Introduction
“What is essential is invisible to the eye”.
At the risk of disappointing St Exupéry, it is not love that we are seeking in this project, but a nearly invisible poison hiding in plain sight.
“What makes the desert beautiful is that somewhere it hides a well…”.
But what happens when that well, which should quench our thirst, now puts both us and ecosystems at risk? Our IGEM Lyon 2025 project addresses the water pollution by PFAS, per- and polyfluoroalkyl substances. These “forever chemicals” hide like secrets in our water.
“You are responsible, forever, for what you have tamed”.
Like the Little Prince’s rose, what we love must be protected. But the IGEM Lyon 2025 team is not looking for a rose, though in this case, we wish we could lock the danger away in a glass bell jar. We are chasing something far less beautiful and far more persistent. Our project aims to detect these invisible pollutants in our drinkable water and, should their concentration be too high, degrade them.
1. What are “PFAS”
PFas are synthetic molecules, mainly made up of carbon and fluorine, a duo so tightly bound, it’s like the Romeo and Juliet of chemistry. That bond is nearly impossible to break down. As a result, these molecules accumulate in water and in our body. Currently, there are no scalable and low energy ways of degrading PFAS… that’s when we come in.
Figure 1 : Molecular structure of PFAS
"Fluorinated substances that contain at least one fully fluorinated methyl or methylene carbon atom (without any H/Cl/Br/I atom attached to it).
e.g. With few exceptions, any chemical with at least a perfluorinated methyl group (–CF3) or a perfluorinated methylene group (–CF2–) is a PFAS.
A chemical or a biological problem: that is the question ?
Interestingly, fluorinated molecules are almost nonexistent in nature. However, in less than a century, human activity has generated over 19 million fluorinated compounds (Barnabas et al., 2022), leaving no time for micro organisms to adapt.
Moreover, when the carbon–fluorine bond is oxidized, the fluoride ion retains its electron due to its extreme electronegativity, making PFAS essentially unusable as an energy source. Since cleaving fluorine molecules offers no obvious survival advantage, evolution had no reason to select enzymes capable of breaking these bonds (Lawrence P. Wackett, 2024). This evolutionary gap explains why PFAS are so persistent in the environment. Degrading them would require the simultaneous emergence of multiple enzymatic and transport functions—an improbable event without strong selective pressure.
In this context, the persistence of PFAS is less a purely chemical problem and more a biological challenge. It is precisely this gap that makes synthetic biology a powerful tool: by designing enzymes and biological systems tailored to break these stubborn bonds, we can overcome natural limits !
Industrial use and environmental persistence
PFAS first appeared in 1938, and by the 1940s they were being produced on a large scale.
Why? Because they repel water and oil,
making them incredibly useful for everything from non-stick pans and waterproof clothing to firefighting foams and food packaging.
But their strength soon became a problem.
Today, they are everywhere. They drift in the air, seep into water and soil, accumulate in wildlife, and even end up in our blood. Some of the most famous, like PFOA and PFOS, were used for decades, while newer compounds like TFA are quietly spreading because they are small, stable, and hard to remove.
Health and Public Health Concerns
A growing body of research is uncovering just how harmful PFAS can be to human health.
The European Food Safety Authority considers the most critical health impact to be
“reduced immune response to vaccination”
, but the list goes on…
- Hormonal disruptions
- Fetal developmental effects
- Liver and kidney damage
- Increased risk of certain cancers, particularly kidney and prostate (ATSDR, 2021)
Some PFAS are toxic even at extremely low concentrations. Reflecting this, the US Environmental Protection Agency (EPA) set a regulatory limit of
4 parts per trillion (ppt)
for PFOA and PFOS in drinking water in April 2024.
In December 2023, the International Agency for Research on Cancer (IARC) classified PFOA as
carcinogenic to humans (Group 1)
and PFOS as “possibly carcinogenic” (Group 2B). Their long biological half-lives,
3.4 years for PFOS and
2.7 years for PFOA
(Rosato et al., 2023), further increase the risk of chronic exposure.
PFAS in France
The Esteban national health study in France revealed a striking reality: PFAS were detected in 100% of the individuals tested , showing that exposure is across all ages and regions. Even with limited monitoring (IGEDD, April 2023), moderate contamination is observed nationwide, emphasizing the urgent need for more comprehensive surveillance.
Figure 2 : PFAS groundwater contamination across Europe. Contamination is particularly pronounced in areas such as Limagne, Alsace, the Rhône Valley, Nord, Seine Valley, Meuse, Moselle, Brittany, and along the Mediterranean coast.
