Integrated Human Practices


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Human Practices Report



Figure 1. Florida Fish and Wildlife Conservation Commission.

The year 2021 was one of the worst for Florida's manatee population. Just over 10% of its population was lost within the calendar year. According to West University of South Florida (WUSF) Public Media, “An unprecedented 937 manatees have died in Florida in 2021, representing 10% of the animal’s population in the state.” (1) This mortality event, as the state of Florida defines it, has continued into 2022. Government, academic, and non-profit researchers have attributed the event to a loss of seagrass, manatees’ primary source of nutrition. Bioexplorer, a software for real-time biophysical data acquisition, claims that "The West Indian manatees mainly feed on seagrasses that grow on the seafloor, leaves, or plant roots.” (2) This devastating loss of seagrass can be directly correlated to the increasing spread of harmful algal blooms (HABs). More recent findings point that the radical increase of HABs can only be attributed to eutrophication. As a result this increase in HABs is causing a rapid decline in aquatic biodiversity. Qiuying Han and Dongyan Liu at the Chinese Academy of Sciences explain that, "The competition for living space and using resources has the most direct effect on seagrass beds when macroalgae are blooming in an aquatic ecosystem. The consequence of macroalgae blooms (e.g., light reduction, hypoxia, and decomposition) can produce significant indirect effects on seagrass beds." (3)


Figure 2. Conceptual diagram of interactive physical, chemical, and biological controls on HAB formation and proliferation along the freshwater-to-marine continuum." Pearl, 2018

The process of eutrophication begins when a body of water is contaminated with a large amount of nutrients, including but not limited to nitrogen and phosphorous. These nutrients promote the growth of harmful algal organisms. Algae has a natural buoyance, so as it grows, it covers the surface of bodies of water. The preferential placing of alage bloom on the surface of water bodies blocks sunlight from reaching the bottom. As a result, plants the live at bottom of aquatic organism are depleted on solar energy. Among these species of plants exists the focus of our project: seagrass, a manatee's main food source. Author Rob Simbeck asserts that, "A manatee regularly eats as much as 10 percent of its bodyweight per day." (5) However, due to the slow regeneration of seagrass, many manatees have been starving and population sizes are declining in record numbers. According to Aarin-Conrad Allen, a Ph.D. student at FIU's Institute of Environment, manatees have begun eating the algae at larger rates. (6) As described by their research, algae serves as a comparable "junk food" to our human diets.

Action has been taken by many activists, researchers, and state officials to prevent further starvation. However, manatees have continued to die at an unprecendented rate. During the winter of 2021 and continuing into 2022, state officials took the extraordinary measure of directly feeding manatees 25 tons of romaine lettuce(7). This action was covered by Curt Anderson in an article published by AP News.

While this make seem like a way that the community set out to help the problem, in reality, it’s like putting a bandage on an amputation. Allowing these hand feedings could easily become more dangerous to their populations with time. The National Park Service explains that, "Feeding or approaching wildlife can cause problems for both humans and wildlife. Wild animals that learn to associate humans with food often become dependent on human-related food and garbage. They lose their wildness, may become unhealthy and often threaten people and property. They may be cute, but feeding them is bad for you and the animals."(8) Soon enough, The State of Florida will have to face that we have let an entire species with no natural predators potentially become extinct because of our own ignorance and greed.

Many locals remain unaware of the atrocity being committed to these gentle creatures. An even more heartbreaking picture is painted when we realize there is an entire generation of manatees being born directly into starvation and habitat loss. Manatees are creatures of habitat with elders teaching young calves migration patterns to find food. Continous swimming with a false promise of food takes a toll on their bodies. Starvation and habitat loss are not the only threats manatees face through their migration, but like these, the other worries are a byproduct that were designed by man. While injuries from collisions with boats or other vehicles are very common, the most common cause of manatee deaths in the State of Florida is related to starvation. To propose a solution to this problem the 2022 FSU iGEM team developed a plan to research the problem; design, build and test our solution to prevent the spread of harmful algae blooms.



Design Thinking Process


Figure 3. The design thinking cycle that the FSU iGEM team follows in the development of its project.

The FSU iGEM team uses the principles of design thinking to guide its project. The four stages of design thinking, or human centered-design, start with conducting empathy-focused research led by our Human Practices sub-team. This is then followed by ideation, led by our Design sub-team. Finally, our Build sub-team take our efforts to build and test our proposed solution. The team overall seeks to complete multiple engineering cycles influenced by our stakeholders to help shape a more perfect solution allowing for all sub-teams to learn from the build and test stages.




