Doctor of Philosophy, University of Toledo, 2023, Chemistry

The formation of cyanobacterial harmful algal blooms (cyanoHABs) in waterbodies, including Lake Erie, has become a global issue. CyanoHABs can generate toxic metabolites, such as cyanotoxins, or beneficial natural products. Microcystins (MCs) are cyclic cyanopeptides that have attracted enormous attention, primarily due to the known human hepatotoxicity and their wide distribution. Anabaenopeptins (APs) and cyanopeptolins (CPs) are two understudied classes of cyclic cyanopeptides that co-occur with MCs in similar abundance and frequency, and their toxicity profiles are yet to be understood. Since they can reach the drinking water systems, more studies are needed to determine whether they should be included in drinking water monitoring and management protocols. Also, their ability to inhibit proteases makes them suitable drug candidates. MC biodegradation is considered an environmentally friendly approach to detoxifying MC-containing drinking water. Detecting and identifying the degradation products of MCs enable their toxicity studies and ensure the safety of human health. Ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry (HRMS) is a gold standard in state-of-the-art compound identification. This dissertation accelerates the studies on cyanopeptides besides MCs and advances cyanopeptide research by incorporating LC-HRMS. It is divided into five main chapters with respect to five research projects I conducted during the last five years. The first project focuses on identifying linear MCs with C-terminal Arg in Lake Erie water samples using UHPLC-HRMS/MS and in-source fragmentation. The novel in-source fragments of linear MCs containing a modified Adda moiety and C-terminal Arg were discovered, and their structures were putatively assigned using HRMS and MS/MS data. The fragment ion at m/z 175.11 was used as the major diagnostic ion to identify whether a MC is linear with arginine at the C-terminus. The distinguishi (open full item for complete abstract)

Committee: Dragan Isailovic (Committee Chair) Subjects: Analytical Chemistry

PhD, University of Cincinnati, 2023, Engineering and Applied Science: Environmental Engineering

Nowadays, water scarcity, deterioration of source water, and the more stringent water quality requirements are the primary driving forces for the development of novel and robust purification processes to make the most use of the limited water resources. Ultraviolet (UV) -based advanced oxidation processes (UV-AOPs) have been intensively studied and have started to be implemented in full scale potable water treatment facilities due to their high efficiency in the degradation of contaminants of emerging concern (CECs). Compared with the well-studied UV/H2O2 process, the UV/chlorine process, by integrating chlorination and UV irradiation, draws increasing attention due to its wider application range and higher cost-efficiency, and has been proposed and implemented as an alternative to UV/H2O2 in pilot and full-scale drinking water treatment facilities. Chlorination is the most widely applied disinfection process. By dosing free active chlorine (FAC) to the water treated by coagulation and filtration, chlorination can inactivate waterborne pathogens and degrade various organic pollutants. However, the CECs degradation by chlorination was not efficient with the practical chlorine exposure (Ct value). Decontamination by chlorination suffers from a ubiquitous, pernicious trade-off: enhancement of chlorination by increasing chlorine exposure by either increasing chlorine dosage or prolonging contact time, leads to unintended toxic consequences, including a greater yield of disinfection by-products (DBPs) and chlorinated transformation products. This dissertation provides a comprehensive assessment of using UV/chlorine as an emerging treatment to overcome this trade-off. Frequent and severe occurrences of harmful algal blooms (HABs) pose a risk to human health due to the production of algal toxins with high hepatotoxicity profiles, especially microcystins (MCs). In this study, the degradation and detoxification of the four most ubiquitous MCs with different amino acid (open full item for complete abstract)

Committee: Dionysios Dionysiou Ph.D. (Committee Chair); Heath Mash Ph.D. (Committee Member); Margaret Kupferle Ph.D. (Committee Member); Soryong Chae Ph.D. (Committee Member) Subjects: Environmental Engineering