Tackling this challenge requires innovative detection, monitoring, and remediation strategies—the very mission at the heart of the FluoroBreaker project.
TFA and PFOA cases
There are more than 4700 known PFAS, obviously we had to make a choice. In our project, we focused on: TFA and PFOA.
PFOA (C8HF15O2, perfluorooctanoic acid) is a well-known long-chain PFAS, historically used by companies like Daikin between 2004 and 2008. In 2023, its use was officially banned in the European Union.
However, despite this ban, it is still detected above safety thresholds in: tap water (0.1 µg/L, EDCH), raw water (2 µg/L, EDCH), and in water discharged by factories such as Arkema and Daikin. Its classification as “carcinogenic to humans” makes it a major challenge.
Some enzymes can partially break down PFOA, but its many strong carbon–fluorine bonds make complete enzymatic defluorination unrealistic. Instead, we focused on improving detection, which is why PFOA is the main target for our biosensor.
TFA (C₂HF₃O₂, trifluoroacetic acid) is a short-chain PFAS that raises particular concern. Its small molecular size makes it highly mobile, which explains why conventional removal methods such as activated carbon or ion-exchange resins are largely ineffective. TFA is also bioaccumulative and has been associated with hormonal disruptions. Alarmingly, it has been detected in 94% of European tap water samples (Moscado et al., 2025).
Although reverse osmosis can separate TFA from water, it produces a concentrated waste stream that remains problematic. Likewise, incineration—often considered the ultimate method for PFAS destruction—can paradoxically generate TFA instead (Ellis et al., 2003). Because of its ultrashort chain and chemical stability, TFA persists even in treated water, while regulatory frameworks often exclude it from PFAS definitions.
From a biotechnological perspective, however, TFA presents a unique opportunity: it contains only three carbon–fluorine bonds, just one more than what natural dehalogenases are known to break. For these reasons, we selected TFA as our primary enzymatic target.
2. State of the Art on PFAS detection and degradation methods
Degradation
Several methods exist to degrade PFAS, each with its own strengths and limitations:
Current solutions are insufficient. Our project aims to develop an enzymatic treatment that destroys TFA at the molecular level. Haloacid dehalogenases (e.g., RPA1163, DAR3835) can break down fluoroacetate (FA) and difluoroacetate (DFA), showing the potential of enzyme-based defluorination (Khusnutdinova, 2023). Oxidative enzymes like laccases and peroxidases , like laccases and peroxidases, can partially degrade PFOA in smaller PFAS, suggesting opportunities to combine enzymatic pathways for more effective PFAS removal.
Detecting PFAS in Water
Detecting PFAS in water is still dominated by gold-standard techniques like
liquid chromatography–mass spectrometry (LC/MS, LC/MS/MS)
or
gas chromatography–mass spectrometry (GC/MS)
(Zhang et al., 2024). These methods are incredibly sensitive and precise, but they come with steep costs: complex sample preparation, long analysis times, expensive instruments, high expertise requirements, and zero portability (Académie des sciences, 2025).
Key parameters in PFAS detection include the
limit of detection (LOD)—how low a concentration can be reliably measured—and
specificity, the ability to accurately identify the target without false positives.
We identified a need for a user-friendly, rapid, and low-cost detection tool.
Where FluoroBreaker Comes In
PFAS pollution is one of the most persistent and challenging environmental issues of our time. To address it, FluoroBreaker focuses on two complementary strategies:
By combining detection and degradation, our goal is to provide an integrated solution for water treatment plants, specifically targeting the two critical PFAS compounds: PFOA and TFA.
The FluoroBreaker Team
Objective 1: Detection
To address our first objective, DETECTION, we are developing a bacterial biosensor. For this, we use a genetically engineered E. coli K-12 strain, designed to respond to PFAS by emitting a detectable light signal.
The study of Wintenberg et al. (2025) exposed E. coli MG1655 to different concentrations of PFOA in order to identify the bacteria's transcriptomic (RNA sequencing) response. We used the results to investigate which genes are up-regulated in response to PFOA exposure. Then, we attach a “light-producing” gene (luciferase) right after the promoter (switch on/off) of the PFAS-responsive genes. This way, whenever the bacteria encounter PFAS, the switch is flipped on and the bacteria start to glow. The whole construct is inserted into a small circular DNA (plasmid), then into the bacteria.
Figure 3: Biosensor principle
Our focus is mainly to develop an accessible and low-cost solution, easy to use on a large scale (shooting for the stars like the Little Prince indeed)!
