HUMAN PRACTICES

PCBs: Long Lasting Toxins

Polychlorinated Biphenyls have a variety of health effects in both humans and animals, and are considered a “forever chemical” which means they will never degrade or leave the environment naturally. PCBs are also a known carcinogen and have been linked to several types of cancer in humans. Animals, when exposed to high levels of PCBs, have sustained damage to the liver, stomach and thyroid gland, as well as developing skin conditions and anemia [7].

There have been many studies conducted supporting the theory that PCBs cause cancer. Studies on animals have proven that PCBs are carcinogens, and further studies on humans have given scientists enough evidence to declare that PCBs are probable human carcinogens [8].

PCBs can target many systems throughout the human body. This includes the endocrine system, nervous system, reproductive system, cardiovascular system, and the immune system [8]. In children, whose brains are still developing, PCB exposure is even more dangerous. It increases the likelihood of neuropsychological deficits, including attention, memory and learning issues [8]. Prenatal exposure is also associated with low birth weight [8]. Because PCBs target many systems in the body, PCB exposure may be associated with conditions such as strokes, hypertension, diabetes, suppressed immune responses and thymic atrophy, though how this occurs is still not well understood [8]. A study conducted in Anniston Alabama found a strong connection between high PCB exposure and hypertension [9]. Some studies have shown associations between PCB exposure and these conditions, and others have shown no association.

PCBs have also been shown to affect the reproductive system in both men and women. In women, PCB exposure is associated with damage to the ovaries, which can cause other problems such as abnormal hormone levels and infertility [10]. In men, PCB exposure is associated with decreased sperm quality and fertility [10].

PCBs in the Environment

After the discovery of PCBs negative health consequences, the compounds were banned in the 1970s and 1980s worldwide[12]. Despite this ban, PCBs continue to circulate in the environment. Eighty percent of global PCB stockpiles have not been destroyed yet and PCBs are still found in legacy products, such as transformers, cable insulation, and some ship paints [12].

Acute levels of PCBs can cause rapid effects however these have typically only occurred in industrial settings prior to awareness of the toxin's dangers. Low to moderate levels of PCBs are a slow-acting contaminant so seemingly healthy populations can still be affected sometime after exposure making cause and effect difficult to isolate precisely. PCBs continue to affect ecosystems in the ocean since they persist in these products and because of the way PCBs can bioaccumulate up the food chain.

What is Bioaccumulation?

In the environment low concentrations of PCBs will bioaccumulate over time. In this process toxins accumulate in a food chain and the animals at the top are affected the most heavily [11]. PCBs are taken up at the bottom of the food chain by small organisms [7]. PCBs are fat soluble and hydrophobic and so bind tightly to organic material and build up in the tissues of the small organisms [11]. Higher trophic level predators consume the smaller PCB contaminated organisms and then lock those PCBs away in their tissue, particularly fat stores.

The concentration of the toxin is highest in the body tissues of the animals at the top of the food chain since they eat the largest amount of PCB contaminated organisms over their lifespan.

Environmental and Societal Harm from PCBs

Marine PCB pollution is particularly problematic. Pollution from the land flows into the sea. Organisms in Washington State's Puget Sound have high levels of PCB in their biomass. This can harm humans directly via consumption and absorption into the body.

In Washington PCBs are the cause of almost 80% of our state's marine fish consumption advisories(rules against consuming or selling specific seafood)[13]. Fishing is a major industry with great significance to various communities in our state. Resident Chinook in the Puget Sound have on average PCB concentrations in their biomass of 130 ng/g, similar levels in soil are considered highly polluted [14]. Many other species of fish and shellfish also have elevated PCBs levels. In this way, PCBs hinder economic profit and recreation by closing off species and areas to fishing activity.

Marine biomass can deliver its high toxin concentrations to humans. Research conducted by Propublica, a media outfit centered on transparency and public accountability, found that high PCB concentrations in fish(particularly salmon) are disproportionately putting Washington State tribal nation members at risk of toxic side effects from PCBs and other pollutants [15].

