why did we establish contact?

We read in the newspaper that there had been an accidental oil spillage in the “Woog”, a small local lake right in the city of Darmstadt. We were sure that this would be the perfect opportunity to learn about the handling of such an accident and to discuss the ways in which our project could contribute to the resolution of this kind of pollution.

What did we learn?

• Contact with the producer of the spilled oil was crucial
• Because such accidents happen so rarely, it was difficult for the Umweltamt to build on previous experiences.
• Mrs. Luebbe gave us valuable contact information to state institutions, who could help us learn more about the action plans in place for dealing with oil spillages.

Reflection

Through our conversation with the Umweltamt Darmstadt we were able to receive first-hand information on how small oil spillages are being handled. We gained insight into the different parties that play important roles in such instances and got a feeling for the diverse impacts caused by an accident like this, ranging from the purely environmental side to societal and financial impacts as well. It also sparked in us the idea to further investigate the action plans provided by state institutions to learn more about the commonly established clean-up methods.

Why did we reach out?

After our insightful talk with Darmstadt’s local Department for the Environment we wanted to find out more about the action plans established to counter aquatic contamination. Luckily, Mrs. Luebbe could give us a contact to the State Department for Agriculture and Environment of Hesse (HMLU), who are responsible for these action plans.

What did we learn?

The HMLU was able to provide us with the local action plans, which gave us great insight into the different parties involved in such an effort. However, since contamination of water with oil is so rare in the state of Hessen, we were not able to receive any new experiences from past contamination. This made it difficult for us to try to identify cases in which our project could have been of substantial help, other than during long term tracking and cleaning.

Why did we take part in Bielefeld-Meetup?

We were very eager to find out what other teams were working on, and which approaches they had taken in order to solve their chosen problems. We also wanted to use this meetup to receive feedback from the judges there to let our project evolve further.

What did we learn?

Initially we planned to immobilize our bacteria on a membrane, where polluted water would flow through (see Figure 1). But we learned from the feedback and the exchange with the judges that our membrane concept probably would not work in our application and would simply be too difficult to realize. They encouraged us to investigate systems with moving balls on which our bacteria would be attached as an alternative solution.

Integration

The input we received in Bielefeld directly influenced the Dry-Lab design team. Following the meetup, we did more research into membranes and other water purification techniques and in the end decided to scrap the membrane entirely from the design of our box. Instead, we focused on maximizing the surface area inside the box and controlling the flow of water to achieve the most efficient configuration for the interior of our box (see Figure 2).

Why did we establish contact?

In an iBiology Podcast [2], Victor de Lorenzo, who is Professor for Molecular Environmental Microbiology at the Centro Nacional de Biotecnología (CSIC), discussed the application of various Pseudomonas strains for bioremediation. Upon further research into his work, we discovered that his research focuses primarily on engineering Pseudomonas putida KT2440 [3]- a versatile soil bacterium renowned for its ability to degrade a wide array of toxic compounds.
By reaching out, we aim to gain a deeper understanding of the use of soil bacteria, particularly Pseudomonas strain, as a chassis for bioremediation. We are keen to explore the potential and challenges associated with utilizing these bacteria in environmental applications.

What did we learn?

Through our online meeting with Prof. de Lorenzo, we gained valuable insight into the application of Pseudomonas in bioremediation. We learned about the SEVA (Standard European Vector Architecture) [4] collection, which offers shuttle plasmids that can be used in both E. coli and Pseudomonas. That would help us to use E. coli for cloning and amplification of the plasmid and Pseudomonas as our chassis.
However, as Dr. Lorenzo pointed out, in our application in rivers, no antibiotically selection pressure would be present and the chassis would most likely lose its genetic modifications on plasmids. To address this challenge, Dr. Lorenzo emphasized that genomic integration would be a reliable method for stabilizing genetic modifications in Pseudomonas putida KT2440 [5].
Furthermore, another potential problem would be the import of PAHs into Pseudomonas due to their low water solubility. That would limit their bioavailability and, consequently, the efficiency of their degradation and such this would be a bottleneck in the degradation of PAHs.
In addition, Prof. de Lorenzo discouraged us to express pyrene degradation enzymes on high levels, as this would be a huge metabolic burden for our chassis. Instead, he suggested to use a low or medium level promoter.

Integration

We integrated the advice and ideas by designing our Operon with a low constitutive Promoter. This choice was driven by the need for a balanced expression system that ensures sufficient but controlled gene expression, therefore minimizing the metabolic burden on the host organism. For the implementation of this operon, we utilized the SEVA (Standard European Vector Architecture) Plasmid 231, generously provided by Prof. de Lorenzo together with strain P. putida KT2440.
We established that for our project the expression of rhamnolipids [6], as advised by Prof. de Lorenzo, might enhance the biodegradation of PAHs and production in our chassis should be tested. In addition, we agreed to integrate pyrene degradation pathway into the host’s genome for final application, to provide stable genetic modifications. In in this way, no antibiotic resistance markers would be present in real-world applications, which is important to prevent potential antibiotic resistance spreading into the environment.
As proposed by Prof. de Lorenzo, we finally analyzed pyrene degradation using HPLC probes from liquid culture medium. See our results page for more information.

[1] United Nations, General Assembly Adopts Resolution Recognizing Access to Clean Water, Sanitation as Human Right, by Recorded Vote of 122 in Favour, None against, 41 Abstentions. [Online]. Available: https://press.un.org/en/2010/ga10967.doc.htm (accessed: Sep. 29 2024).
[2] Victor de Lorenzo, Bioremediation: Cyborg-ization of Soil Bacteria for Smart Degradation of Environmental Pollutants. [Online]. Available: https://www.ibiology.org/ibiology_podcasts/victor-de-lorenzo-bioremediation-cyborg-ization-of-soil-bacteria-for-smart-degradation-of-environmental-pollutants/ (accessed: Sep. 29 2024).
[3] E. Martínez-García and V. de Lorenzo, "Pseudomonas putida as a synthetic biology chassis and a metabolic engineering platform," Current opinion in biotechnology, vol. 85, p. 103025, 2024, doi: 10.1016/j.copbio.2023.103025.
[4] E. Martínez-García et al., "SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes," Nucleic acids research, vol. 51, D1, D1558-D1567, 2023, doi: 10.1093/nar/gkac1059.
[5] S. Köbbing, T. Lechtenberg, B. Wynands, L. M. Blank, and N. Wierckx, "Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis," ACS synthetic biology, vol. 13, no. 7, pp. 2060–2072, 2024, doi: 10.1021/acssynbio.3c00747.
[6] R. Posada-Baquero, M. Grifoll, and J.-J. Ortega-Calvo, "Rhamnolipid-enhanced solubilization and biodegradation of PAHs in soils after conventional bioremediation," The Science of the total environment, vol. 668, pp. 790–796, 2019, doi: 10.1016/j.scitotenv.2019.03.056.