Human Practices

Figure 2: A map of sites tested for potential PFAS contamination in Arizona. Gray triangles represent data from EPA UCMR 3 2013-2015. Gray circles represent the ADEQ Drinking Water Data from 2018-2022. Blue circles represent the most recent ADEQ Drinking Water Data from 2023. (4)

In light of these substantial health concerns, at the beginning of 2023, the U.S. Environmental Protection Agency (EPA) took a significant step by introducing a proposal for a national drinking water standard. This proposed standard aims to limit six specific PFAS chemicals, namely perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), hexafluoropropylene oxide dimer acid (HFPO-DA), perfluorohexane sulfonic acid (PFHxS), and perfluorobutane sulfonic acid (PFBS) (5).

The EPA's proposal has not been enacted into law yet, so the regulatory landscape for PFAS varies from state to state. At present, Arizona has yet to implement specific state regulatory limits for these substances (6). In response to this pressing issue, the Arizona Department of Environmental Quality (ADEQ) began conducting targeted water testing as far back as 2018 to gain insights into the prevalence and impact of PFAS within the state (7). In their initial assessments from 2018, the ADEQ determined that widespread contamination of PFOA and PFOS was not a pervasive concern. Data collected from drinking water wells revealed that 81.7% showed no detectable levels of PFAS, 12.8% exhibited PFAS concentrations below the EPA Health Advisory, and only 5.5% exceeded the EPA Health Advisory levels (8). However, recent developments have brought to light a more alarming situation. The ADEQ has reported an unsettling number of 57 sites with significantly elevated levels of PFAS contamination in public water systems throughout various counties in Arizona, including Yavapai, Yuma, Gila, Maricopa, Pima, and Pinal. Of particular concern is that these counties are witnessing PFAS contaminant levels surpassing the recently reduced EPA regulatory limits, which now stand at 0.004 ppt for PFOA and 0.02 ppt for PFOS (9).

On top of this, recent developments have cast a spotlight on an increasingly concerning situation in Arizona, specifically related to the use of Aqueous Film Forming Foam (AFFF). This firefighting foam remains a significant source of PFAS pollution in water and soil, and its widespread utilization within military installations, airports, and local fire departments has only amplified the PFAS issue (10). In a disconcerting turn of events, the Luke Air Force base site in Glendale, Arizona publicly revealed in 2021 that their supply wells contained PFAS levels exceeding the 2016 EPA Health Advisory Level of 70 ppt (11). Moreover, multiple Army installations in Arizona have detected PFAS concentrations exceeding 1 ppt in their drinking water supplies, with concerning figures such as 66.6 ppt at Yuma Proving Ground, 39.17 ppt at Silverbell Army Heliport, 31.26 ppt at Camp Navajo, and 26.5 ppt at Fort Huachuca (12). These unsettling findings underscore the pressing need to address the PFAS issue in both military and civilian contexts, with significant implications for the protection of public health and the environment.

During our initial research, we discovered that in 2020 and 2021, the United States Air Force Academy (USAFA) iGEM team had undertaken a similar challenge to address the issue of PFAS contamination. Their approach to the problem involved initially collecting soil samples from the Colorado Air Force base, which were likely heavily contaminated with PFAS due to the use of AFFF (13). These samples then underwent screening in search of microbes adapted to thrive in PFAS-laden conditions as they could potentially be harboring genes that enabled them to metabolize PFAS. They singled out a novel strain of Delftia acidovorans as a promising candidate due to its possession of genes associated with several dehalogenases, suggesting their potential to break down PFAS compounds. During their first year of the project, they successfully identified two haloacid dehalogenases, referred to as DeHa 1 and 2. The subsequent year, they furthered their research and identified three additional potential dehalogenases, known as DeHa 3, 4, and 5, through genome sequencing of their Delftia acidovorans strain. Notably, when expressed in E. coli, DeHa 1 and 5 exhibited defluorinating activity, cleaving the terminal trifluoromethyl group of PFOA. Their remarkable success in genetic engineering led us to believe that they held valuable insights that could inform our own project.

