Search

Cyanobacterial harmful algal blooms are ubiquitous globally and can have enormous impacts on the environment, economy, and human health. These blooms have the potential to produce toxins such as microcystin (MC), cylindrospermopsin (CYN), and saxitoxin (STX). Currently, the standard method for toxin testing is by an Enzyme Linked Immunosorbent Assay (ELISA). This method is time consuming; LightDeck Diagnostics has developed a rapid and portable multiplexed assay to simultaneously test for MC, CYN, STX as an alternative to ELISA testing. During the summer of 2021, LightDeck’s duplexed MC-CYN assay was successfully field validated in Sandusky Bay. A method of portable lysis was also tested but is undergoing further development prior to being used in additional testing. During the summer of 2022, a triplexed version of the LightDeck cartridge (MC-CYN-STX) was field validated in both Sandusky Bay and Springbrook Lake in Whitehouse, Ohio. The STX assay successfully detected toxin, however the triplexed MC assay did not perform as well as the previous duplex formulation. Additionally, toxin concentrations were correlated with water quality parameters and nutrients in both lakes to investigate potential environmental drivers of toxicity during bloom events. The LightDeck assay was also used as part of a research cruise in the Winam Gulf, Kenya. Though ELISA remains the gold standard for toxin detection, the LightDeck assay could be a valuable tool in monitoring and reporting toxin in water bodies that people interact with, whether they be recreational lakes or water that the community relies on for daily functions as demonstrated in the case of the Winam Gulf.

The objective of this project was to determine how the mortality rate of a test organism,Ceriodaphnia dubia (C. dubia), may change with increased surface water temperature whenexposed to the toxin released by a cyanobacterial species, Microcystis aeruginosa, as well as acommon algaecide, hydrogen peroxide. In-vivo toxicity tests with C. dubia, an aquatic indicatorspecies, were performed with both the cyanobacterial lysate and algaecide at water temperaturesof 25°C and 27°C to better understand how temperature affects the toxicity of the algaecide andthe cell lysate as indicated by the mortality of the test organism. The LC50 for organisms at 25 and27°C exposed to hydrogen peroxide was 3.63 mg/L and 2.99 mg/L, respectively. The LC50 fororganisms at 25 and 27°C exposed to cell lysates was 1400 µg/L and greater than 1700 µg/L,respectively. These results indicate that an increase in surface water temperature may increase thetoxicity of algaecide to test organisms, but cell lysate toxicity may not change significantly. In theface of global warming and the concomitant increase in surface water temperature, thecombination of algaecide and more toxic harmful cyanobacterial blooms may cause adverse effectson aquatic ecosystems. Nevertheless, additional testing is needed to determine whether there is asignificant difference between temperature increase and cell lysate toxicity to test organisms.

Previous studies on harmful algae blooms (HABs) and cyanotoxin contamination in Lake Erie mainly focused on surface water bodies, and the knowledge of groundwater contamination caused by cyanotoxin migrated from HABs polluted lakes remains notably limited. Following a recent proof-of-concept, MODFLOW-based modeling study (Abesh et al., 2022), this study aimed to advance our understanding of such a contamination issue using a newer generation hydrologic model, i.e., HydroGeoSphere (HGS). Specifically, a 3-D HGS model was developed to simulate pumping-induced reverse groundwater flow from Lake Erie to coastal aquifers. The model domain, encompassing South Bass Island, its aquifer, and the surrounding lake, was discretized into 15 vertical layers, consisting of 7,543 2-D triangular elements each. The model was applied to explore how different hydrogeologic settings (including fracture configuration, lake levels, hydraulic conductivity, dispersivity, and reactions coefficients) would impact the transport processes. Simulation results show that fractures along longitudinal flow directions and in the forms of a connected matrix, high lake levels, high dispersion, low hydraulic conductivity, and low retardation factors would facilitate such movements. We estimated the arrival time of microcystin and identified pumping strategies for management and decision-making on drinking wells. Based on simulation results, wells closer to the lake with higher pumping rates would reach EPA's health advisory (HA) levels faster. For example, a water well 25 m away from the coast could exceed HA levels for infants-children (0.3 ug/l) and adults (1.6 ug/l) in about 47 and 115 days, respectively, if pumping at 200 m3/d. At the same rate but 50 m away from the lake, it would take 50 days to reach the infants-children level, but hard to reach the adult level. Pumpingivwells over 75 m away from the lake either took very long days or did not reach HA levels and hence seemed safe from microcystin contamination. However, unfavorable hydrogeologic conditions (e.g., fractured geology, increasing lake levels due to climate change, and higher microcystin concentrations caused by severe HABs events in the lake) could cause uncertainties in microcystin transport and threaten nearby wells. Therefore, we strongly recommend that drinking wells along the Lake Erie coast should be regularly monitored and tested for the presence of microcystin.

