Why PFAS
What do waterproof clothes, non-stick pans, and fire-fighting foams have in common? They all contain substances that confer water resistance and oil-repellent properties. These compounds, better known as PFAS, are a broad class of molecules characterized by strong carbon-fluorine bonds, making them highly effective surfactants and surface protectors. Since their invention, PFAS have been widely exploited in countless fields of applications.
PFAS release in air, soil, and water has been linked to a wide plethora of
health risks. The Veneto region in northern Italy, home to UniPadua University,
has been severely affected by PFAS pollution.
Since PFAS contaminated sites were unraveled in Veneto, the issue became closer
and closer to us. After looking around, we realized that this issue was not just ours:
PFAS contamination is spread all around the world, calling for action on a global level.
After careful investigation and countless interactions with society and the community,
we understood that the field where our intervention would be effective the most was
undoubtedly groundwater. Therefore, we decided to start from our neighborhood with the
aim of creating a system fit to the necessities of our community, that could be then
scaled up and adjusted to the morphology of any ground water treatment facility. Current
technologies are unable to fully contrast PFAS presence and degradation, highlighting even
more the necessity for innovative ideas. The issue of PFAS pollution could be addressed under
several aspects, and we decided to tackle as many as possible, in order to gain a comprehensive
understanding of the problem and to respond with a powerful, resourceful solution.
The System
SurPFAS derives from “surface”, as we intend to surface-express PFAS degradative enzymes
on the outer membrane of Escherichia coli. Our system will present two E. coli populations,
exposing two different enzymes, carrying out a chain reaction that culminates
with PFAS degradation.
Once PFAS are absorbed by filters in ground water facilities, a
chemical regeneration
process allows for PFAS desorption. The gathered PFAS will now meet our engineered bacteria
in a
bioreactor
, where the degradative reaction will take place. The surface-expressed enzymes
are selected through meticulous
bioinformatic research
, and then cloned into E.coli chassis.
The whole process, from PFAS adsorption in ground water to the degradation stages in the bioreactor,
will be monitored through a
sensor
that exploits principles of impedance and spectroscopy
to obtain a real-time qualitative and quantitative analysis of PFAS occurrence.
Biological degradation is just one of the many tools entailed in the SurPFAS system, alongside with chemical absorption and desorption in PFAS filtering, spectroscopic and electronical detection of PFAS molecules, and engineering of a functioning industrial pipeline. This multidisciplinary approach allows SurPFAS to be a project strongly linked to reality, essential in the fight against PFAS.
A Bioremediation Approach
Although PFAS do not naturally occur in nature, it has been observed that several
microorganisms can endure high PFAS concentrations and, in some cases, even exploit
it as a nutriment source. Microorganisms can process pollutants in a passive or active
manner: bioaccumulating the contaminants or actively breaking them down.
Scientific literature and even previous iGEM projects, provide some examples of
bacteria, algae, fungi and plants capable of passively or actively dealing with PFAS.
So, obviously, they soon became our natural allies. In particular, two bacteria
caught our attention:
- Delftia acidovorans, a bacteria that has been detected in PFAS-contaminated sites by the USAFA iGEM team 2021 [3]. The team also managed to isolate a series of dehalogenase enzymes that may be responsible for PFAS defluorination
- Synechocystis sp., a cyanobacteria that provided the most surprising results among other PFAS-resistant organisms. A study conducted in our University [2] shows how Synechocystis is not only able to survive in high-PFAS concentrations, but can also degrade some kinds of PFAS molecules.
Enzymes: Laccases and Dehalogenases
Once selected our microbial helpers, we then identified proteins that may assist PFAS
breakdown. Several scientific papers often indicate two classes of enzymes putatively
responsible for PFAS degradation: dehalogenases and laccases, both detected in
PFAS-resistant microorganisms.
Dehalogenases possess the ability of removing halogen atoms from an organic scaffold.
Experimental evidence shows potential defluorination power against fluorine atoms bonded
to the carbon skeleton in PFAS, that make for the high toxicity of these compounds.
Laccases, on the other hand, are a well-known enzymatic class, broadly implemented in
numerous bioremediation experiments. When it comes to PFAS, some studies propose how
laccases may help in the fragmentation of the carbon chain, producing smaller precursors.
For both classes, a detailed
bioinformatic analysis
was carried out in order to detect
the best candidates for each family, investigating within the genome of PFAS-resistant microbes.