Stakeholder Engagement


Figure 4. A visual depiction of the institutions and organizations our team reached out to for our research.

To give the team direction at the beginning of the empathy stage, the team developed four stakeholder maps laying out what individuals and organizations needed to engage with to gather a holistic perspective on the problem of HABs and how they relate to the manatee mortality event. The maps represented stakeholders in four sectors: academia, government, past iGEM teams, and non-governmental organizations. The identification of stakeholders gave the team structure to its initial empathy research efforts. Through this process of research, we have met very knowledgeable individuals, among them existing professors, activists, reporters, rangers, and enthusiasts. We continued this track whilst constantly identifying where each stakeholder belonged within the stakeholder map.





Figure 5. FSU iGEM team members sharing their academic stakeholders map.

➤ The academia team discovered different papers written about HAB’s and professors/institutions who have specialized on the topic at hand.

➤ The Government team did their investigations on different gov. funded research, organizations, tasks forces. Among their research, they found the FDEP to play a large role on sustainability against the issue. The biggest help we’ve found came from Max Chesnes’ contact in the St. John River Water Management district who explained the situation in the Indian River Lagoon and what they have been doing to stop the spread. Lastly, the FWC also gave us perspective on the issue.

➤ The Past iGEM Projects team came up a bit short with research on past iGEM’ s doing work on this specific topic. Whilst it became good news for our creativity/uniqueness, the situation became worrisome due to lack of attention being received.

➤ The NGO’s team found different organizations who mainly focused on taking care of troubled sea cows, but not much being done for the spread of HAB’s. Some activism and clean-ups came relevant but not enough to mitigate the problem. MOTE was one of the large organizations found most useful in the situation as well as the NRDC.


Figure 6. Environmental journalist, Max Chesnes, provides the team with an overview of the unusual mortality event the manatees were facing.



Partial List of Stakeholders Interviewed


  • Dr. Melvin Bolton: Research Plant Pathologist with the U.S. Dept. of Agriculture-Agricultural Research Service and Dr. Bolton's research has also proven invaluable, in addition to a generous donation from his cercosporin DNA library.
  • Dr. Jennifer Toyoda: MOTE Marine Laboratory & Aquarium Provided insight into potential mechanisms for cercosporin deployment, effects of toxicity besides lethality, weight of evidence for gaps in cercosporin literature, and justification of usage.
  • Manatee Lagoon: An educational facility whose mission is to inform the public about Florida's official marine mammal, but also inspire them to help preserve the state's environment. The facility is located in the Indian River Lagoon. Their staff gave us an overview of what was happening locally and what is being done in the non-profit sector.



    Understanding the Source of the Problem


    As we began our research, it was clear that the hardest-hit manatee population by the mortality event resided in the Indian River Lagoon (IRL). The IRL ranges through six counties on the central Florida coast. The Guardian posted an article that claims, "Deaths have been particularly high in the Indian River Lagoon on Florida’s east coast. One of the most biologically diverse estuaries in North America, it supports about a third of U.S. manatees. More than 30% of manatee deaths last year occurred here." (4) They go on to explain how the excess nutrients are fueling the fire. According to Frontiers in Marine Science, in the last ten years we have lost more than 58% of our seagrass coverage. One way activists have helped the problem is through seagrass replanting. This seemed like a great idea but unfortunately, with the algae still covering the surface and manatees over-grazing these new fields before they have a chance to root, the sea grass has no opporunity to survive for the long term. Still feeling dissatisfied with the amount of information on the subject, our team began reaching out to different stakeholders and professionals so that we could form a proper hypothesis. One of our first points of contact was Lucas Davis, a wildlife biologist at the U.S. Fish and Wildlife Service. He was able to explain the day-to-day life of a wildlife biologist and took us on a first-person journey describing exactly what a manatee rescue incorporated. He finished with explaining how our next point of contact should be the individuals responsible for taking care of the IRL. The entity with this authority operates out of Putnam County and is responsible for overseeing the inflows and outflows of the IRL. This organizations being the St. John River Water Management District. After reaching out, this organization was able to provide us with the following graphs describing the growth in algae through a 30 year period:

    Figure 7. Image maps provided by St. John River Water Management District.