Two research areas in the field of biomedical engineering that have progressed significantly and garnered a great deal of interest in recent years are electroporation and biosensors. Electroporation has found wide usage in microbiology and cellular manipulation. Conventional or “bulk” electroporation (BEP) is the most commonly employed electroporation technique, and while it is easy to use and able to transfect a large cell population, it suffers a variety of drawbacks such as high voltage levels (resulting in low transfection efficiency due to cellular damage) and non-uniform biasing applied to the target cell population. 2D Micro-electroporation (MEP) and its successor 2D nano-electroporation (NEP) allow for control over delivery dosage and uniform biasing of target cell populations, but at low throughput. The first portion of this Ph. D. research uses Bosch etching process, which is optimized and characterized with respect to etch rate, etching parameters, and feature size to create a 3D NEP silicon platform that conserves the best attributes of both 2D NEP and BEP. Before device fabrication, a Bosch etching process is optimized and characterized with respect to etch rate, etching parameters, and feature size. The 3D NEP device is evaluated to ensure it possesses the “hallmark” benefits of NEP such as dosage control, good transfection uniformity, and high cell viability in a high throughput system. Simulations are performed and corresponding experiments are run to determine the ideal voltage for optimizing these parameters. As a result of this research the following have been achieved, (1) a tracked-etched membrane (which suffered from randomized nano-channel locations and thus poor throughput) is no longer the only available 3D NEP-style system. (2) The design and creation of this 3D NEP device allows the user to have dosage control, high cell viability, and uniform transfection for a large cell population. (3) A high-throughput 3D NEP system is now available for the transfection of delicate cell types that do not respond well to conventional electroporation or other cell transfection approaches. (4) While previous 2D NEP devices were limited to in vitro applications due to low throughout and the device layout itself, the 3D NEP chip’s design and high throughput capability allows it to be utilized for in vivo applications and by extension, open entirely new avenues for the usage of NEP. This includes, but is not limited to, the delivery of transcription factors into living organisms and the ability to control therapeutic dosages. Indeed, this has already been used for successful in vivo delivery of transcription factors to enhance wound healing in a murine model. Lastly, (5) the well-characterized Bosch etching process allows the devices to be easily replicated and produced by other audiences unfamiliar with the process. The methodology herein also allows straightforward tailoring of the recipe in the event of shifting machine conditions. The second part of this research investigates an aluminum gallium nitride/gallium nitride (AlGaN/GaN) heterostructure field-effect transistor (FET) platform for biosensing applications. Over the years, many semiconductor-based biosensors have been employed for the detection of a variety of analytes such as proteins, DNA, and bacteria. More recently, amidst growing concerns of algal blooms and by extension, their associated toxins in drinking water supplies, there is a critical need of sensors that are easy-to-use for rapid and sensitive detection of cyanobacterial toxins produced from harmful algae blooms (HABs). To address this, an AlGaN/GaN immunologically modified field effect transistor (immunoFET) is developed for the detection and quantification of algal toxins, specifically microcystin-LR (MC-LR) and saxitoxin. Herein, a four-parameter logistic model is used to relate the device output (normalized current) to microcystin-LR concentration, and subsequently, compare the accuracy, precision, and limit of detection to the `standard’ ELISA and other USEPA (United States Environmental Protection Agency). As with any new technique, it is prudent to both compare and validate it against accepted existing methodologies. The final aim is to create a “point-of-care” (POC) immunoFET system that is able to quantify toxin concentrations in a given aqueous sample in real-time without laboratory processing. Many sensors reported in the literature have an array of desirable characteristics, such as high sensitivity, fast response time, excellent precision, and point-of-care diagnostic capabilities, but few reported devices excel in all four areas. In addition, even fewer show the ability to detect multiple toxins with high sensitivity in real-time within a variety of sample solutions. As on-site detection of cyanotoxins requires the sensor to operate in a variety of aqueous samples, 1/f noise investigations are performed to determine the effects of ionic strength on device signal-to-noise ratio. The following are determined to serve as guide both for this research and for other individuals performing biosensing measurements. (1) The sub-threshold regime is compared to the saturation regime with respect to signal-to-noise ratio (SNR) and it is determined that the sub-threshold regime has higher SNR by approximately two orders for these devices. (2) When dealing with ionic strengths above ~9×1014/ml, the characteristic frequency exponent β begins increasing from 1 to 2 with increasing ion concentration if the gate voltage is sufficiently high (|Vg| > 1). Thus, when performing biosensing measurements, it would be prudent to keep ionic strengths and/or gate biases low if possible. These findings allow for the optimization of measurements conditions and combined with the fabricated AlGaN/GaN FET sensors, results in a cyanotoxin biosensor platform with the following benefits – (a) label-free detection, (b) real-time measurement results within 12-13 minutes, (c) a limit of detection (LOD) determined to be 153 fg/L for microcystin-LR, (d) validated against ELISA, and (e) CV (coefficient of variation) values ranging from 2.53% to 12.12% with only one measurement condition (17.25%) exceeding the USEPA 15% threshold. For saxitoxin, a normalize device response of 0.3 is observed for 1 pg/L saxitoxin in phosphate buffered saline (PBS) These attributes make these sensors comparable to the best reported devices in the literature and the only devices (to our knowledge) shown to successfully detect two types of cyanobacterial toxins (microcystin-LR and saxitoxin) in multiple sample solutions (water, PBS, and murine serum). While sensitivity is reduced for detection in murine serum for both toxins, the devices show successful response down to 1 ng/L concentrations. This concentration is lower than ELISAs LOD and makes these devices suitable for cyanobacterial toxin detection for clinical applications when toxin exposure is suspected. Lastly, the devices are made to be compatible with a microfluidic, portable platform prototype so that with further optimization, these sensors could offer unprecedented diagnostic capabilities for the detection of cyanobacterial toxins on-site with similar accuracy and significantly higher sensitivity compared to ELISA.