Master of Science, The Ohio State University, 2023, Civil Engineering

Microcystins (MCs), a category of cyanotoxins produced by some species of the photosynthetic heterotrophic cyanobacteria, are the most studied, most associated with toxicity-related events, and most monitored in water. Water treatment plant (WTP) residuals are the solid byproduct of water treatment and consist of solids removed from physical and chemical processes and have been shown to contain cyanobacterial cells removed by conventional treatment processes. However, MCs in WTP residuals are not well understood. Although quantification methods of MCs for water samples have been adapted for solid matrices, their suitability for understanding the level of microcystins within a WTP residual sample is not well understood. Additionally, due to the inherent high variability within WTP residuals across the United States, these methods may not be optimal for every sample. Extraction and quantification methods were investigated to assess MCs in varied WTP residuals. Additionally, natural degradation observed in a utility's storage lagoon and was investigated to determine the impact of physical, chemical, and biological treatments on MC concentration in high-biomass residuals. This study demonstrates that residuals of various characteristics across the United States contain MCs, and that in some sample's extraction method (particularly methanol) and quantification method (particularly UPLC-PDA) increased measured MC, no single methodology is optimal for extracting and quantifying MCs across various residuals.

Committee: Natalie Hull (Advisor); Linda Weavers (Committee Member); John Lenhart (Committee Member) Subjects: Civil Engineering; Environmental Engineering; Environmental Science; Water Resource Management

Master of Science (MS), Bowling Green State University, 2022, Biological Sciences

Chautauqua Lake is a eutrophic lake located in western New York that is frequently impaired by cyanobacterial harmful algal blooms (cHABs). cHABs generally occur in systems with high nutrient concentrations, but factors including lake morphology and temperature also contribute to growth and development of cyanobacteria. cHABs often result in production of hepatotoxins (e.g., microcystins) and neurotoxins (e.g., saxitoxins) that are harmful to living organisms. This study focuses on examining the environmental drivers of toxin production in Chautauqua Lake through nutrient addition experiments to determine which conditions favor toxin production. Findings will help in lake management efforts, specifically by reducing toxin concentrations. This project consists of long-term nutrient diffusing substrata trays deployed at the benthos and short-term nutrient amendment bottle experiments. The trays were deployed in two-week to month long periods from July to October 2020, and the bottle experiments were performed in 2020 and 2021. It was found that nutrient limitation in Chautauqua Lake changes through the summer season corresponding to taxa present in the bloom. Overall, when Gloeotrichia was the dominating taxon in 2020, nutrient additions did not influence toxin concentrations, mcyE copies, or sxtA copies in the water column. In contrast, 2021 data demonstrated phosphorus (P) limitation. However, when Microcystis was the dominating taxon in the bloom, nitrogen (N) limitation was observed in the water column in both years. The benthos in 2020 exhibited changing nutrient limitation throughout August to October, where data suggest P may be limiting chlorophyll-a in August through September, and in September through October data suggests dual N and P limitation.

Committee: Christopher Ward Ph.D. (Advisor); Dianne Greenfield Ph.D. (Committee Member); George Bullerjahn Ph.D. (Committee Member) Subjects: Ecology; Environmental Science; Freshwater Ecology; Microbiology

Doctor of Philosophy, University of Toledo, 2021, Chemistry

Microcystins (MCs) are secondary metabolites generated by cyanobacteria, which can be present in drinking water sources during harmful algal blooms (HABs). MCs are potent liver toxins that inhibit the function of protein phosphatases 1 and 2A (PP1 and PP2A) by binding to the enzymes' active sites. Large MC doses lead to acute liver failure, but prolonged exposure to low levels of MCs may be more prevalent and pernicious. The effects of such exposure in humans are not well understood and are generally extrapolated from animal models. Further complicating their study, over 275 MC congeners have been discovered, though many remain unidentified. This dissertation advances MC research by describing novel extraction and data analysis methods coupled with liquid chromatography-mass spectrometry (LC-MS) for quantitative and qualitative analyses of MCs. A method was developed to extract and quantify MCs from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using LC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) and the MC-LR cysteine adduct (MC-LR-Cys), which can be created from MC-LR in vivo by the glutathione detoxification pathway, from spiked liver tissue. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 μg or 100 μg of MC-LR per kg body (open full item for complete abstract)

Committee: Dragan Isailovic (Advisor); Jon Kirchhoff (Committee Member); David Kennedy (Committee Member); Peter Andreana (Committee Member) Subjects: Analytical Chemistry; Biochemistry