As such, in early April, we arranged a meeting with the team's research advisor, Dr. Jordan Steel. Following our discussion with Dr. Steel, we solidified our decision to continue using E. coli as our preferred organism. This decision was based on their successful expression of dehalogenases in E. coli, demonstrating E. coli's ability to interact with PFAS chemicals and partially break down a portion of its structure. Additionally, influenced by USAFA's success with dehalogenases, we decided to also use defluorinating enzymes in our approach to PFAS degradation, and we chose to test both DeHa 1 and 2 for our defluorinating construct. Although we also wanted to test DeHa 3, 4, and 5, their sequences were unfortunately not available on the iGEM Parts page, preventing us from pursuing testing of these enzymes. Furthermore, our meeting with Dr. Steel revealed a crucial detail - the enzymes they had expressed were only monomers, whereas the fully functional enzyme was actually a dimer. They suspected that the presence of a His-Tag was preventing the expression of the dimer structure. This crucial piece of information guided our experimental design, leading us to incorporate a step in the creation of our defluorinating construct to remove any His-Tags, thus ensuring that they would not interfere with the defluorinating enzymes we expressed. These valuable insights from Dr. Steel and the USAFA iGEM team not only provided us with a strong foundation for the development of our experiments but also enriched our project's design, setting the stage for a promising journey towards addressing the complex issue of PFAS degradation.

In the early stages of our project, we also had the privilege of engaging in insightful discussions with two PhD students from the Rittmann Lab. Dr. Bruce Rittmann, a Regents Professor at ASU's School of Sustainable Engineering and the Built Environment, is known for his pioneering research in the development of microbiological systems with a dual focus on harnessing renewable resources and mitigating environmental pollution (14). A standout facet of his research involves the application of hydrogen-fed biofilm reactors equipped with a palladium catalyst for the decontaminatioin of water. The two PhD students we conversed with were applying this unique approach to tackle the formidable challenge of PFAS contamination.

Our meeting with them provided a remarkable opportunity to delve into their current strategy for breaking down PFAS and to gain valuable insight into the challenges they encountered with their approach. One salient point of discussion revolved around the environmental and financial ramifications of utilizing palladium as a catalyst, which is a significant concern given its non-renewable nature. What particularly captured our attention was their innovative utilization of a biofilm derived from sludge sourced from a water treatment plant. This biofilm was a thriving ecosystem housing a diverse array of microorganisms. This aspect caught our attention as they fed these microorganisms the byproducts of the defluorinating reaction catalyzed by palladium, essentially creating a microbially-driven, two step system to break down PFAS chemicals. This approach leveraged the natural metabolic capabilities of diverse microbial communities to enhance PFAS degradation.

The substantial monetary and environmental costs associated with palladium catalysts raised concerns that conflicted with our environmentally conscious objectives. Consequently, this cemented our decision to pursue a synthetic biology approach to the issue. It became evident that over the long term, employing a biological organism held the promise of being the most effective and efficient solution, reinforcing our choice of E. coli, which had already shown promise in breaking down PFAS.

Their intriguing two-step approach, capitalizing on the synergy between diverse microbial communities and defluorinating reactions catalyzed by palladium, significantly broadened our perspective and prompted us to consider a novel strategy. This insight led to the idea of combining our defluorinating construct with a metabolic construct, particularly one housing beta-oxidation enzymes. This strategic combination held the potential to enhance E. coli's PFAS degradation capabilities. Since PFAS compounds are structurally similar to fatty acids once the fluorines have been removed, this approach could facilitate their transformation into smaller, non-toxic molecules that would readily degrade in the environment, presenting an exciting avenue for exploration. This collaborative experience underscored the importance of learning from and collaborating with experts in the field, enriching our project's sophistication and enhancing its potential impact.

Theoretical Project Implementation

Once our innovative project solution is realized, we will be able to completely degrade PFAS contamination. Our engineered E. coli strain, Beta-FluorinX, carrying both a defluorinating construct and a metabolic construct housing beta-oxidation enzymes, holds the potential to usher in a new era of environmental remediation and public health.

In an ideal scenario, Beta-FluorinX could be incorporated either as a bio-membrane for water filtration or as an additive in the activated sludge process. As contaminated water passes through either system, Beta-FluorinX will effectively break down the PFAS. Subsequently, the water will undergo UV sterilization and a secondary membrane filtration to remove any remaining bacteria. Along the water filtration line, testing stations will ensure the absence of bacterial and chemical contaminants. Although this water treatment process is relatively slow, it guarantees clean water and prevents continued PFAS contamination in the environment. Furthermore, the process can be seamlessly integrated into existing water treatment plants as they would only need to add the safety measure of scanning for bacteria.

The successful implementation of our solution promises multifaceted benefits, starting with the effective removal of PFAS contaminants from various water sources. PFAS compounds have posed a persistent and pervasive threat to ecosystems and human health because of their resistance to degradation and propensity to bioaccumulate. By effectively eliminating these contaminants, we have the opportunity to halt the ongoing biomagnification of PFAS in the food chain. This development is particularly crucial as PFAS can infiltrate various food sources, eventually making their way into the human body through consumption.