    At this point in our research, our team felt confident enough with the evidence accumulated to begin formulating a plan of action to tackle the issue at hand. We began our pathways with unique solutions that properly incorporated everything from biomedical engineering to even mechanical engineering. After a meeting with journalist and reporter, Max Chesnes, he was able to share different implemented solutions used in the industry today. This included the flocculation of the algae. According to Science Direct, "Flocculation is a process by which a chemical coagulant added to the water acts to facilitate bonding between particles, creating larger aggregates which are easier to separate. The method is widely used in water treatment plants and can also be applied to sample processing for monitoring applications."(10) After this substance bonds all the algae together, it sinks and accumulates at the bottom of lake floors creating muck. The biggest problem with this solution is collection of said muck. Not only is this a very difficult task because of the magnitude of the blooms, but once collected we inherit the new problem of proper disposal. He mentioned that most of the time, the muck is laid on nearby islands to let dry, destroying the small ecosystems by further interfering. Another solution our team found was published by the South Florida Water Management District. This solution being the immediate use of Hydrogen-Peroxide to target emerging blooms.



    Economic Impact on Florida


    The state of Florida currently has one of the most prosperous economies in the US and across the globe. With a GDP that surpassed $1 trillion dollars in 2021, our government aspires to make the top 10 list (11) of worldwide economies by the year 2030. With its amusement parks, warm weather and spectacular beaches, the number one source of income for the state is tourism. However, with the HABs outbreaks surrounding the coast and seeping into our lagoons, the future of the economy is looking murky. In a state where 25.5% (12) of tourists book their trips primarily for the beaches, the water quality is of utmost importance; and yet the problem has escalated so greatly that with the last red tide event between 2017 and early 2019, Gov. Rick Scott was forced to declare a state of emergency. (13) We are also aware that with each wave, these algal blooms are worsening, becoming more frequent, toxic, and lasting longer than ever. (14) This problem is not only detrimental to the swimmers and the sightseers, but to the wildlife as well. In order to protect Florida’s economy and ensure its success in the future, we must preserve our waters. That is why FSU’s iGEM team has dedicated the last year towards developing a product that will degrade these algal blooms and allow for clearer waters. Not only would this create safer waters for tourists, but it would allow sunlight through the waters to support a healthy underwater ecosystem.

    These algal blooms have wreaked havoc on many other markets as well, ranging from real estate on the coast, to the commercial fishermen out at sea. Properties near the water have lost up to 40% (15) in value due to such water quality issues, while fishermen have been losing their money makers to dead zones caused by HABs, which costs them $18 million a year on average. (16) An early, highly conservative estimate on the annual effects of HABs on Florida’s economy was placed at $50 million; (17) in reality, it is certainly much higher. Without intervention, this problem will continue to escalate, thus hindering Florida’s economy and the wide range of afflicted industries. It is for these reasons that the algaecide industry is projected to boom over the course of the next decade. The economy has demonstrated a colossal demand for a cure to HABs. Cercosporin, with its natural process of algae extermination, potentially poses a disruption in the market, fulfilling just what Florida needs to remain on course in actualizing our goal of reaching top 10 economies in the world.


    Our Solution Overview


    Current State of Algaecides


    Several algaecides were investigated in addition to cercosporin. Among them were hydrogen peroxide, copper sulfates, heavy metal salts, sodium percarbonate, sodium hypochlorite, magnesium hydroxide, benzalkonium chloride and sterol surfactants (18-23, 43). Many of these solutions are indiscriminate in their effects and result in secondary pollution, motivating the development of more innovative solutions, including the thioacetamide, Q2 (35), plant derived alkaloids (36) and more selective reactive oxygen species (33,34) like the one proposed here (25). Given the available options, our primary concerns were specificity, secondary toxicity, long term accumulation, algal resistance, changes to water quality, and activation. All things considered, we found cercosporin to be among the more promising algaecidal solutions, warranting further development.


    Cercosporin as a Solution


    Cercosporin is a light activated reactive oxygen species producer best known for its role in crop blights, whereby pathogenic fungi of the genus Cercospora synthesize the compound to lyse cell membranes of host crops for nutrient absorption (26), resulting in hundreds of millions of dollars in damages every year (24); consequently, research efforts have historically focused on the molecule within the context of agricultural loss. However, more recent research has revealed that cercosporin and similar molecules known as perylenequinones have properties of interest for a multitude of applications, including anticancer therapeutics, antimicrobial agents, and ironically, pesticides and fungicides (27). That is, the same compound that researchers, government agencies, and farmers are so desperately trying to mitigate to control crop blights can be used to eliminate pests and non-resistant fungi. Both the appropriate dosage and context dependence of cercosporin toxicity are critical to its application as a reasonably selective toxin and algaecide.