Master of Science, University of Toledo, 2020, Chemistry

Microcystins (MCs) are widespread cyanotoxins present in water sources contaminated with cyanobacteria during harmful algal blooms (HABs), and are compounds of concern due to their hepatotoxicity. Despite the regulation of their allowed concentration in drinking water, humans could be exposed to MCs by water or food consumption, or during recreational activities in affected bodies of water. Therefore, the studies of mechanisms of MC activity, transportation and detoxification pathways are important for the better understanding of both their chronic and acute toxicity. Also, quick and reliable methods of water analysis are needed in order to determine the presence of the toxins and prevent human exposure. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a valuable technique for the visualization of analytes in biological tissues and other media. Herein, MALDI-MSI was applied to investigate the distribution of MC-LR and its metabolites in liver tissue sections obtained from mice exposed to MC-LR. The study was directed on the effect of the chronic MC-LR toxicity below the no observed adverse effect level (NOAEL) on control mice and mice used to model non-alcoholic fatty liver disease (NAFLD). Limits of detection (LOD) of MC-LR with MALDI-MS were determined for on-plate and on-tissue MALDI-MSI experiments. MALDI-MS imaging procedures were also optimized. Different variations of the matrix deposition and sample preparation were examined, and the most effective MALDI-MSI protocol was used for imaging of MC-LR in the liver tissue of the studied mice. MCs are produced within cyanobacterial cells, and their release into water could happen during cell death or water treatment procedures. Thus, cell lysis and MC release and extraction have to be studied and evaluated. At the same time, monitoring of the extracellular MCs in the water sources is an important task and should be rapid and reliable. In the second project, high-pe (open full item for complete abstract)

Committee: Dragan Isailovic (Committee Chair); Eric Findsen (Committee Member); Terry Bigioni (Committee Member) Subjects: Analytical Chemistry; Chemistry

Master of Science, The Ohio State University, 2020, Environmental Science

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 co (open full item for complete abstract)

Committee: Jiyoung Lee (Advisor); Nicholas Basta (Committee Member); Jay Martin (Committee Member) Subjects: Biology; Climate Change; Environmental Health; Environmental Science; Microbiology; Public Health

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Sandusky Bay and Lake Erie are plagued with harmful algal blooms every summer. Sandusky Bay is a drowned river mouth that is very shallow and turbid and is dominated by Planktothrix agardhii, while Lake Erie is dominated by Microcystis aeruginosa. Both species of cyanobacterium are non-diazotrophic and produce microcystin, a hepatotoxin. A competition experiment was conducted culturing both species alone and in coculture at nitrogen (nitrate) replete, nitrate restricted, and nitrogen-free environments. Planktothrix grew better alone at nitrogen restricted medium than in co-culture with Microcystis. In coculture, Microcystis was dominant over Planktothrix however, that dominance decreased as nitrogen was reduced in each treatment. In the nitrogen replete environment, the coculture produced significantly more toxin than the monocultures and in the no nitrogen environment the Planktothrix monoculture produced more toxin than the Microcystis monoculture or the coculture. The community composition in Sandusky Bay was monitored over the winter and spring months (January-April) to see how it changed as time progressed. Nutrient amendment experiments were also conducted adding nitrate, phosphate, and a combination of nitrate and phosphate to stimulate growth and identify any possible nutrient limitations. The initial community yielded low cell densities until the temperature increased and cell abundances followed shortly thereafter. Planktothrix dominated over the winter followed by a transitional period of cryptomonad and diatom dominations before transitioning back to Planktothrix. Both nitrate and phosphate were limiting Planktothrix growth in the spring, while nitrate alone was limiting the overall community.

Committee: George Bullerjahn PhD (Advisor); Timothy Davis PhD (Committee Member); Robert McKay PhD (Committee Member) Subjects: Biology; Environmental Science; Limnology; Microbiology

Master of Science, The Ohio State University, 2019, Public Health

Cyanobacteria present significant public health and engineering challenges due to their expansive growth and potential synthesis of microcystins in surface waters that are used as a drinking water source. Eutrophication of surface waters coupled with favorable climatic conditions can create ideal growth environments for these organisms to develop what is known as a cyanobacterial harmful algal bloom (cHAB). Development of methods to predict the presence and impact of microcystins in drinking water treatment systems is a complex process due to system uncertainties. This research developed two predictive models, first to estimate microcystin concentrations at a water treatment intake, second, to estimate the risks of finished water detections after treatment and resultant health effects to consumers. The first model uses qPCR data to adjust phycocyanin measurements to improve predictive linear regression relationships. Cyanobacterial 16S rRNA and mcy genes provide a quantitative means of measuring and detecting potentially toxic genera/speciess of a cHAB. Phycocyanin is a preferred predictive tool because it can be measured in real-time, but the drawback is that it cannot distinguish between toxic genera/speciess of a bloom. Therefore, it was hypothesized that genus specific ratios using qPCR data could be used to adjust phycocyanin measurements, making them more specific to the proportion of the bloom that is producing toxin. Data was obtained from a water treatment plant (WTP) intake at Tappan Lake, Ohio, a drinking water source for the Village of Cadiz. Using Pearson correlations and linear regressive analysis, it was found that adjusted phycocyanin, based on Planktothrix 16S and Planktothrix mcyE gene abundance ratios, exhibits improved correlation with microcystins. Furthermore, the analysis demonstrated the practicality of the adjustment in turning negative correlations between phycocyanin and microcystins to positive. More data from other water systems are need (open full item for complete abstract)

