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Background

[Introduction to microcystin]

Microcystis aeruginosa is a species of freshwater cyanobacteria that secretes microcystin, a potent hepatotoxin and carcinogen [1]. The accumulation of microcystin in crops, livestock, potable water, and recreational water has been associated with detrimental health issues. Exposure to microcystin via ingestion or physical contact with contaminated water can cause symptoms including abdominal pain, nausea, vomiting, headache, diarrhea, sore throat, pneumonia and in severe cases, liver failure and death [2]. In addition to the evident health implications, microcystin has been shown to have allelopathic properties which can disrupt local ecosystems by inhibiting growth in M. aeruginosa competitors like green algae [3]. This allows the cyanobacteria to dominate the phytoplankton community, threatening aquatic biodiversity.

[Microcystin]

Cyanobacteria like M. aeruginosa commonly form dense mats in eutrophic water bodies. Nationally, cyanobacteria blooms, colloquially known as harmful algal blooms (HABs), have been detected in all 48 contiguous states. At the end of its life cycle, M. aeruginosa cells lyse and release toxins like microcystin, which has been associated with human and animal illness in at least 43 states [4,5]. A multitude of industries, including seafood, restaurant, agricultural, and tourism, are severely susceptible to HABs, experiencing economic losses of about $82 million USD each year [6] . When microcystin-contaminated water is used for crop irrigation, these toxins can diminish nutritional quality, pollute surrounding soil and plants, limit plant growth, and decrease production yield [1]. HABs tend to form due to factors including high surface water temperatures, extreme weather, slow water circulation, wind currents, and water currents [6,7]. The growth of HABs has also been linked to an accumulation of nutrients such as carbon, nitrogen, and phosphorus that may come from local farmland and industrial runoff, which causes the algae to be overfed and overgrown. Recent studies that measured microcystin levels found 4,400 lakes in America that exceeded the EPA recommended recreational water quality standards [8].

Inspiration: Why?

The City of Watsonville is an agricultural community in the heart of the Central California Coast. Just 30 minutes away from Santa Cruz, Watsonville is a major contributor of local and national produce. Its food processors freeze and distribute more fruits and vegetables than any other single area in the United States [9]. Watsonville is also home to Pinto Lake, which attracts over 100,000 visitors. The lake covers 110 acres and is one of the few natural fresh water lakes of its size on the Central California Coast. Pinto lake harbors a diverse array of species, including 133 different bird species and fish such as bass, trout, crappies, bluegill and catfish [10]. Prior to _____, Pinto Lake was utilized for crop irrigation, but farmers in the Watsonville area were required to shift to other water sources due to dangerously high microcystin levels [11]. Today, Pinto Lake is mainly recreational, allowing visitors to engage in activities such as fishing and boating. Pinto Lake has struggled with HABs since the 1980s, but in recent years, microcystin levels have posed greater environmental and public health concerns [12]. At times, elevated levels of the toxin have led to the lake closing to the public. Previous research has suggested that over the years, excess nutrients from agricultural runoff and septic system leaks have accumulated at the bottom of the Pinto Lake. The buildup of carbon, nitrogen, and phosphorus provides the ideal habitat for M. aeruginosa to grow and thrive. In late summer and early fall, microcystin samples from Pinto Lake have been observed to be three million times greater than the toxin exposure limit in California [13,11]. In 2017, in an effort to address this issue, the City of Watsonville treated Pinto Lake with 118,000 gallons of alum and buffer, which are commonly used in water treatment processes. Alum, also known as aluminum sulfate, works by binding phosphorus, making it unavailable for use by cyanobacteria [14]. While this treatment was successful, resulting in a 93.5% reduction of the nutrient-load in the lake, it was costly at $395,000 and not a permanent solution. Recent climate patterns have contributed to a rapid rise in excess nutrients, increasing HABs formation up to five blooms every 15 days [15].