Cyanobacteria harmful algal blooms (Cyano-HABs) are increasing in frequency and cyanotoxins have become an environmental and public concern in the U.S. and worldwide. In this dissertation, a thorough literature search was undertaken, and the state of knowledge is presented. The majority of reported studies and developments of electrochemical affinity biosensors for cyanotoxins are critically reviewed and discussed. Essential background information about electrochemical biosensors is combined with the rapidly moving development of electrochemical biosensors for these toxins. Current issues and future challenges for the development of useful electrochemical biosensors for cyanotoxin detection that meet the demands for applications in field freshwater samples are discussed.Electrochemical impedance spectroscopy (EIS), an extremely sensitive analytical technique, is a widely used signal transduction method for the electrochemical detection of target analytes in a broad range of applications. The use of nucleic acids (aptamers) for sequence-specific or molecular detection in electrochemical biosensor development has been extensive, and the field continues to grow. While nucleic-acid based sensors using EIS offer exceptional sensitivity, signal fidelity is often linked to the physical and chemical properties of the electrode-solution interface. The goal of this study is to improve our understanding of the effect of multiple factors on EIS signal response and to optimize the experimental conditions for development of sensitive and reproducible sensors. The presented data in this dissertation demonstrate a need for rigorous control experiments to ensure that the measured change in impedance is unequivocally a result of a specific interaction between the target analyte and nucleic recognition element.We demonstrate the successful development of an electrochemical aptamer-based sensor for point-of-use detection and quantification of the highly potent microcystin-LR (MC-LR) in water. The sensor uses hexaammineruthenium(III) chloride ([Ru(NH3)6]3+) as redox mediator, because of the ability of the positively charged (3+) molecule to associate with the phosphate backbone of nucleic acids. We quantitatively measure the target-induced displacement of aptamer associated, or surface confined, [Ru(NH3)6]3+ in the presence of MC-LR. Upon the addition of MC-LR in the water, surface-confined [Ru(NH3)6]3+ dissociates resulting in less faradaic current from the reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+. As a result, square wave voltammetry (SWV) recorded substantial signal suppression within 15 min of target incubation. The sensor showed a calculated limit of detection (LOD) of 9.2 pM in water. Also, the sensor performed well in drinking water samples. These results indicate a sensor with potential for fast and specific quantitative determination of MC-LR in drinking water samples.