Committee: Mark Weir (Advisor); Jiyoung Lee (Committee Member); Allison MacKay (Committee Member) Subjects: Environmental Health; Public Health

Master of Science, University of Toledo, 2018, Chemical Engineering

Harmful algal blooms can release a variety of toxins. These include the myriad microcystin variants that are hepatotoxins of which microcystin-LR (MC-LR) is the most common. Conventional water treatment processes such as coagulation, flocculation and sedimentation are not effective at MC-LR removal. Potential techniques to remove MC-LR include activated carbon adsorption, ozonation, biosorption, nanofiltration, reverse osmosis (RO) and chlorination. The ability of RO to remove MC-LR has raised questions among vendors of home point-of-use systems for drinking water treatment as to the efficacy of removal. This study evaluates the performance of several commercial RO membranes. The water permeability and salt rejection of the membranes are characterized in addition to MC-LR rejection. All membranes remove MC-LR to non-detectable limits. Membranes are subject to accelerated aging by exposure to elevated chlorine concentrations for various times. Upon exposure to chlorine, the membranes either exhibit a continuous increase in hydraulic permeability or slight decrease followed by an increase. Salt rejection does not change in those membranes which show an initial hydraulic permeability decrease until the permeability starts to increase. AFM imaging indicates membrane surface roughness decreases upon chlorine exposure for the membranes that exhibit an initial permeability decrease. MC-LR rejection does not change with chlorine exposure for most membranes. Rejection remains nearly 100% despite dramatic increases in hydraulic permeability.

Committee: Glenn Lipscomb (Committee Chair); Youngwoo Seo (Committee Member); Maria Coleman (Committee Member) Subjects: Chemical Engineering

MS, University of Cincinnati, 2009, Engineering : Environmental Engineering

Cyanobacteria are prokaryotic microorganisms that are present in many environments. The presence of certain genera of cyanobacteria in aquatic systems is of great concern due to potential toxin formation and release. Cyanotoxins have been shown to have undesirable health impacts ranging from acute (skin irritation, gastrointestinal, and neurotoxic) to chronic (liver damage, kidney damage, and possible carcinogenic) effects. The toxins exist in different structural forms, such as alkaloids (anatoxin-a, saxitoxins, cylindrospermopsin) and cyclic peptides (microcystins, nodularin). Current knowledge and occurrence data of cyanotoxins have led to increased regulatory attention for limiting concentrations in water.The purpose of this paper is to review available literature and knowledge on the potential of drinking water treatment processes to remove cyanotoxins. The main mechanisms of contaminant removal in water are: physical (sedimentation, filtration, flotation, adsorption), chemical (oxidation), and biological. These processes may work singularly or in conjunction to accomplish reduction of the target compound. For cyanotoxins, removal efficiency is complicated by the fact that the toxins exist in two forms: intracellular and extracellular (dissolved). If contained within the cell, physical removal processes would likely be the best form of treatment. Once released from the cell, dissolved toxins may require additional treatment.Evaluation of the literature reviewed indicates that many common drinking water treatment processes are capable of removal or degradation of cyanotoxins. A key point when considering treatment for cyanotoxin removal is the form of the toxin (intracellular or extracellular) with respect to the treatment process being utilized. Physical removal of particulate cyanotoxins, either by sedimentation, floatation, or filtration, has been shown to be successful since cyanobacterial cells are generally well removed. However, sedimentation and filtrati (open full item for complete abstract)

Committee: Dionysios Dionysiou PhD (Committee Chair); Armah Del la Cruz PhD (Committee Member); George Sorial PhD (Committee Member) Subjects: Civil Engineering; Environmental Engineering