Preventing the continued bioaccumulation of PFAS is not only an ecological imperative but also a critical step towards safeguarding human health. These resilient chemicals have been linked to a wide range of adverse health effects, so by putting a stop to the bioaccumulation process, we are laying the foundation for reducing the risk of individuals developing diseases associated with PFAS exposure.

The impact of our project extends far beyond Arizona. The ubiquity of PFAS contamination in water sources is a worldwide concern, affecting communities and ecosystems on a global scale. Our solution has the potential to serve as a blueprint for addressing PFAS contamination not only in Arizona but also in regions around the world facing similar challenges. By mitigating the health risks associated with PFAS exposure, we are contributing to improved health outcomes for people everywhere. Thus, the successful implementation of our project, which harnesses the power of engineered E. coli to degrade PFAS contamination, has the potential to revolutionize environmental and public health efforts.

PFAS Heatmap

In order to create a comprehensive heatmap of PFAS contamination levels across Arizona, we embarked on a water sampling mission. Our samples were drawn from a diverse range of sources, including the ASU campus and Tempe Town Lake. Moreover, in a collaborative effort, we forged a partnership with the Salt River Pima-Maricopa Indian Community (SRPMIC), which allowed us to obtain water samples from three previously unsampled water wells, significantly expanding the scope of our project. To achieve accurate PFAS level analysis of our various collection locations, our team coordinated with the Mass Spectrometry Facility situated within ASU's Biodesign Institute to make use of Liquid Chromatography Mass Spectrometry (LC-MS) technology.

During this process, however, we encountered a substantial challenge concerning usage of the LC-MS machine. We faced a lack of control over its scheduling and availability, so the necessary columns required for our PFAS analysis were not available within the timeframe of our project.

Despite this hurdle, our unwavering commitment to PFAS awareness and our collaborative work with SRPMIC marked significant milestones in our ongoing mission to comprehensively understand and address PFAS contamination in the state of Arizona. These experiences have only served to strengthen our resolve to continue advancing our research and engagement in the fight against PFAS contamination. We are steadfast in our plans to analyze our water samples as soon as the LC-MS machine becomes available, as we remain dedicated to the pursuit of PFAS awareness and understanding in our region.

Figure 3: A heatmap of PFAS contamination levels in Arizona in parts per trillion (ppt).

A Triangulated Irregular Network (TIN) is employed to estimate the spatial distribution of Per- and Polyfluoroalkyl substances (PFAS) across Arizona. Utilizing a set of irregularly distributed data points, each with associated PFAS concentration values, TIN generates non-overlapping triangles to create a three-dimensional representation of PFAS concentration over the area. The vertices of the triangles represent specific data points, and by interpolating between these vertices, an estimation of PFAS concentration across different locations within Arizona can be derived.

The concentration of PFAS at each point was determined using data from the Arizona Department of Environmental Quality (ADEQ) using well water screening data obtained directly from the ADEQ. The sample dates ranged from 2014 to 2023 and and included a sample test from Perfluorooctanesulfonic Acid (PFOS), Perfluorohexane Sulfonic Acid (PFHxS), Perfluoroheptanoic Acid (PFHpA), Perfluorobutanesulfonic Acid (PFBS), Perfluorohexanoic Acid (PFHxA), N-deuterio methyl perfluoro-1-octanol sulfonamido acetic acid (PFHxSNEtFOSAA), N-Methyl Perfluorooctane Sulfonamido Acetic Acid (NMeFOSAA), and Perfluorononanoic Acid (PFNA).

We chose to stratify the data by wells with confirmed positive PFAS detection because the lab reporting limit, or how much PFAS must be present in order for their test to identify and report the concentration, is often significantly higher than the proposed EPA limit, leading to a significant amount of false negatives, thereby significantly underestimating the actual values. This approach is logical from both environmental and public health advantages. Severe contamination in a single well likely extends beyond its immediate vicinity due to the inevitability of PFAS integrating into the food web, regardless of PFAS contamination in nearby wells. This scenario holds true even in the absence of direct human interaction; for instance, a well with high PFAS concentration can impact the food chain adversely as birds, rodents, and livestock consume the contaminated water. Contaminating the food web could eventually affect human populations as well as the ecology. Therefore while this heatmap may result in a more liberal interpretation of contamination this stratification was deemed necessary to ensure an accurate and relevant portrayal of the data.

While most heatmaps utilize kernel density for the methodology of the heatmap, this was deemed less useful for the visualization due to two factors. First, the large cluster of high PFAS concentrations in the south-eastern portion of the map correlate with some of the most populated areas of Arizona. This could lead to a sampling bias which causes the additive effect of the clustered kernels to predict values of PFAS into the millions of parts per trillion, which is a discrepancy from the measured values at the waterwells. Second, these extreme values made it nearly impossible to display the high estimated concentration in the south east while still displaying contamination in the midwestern area that exceeded EPA guidelines. Thus the quantitative and qualitative aspects of the heatmap were misleading when using kernel density.