    The proposal of a reactive oxygen species as an algaecide is sure to raise concerns given the seemingly indiscriminate nature of ROS and their direct application into the environment; however, existing literature and a careful examination of the problem at hand lend support in favor of our proposal. The advantages of cercosporin use include light activation, preferential membrane binding and water insolubility, rapid response, reasonable rates of degradation, a lack of secondary pollution, and potential use in conjunction with other algal bloom solutions - all of which are outlined in the following sections.


    Algaecide Use and Selectivity


    Returning to the 2022 paper published by researchers at Jiangnan University that motivated our project, four fungal perylenequinones were tested for their ability to inactivate Microcystis aeruginosa, a cyanobacteria responsible for harmful algal blooms. Of those compounds, cercosporin showed highest degree of inactivation at a 20 µM concentration, with just over 50% of the algae in culture eliminated after 12 hours in the presence of light, around 75% eliminated after 24 hours, and almost complete elimination after 36 hours; photoactivation also occurred in the absence of sunlight - although to a lesser extent (25). This indicates that cercosporin itself and not just the reactive oxygen species it produces can lyse cell membranes – contributing to the necrosis like cell death observed.


    Figure 8. Shows the candidate compounds used in this study(A), along with the survival rates of M. aeruginosa over time in response to different perylenquinone treatments (B), cercosporin concentrations (C) and cercosporin under different levels of light exposure (D). Taken from (25).


    Figure 9. Shows the degradation of M. aeruginosa treated with 20µM cercosporin under light conditions (A,B), and in the dark (C,D). Taken from (25).


    Furthermore, the compound is insoluble (25,26) and has been found to preferentially bind to cell membranes (24,25) as well as degrade pigments required for photosynthesis, such as chlorophyll and carotenoids (25), resulting in a more targeted toxicity and inactivation in the case of algal blooms covering the surface of water.

    The photoactivation requirement for ROS production and the bulk of cercosporin’s cell lysing activity (24-27) is also worth mentioning since its toxicity upon ingestion is minimal in cases where the molecule is shielded from light. A striking example of this comes from an early paper on cercosporin, where mice dosed and continuously irradiated over the course of 24 hours died, but mice given 10 times the dosage in the dark were asymptomatic, with a much higher amount of the molecule being excreted (28). Since then, cercosporin (29) and other perylenequinones (30,31) have garnered interest for their applications in photodynamic tumor treatment, which given its nature as a therapeutic requires that toxicity be tolerable and preferential toward tumor cells. While this last example illustrates the specificity of cercosporin within the context of eradicating tumor cells, it is also meant to draw a parallel to specific toxicity at an ecological level, where the goal is to eradicate HABs while minimizing secondary impacts.



    Degradation and Ecological Impact


    Furthering our discussion of photoactivation, the first paper mentioned (25) also reports a rapid degradation of cercosporin under light conditions, with the highest amount occurring within 12 hours, shown in figure 6. Another study screening bacteria for the ability to degrade cercosporin revealed that 40 of the 244 isolates assessed were able to degrade over 85% of cercosporin present into a nontoxic compound, despite most of the isolates having no known interaction with cercosporin in their natural habitats (32).


    Figure 10. The extracellular (B) and intracellular (C) concentrations of cercosporin in both light and dark conditions. The combined data suggest degradation in the presence of light and cultured algae. Borrowed from (25).


    Despite evidence pointing to its degradation, the ecological toxicity of purified cercosporin has not been well documented (44-48) beyond basic model organisms and crops. For this reason, toxicity can only be approximated using data from the broader class of perylenequinones mentioned earlier, which have a longer history of consideration for biocontrol applications.


    Figure 11. Some representative perylenequinones including cercosporin, all of which are light activated ROS producers. Borrowed from (30).


    Among the most tested for biocontrol applications is elsinochrome A, which has demonstrated preferential damage to fungi and bacteria, with minimal damage to plants and rapid degradation when properly dosed as purified extract rather than in the context of invasion and secretion by fungi (37). The planktonic crustaceans Artemia salina (brine shrimp) and Daphnia magna (water flea) have also been tested for survival as an accepted readout of ecological damage and showed no lethality to 15 minute light exposure following incubation in the dark (37), but were sensitive to prolonged exposure to the toxin, with lethal doses shown in figure 8 (38).