Our surface water drinking water sources are increasingly hosting “harmful algal blooms” of cyanotoxin-producing cyanobacteria, which presents a challenge to public water systems. The most common toxin class, microcystins, are liver toxins which can cause a range of symptoms from abdominal pain and headaches to internal bleeding and organ failure. Conventional treatment (coagulation, flocculation, sedimentation, and filtration) can remove cyanobacteria, but does not address extracellular toxin. Following two instances of `do not drink’ advisories in Ohio in 2013 and 2014, the Ohio EPA requires public water systems to develop and submit treatment optimization protocols when microcystins are detected in raw water above 1.6 µg/L. Water utilities and regulatory agencies are, therefore, seeking effective and implementable treatments to optimize cyanotoxin removal and destruction.Although many advanced processes are being explored, utilities without the ability to make large capital investments are focusing on optimizing existing adsorption processes. Granular activated carbon (GAC) is an increasingly utilized unit process, often installed to meet disinfection byproduct regulations, which can also reduce the extracellular toxin concentration to below health affect levels. Most quantifications of the capacity and kinetics of MC-LR onto activated carbon were not conducted under matrix conditions or experimental designs that are most relevant to GAC contactors. These contactors differ from PAC applications in that they are typically installed after conventional coagulation/flocculation/sedimentation processes at different pH and natural organic matter (NOM) conditions and operate under different kinetics. Therefore, this study specifically examines the congener microcystin-LR (MC-LR) and developed equilibrium isotherms and rapid small scale column test (RSSCT) breakthrough curves for a variety of GAC products under different matrix conditions. Equilibrium isotherms were developed for six different GAC products from four different carbon source materials (hardwood, lignite, bituminous coal and coconut) both with and without the presence of NOM. Experiments were conducted in model water with an initial concentration of 50 ug/L of MC-LR and 5mg/L of Suwanee River Fulvic Acid. Further, a series of proportional diffusivity rapid small scale column tests (PD-RSSCTs) were designed to compare MC-LR and NOM breakthrough for three different GAC source materials (bituminous coal, lignite and hardwood) and at a pH range of relevance to GAC unit processes (pH = 7.8 and 9.5). PD-RSSCT were conducted in a 0.38cm inner diameter column with GAC bed depths packed to 1.5 cm. Model water with ~100ug/L of MC-LR and 5mg/L of Suwanee River Fulvic Acid was fed at ~0.32mL/min to generate a full scale empty bed contact time of 15minutes. From the results it appears that material type does have an effect on the adsorption capacity; however, other factors such as activation method likely also have an influence. The addition of 5mg/L Suwannee River Fulvic Acid as competitive NOM decreased the maximum adsorption capacity for the GAC products tested as expected. MC-LR breakthrough exceeded the Ohio EPA action level of 0.3 µg/L MC-LR for bituminous coal, lignite and hardwood GAC in 1000, 9700, and 9800 treated bed volumes respectively, and the MC-LR breakthrough curve was most gradual for the wood based GAC and steepest for the bituminous coal based GAC. The varying pH trial was conducted on the bituminous product only and the volume treated prior to breakthrough for MC-LR was similar at the 1.6 µg/L MC-LR Ohio EPA action level (1600 to 1900 bed volumes). However, the slope of the pH 9.5 curve was more gradual than the pH 7.8 curve likely due to decreased interaction between the GAC and competitive NOM. Any interpretation of these reported full scale breakthrough times must consider that this study was conducted at an unrealistically high influent MC-LR concentration (100µg/L) in order to obtain quantifiable data. These findings indicate that the most commonly used GAC media in Ohio (bituminous coal) is not as effective as other source material carbons for removal of MC-LR and that some lignite products may be comparable with wood. However, this cannot be extrapolated to its suitability for other contaminants of concern such as MIB, geosmin, disinfection byproduct precursors or other organic molecules, and further, there are operational reasons for public utilities to choose a particular manufacturer.