TIN avoids these factors and allows for a more useful quantitative view of the heatmap. TIN doesn't utilize kernels when determining the estimated value of the area of interest meaning that each point will estimate a PFAS value that will stay within the range of concentration in the given dataset. Not only does this avoid sampling bias because each triangle is non overlapping, but also allows for the severely high concentration of PFAS in the southeastern parts of Arizona to be represented while still showing that the areas including lower density clusters of data points, such as those represented in the western part of the state, still show that they are estimated to be above the EPA proposed limit. Areas with low densities of data points however are likely to be less accurate than high density areas.

The classification breakpoints were chosen according to the following rationale. 0-1PPT represents a value that is below the EPA proposed MCL. While no concentration of PFAS is safe, it is generally accepted by most public health and environmental health entities that concentrations 1PPT and lower is ideal. 10PPT and 100PPT breakpoints allowed for a graphical representation of relatively low classifications that were not too visually cluttered. The 500; 1,000PPT; 2,000PPT; 5,000PPT; 10,000PPT; and 20,000PPT breakpoints were determined to be classifications that allowed for a visualization of the quick ramp to high concentrations of PFAS in the southeastern part of the state, while reducing visual clutter. While the max city PPT is 122,642PPT no further classifications were made as 20,000PPT is already 5,000 times the EPA proposed MCL and no logical breakpoints could be created that would show the jump in a way that wouldn’t visually clutter the heatmap in a way that would compromise the approachability to a general audience.

Finally, it is important to note that while the EPA proposed MCL is 4PPT, there are currently no laws or regulations on PFAS production or use that incentivise this value. This could account for city averages PFAS concentrations being estimated to be over 30,000 times the proposed EPA regulation.

In response to the insights garnered from our survey results, we made the decision to host an advocacy and awareness event dedicated to PFAS, with a specific focus on engaging college students. This event served as an essential platform to disseminate critical information about PFAS and raise awareness among the 17-26 year old demographic.

Our target audience included members of ASU's Next Generation Service Corps (NGSC), a dynamic organization committed to nurturing leaders who are poised to drive positive impacts both locally and globally, including in the realm of sustainability. During the event, we delivered a comprehensive presentation that covered the foundation aspects of PFAS, including its background as well as the associated dangers to humans, animals, and the environment. Moreover, we delved into notable PFAS-related cases, such as the DuPont PFAS scandal, which was the subject of the documentary “The Devil We Know” (15, 16).

As the evening unfolded, we encouraged active participation from the audience, engaging them in discussions about potential solutions to the complex PFAS problem. The audience further participated by partaking in an engaging and informative quiz. The level of interaction and the enthusiasm displayed by the attendees were remarkable and reinforced the importance of our mission to raise PFAS awareness.

Crucially, the feedback we received was overwhelmingly positive and encouraging. The event served as a catalyst for us to continue our commitment to PFAS awareness initiatives in the future. These events are instrumental in fostering greater understanding and facilitating collective efforts to address the multifaceted challenges posed by PFAS contamination. Through our continued endeavors, we aim to empower individuals with knowledge and tools to actively contribute to the mitigation of PFAS-related issues in their communities and beyond.

You can find a link to the presentation here.

How can we apply this research to PFAS?

While Dr. Mana and Dominic's expertise primarily lies in diet-related aspects of cancer research, they were still able to offer some intriguing theoretical considerations on how we might apply our project’s results to address the PFAS issue with the human microbiome.

Given that PFAS are chemically similar to lipids, one pertinent avenue of exploration would be to understand how they interact with lipid trafficking into cells, where they accumulate, and how they potentially compete for cellular resources. If PFAS displaces usable fatty acids, it's essential to decipher the consequences for cellular energy production, including potential alterations in the chemistry of electron donors like FADH2 and NADH. Moreover, introducing new biological materials, even if "similar," that do not provide an equivalent degree of nutrition could have detrimental effects on the microbial milieu, particularly within the microbiome, which plays a critical role in breaking down food for cells. As such, they also proposed blocking PFAS's entry into cells as a promising avenue for research.

These insights are invaluable for our research team as we chart our project's future directions with a keen eye toward maximizing its potential benefits for human health. By exploring how the results from our own research project can be applied in the context of the human microbiome, we aim to contribute to addressing the pressing issue of PFAS contamination and its potential impacts on human health and well-being.