Introduction

In the 1950s, humans started using a harmful group of chemicals called per- and polyfluoroalkyl substances (PFAS).

It has been used in products like non-stick pans, raincoats, and fire-fighting foam because of their water- and oil-repellant properties, and their chemical stability¹. Because of their longevity, they travel from waste products into the environment and eventually seep into the ground and accumulate in humans and animals.

At vitroFAS, we aim to improve the health of humans and the planet by degrading PFAS. We do this by enhancing PFAS degrading enzymes for waterworks to implement in their filtration of drinking and wastewater.

Our First Contact with PFAS

Back in 2021, major Danish news outlets published stories about PFAS contamination on firefighting training grounds². This was the first time our teams members heard about PFAS. These stories directed us toward the topic of PFAS.

After discussing the issue with professionals in the field of clean water we grew to understand that the PFAS problem caused a major inconvenience to Danish Water treatment facilities around the country. The chemicals have now infiltrated the groundwater, the source of Denmark’s drinking water. This is unfortunately not only an issue in Denmark it is a worldwide problem³.

The Destructive Effects of PFAS

Health issues such as a weakened immune system, endometriosis, various disruptions of liver function, reduced fertility, and increased risk of certain cancers are just some of the complications PFAS are complicit in^4,5.

PFAS do not only impact humans; it also finds its way into the ocean, affecting underwater ecosystems, and seeps into the soil, where it can harm farm animals and wildlife. Studies have shown that PFAS are harmful to humans. Their ecological effects remain widely unknown, even though PFAS contamination has been documented in more than 330 animal species – from polar bears to dolphins and even pandas⁶.

All of these consequences call for action.

A Global Concern

The European Union has been working on determining various regulations concerning PFAS, like the Persistent Organic Pollutants (POPs) Regulations. In 2001 the Stockholm Convention was signed by 182 countries. Here they opted to protect human health and the environment from POPs. In 2009 the PFAS molecule Perfluorooctane sulfonic acid (PFOS) was added to the list of restricted pollutants and in 2019 Perfluorooctanoic acid (PFOA) was added⁷.

Additionally, there are limits to how high the concentration of PFAS are allowed to be in drinking water. In the European Union, the limit is 0.5 µg/L⁸ and in Denmark, it is set to 0.002 µg/L⁹. Waterworks finding concentrations above this limit must shut down or dilute the contaminated water.

These restrictions are a step in the right direction, though years of PFAS production has already caused damage. Therefore, to fix the current contamination, we focused on cleaning our drinking- and wastewater to protect citizens and wildlife from consuming the harmful ‘forever chemicals’.

To get the most out of our efforts, we turn our focus to one of the most widespread types of PFAS: Perfluorooctanoic acid (PFOA). Together with Perfluorooctane sulfonic acid (PFOS), it is the most produced and widespread PFAS molecule. PFOA is a long-chained substance. The long chain increases the bioaccumulation in humans and wildlife¹⁰. PFOA specifically is, besides the health risks listed above, linked to reduced fertility, complications during pregnancy, increased risk of testicular cancer, and more¹¹. Because of these risks, it is of great interest to get rid of PFOA.

The vitroFAS Solution

Current efforts in removing PFAS from water bodies include^12,13:

• Incineration at 980-1200 °C, which is very inefficient and costly in terms of energy consumption.
• Filtering PFAS, which does not eliminate it, but only extracts it from the water.

Bioremediation is a promising approach to eliminating PFAS from our environment but currently, it is a very slow and time-consuming process. Our team builds upon the discoveries of previous iGEM teams – USAFA20 and USAFA21. The two teams found a set of hydrolytic dehalogenases (DeHa) in Delftia acidovorans, capable of degrading PFOA, by cleaving fluoride from the long carbon chains and by cleaving the trifluoromethyl group (-CF3) of the carbon chain^14,15.

We are optimizing the effectiveness of the enzymes’ ability to cleave fluoride and the trifluoromethyl group (-CF3) from PFOA. We use error-prone PCR to induce random mutations in the gene sequences encoding each DeHa.

To find and select the most efficient enzymes after optimizing, we have constructed a plasmid BBa_K4868001 that carries two major features:

1. A red fluorescent protein (RFP) expressed under an IPTG-inducible T7 promoter. This RFP gene can be cut out via Golden Gate cloning to be exchanged with a DeHa gene.
2. A fluoride-responsive riboswitch, that when bound by fluoride induces expression of green fluorescent protein.

To select enzymes with higher catalytic activity, we use a combined approach of Fluorescence-activated Cell Sorting (FACS), and 96-well fluorescence plate reader screening. We also characterize the enzymes in vitro with F-NMR and by using a fluoride probe to measure fluoride release from degradation.

In summary, when cultured with PFOA, cells with enzymes of higher activity will be more fluorescent.

The DeHa mutant plasmids were sequenced to reveal the underlying mutations of the improved enzyme, and the protein was isolated via its His-tag, to determine the enzymatic activity in vitro.

To complement the work performed in the laboratory and to excel the project even further, we performed various computational investigations. These involve predicting the tertiary structure of the DeHas, performing docking simulations with PFOA and thousands of other PFAS molecules, and inducing mutations to investigate the effects of these on the DeHas ability to bind PFOA.

Implementation Into the Real World

We have researched the implementation of our purified enzymes: vitroZymes.

The vitroZymes will be used to degrade PFAS in highly concentrated PFAS water. This synthetic biology solution is an alternative to the current unsustainable process of incineration.

Recently a waterwork in Denmark started using an ion exchange resin filter to catch PFAS in their water¹⁶. This filter has been effective in removing PFAS from drinking water, however, once used, it cannot be reused. The resin filter has a byproduct of highly concentrated PFAS contaminated water. We envision that the integration of our vitroZymes in the water treatment process ensures efficient removal and degradation of PFAS from drinking water and wastewater. As a result, we reduce the pollution of PFAS in the environment.

Conclusion

We have observed that PFAS is a long-term issue, which is impacting human and planet health. We, vitroFAS, are addressing this problem through the application of synthetic biology. Using optimized dehalogenases, we are degrading one of the most prevalent PFAS molecules, PFOA! Consequently, we intend to integrate our vitroZymes into PFAS filtration systems to address this challenge.