Blue-green algae, also known as cyanobacteria, are photosynthetic bacteria found naturally in marine, freshwater, and estuarine ecosystems. An increase in nutrient input and changes in the climate have contributed to the proliferation of cyanobacteria, forming harmful algal blooms, or HABs. In addition to the ecosystem effects, loss of recreational lake use, and economic impact, a serious issue associated with these blooms is the production and release of cyanotoxins. While some factors that influence the proliferation of HABs have been identified, how these factors affect blooms and the resulting production of toxins are still unknown. In order to investigate the environmental factors and nutrient concentrations that influenced the changes of total Microcystin (MC) in an inland reservoir, high frequency environmental data and discrete water sampling were compared to toxin analysis for the 2015 and 2016 sampling season at William H. Harsha Lake in Clermont County, Ohio. For toxin determination, Enzyme-Linked Immunosorbent Assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used. In-vitro spectrophotometric analysis was used alongside in-vivo relative fluorescence for pigment analysis. Nutrients were determined by Lachat Quikchem. Weather and water quality observations were recorded by a weather station and YSI sonde. Methods for toxin determination and pigment analysis were compared. Correlations between environmental parameters and MC concentrations were also investigated. Data suggested that multiple methods of chlorophyll-a analysis do not provide comparable results. In-vivo readings did not show a significant correlation with toxins, while in-vitro analysis did. The collection effort and processing time necessary for in-vitro analysis does not provide the immediate knowledge that is necessary for decision making and management actions. The use of real-time pigments such as phycocyanin provides a real-time alternative for Harsha Lake. Results also showed that MC concentrations were significantly correlated to parameters such as in-vivo phycocyanin (Lr=0.455, p=0.0003), in-vitro chlorophyll-a (Lr=0.481, p

Cyanobacteria are ubiquitous in aquatic ecosystems across the earth. In many environments they are present at low abundances, however under certain environmental conditions cyanobacteria bloom and become one of the dominant organisms in an waterbody, degrading aquatic food webs and water quality. Cyanobacteria evolved over 2 billion years ago, and cyanobacterial harmful algal blooms (cyanoHABs) have been documented for decades. Of particular concern is the production of cyanotoxins, secondary metabolites toxic to humans and other organisms, by certain strains of cyanobacteria. Most research of cyanoHABs has been of planktonic blooms in lakes or estuaries, and cyanotoxin production by benthic cyanobacteria in rivers has been more recent, but in many rivers benthic cyanobacteria are the primary source of cyanotoxins. With field surveys and monitoring, manipulative field experiments, and genome-resolved metagenomics, this dissertation investigated the ecology of benthic cyanobacteria in the Eel River, California.Like most coastal rivers in Northern California, the Eel River is in a Mediterranean climate. During the seasonal summer drought rivers become shallow, slow flowing, and warm, excellent habitat for benthic algal production. Non-toxic benthic algae are consumed by vertebrate and invertebrate grazers and are foundational to the aquatic summer food web. However, these benthic algal assemblages can tip towards toxicity when cyanobacteria begin to dominate the assemblage. Over a dozen dogs have died in the Eel River since the year 2000 due to ingesting cyanobacteria. Neighboring watersheds have also experienced dog deaths from cyanotoxin poisoning, and benthic cyanobacterial mats are an increasing public health concern in multiple Northern California rivers. Prior to this dissertation there were few data on the distribution and ecology of benthic toxigenic cyanobacteria in California rivers. During the summers of 2013-2015, I documented spatial and temporal patterns of cyanobacterial occurrence and cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a produced by benthic cyanobacteria. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 cyanobacterial mat samples were collected from 31 sites to measure intracellular anatoxin-a and microcystins. Anatoxin-a levels were higher than microcystin for both SPATT and cyanobacterial mat samples. Species of benthic Anabaena and Phormidium were frequently found to produce cyanotoxins in the Eel watershed. Of the benthic mats sampled, 50% had detectable anatoxin-a (mean µg g-1 DW= 1.71, max= 70.93), while 24% had detectable microcystins (mean µg g-1 DW= 0.067, max= 2.29). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence and anatoxin-a production by benthic cyanobacterial mats in a North American watershed.Field experiments were used to study the buoyancy of benthic Anabaena spp. mats to understand implications for Anabaena dispersal. Experiments addressed oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4 days, while in low light mats begin to sink in