The environmental impacts of PCBs are significant and quantifiable. New research published in the journal Science suggests more than half of the world's killer whale populations could become extinct in 30 to 50 years due to legacy PCB pollution [12]. Mother orcas pass these pollutants along to their young during birth or through breast milk to their children which could affect early development [12]. PCBs are believed to be altering orca behavior, damaging their immune systems, and harming reproduction and fertility[12].

The level of this harm is such that many populations of killer whales may go extinct in the next few decades [12]. Top researchers suggest that PCBs remain the number one pollutant of concern at the top of the food chain for wildlife in the northern hemisphere [12]. PCBs threaten the health of apex and higher level ocean predators and thus, undermine the resilience of ocean environments more broadly.

Developing Our Project

Our Biphenyl project was first inspired by an article on unintentional PCB removal that had occurred at a wastewater treatment plant (WWTP) along Chesapeake Bay, a water body with areas of high PCB contamination[18]. The WWTP had installed a nutrient pollution removal system, which uses a large biofilm system to trap nitrogen and phosphorus pollution[18]. In addition to removing those chemicals the biofilm could remove 90-95% of PCBs in the wastewater [18].

This biofilm wasn't equipped to break down the PCBs however. So while it did trap and remove the pollutants, it left behind PCB contaminated biomass. Our team saw this limitation and set out with the goal of designing a system that would use the ability of biofilm to collect PCBs and absorb PCBs and combine that with natural methodologies to destroy them.

At the Black River WWTP, a nutrient removal system was installed and the plant was measured to remove 97% of the PCBs from the influent wastewater [18]. After seeing this our team was inspired. PCB contaminated water could be run through a biofilm and the PCBs captured. Then by transforming cyanobacteria to express metabolites for PCB degradation, we could in theory break down the accumulated pollutants.

The biofilm credited with removing PCBs in the first study could be one way to implement our idea. However we spoke to Corinne Klohman a former researcher in the Gomez-Padillo lab at the University of Washington to understand the potential of using macroalgae, not just microalgae towards this solution. She noted that restoration efforts of marine habitat have been slow. One potential carrier for PCB metabolites we were interested in was eelgrass, a species of perennial marine plant that we hoped could then be planted in areas with heavy PCB contamination. However Ms. Klohman explained to us the seagrass restoration has frequently failed for a mix of factors that lead to the seagrasses dying soon after introduction.

This led us to focus instead on designing a biofilm product made of cyanobacteria similar to that occurring in the wastewater treatment plants. Discussion with Dr. Joel Baker, Science Director of the Center for Urban Waters, who has done previous work on PCBs and a similar class of compounds called PBDEs (polybrominated diphenyl ethers), allowed us to identify some of the major obstacles. He confirmed what we suspected: the cells in our biofilm expressing the biphenyl degradation pathway and or the chlorine dehalogenase would lose out in competition with more efficient unmodified microbes in a non sterile environment. This leads to several additional problems covered more extensively in the implementation section of our wiki.

The next major shift in our project design occurred after speaking to Professor Stuart Strand at the UW's Department of Civil and Environmental Engineering. He explained that the BPH pathway occurs in nature quite regularly. The bottleneck to PCB degradation was actually the dehalogenase's removal of chlorine from the biphenyl. With the chlorines removed natural bacteria would (relatively quickly) degrade the remaining biphenyl which is also toxic but less so than PCBs.

This input led us to dramatically shift the design of our project from expressing the BPH pathway to focusing on the dehalogenase pathway. Professor Strand explained the main factoring in delaying PCB degradation was that dehalogenation of the chlorines typically occurs in anaerobic conditions while the Bph pathway must be aerobic. With this knowledge we reviewed our team's plan and made adjustments.

At the same time as the Human Practices subteam was discovering this, our protein modeling subteam was attempting to optimize a aerobic dehalogenase for PCBs based on data from the 2015 paper Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation [19]. Knowing Bph's high frequency of natural expression we shifted our project towards dehalogenation. With input from the Human Practices team, our Wet Lab subteam adjusted from expressing the Bph pathway in cyanobacteria to the target of expressing protein modeling's designed dehalogenase instead.

