Harmful algal blooms (HABs) formed by cyanobacteria are found in many water ecosystems and can disrupt water usage and damage the natural environment. These cyanobacteria often produce various toxins, such as the hepatotoxin microcystins (MC), that directly threaten public health. HABs are increasingly becoming more intense and frequent in the United States and around the world due to eutrophication and climate change. These harmful cyanobacteria blooms and toxins present in source water for drinking water treatment plants are usually removed to provide safe drinking water for communities. Water treatment plants produce daily waste called water treatment residuals (WTR) that can contain the removed cyanobacteria and their cyanotoxins. As current WTR disposal methods, such as storage in lagoons or waste disposal areas, become more difficult to sustain environmentally and economically, agricultural land application has become a beneficial future disposal option. However, WTR-applied lands, some of which may be used for growing crops meant for human consumption, may contain these cyanobacteria and their cyanotoxins from the WTR and pose a public health threat. Little is known about the comprehensive microbiome profile of WTR, especially key community players, such as cyanobacteria, cyanophage, and symbiotic bacteria (potential toxin degraders) present in bloom ecosystems. Feasible biocontrol solutions for reducing both cyanobacteria and cyanotoxins in this lesser known environmental matrix are necessary for protecting human health and the environment.
Chapter 1 is a literature review that highlights the current knowledge around cyanobacteria, cyanotoxins, and the role climate change is playing in the proliferation of the two, particularly in the Midwest and Lake Erie. Insight into Lake Erie-specific public health concerns, with unique environmental matrices becoming new exposure pathways for toxin, as well as the current knowledge gaps, are included. Lastly, HAB controls, including using cyanophage and toxin-degrading bacteria, and feasibility of implementation are discussed.
In order to implement HAB controls such as cyanophage and toxin-degrading bacteria, the ecosystem they inhabit must be well understood. In Chapter 2, cyanophages from WTR of a HAB-affected inland lake in Ohio that infect the toxic bloom-forming filamentous cyanobacteria Planktothrix agardhii, the most predominant genus during bloom seasons in inland lakes in Ohio, were isolated and characterized. The main objectives were to 1) morphologically and phenotypically characterize lytic phages from WTR, 2) examine host-phage interactions using fluorometry and microscopy, and 3) conduct a metagenomic analysis of the microbial community present in this Lake Erie microcosm. Visual and microscopic changes observed in Lake Erie Planktothrix cultures with cyanophage infections included shearing and shriveling of filaments, reduced clumping, and changes in buoyancy. Photosynthetic pigmentation, a proxy for cyanobacteria biomass, was more apparent for the host infected with phage, which was an unexpected result. Nineteen lytic and lysogenic phages and seven bacteria genera other than Planktothrix were identified. Viral genomes were both tied to microbial hosts and successfully annotated with important phage proteins. Microbial metabolic pathways associated with survival in this microcosm were highlighted, such as genes associated with photosynthesis, competency, environmental stress, phosphorus uptake, etc. This is the first study to isolate and characterize cyanophages from WTR infecting the filamentous Planktothrix and provides insight into the potential application of host-specific phage to reduce cyanobacteria populations and their toxins.
When investigating biocontrol solutions for cyanobacteria, the main concern is releasing large amounts of intracellular toxin into the environment. Because of the successful isolation of phages from WTR, there was interest in isolating other microbes from this concentrated matrix. Microcystin-degrading bacteria (MCDB), ubiquitously present bacteria present in bloom areas, naturally degrade MC and can help as a second step in this biocontrol for toxin reduction. Chapter 3 details the successful isolation and characterization of four MC-LR degraders from WTR of a HAB-affected inland lake in Ohio. The main objectives were to 1) characterize MCDB isolated from WTR from HAB-affected areas, 2) quantify MC-LR degradation by the isolates, and 3) further investigate MC-degradation pathways in bacteria isolated from western Ohio. The bacteria isolates were identified to the genera Bacillus and Paenibacillus. Three of the isolates successfully degraded 60% of a 50 ppb solution of MC-LR over five days, although no intermediates were confirmed. These bacteria lacked the three enzymes from the mlr-operon, a well-understood degradation mechanism, but a fourth protein, mlrD, was detected. This study is the first to isolate MCDB from WTR, and the outlined methods may encourage many future studies to isolate more degraders from this matrix.
After the investigation of the MCDB and their degradation capabilities, there was interest in understanding the possible application of MCDB to WTR that may be used for agricultural amendments, since such solutions have not yet been achieved. A quantitative microbial risk assessment (QMRA) model was implemented to test how MCDB application could influence health risks to different populations based on the types of WTR applied. The objectives of Chapter 4 were to 1) simulate and compare health risk associated with the application of MC-containing WTR (from low bloom and high bloom areas) applied to agricultural lands growing crops meant for human consumption, and 2) compare the health risks of untreated high bloom WTR and treated WTR to evaluate treatment effectiveness. The model concluded that risks were two-log higher for crops grown in high bloom-affected WTR compared to low-bloom (p-value < 0.001). With MCDB treatment prior to application, health risks were reduced by one-log compared to the high bloom-affected untreated WTR (p-value < 0.001). This model not only quantified risks associated with interactions with this matrix, but also confirmed that this is a possible exposure pathway for MC. Models like this can be utilized for various environmental matrices, informing accurate policies prior to actual land application experiments.
In summary, this thesis addressed the HAB crisis in Lake Erie and the Ohio River watershed region and the concern of emerging transmission pathways for related toxins and highlighted the numerous knowledge gaps associated with these blooms. New isolation and characterization techniques were utilized in many of these studies, and can inform future studies in this field. The hope of this research is to optimize the possibility of using such microbial agents as true biocontrols that would not only reduce harm to the environment but can also reduce public health risks to both cyanobacteria and their toxins. As climate change impacts continue to worsen, biocontrols like these are needed now more than ever before, and continuous study of the microorganisms can encourage their use for effective and safe changes. Future studies would be to complete field applications of WTR in a controlled setting, and introduce the proposed biocontrol solutions, cyanophage and microcystin-degrading bacteria as a non-chemical based, sustainable alternative solution.