Goals

As a primarily low-income community, it is critical that we preserve free, safe, and accessible outdoor opportunities in Watsonville. Our team aims to create a safe, long lasting, and cost effective solution to treat future HABs in Pinto Lake. We will produce a repeatable spot treatment to apply to HABs in Pinto Lake, allowing lake officials to effectively neutralize microcystin. We propose that we can reduce microcystin levels using single chain variable fragments (scFv) that are expressed in selective phagemids. Single chain variable fragments are short engineered antibody fragments that can selectively bind to a wide variety of specific targets, including toxins such as microcystin. The delivery phagemid is a genetic vector that combines features of both bacteriophages and plasmids. Phagemids have the ability to infect bacterial cells, reproduce inside them, and ultimately cause lysis. Using the components described, our goal is to produce a cost effective and flexible treatment approach to reduce microcystin levels in eutrophic bodies of water.

Approach

We will be using Escherichia coli (E.coli) to develop a proof of concept of our phagemid expressing ScfVs that bind and neutralize microcystin. Gibson Cloning will be employed to engineer a phagemid that will produce the T7 bacteriophage and ScfVs without the need for traditional restriction enzyme based cloning or ligation steps. To test the viability of our new phage we will run a plaque assay on our E.Coli. Additionally, we will also run a binding assay to ensure our ScfVs'binding affinity is sufficiently high. Once we have successfully integrated and confirmed production of our antibody fragments we will utilize High-Performance liquid Chromatography (HPLC) to measure levels of microcystin before and after deployment of our antibody fragments.

Future

After establishing a proof of concept using E. coli, moving our mechanism into a viable cyanophage would be the next step. Repeating the protocol in a cyanophage capable of infecting microcystis allows for the testing and neutralization of microcystin directly from lake samples. We plan on obtaining cyanophages from other research institutions or isolating our own from blooms found in the lake. Capitalizing on the poor fitness cost of the plasmid design, the treatment could be used as a spot treatment with low risk of proliferation across the entirety of Pinto Lake. Our solution provides a road-map to a cost-effective and environmentally friendly treatment that could be scaled to the increasingly concerning microcystin issue worldwide.

  1. R. Melaram, A. R. Newton, and J. Chafin, “Microcystin Contamination and Toxicity: Implications for Agriculture and Public Health,” Toxins, vol. 14, no. 5, p. 350, May 2022, doi: 10.3390/toxins14050350.
  2. “Facts about Cyanobacterial Blooms for Poison Center Professionals | CDC,” Nov. 28, 2022. (accessed Jun. 28, 2023).
  3. I. Teneva, V. Velikova, D. Belkinova, D. Moten, and B. Dzhambazov, “Allelopathic Potential of the Cyanotoxins Microcystin-LR and Cylindrospermopsin on Green Algae,” Plants Basel Switz., vol. 12, no. 6, p. 1403, Mar. 2023, doi: 10.3390/plants12061403.
  4. N. O. and A. A. US Department of Commerce, “Why do harmful algal blooms occur?” (accessed Jun. 28, 2023).
  5. O. US EPA, “Background of the National Aquatic Resource Surveys,” Sep. 04, 2013. (accessed Jun. 28, 2023).
  6. T. Le, “Microcystis: Toxic Blue-Green Algae,” Office of Environmental Health Hazard Assessment (OEHHA), Feb. 2009.
  7. C. Joab and G. Chetelat, “Nonpoint Source 319(H) Program Cyanobacteria and Harmful Algal Blooms Evaluation Project Harmful Algal Bloom Primer,” California Water Boards, Nov. 2019.
  8. “Cyanobacteria in Sacramento Region Waterways,” Sacramento Environmental Commission. (accessed Jun. 28, 2023).
  9. G. Alberola, M. Gehrke, R. Ketley, M.L. Huertos et al., “Pinto Lake Pilot Treatment Project | Final Report,” California State University Monterey Bay, Nov. 1, 2013.
  10. P. Osmolovsky, “Progress Report to Support Development of Total Maximum Daily Loads Addressing Nutrients and Algal Toxins in Waterbodies of the Pinto Lake Catchment,” California Environmental Protection Agency State Water Resources Control Board, Nov. 2015.
  11. “Increase in Algae Blooms a Concern at Once-Toxic Pinto Lake,” Good Times, Nov. 30, 2022. (accessed Jun. 28, 2023).
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