This left us with a final target design of a cyanobacteria based biofilm that would express aerobic dehalogenation protein for PCB degradation. Our team's approach targeting dehalogenation in an aerobic setting instead of the Bph pathway is novel as far as we can tell from reviewing other literature. From there we needed to figure out how this could be implemented and to mitigate potential problems.

Uh-OH!

One potential challenge to our project could be the inadvertent generation of hydroxylated PCBs or OH-PCBs. These are PCBs which in the process of being broken down have had a hydroxyl group added on [20]. -OH PCBs are concerning because the hydrophilic nature of the -OH group makes these side products much more water soluble and easier to spread as well as more willing to bind to organic matter [20]. According to Dakhal et. al (2018) "OH-PCBs are environmentally stable and may be more toxic by some measures" [20]. Inadvertent generation of these compounds is a risk that must be monitored in PCB bioremediation projects.

Implementation

Since our project was inspired by a study of wastewater treatment plants we looked to wastewater treatment for ways to implement our design. Our initial research brought two major constraints to the forefront:

1. How to supply sufficient amounts of PCBs for destruction to make our methodology justifiable?

2. How to prevent displacement of dehalogenase producing microbes by natural competitors?

These core problems are generally the main limits of PCB bioremediation. While PCB pollution is widespread it often isn’t present in extremely high concentrations and is usually dispersed over a wide area. Consistent water flows from treatment plants continually bring in new material for detoxification which seemed a promising start.

In our attempt to locate areas for implementation we considered a variety of methodologies and sources. In addition to wastewater treatment plants we looked at stormwater, as well as PCB contaminated groundwater flows. We also examined the mechanism of protein secretion as an ideal modification to our biofilm design. We talked to experts representing different groups and interests at several PCB contaminated sites in Washington including: the Spokane River Valley and the Lower Duwamish River.

As part of the process of identifying PCBs sources we attended the virtual PCB Symposium hosted by the Cross Contaminant Working Group on June 15, 2023. The focus of the three presentations was on "Source Identification and Tracking". From the case studies presented we found that in some test cases the PCB hotspots can be very heterogeneous in their distribution of toxins.

From those presentations and after following up later in a meeting with Michael Jeffers, who attended the symposium, we found that a major limitation of PCB remediation and research is the high cost of testing for PCBs. Mr. Jeffers mentioned that testing fees routinely run into the thousands of dollars, and as a result, this seems to be an area that could be promising for a future biological assay.

Mr. Jeffers also mentioned that in addition to large industrial sites, large amounts of PCB pollution can come from building materials in older buildings such as window caulking and paint. He also noted their presence in everyday materials such as dyes, inks, pigments, and lights.

When we met in-person at the University of Washington-Seattle, he pointed out multiple campus buildings made with PCBs materials. These commonly found PCBs may not be harmful if they are simply within building materials, but storms or building maintenance can release the PCBs into the surrounding environment [21]. Knowing that it was unfeasible to address all these distributed sources of PCB pollution, our team felt that targeting the toxins after they began to flow through the environment in stormwater and wastewater was a better solution.

Michael Jeffers and our Human Practices Team Member Akira Morishita who organized the meeting.

Mr. Jeffers showed us an area of campus with PCB removal in progress, the film below is set in hope of catching falling fragments of material containing PCBs.

Preventing Contamination

To prevent natural microbes from outcompeting our modified ones we began by trying to find a way to isolate the biofilm from other species contamination. One option we hoped would give our biofilm a fighting chance would be to implement it as an additional step in the wastewater treatment process after the wastewater has been treated to remove various microbes.

Speaking to Professor Stuart Strand, however, he noted three major obstacles to this proposed plan. First, the chlorine used in WWTPs to kill microbes would likely also kill our biofilm. Even if it didn't, chlorine treatment wouldn't remove all microbial life. Spores would survive the treatment and then grow in our biofilm displacing the modified species. On top of this the effluent water would then need to be chlorinated again which would not be cost effective for the WWTP. This feedback led us to seek other methods of implementation and suggested some inevitable limitations.