    Figure 12. Lethal concentrations of irradiated elsinochrome A in D. magna (a) and A. salina (b). Converted to µM concentrations for the sake of consistency - D. magna exhibits an LC50 of approximately .53 µM at 24 hours and .40 µM at 36 hours. A. Salina shows an LC50 of approximately 36 µM at 24 hours and 20µM at 36 hours. Borrowed from presentation (39), which was adapted from (38).


    While this does provide some indication of the level of damage that can be expected from perylenquinones in smaller, developmentally sensitive model organisms, we must exercise caution in our extrapolation of existing data. Elsinochrome A is being used as a proxy for cercosporin, and model crustaceans as an estimate for whole ecosystem toxicity.

    Toxicity assays in a variety of organisms at high resolution are necessary to holistically determine effects on survival and health, and while the use of elsinochrome A as a proxy is reasonable given its similarity to cercosporin, the two are not identical. Perylenquinone structure dictates ROS generation, lipophilicity, and specific binding (39) among other factors, and these structural differences can prove significant as we have already seen in the case of algal toxicity.

    In conclusion, while the degradation of cercosporin occupies a reasonable timeframe in existing literature, more resolute testing of environmentally representative organisms must be conducted to fully justify its application.



    Multistep Approach


    Given the limitations of any particular solution, the most reasonable approach in mitigating algal blooms is a combinatorial approach such that weaknesses from one solution will be compensated for by others – often referred to as a “Swiss cheese model” in risk analysis and engineering, depicted in figure 9. Responsible solutions that would ideally be used in conjunction with cercosporin include the bioretention of fertilizer runoff, predictive algal bloom modelling (41,42), public outreach and education, political activism, collaboration with local law enforcement, barley straw usage to prevent algae growth (40), mechanical removal of algae from bodies of water, and additional chemical treatments and/or genetic engineering of bacteria to treat water impacted by algal toxins, as seen in the work of previous iGEM teams.


    Figure 13. Illustration of some solutions to be used in conjunction with algaecides to promote responsible environmental management.



    Reactive and Preventative Solutions


    A representative example of a preventative solution is bioretention. Bioretention introduces a way to prevent HAB inciting nutrients such as nitrogen and phosphorous from entering large bodies of water. We hope to design a mechanism at some point in the future that will create a barrier capable of catching nutrients from fertilizer runoff, which can then be reintroduced into the arable land they came from. We have concluded that these filters will only have to be replaced every 10 years and that their replacement would coincide with constant monitoring and upkeep. To implement this solution, we would begin with land generating agricultural runoff. First, we would dig up the soil, about 6-10' in depth, and lay down a layer of biochar acting as a filter. Next, the layer would collect nutrients and pump them into a reservoir containing algae for its nutrient retention capabilites. Lastly, the waste would collected and reused as fertilizer. Given the envrionmental benefit coupled with reduced cost in the face of rising fertilizer prices, we are hoping farmers would be willing to implement our solution.

    It is also worth justifying the use of cercosporin as a predominantly reactive solution (although it could be proactive to some extent given reliable HAB predictions). While preventative solutions alone would be ideal, the world we live in is far from it. This is not meant to discourage the implementation of preventative solutions, but rather to provide justification for more reactive solutions that are often shunned. Whether the focus is on the impact of HABs on manatees or the broader implications of climate change, sometimes idealizations remain just that, and are championed at the cost of ignoring lower hanging practical solutions. Inaction at critical junctions in the timeline of environmental impact is costly, whether at population bottlenecks for keystone species or tipping points for the ecosystems they occupy. Any environmental treatment will come at a price, economic or environmental - the question is whether we can justify and responsibly implement the solutions available to us. With further testing and optimization, we are hoping cercosporin could be an innovative solution that provides a net environmental benefit.



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    Additional Image References


    Figure 1. https://wusfnews.wusf.usf.edu/environment/2022-03-10/mixed-grades-for-ailing-indian-river-lagoon-and-dying-manatees-in-new-report-card
    Figure 2. https://www.mdpi.com/2072-6651/10/2/76/htm
    Figure 3. https://www.innovation.fsu.edu/design