Soon enough, the essential (PFAS) will not be so invisible to the eye anymore…
The goal is also to generate our own RNA sequencing data by analyzing E. coli response upon TFA exposure. The results will allow us to find genes that react differently to TFA compared to PFOA, and design a TFA biosensor detection kit.
Objective 2: Degradation
The goal is to design an enzyme capable of breaking down TFA in reverse osmosis concentrates, which are particularly enriched in PFAS. By “breaking down” we intend full mineralization of TFA into harmless end-products (such as fluoride ions and CO₂).
Beyond enzyme optimization, we also aim to pioneer the concept of an enzymatic bioreactor for water treatment — a system that does not yet exist in current plants — where contaminated water can flow through while preserving enzyme efficiency and stability.
Several bacterial strains have shown ability to interact with fluorinated compounds. For example, Acidimicrobium sp. strain A6 was reported to slowly reduce PFOA concentrations (Huang & Jaffé, 2019). However, bacterial degradation presents major drawbacks: reactions are extremely slow, and degradation products are uncontrolled—sometimes as toxic as, or even more toxic than, the original compounds. For these reasons, we decided to focus on an enzymatic strategy.
Enzymes are chemically active proteins that can be optimized for efficiency, specificity, and safety. The literature (Wackett, 2025) already highlights several enzymes capable—or potentially capable—of breaking C–F bonds, making them promising candidates for engineering:
Dehalogenases emerged as the most promising enzymes for defluorination, as they can act on small fluorinated substrates such as fluoroacetate (FA) and difluoroacetate (DA), structurally similar to TFA. Within this group, haloacid dehalogenases (HADs) are particularly attractive since they are well characterized and suitable candidates for engineering toward PFAS degradation.
Our research focuses on two haloacid dehalogenases:
These enzymes share the same catalytic mechanism:
- ➤ The reaction involves a conserved Asp–His–Asp catalytic triad (Chan & al, 2011).
- ➤ The aspartate residue performs an SN2 nucleophilic attack, displacing fluoride.
- ➤ The resulting covalent intermediate is hydrolyzed by a water molecule, activated by the histidine base, with assistance from the second aspartate residue.
Figure 4: Dehalogenase enzymatic reaction
TFA, however, is significantly more stable and sterically different, with three carbon–fluorine bonds. Its active site chemistry lacks the necessary adaptations to destabilize multiple C–F bonds within such a compact structure.
Overcome TFA Degradation: The Aromatic Ring Strategy
To enable enzymes to degrade TFA, we consulted experts in fluorine chemistry and explored potential strategies.
One promising hypothesis is that attaching an aromatic ring to TFA could delocalize electrons and weaken the carbon–fluorine bonds, thereby lowering the energy required for bond cleavage. However, this approach remains theoretical, and we cannot guarantee its effectiveness.
Interestingly, we identified an enzyme, lipase SpL, that has been reported to catalyze the addition of a halogenated aromatic ring to carboxylic acids (Zeng et al., 2018). This reaction involves the formation of an amide bond between a carboxylic acid and an amine.
Figure 5: Aromatic ring addition by lipase SpL.(article) The enzyme catalyzes amide formation from a carboxylic acid and an amine (adapted from Zeng et al., 2018).
We aim to exploit the following reaction and monitor its progress using HPLC-UV:
Figure 6: Aromatic ring addition to TFA by lipase SpL
The reaction product, N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide, is significantly larger than the original TFA molecule. A potential drawback of this increased size is reduced accessibility to the enzyme’s catalytic site, thus preventing the degradation reaction from occurring.
Our molecular docking simulations with YASARA confirmed this concern: the cycled TFA preferentially binds outside of the catalytic site, whereas TFA is able to travel through the enzyme’s tunnel and reach the catalytic center (Farallajolahi et al., 2024).
To overcome this limitation, our final strategy was to improve the binding energy between the enzyme and the cycled TFA using AI-assisted docking.
This approach allowed us to identify mutations that improved the theoretical affinity of the dehalogenase for N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide.
Complementary Docking Experiments
Beyond the aromatic ring strategy, we also used molecular docking to improve the affinity of our dehalogenase for several other fluorinated substrates:
- Fluoroacetate (FA)
- Difluoroacetate (DFA)
- Trifluoroacetate (TFA)
- TFA with halogenated ring (new compound via lipase SpL)
- PFOA and its degradation product (1,1,1-trifluoro-2-butanone)
This allows us to have alternative approaches (Plan B, C, D…) in case the main strategy doesn’t work. To learn more about our docking methods, go check our model page!