Any biofilm based PCB degrading microbes will likely need to be applied and exchanged or replenished in some way at regular intervals due to inevitable contamination with foreign microbes regardless of how it was implemented. This means, ideally, our biofilm product should be exchangeable or replaceable as well as standardizable. It also means in a WWTP our biofilm would need to be applied before chlorination and replaced relatively frequently.

Understanding the Wastewater Treatment Process

After getting feedback from initial interviews, we spoke to a few wastewater experts (Amy Sumner and Jeff Donovan) from the city of Spokane and Spokane County, another area in Washington that has dealt with high amounts of PCB contamination. Their inputs on the specifics of wastewater treatment gave us more context for how our biofilm idea could fit into these systems.

The initial step in the filtration process is screening large items such as towels or bottles. The water is then moved into another tank where sand, gravel, and other grit are subsequently removed [22], [23].

After these preliminary steps, the water undergoes a primary clarification process. First, the water flow slows as the water sits in settling tanks. In these tanks, waste particles and some nutrients like phosphorus settle to the bottom, and other materials like oil or soap float to the top. These organics are removed and put through solids treatment.

The next step of water treatment is the secondary treatment. In both Seattle and Spokane facilities, this is accomplished by an activated sludge process in aeration basins [22], [23]. The sludge consists of mostly aerobic bacteria and small amounts of other microorganisms [24]. By pumping the aeration tanks containing the sludge with a steady air supply, the bacteria can consume the remaining organic waste (such as ammonia) from the influent water, growing the amount of biosolids. This step is crucial because it decreases the biochemical oxygen demand, which measures the amount of oxygen organisms in water would use to break down organic waste in a sample of treated wastewater (i.e., lower results mean cleaner water) [25]. The sludge plus wastewater is then sent to a clarifier settling tank wherein the biomass settles to the bottom. While most of the settled-out biosolids remain a part of the secondary treatment process, a portion of the biosolids are sent to solid treatment [22], [23].

After these primary and secondary clarification processes, wastewater is chlorinated and sent out again. However, some wastewater treatment facilities, such as the Spokane County Regional Water Reclamation Facility, have added tertiary filtration technologies which have improved PCB removal efficiency from wastewater [22].

These tertiary membranes ultrafilter the water using a 0.1-0.5 micron filter, which separates pathogens and retains organics from the influent water [29]. These tertiary filtration systems were found to remove between 95 and 99% of PCBs between Spokane’s influent and effluent water (from 12000 pg/L to around 250 pg/L) [30].

Despite this progress in wastewater treatment, there are several challenges in widespread implementation of tertiary filtration for PCB treatment:

1. While tertiary filtration systems are effective at filtering PCBs, they are also expensive to implement. Membrane bioreactor implementation in Spokane cost $126 million dollars to implement, with experts like Jeff Donovan expecting additional required replacement costs of around $20 million dollars every decade [31].
2. Federal regulations under the Toxic Substances Control Act continue to allow incidental PCBs as byproducts in other industrial processes like inking and dyeing. These unintentionally produced PCBs primarily include lower molecular weight (i.e., less substituted) congeners like PCB-11, which are less effectively removed by the tertiary membrane filtration process [30].
3. PCBs are water-insoluble, meaning a large amount of the PCBs are bound to biosolids filtered out of wastewater. Biosolid PCB testing, while not a big focus of regulatory efforts, shows that biosolids hold most of the removed PCB concentration from influent to effluent water. As these solids are often used for fertilizers, leaching of PCBs could present a risk to soil as they can transfer deep into the soil and potentially contaminate groundwater [32].

While discussing the implementation of our biofilm and addressing these issues, we synthesized our conversations with Professor Strand, Amy Sumner, Jeff Donovan, and Bruce Nairn, who works with King County’s Wastewater Treatment Division. Given that chlorination would kill the biofilm, if our biofilm was implemented in WWTPs, it would need to be before disinfection/chlorination. In addition, both primary and secondary treatments settle out biosolids. We thought of implementing the biofilm at some point after secondary treatment and before disinfection. While we first focused on the wastewater itself, learning that PCBs are mostly found in the biosolids led us to consider implementing the biofilm in the solids treatment process.