Chassis choice: Escherichia coli
We decided to clone the degradative enzyme discovered in those bacteria in Escherichia coli, since it seems the perfect fit for this role: extensively studied, relatively easy to transform, engineer and cultivate, it constitutes the perfect microorganism for our purposes. Obviously, we first had to investigate whether E. coli could keep up with high-PFAS environments, therefore we carried out several growth tests to assess the top PFAS concentration E. coli can be exposed to, without overly affecting its activity. Given the current state of legislation over the release of engineered biological material in the environment, we opted for a biotechnological degradative system restrained to the controlled environment of a bioreactor . Here, our carefully-engineered bacteria will encounter PFAS gathered from ground water filtration, and the breakdown reaction will take place. The bioreactor will guarantee the optimal conditions for E. coli activity and increase the reaction yield while addressing sanitary and environmental safety precautions.
Challenges of PFAS degradation - Our solution
Our idea is to merge the activities of laccases and dehalogenases in order to achieve a series of chain reactions that will ultimately tear apart any kind of PFAS molecule. Laccases will fragment PFAS carbon chains into small precursors and these will be able to fit into the active site of dehalogenases. Indeed, the two enzymes cannot carry out PFAS total breakdown by themselves. The fragmentation activity of laccases might lead to the production of smaller precursors that can still be considered PFAS compounds, still harmful, but more difficult to detect and contain. As far as dehalogenases are concerned, the narrow dimensions of the binding fold.
We also intend to express laccases and dehalogenases on the outer membrane of Escherichia coli. This way, PFAS and their intermediates will get directly in touch with both enzymes without having to enter and exit the cell, allowing for much more rapid reactions, and fluorine will never be released in the intracellular space. Aiming to avoid metabolic burden, our system will present a population with two different E. coli strains: one exposing a laccase on its surface and the other similarly presenting a dehalogenase.
In order to achieve expression and transportation of the exogenous protein to the cell surface, a construct was developed and cloned in E. coli. The modularity of this composite part allows for characterization of both intra and extracellular expression, thanks to the presence of specific enzymatic sites that enable the optional addition of a membrane anchor, through simple digestion and ligation. What’s more, the design of the construct is meant to make the enzymatic sequence that features the part, interchangeable with different enzymatic sequences, eliminating the costs of the synthesis of a different construct for every enzymatic characterization.
The cooperation between two enzymes and their extracellular expression will give a new lease on PFAS destruction, one that considers the implementation of many biological tools to reach a common goal.
Filtering and sensoristic system
After consulting experts and water treatment facilities, we were able
to outline what the principal challenges regarding the current PFAS
purification techniques are. Currently in Veneto, PFAS are retained from
ground water through granular active carbon filters that get replaced after a
certain period of time. PFAS are then detached from these substrates through thermic
treatments, that are also supposed to breakdown PFAS molecules. Unfortunately, several
drawbacks arise from this method, such as the modification of the original characteristics
of the filter, which complicates their re-use. Scientific evidence also shows how thermic
treatment of PFAS could simply transfer the compounds from the filter to the fumes, without
even reaching a complete degradation. Once released, those might contaminate the
environment nearby the facility.
The second main bottleneck is detection: at the moment it is only possible to
detect PFAS through traditional analysis like mass spectroscopy and chromatography.
These measurements are expensive and time consuming and need sampling, whereas a real-time
detention technology has not been developed yet.
Our project had to address these weaknesses, therefore we have developed a system meant to optimize
the already-existing anti-PFAS technologies and introduce some key innovations that make our
system more convenient, sustainable and, overall, unique.
To do so, we have actively researched and tested different kinds of
filters
and regeneration solutions. We even started to develop a new
PFAS sensor
, based on surface-enhanced raman and impedance measurements. Check out our
Plant Design
page to learn more!
SurPFAS - A Project for People
PFAS pollution is much more than a serious environmental issue. To us,
PFAS represents a plague that led to terrible repercussions on our community and forever
impacted our territory. We needed a project that could reflect this, therefore we didn’t
stop at the biological component of PFAS degradation, but we dug deeper, in the hope of
understanding the issue from more than one perspective.
Thanks to
interactions
with society, water facilities and experts of various fields,
we managed to design, develop and test a system that could address PFAS degradation in
ground water from head to tail, starting with PFAS detection, passing through its
filtration and gathering, ending with PFAS enzymatic breakdown. Our interpretation
of synthetic biology goes beyond the molecular biology laboratory, as we implement
concepts of chemistry and electronics in the development of a solid, comprehensive
system. Our efforts enabled us to investigate avant-garde solutions for PFAS absorption,
desorption and detection, generating a complementary pipeline that culminates with
PFAS biological breakdown. With these fundamental components, our project will gain
applicability and strategic placement in the water treatment industry on the territory, check out our
Entrepreneurship page to
learn more about the market
analysis of our water purification service.
References