From the Lab to the Market: The Enzymatic Bioreactor
If successful, the optimized enzyme will be produced by bacteria in a bioreactor, immobilized, and subsequently integrated into a membranar bioreactor (MRE) system
Figure 7: Enzymatic Bioreactor Diagram
The bioreaction will be placed post reverse osmosis. Indeed, Reverse osmosis (RO) removes >90% of the total contaminants in water. By using our enzymes post-RO, we treat the portion of the water stream that contains the overwhelming majority of PFAS. Instead of trying to process the entire volume of water entering the plant, we only need to act on this smaller, concentrated fraction. This makes treatment more efficient, cost-effective, and technically feasible.
Chassis
We used only one bacterial chassis for the wet lab experiments: competent E. coli dH5α.
Conclusion
Together, our two modules (one to reveal, one to neutralize) create a complete bio-based tool to help communities reclaim their water from invisible threats. A PFAS concentration detected to be higher than a certain threshold can be verified again after the degradation by the enzymes, all thanks to our biosolution!
The Driving Force Behind Our Project
A picture of Lyon
This project is the result of collaboration between students from Université Lyon 1, ENS Lyon, and INSA Lyon. Beyond our passion for science, we were motivated by a shared goal: addressing both a local and international issue.
Lyon and its surroundings, especially Pierre-Bénite, are increasingly affected by PFAS pollution due to the massive release of companies like Daikin and Arkema. There is still no durable solution for detecting or degrading these compounds.
The Arkema Controversy
In 2023, forty-one municipalities filed a complaint against Arkema for “endangering human life,” “ecocide,” and “pollution.” The company, producer of Kynar (PVDF), allegedly released PFAS for decades. Internal documents revealed that the company was aware of PFAS risks since at least the late 1990s.
Even today, local water remains contaminated, with PFAS detected in employees and discharged into the Rhône. Among 26 employees tested for Capstone, the compound was detected every time.
In early 2024, the most exposed worker had 277.5 µg/L of PFNA — 345 times higher than the French average (Rosso, 2024). Contaminated waters are directly discharged into the Rhône, representing a monthly discharge of 4–5 kg of PFAS.
For more details and local context, see our HP page.
- Barnabas, S. J., Böhme, T., Boyer, S., Irmer, M., Ruttkies, C., Wetherbee, I., Kondic, T., Schymanski, E. L., & Weber, L. (2022). OntoChem PFAS CORE and Patent Files for MetFrag [Base de données]. Dans Zenodo (CERN European Organization for Nuclear Research). https://doi.org/10.5281/zenodo.6034586
- Buck, R. C., Franklin, J., Berger, U., Conder, J. M., Cousins, I. T., De Voogt, P., Jensen, A. A., Kannan, K., Mabury, S. A., & Van Leeuwen, S. P. (2011). Perfluoroalkyl and polyfluoroalkyl substances in the environment: Terminology, classification, and origins. Integrated Environmental Assessment And Management, 7(4), 513-541. https://doi.org/10.1002/ieam.258
- Colosi, L. M., et al. (2009). Peroxidase-mediated degradation of perfluorooctanoic acid. Environmental Toxicology and Chemistry, 28(2), 264-271. https://doi.org/10.1897/08-282.1
- De Sécurité Des Aliments, A. E. (2025, May 23). Substances per- et polyfluoroalkylées (PFAS). Autorité Européenne De Sécurité Des Aliments. https://www.efsa.europa.eu/fr/topics/per-and-polyfluoroalkyl-substances-pfas
- EFSA - 52024XC04910 - EN - EUR-LEX. (n.d.). https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A52024
- Evich, M. G., et al. (2022). Per- and polyfluoroalkyl substances in the environment. Science, 375(6580), eabg9065. https://doi.org/10.1126/science.abg9065
- Farajollahi, S., et al. (2024). Defluorination of Organofluorine Compounds Using Dehalogenase Enzymes from Delftia acidovorans (D4B). ACS Omega, 9(26), 28546-28555. https://doi.org/10.1021/acsomega.4c02517
- Hu, M., & Scott, C. (2024). Toward the development of a molecular toolkit for the microbial remediation of per- and polyfluoroalkyl substances. In P. I. Nikel (éd.), Applied and Environmental Microbiology, 90(4), e00157-24. https://doi.org/10.1128/aem.00157-24
- Huang, S., & Jaffé, P. R. (2019). Defluorination of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) by Acidimicrobium sp. Strain A6. Environmental Science & Technology, 53(19), 11410-11419. https://doi.org/10.1021/acs.est.9b04047