The biosolid sludge is a very promising implementation area since the fluidity of the sludge slurry would help PCB-contaminated products pass through the biofilm considering the solids treatment involves thickening, digesting, and finally dewatering. Due to the high heat and anaerobic nature of the digestion process as well as the cakey, less liquid nature of the dewatered product, it seems reasonable to add our biofilm either before or after the initial thickening step. More research on the ideal percentage of solids for our biofilm product and replacement requirements would help inform the exact point of the solids treatment process our biofilm would be most effective.

Stormwater Implementation

Early during our project, we heard from our Wet Lab team as well as Professor Joel Baker that the concentrations of PCBs within most wastewater systems were relatively low. When we mentioned this in our interview with Dr. Andy James a few weeks later, he suggested that stormwater could be a significant source of PCBs, which was corroborated by Jenee Colton, a toxics management supervisor, who informed us that stormwater contributes the largest total quantity of PCBs into our local waterways [33].

Though wastewater and stormwater are both forms of unclean water, their sources and treatment processes vary. Wastewater originates from within residences and some commercial settings, and includes water used for bathing, household toilets, and laundry [34].

On the other hand, stormwater consists of water that washes into outdoor storm drains found on streets, industrial areas, or parking lots [35]. These systems primarily work to divert rainwater during storm events and thus flow directly into local bodies of water without treatment. In addition to rainwater, these systems can also transport harmful contaminants such as PCBs, which will move directly into rivers or lakes and harm marine life.

Though wastewater and stormwater do not interact in most water treatment systems, wastewater and stormwater merge into one pipe in combined sewer systems [36]. Though this water is normally treated at wastewater treatment facilities, if a large storm event leads to water levels beyond a facility’s capacity, this water moves through Combined Sewer Overflows (CSOs), which flow directly into water bodies [36]. This leads to wastewater, which is normally treated, to flow untreated into rivers or lakes.

PCBs in runoff tend to bind to sediment particles due to their insolubility in water [38]. This means these solid particles contain higher concentrations of PCBs than the surrounding liquid, so in the implementation of our project, we would need to isolate the sediment into a slurry-like mixture containing higher concentrations of PCBs. This could then be treated via biofilm similar to how we proposed to apply our project in wastewater treatment. By using this method to concentrate the PCBs within stormwater, we hope to eliminate a major source of PCB contamination into the environment.

Legal and Regulatory Context of PCBs

The Toxic Substances Control Act (TSCA) banned the manufacturing of PCBs in the United States in 1979 [39]. Between the 1930's and 1970s an estimated 1.3 million tons of PCBs were manufactured and then dispersed into a variety of industrial, building and household products [1]. Legacy PCBs produced in the time before the ban makes up the majority of PCB pollution worldwide [1]. This PCB ban also included multiple exceptions for inadvertent production which can research 50ppm [40].

In Washington State, the standard for acceptable PCB levels in the environment is 7 parts per quadrillion [41]. This standard is significantly lower than United States federal limits on PCBs [41]. As Ms. Sumner and Mr. Donovan informed us, this standard is so low that it actually can't be measured effectively with current EPA approved methods of measurement. Current efforts to remove PCBs are limited to targeting sources of PCBs and areas with extensive and concentrated pollution for removal. We hope our project could help Washington more effectively meet this standard.

In WA, a lot of the current solutions to reducing the accumulation of PCBs are centered around regulating building materials and dyes, especially policies surrounding schools and light fixtures [42]. One of the most polluted areas in Washington is the Spokane River, which is still subject to PCB pollution from both historical waste and inadvertent production of PCBs through current legal industrial practices [43].

As we looked more into the implementation process for our project, we realized that we would probably run into laws and regulations that we would need to address, work around, or adjust our project to in order to meet specific regulatory requirements. In order to get more information about this, we spoke to Professor Wildermuth, a professor in the University of Washington’s law school.