Eutrophication, a process that occurs due to excessive accumulation of nutrients such as nitrogen and phosphorous is primarily from anthropogenic nitrogen and natural occurrences. This phenomenon causes cyanobacterial overgrowth which can potentially lead to toxic algal blooms that affect public drinking water sources and recreational usage. An immediate need to detect bloom forming cyanobacteria in freshwater bodies early hand is critical to implement prevention strategies. These microorganisms contain phycobiliproteins such as phycoerytrhin, and allophycocyanin as part of the phycobillisome that allow autofluorescence. In this study, 36 freshwater bodies from 14 New Jersey counties were collected and processed for flow cytometric analysis for forward- scatter, phcyoerthrin and allophycocyanin parameters. Pure cultures of Synechococcus sp. IU 625 (S. IU 625), Cylindrospermum spp. and Microcystis aeruginosa (M. aeruginosa) were utilized as references. Polymerase chain reaction (PCR)-based assay was performed for the validation of the flow cytometric analysis. The results revealed 17 out of the 36 sites showed all three reference species and their related. 6 waterbodies showed Cylindrospermum like species, 7 waterbodies showed Microcystis and Cylindrospermum like species, 4 waterbodies showed Microcystis and Cylindrospermum like species and 2 sites showed Microcystis like species. PCR results validated these results by showing positive results for phytoplankton, cyanobacteria and Synechococcus. Flow cytometry has high potential for the rapid detection of cyanobacteria in live form due to their autofluorescence properties from the phycobilisome.

Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored.

The cyanotoxin β-methylamino-L-alanine (BMAA) has long been associated with the elevated incidence of amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). BMAA has been shown to be produced across the cyanobacterial order and its detection has been reported in a variety of aquatic and terrestrial environments worldwide, suggesting that it is ubiquitous. Various in vivo studies on rats, mice, chicks, monkeys and zebrafish have shown that BMAA can cause neurodegenerative symptoms such as ataxia and convulsions, as well as disruption to neural development. Previous in vitro studies using mice, rats and leeches have shown that BMAA acts predominantly on motor neurons. Observed increases in the generation of reactive oxygen species (ROS) and Ca2+ influx, coupled with disruption to mitochondrial activity and general neuronal death, indicate that the main mode of activity is via excitotoxic mechanisms. Data presented here indicate that BMAA induces increased intracellular Ca2+ influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of ROS in human neurons. The amelioration of LDH release in the presence of the N methyl-D-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Immunocytochemical stains show that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors. Rat olfactory ensheathing cells (OECs) were also challenged with exogenous BMAA resulting in elevated cell death. Significant increases in Ca2+ influx, enhanced production of ROS, and disruption to mitochondrial activity were observed in OECs. This is the first study investigating BMAA toxicity using pure glial cells. These findings align BMAA with all proposed mechanisms of degeneration in ALS, those being non-cell autonomous death, excitotoxicity and mitochondrial dysfunction. Additionally, novel genes and proteins have been identified that were differentially expressed in BMAA treated OECs. Some of the identified genes/proteins of interest have previously been linked to Alzheimer's disease. Other genes/proteins identified have reported functions in the regulation of mitochondrial activity and cell cycle and apoptosis. Cell cycle analysis of OECs treated with BMAA resulted in cell cycle arrest at the G2/M phase.

