The issue

PFAS: new toxic chemicals

PFAS (for per- and polyfluoroalkyl substances) are a family of synthetic chemicals that have been part of our daily lives for over 60 years. Found in waterproof clothing, food packaging, non-stick coated cookware, firefighting foams… these substances are the perfect candidates for many industrial applications, as they are extremely hydrophobic, lipophobic and heat resistant.

Their exceptional chemical stability is due to their specific structure: a carbon chain bonded to fluorine atoms, as represented on Figure 1.

Carbon chain Fluorine atom C-F bond Functional group

In the PFAS family, the length of the carbon chain can vary from 1 for short-chain PFAS to more than 10 carbon atoms for long-chain PFAS. The longer the carbon chain, the more fluorine atoms are bonded. Fluorine is part of the halogen family, as well as chlorine, bromine or iodine. It is the most electronegative element on the periodic table. This is the feature of PFAS that confers their extreme stability. Due to the high electronegativity of fluorine, this bond is the strongest in organic chemistry and requires a considerable amount of energy to break, with a bon dissociation energy of 439 kJ/mol (O’Hagan, 2008). The chemical function at the head of the PFAS defines its nature. The most common ones are carboxylic acid (-COOH) and sulfonic acid (-SO3H) groups.

Unfortunately, their advantage comes at a cost, and PFAS are now nicknamed "forever pollutants”. Why? Because PFAS are released into the environment during their industrial production or during their use in everyday life. They are so stable that they do not break down and persist around us.

PFAS have been detected in urban areas like Toulouse, our city, but also at a global scale in remote regions like in the Arctic (Routti et al., 2019) and the Himalayas (Miner et al., 2021), as well as in groundwater (Ackerman Grunfeld et al., 2024) and wildlife blood samples (Routti et al., 2019; Routti et al., 2015; Birgersson et al., 2021) (for more information about the contamination scale, go to HP page).

PFAS are not only persistent in the environment: they also accumulate in living organisms, including humans (Pérez et al., 2013). Chronic exposure to certain PFAS has been associated with a range of adverse health effects (EFSA CONTAM Panel et al., 2020), including endocrine disruption, immunosuppression, developmental toxicity, and an increased risk of certain cancers. Their bioaccumulative nature raises serious concerns about long-term impacts on human health (for more information about health effects, go to HP page).

Our motivation

A rising concern in France

PFAS have raised serious concerns in France for several years now. In 2025, concrete actions were taken to limit their use and the exposure of the population to these chemicals. In addition to the European rules already monitoring the use of specific PFASs (like PFOS and PFOA), the French legislation built a National Plan of Action against PFAS, which was implemented in February 2025 (LAW n° 2025-188 27/02/2025). With this initiative, France becomes one of the first European countries planning to ban PFAS in cosmetics and textile, by January 2026.

But contamination is already all over the country, all over Europe, and PFAS production itself is still not forbidden. To understand the scale of this contamination and what it involves, the French journal Le Monde and 29 other collaborators estimated the decontamination cost of PFAS in Europe (Le Monde, 2025).

It stands at 100 billion euros a year.

Surveillance also uncovered alarming hotspots in France threatening the health of inhabitants and local communities. We knew from the start as an iGEM team that we wanted to act for people and for the environment. Guided by the power of biotechnology and the urgency of the problem, our choice was made, and PFAway was born to fight back forever pollutants.

Our solution

PFAway: decontaminating our environment with a fully integrated solution

We present PFAway: an encapsulation bead system represented in Figure 2.

The beads support two bacterial strains designed to remediate PFAS-contaminated water through three complementary mechanisms:

• Degrading long-chain PFAS
• Defluorinating short-chain PFAS
• Enhancing the resistance of our bacterial chassis to fluoride ions

1. Degrading long-chain PFAS

Degrading long-chain PFAS is the first step of the process, using a newly described bacterium: Labrys portucalensis F11. This strain has the capacity of degrading long-chain PFAS into short-chain PFAS, like trifluoroacetic acid (TFA, three fluor-carbon bonds) and perfluoropropanoic acid (PFPrA, five fluor-carbon bonds) (Wijayahena et al., 2025) (Figure 3).

After shortening the PFAS chains, the next step is defluorination.

2. Defluorinating short-chain PFAS

This is the core part of PFAway: enabling the enzymatic defluorination of short-chain PFAS, such as PFPrA and TFA released by L. portucalensis. As a starting point, a defluorinating enzyme is used: the dehalogenase 2 (DeHa2) (D90423.1) from Delftia acidovorans. This enzyme was used in previous iGEM projects (USFA 2020, USAFA 2021, SDU Denmark 2023, Uni-Padua-IT 2024) and described in the literature as being active on mono and difluorinated compounds (Farajollahi et al., 2024; Dodge et al., 2024).

Our goal is to improve the activity of DeHa2 so that it can be more efficient on poly-fluorinated short-chain PFAS such as TFA and PFPrA. To this end, we used a directed evolution approach.

Directed evolution (Figure 4) aims at accelerating the discovery of proteins with improved or novel functions by iteratively introducing genetic variation and screening for the desired variants. To do so, we applied two innovative techniques: orthogonal replication for random mutagenesis of DeHa2 in vivo coupled to fluorescence-based screening of the gene variants with a fluoride-responsive aptamer.

+ Directed evolution on DeHa2

Orthogonal replication (ORep) is a novel in vivo mutagenesis system that uses intracellular resources to efficiently generate a library of a user-defined gene without mutating the native genome nor interfering with the host replication machinery. It has recently been implemented in E. coli (Tian & al. 2024). In this work, an E. coli strain expresses an error-prone DNA polymerase that specifically replicates a linear DNA plasmid flanked by short inverted terminal repeats (ITRs). The linear replicon encodes the user-defined gene that is replicated at high mutation rate, while the genomic mutation rate is unaffected. Therefore, ORep is invaluable to accelerate the evolution of chosen proteins. This technique is represented in Figure 5.

In our case, the dehalogenase gene is inserted in the linear replicon and mutated in vivo at high rate to generate many variants that are then screened by fluorescence using a fluoride-sensitive biosensor. For more details on this technique, please visit our Design page.

The DeHa2 variants displaying the best activity on TFA and/or PFPrA will then be selected for expression in Pseudomonas putida KT2440, for defluorination of short-chain PFAS released by L. portucalensis.

3. Enhancing resistance to fluoride anions

Defluorination of short-chain PFAS will generate fluoride ions (F^-) in the cells. Fluoride ions are known to be trapped in the intracellular matrix due to the weak acid accumulation effect (Ji et al., 2014) and to inhibit essential metabolic enzymes (Johnston and Strobel, 2020). Therefore, it is critical to enhance the stress resistance of our bacterial chassis to F^-, especially when it comes to degrade poly-fluorinated compounds with the engineered dehalogenase.

In Pseudomonas putida KT2440, FluC (UniProt: Q88FT1) has been identified as the principal fluoride resistance factor. This homodimeric, transmembrane protein acts as a fluoride-specific exporter (Calero et al., 2022). Based on this finding, we selected FluC for overexpression in our chassis strain to improve its tolerance to elevated intracellular concentrations of F^-.

F^- released in water will finally be captured and removed from the decontaminated water. See details in our Entrepreneurship page.

Conclusion

PFAway offers a complete degradation process with two strains embedded in encapsulation beads (Figure 2). Our integrative solution will enable the breakdown of PFAS and pave the way for water decontamination. In addition, we implemented an in vivo mutagenesis technique based on orthogonal replication and microfluidic screening, which will be an invaluable tool for future iGEMers.