He told us that we would most likely not face any issues from environmental law, as federal and state laws only set minimum water quality standards, but we might face issues from utility laws. In Washington State, where we are hoping to implement our project, there is a standard called AKART. This is an acronym that means all known, available and reasonable methods of prevention, control and treatment [44]. All wastewater treatment facilities must obtain a NPDES permit under this standard. The “reasonable” section of this law is where our project could run into trouble. If our product is too expensive, wastewater treatment plants might decide the benefit of cleaning up small amounts of PCBs is not worth the cost.

Because utility companies are controlled monopolies, they are required by law to keep costs reasonable [45]. In order to combat this, we would need to make sure that our project is as cost efficient as possible, easy to install and does not require special handling, training, or processing. This will help to mitigate the risk of lawsuits over high utility prices and increase the likelihood that public utilities will implement our project. Doing public service announcements, ads, or other marketing strategies around the dangers of PCBs even in very small qualities will help to sway the public in support of our project and put pressure on local and state governments to implement this solution.

Professor Wildermuth also suggested that due to the genetically modified nature of our project, we might face public backlash from people who feel that GMOs are unsafe and do not want them introduced into the water supply. Currently there are no major laws that outlaw or restrict GMOs in the context that we are using them in, but Washington State does have a history of backlash towards GMOs. In 2013 there was an initiative to require labeling of GMOs on any product containing GMOs, but this was defeated [46]. In order to combat this, it will be important to reassure the public that GMOs in our specific application are safe and that our biofilm doesn't pose a risk to human or environmental health.

Marketing and Economics

The final product our team envisioned after implementation research has a couple key traits:

1. It would treat a constant water/fluid flow.
2. The biofilm would only be designed to express the dehalogenase protein in order to jump start natural breakdown of the biphenyl.
3. Maintenance of a biofilm would be required and if the influent wasn't sterile it would need to be replaced semi-regularly to ensure the dehalogenase expressing cyanobacteria aren't displaced by microorganisms in the solution.

So far we've discussed wastewater treatment plants as a primary opportunity for implementation. However if the cost of implementing our envisioned biological system is too high then it cannot be adopted. Therefore we conducted research into potential markets to understand obstacles to commercial and industrial application of our design.

Washington State has recently begun requiring that wastewater treatment plants(WWTP) begin building capacity to remove nutrient pollution from their effluent- in particular nitrogen [47]. This is most often accomplished by using a biofilm.

We had hoped at the beginning of our project that our project could easily be incorporated into that capacity expansion. Either by adding our cyanobacteria to biofilm directly or having them as a secondary step. Heather Wyatt-Goodellows and Andrew Jones at the Washington Department of Health informed us this approach is sometimes tried in septic tanks(another waste disposal method) and is not effective.

The nutrient rule from Washington State's Department of Ecology requires 58 WWTPs along Washington's Puget Sound to limit their nutrient pollution release into the environment [47]. Several plants will be required to install nutrient removal systems. This spending creates a potential market for biofilms if dehalogenation capacity could be merged with nutrient removal.

Our system would provide added value by also breaking down PCBs from the system. The aerobic dehalogenase our Modeling and Wet Lab teams have been studying could also be expressed in forms to degrade other halogenated toxins. Dioxins which also have chlorine substituents on aromatic carbon rings could be targeted. With extended research dehalogenation could also be tailored to other similar compounds like PBDEs, which have aromatic rings substituted with bromine.

We compared the price of nutrient removal capacities to understand how much installing a biofilm to degrade PCBs could cost. The Lott Treatment Plant in Olympia, recently installed a biological nutrient removal system with a total cost of 29 million USD [48]. Their first installation of nutrient capacity in 1994 cost 30 million USD. For comparison the previously mentioned City of Spokane's membrane filtration system cost four times more. The total cost of 126 million was just for membrane capacity. Membrane filtration captures around 99% of PCBs in solution. For comparison biological systems not designed to degrade PCBs have been documented capturing between 90-95% of PCBs[18]. Our system would be designed for PCB capture and degradation so would likely achieve greater efficiency. This suggests potentially comparable degradation rates for significantly less cost.