Inland lakes are prone to contamination from a variety of sources within their watersheds. The changing environment can influence transport and fate of fecal indicators and may also influence the growth of harmful cyanobacteria, thereby occasionally creating health-related water quality concerns for recreational water users. To date, epidemiological and limnological studies pertaining to fecal indicators and harmful cyanobacteria have been limited with respect to inland U.S. lakes. The primary goals of this dissertation were to (1) evaluate illness risks associated with the fecal indicator E. coli, and (2) evaluate predictive tools potentially useful for the rapid prediction of E. coli densities and health-related concentrations of cyanotoxins in inland Ohio lakes. Through an epidemiological study and the collection of water quality data, predictive models for human illness and water quality advisories were developed. The relationship between water quality indicators and reported adverse health outcomes among users an inland Ohio beach were examined. Human health data collected via a prospective cohort study over 26 swimming days during the 2009 swimming season at East Fork Lake demonstrated that wading, playing or swimming in the water was found to be a significant risk factor for GI illness (adjusted odds ratio (aOR) of 3.2; CI = 1.1, 9.0). Among water users (n = 806), E. coli density was associated with elevated GI illness risk where the highest E. coli quartile was associated with an aOR of 7.0 (CI = 1.5, 32). Upon observing a significant illness association with E. coli densities among swimmers, the need for rapidly estimating E. coli densities was determined to have merit. Current approaches for quantifying E. coli densities rely on culture-based methods that require 18 or more hours to obtain a result. Using rapidly measured water quality parameters (e.g., total phosphorus, secchi depth, chlorophyll A), univariable models for rapidly estimating health-related E. coli densities were developed and considered for inland Ohio lakes using 182 beach water samples collected from seven Ohio lakes. Univariable logistic regression revealed that deviations in lake-specific water quality as measured by total phosphorus (p < 0.001), phycocyanin pigment (p = 0.018), and trophic state index (TSI) (p = 0.006) were predictive of E. coli levels exceeding recreational water quality criteria. Using the same samples, models were constructed for estimating cyanotoxin concentrations. Microcystin levels exceeding the 4 micrograms/L low risk threshold set by the World Health Organization were detected by ELISA in 48 of 182 (26.4%) samples. A multivariable logistic regression model using practical and real-time measures of in vivo phycocyanin and secchi depth was constructed to predict beach conditions exceeding the low risk threshold for microcystin. The model (p = 0.030) predicted microcystin levels >4 micrograms/L with acceptable discrimination as indicated by the area under the ROC curve (0.795). This study indicates a significant health risk for inland beach users and demonstrates the potential to predict health-related hazard levels using practical real-time measures are possible, enabling opportunities for interventions that protect public health.

Cyanobacterial blooms are of great concern to the drinking water treatment industry due to their capacity to produce microcystins and other cyanotoxins that are deadly to humans, livestock, pets, and aquatic life at low doses. Unfortunately, the strategies currently employed for cyanotoxin detection involve laborious analyses requiring significant expertise or bioassay kits that are subject to numerous false positives and negatives. These methods are incapable of providing rapid, inexpensive, and robust information to differentiate between the >80 cyanotoxin variants potentially present in an aqueous sample. The use of Raman spectroscopy for identification and quantification of the ubiquitous cyanotoxin microcystin-LR (MC-LR) was examined. Raman spectra readily reflect minute changes in molecular structure, spectra can be collected through water or glass, portable Raman spectrometers are increasingly available, and through surface enhanced Raman spectroscopy (SERS) it is possible to achieve femto or picomolar detection limits for a variety of target species. Drop coating deposition Raman (DCDR) was successfully implemented for quantitation of 2-100 ng of MC-LR deposited in 2 ?L of aqueous sample, even without the use of a specifically designed DCDR substrate or Raman signal enhancements. Reproducible MC-LR Raman spectra were observed for both fresh and aged DCDR samples, and the MC-LR Raman spectrum remained identifiable through a matrix of >80% DOM by mass. DCDR methods show tremendous potential for the rapid, simple, and economical detection of cyanotoxins in environmental matricies at environmentally relevant concentrations.

Toxin synthesis in bloom-forming cyanobacterial communities is a dynamic process that responds to changes in the environment, such as light, nutrient availability and oxidative stress. Our limited understanding of the factors that drive bloom formation, and the increased incidence of cyanobacterial blooms worldwide present a significant challenge to the water industry. The production of the hepatotoxic non-ribosomal peptide microcystin has been proposed to be advantageous to toxic cells and appears to be regulated by nitrogen- and iron-stress responsive proteins at the transcriptional level. In this study, protein expression in the unicellular microcystin-producing Microcystis aeruginosa species was investigated in nutrient-replete and nutrient-stress conditions. The proteome of M. aeruginosa was highly strain-specific, with protein expression changes accumulating during prolonged growth in laboratory culture. Microcystin synthesis was found to be a dynamic trait in toxigenic, but inactive M. aeruginosa strains, and the reversal of one such previously non-toxic strain to toxicity was reported. Transcription of the mcyA gene involved in microcystin synthesis was also identified in both toxic and non-toxic strains and suggests that regulation of toxin synthesis occurs at the post-transcriptional level. In nutrient-replete conditions proteins differentially displayed between toxic and nontoxic M. aeruginosa were targets of the global nitrogen control transcriptional regulator NtcA and were involved in carbon fixation, nitrogen metabolism and redox balance. The iron starvation response in M. aeruginosa appeared to result from differential transcription of ferric uptake regulators (Fur), which affected processes involved in iron uptake and modification of the photosynthetic machinery. Iron, but not nitrogen stress, induced microcystin synthesis and prevented chlorosis in the model toxic PCC 7806 strain. In nitrogen-limited growth, the protein expression profiles of M. aeruginosa supported an active involvement of NtcA in the stress response of non-toxic strains. These results suggest that NtcA metabolic control is affected by microcystin synthesis and the nutrient status of the cells, leading to differences in the C:N ratio in toxic and non-toxic cells. This could underpin the differential response of strains of M. aeruginosa to changes in the environment. Further global expression studies of hepatotoxic cyanobacteria are required before reliable bloom control protocols can be established.

Cyanotoxins, the group of toxic chemicals produced by the blue-green algae or cyanobacteria that can proliferate in fresh and salt-water, cause a range of harmful health effects including skin rashes, flu-like symptoms, nausea, diarrhea, tingling and nerve damage, liver damage, tumors, and death ([1, 2]). Although cyanobacteria are one of the oldest organisms on the planet, anthropogenic development (e.g. dams, water diversions, nutrient rich runoff from intensive agriculture, decreased impervious area from urbanization, etc.) has caused many watersheds to lose substantial water volume, suffer tremendous inputs of nutrients and other organic and inorganic pollutants, increase in temperature and overall become more suitable for the proliferation of harmful blooms of cyanobacteria. Cyanobacteria blooms are now increasingly prevalent in freshwaters as eutrophication becomes ever more common with human stressors, and climate change is likely to only further exacerbate this problem. Public health professionals are dependant upon early and dependable information on the presence, concentration, and location of cyanobacteria blooms in order to inform the public and reduce potential exposure. This research project evaluated the efficacy of remote sensing data to provide this kind of surveillance and early detection for characterizing the presence of toxic algae in freshwater systems. It explored the relevance of specific remote sensing techniques to freshwater cyanobacteria bloom identification. The various remote sensing platforms available for this kind of research vary in cost, swath coverage, spatial scale and spectral resolution. For this study, three different remote imagery platforms were compared in terms of their ability to identify surface blooms and to distinguish gradients in cell density or bloom intensity. This exploration of the application of remote sensing used a hyperspectral airborne sensor with high spatial resolution (SpecTIR), a multispectral satellite image also with high spatial resolution (IKONOS), and a lower spatial resolution multispectral satellite image (Landsat). Water sampling data (algal pigment concentrations, turbidity, transparency, and temperature) from known blue-green algae blooms dominated by microcystis aeruginosa on the Iron Gate and Copco Reservoirs on the Klamath River were used to evaluate and classify these different images.This research successfully used both satellite and airborne remotely sensed data to visualize where the medium or high density sections of the bloom were located, to quantify the intensity of the bloom in terms of area impacted, and to compare the intensity of the bloom at different dates in time. Remote sensing can provide a synoptic overview of the entire system, making it possible to truly assess relative bloom intensity. Furthermore the results indicate that when given the choice, the investment in higher spectral resolution should be chosen over higher spatial resolution as the former appears to provide more benefits in cyanobacteria detection.