We looked at two case studies of industrial sources of PCB pollution in the Spokane River Valley. Inland Empire Paper and Kaiser Aluminum are both sources of PCB pollution into the water basin. How these PCBs originate and are dispersed is significantly different between the two sites and both highlight different application needs our biofilm could fill.

Kaiser Aluminum produced large amounts of PCBs pollution during manufacturing in the 1940's [49]. These legacy PCBs were released into the earth. From there they have entered the groundwater system which has created underground plumes of heavily PCB contaminated water and soil. This liquid can reach concentrations of PCBs higher than 3,000 ng/L, which is very high in the context of PCBs. Kaiser has tested multiple technological methods to remove this contaminated groundwater in order to comply with state law.

The company tested two notable systems. One was a biological system that relied on a suspended algae solution through which PCB contaminated groundwater was run. Another technology known as the UV light and advanced oxidation process (UV/AOP)[49]. Kaiser estimated the average efficiency of the system at removing PCBs from groundwater to be around 80%. However their system used a solution of suspended cells, while our biofilm system forces PCBs into contact with organic materials and so should have a greater efficiency rate.

However Kaiser's UV/AOP system which uses no biological systems but instead chemically destroys the PCBs had a removal efficiency around 94.6%[49]. Kaiser also estimated that scaling the UV/AOP system up would be cheaper than scaling the biological system. Biological was estimated to be 3.4 million USD in initial costs while UV/AOP would be around 1.9 million USD. The maintenance costs were projected to be similar. 94.6% is within the range from previous studies finding PCB capture by WWTP biofilm at between 90-95% [18].

The major differences between this comparison study and our project is how the presence of the dehalogenase would affect our systems efficiency and ability to actually degrade PCBs. This study doesn't factor in that the biofilm also produces biomass over the time which can generally be sold as a soil additive if the PCB concentration is less than 50 ppm. Our project also has the potential to be adapted to breakdown dioxins and other halogenated chemicals. From this it seems that PCB contaminated groundwater is another area our biofilm system could be applied.

We also looked into Inland Empire Paper Co(IEP), which is the second contributor of PCBs to the Spokane River watershed although its PCB load is significantly less than Kaiser's. Inland Empire Paper's PCBs originate during their paper recycling process from processing papers with PCB containing inks. We spoke to Doug Krapas, the Environmental Manager at IEP to understand their approach. He explained IEP uses a membrane filtration system to capture PCBs and then incinerates them. IEP experiences an approximately 99% capture rate, this is the same rate that Jeff Donovan told us Spokane gets via their membrane system. Mr. Krapas told us the PCBs that make it through IEP's membrane system tend to be mono- and di-substituted chlorines.

Mr. Krapas mentioned that similar to Kaiser, IEP had also contracted with CLEARAS to test a biological PCB degradation system. He also noted that in IEP's use of biological systems that their paper waste doesn't have a significant nutrient presence and so their systems required amendment with various biologics. This points to an additional challenge to applying the biofilm outside of WWTPs in that most industrial supply chains would need nutrient additives to sustain a biofilm.

Kaiser Aluminum and IEP's study of biological PCB remediation is a promising sign in judging general corporate interest in biological methods of PCB removal. If a system can have promising results it seems there is sufficient interest from PCB producers to test their implementation and adopt them if they are cost effective.

One potential method of implementation would be to produce the dehalogenase as secreted protein in a way that maintains its functionality. While this is currently beyond our capabilities there is evidence that if it could be executed it would have a large potential market. When we discussed an additive dehalogenase with Mr. Krapas, he agreed that a product in that form would be more interesting to companies like IEP.

In summary there appear to be a wide range of potential markets for biofilm based PCB remediation from industrial systems to groundwater treatment as well as WWTP. There is likewise a wide range of further research to be done. Our team only looked shortly at genetically modified kelp and seagrass as a vector to destroy PCBs from and there has been significant research into using plants to degrade PCBs. We also weren't able to fully survey the variety of halogenated pollutants outside of PCBs that an aerobic dehalogenase could be applied to. There is much potential promise for PCB bioremediation.

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