Indiana University-Purdue University Indianapolis (IUPUI) / This project examines the impact of confounding variables that have limited the accuracy of remotely predicting phycocyanin in three Indiana drinking and recreational water reservoirs. In-situ field reflectance spectra were collected from June to November 2006 over a wide range of algal bloom conditions using an ASD Fieldspec (UV/VNIR) spectroradiometer. Groundtruth samples were analyzed for chlorophyll a, phycocyanin, total suspended matter, and other water quality constituents. Previously published spectral algorithms for the detection of phycocyanin were evaluated against lab measured pigment concentrations using linear least squares regression. Algorithm performance varied across study sites (best performing models by reservoir resulted in r2 values of 0.32 to 0.84). Residuals of predicted versus measured pigment concentrations were analyzed against concentration of potential confounding water constituents. Residual analysis revealed optically active constituents contributed between 25% and 95% of original phycocyanin model errors. Inclusion of spectral variables into models to account for significant confounders resulted in improved spectral estimates of phycocyanin (r2 = 0.56 to 0.93).

Geosmin is a strong musty-flavored organic compound that is responsible for many taste-and-odor events in surface drinking water sources like lakes and reservoirs. The taste threshold of geosmin for humans is lower than 10 ng/L. Traditional treatment methods will not remove geosmin to this level. Additional water treatment methods must be implemented to successfully remove the geosmin and its associated flavor and odor from drinking water. Furthermore, geosmin is produced by cyanobacteria somewhat sporadically, so it is difficult to predict when taste-and-odor events are going to occur. The difficulty involved with predicting geosmin events has led most water treatment facilities to adopt reactive approaches towards geosmin treatment; these facilities typically treat for geosmin in response to complaints of an earthy off-flavor in the drinking water. This reactive approach causes issues with consumer confidence, as the flavor of the water is one of the only metrics a consumer has for judging the safety of his or her water. To enable proactive treatment of geosmin from water, more sensitive methods for geosmin detection or taste-and-odor event prediction must be developed.This study investigates the use of quantitative Polymerase Chain Reaction (qPCR) for the early detection of geosmin-producing cyanobacteria. qPCR can detect geosmin-producing cyanobacteria via their DNA. I developed a qPCR assay for this study that is capable of sensitively detecting multiple strains of the geosmin-producing Nostoc genus. The developed assay showed high sensitivity, demonstrating the possibility for its use in detecting low concentrations of geosmin-producing cyanobacteria before detectible levels of geosmin have been produced and released into the water. Through further sequencing of more geosmin-producing genera and species, the methodology outlined in this research could be applied to develop the tools necessary to predict taste-and-odor events caused by geosmin-producing cyanobacteria.

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Cyanobacterial algal blooms have been increasing in frequency and severity over the past few years in Ontario. Depending on the presence of toxigenic Cyanobacteria, these blooms have the potential to release toxins into the water, posing a public and environmental risk to humans and animals. Although traditional methods of studying Cyanobacteria provide important information regarding the microbial community, metagenomic sequencing allows for a more comprehensive examination of microbial diversity and functional capacities as limitations in cultivating organisms is circumvented. Therefore, to gain insight into the community composition of freshwater blooms and to compare them to non-bloom sites within Ontario, we collaborated with the Ministry of the Environment and Climate Change (MOECC) to undergo a high throughput DNA sequencing approach for a comparative metagenomic analysis. In 2015, 108 bloom and non-bloom samples were collected and sent for 16S rRNA sequencing and a subset of these were chosen for shotgun metagenomic sequencing. Our study focuses on comparing community structure and functional differences that may exist between bloom and non-bloom sites as well as analyzing differences in cyanobacterial communities across bloom sites. Our findings reveal differences in the microbial communities between these two environments. At the functional level, large-scale functionalities were conserved across the two groups but differences in specialized functions were revealed. Overall, our results show that metagenomics is a powerful tool for delineating functional and taxonomic analysis of bloom and non-bloom sites across Ontario. The second part of this work studied the utilization of the molecular marker, Conserved Signature Proteins (CSPs), as a valid method for identifying Cyanobacteria to facilitate the problem of cyanobacterial taxonomic classification. It was found that CSPs proved to be reliable in identifying Cyanobacteria within environmental samples when compared to amplicon and shotgun metagenomic sequencing approaches. / Thesis / Master of Science (MSc)

Phytoplankton populations are integral to the structure and function of aquatic ecosystems, and phytoplankton are an excellent study system for exploring ecological questions. Reservoirs often exhibit high horizontal (inflow to dam) and vertical (surface to sediments) environmental heterogeneity, which plays a large role in determining phytoplankton population dynamics. In this thesis, I explore how three bloom-forming phytoplankton taxa, the dinoflagellates Peridinium and Gymnodinium, and the cyanobacterium Planktothrix, respond to horizontal and vertical environmental gradients, respectively. First, I monitored recruitment, or the process of leaving the sediments and entering the pelagic life stage, of dinoflagellates across a horizontal reservoir ecosystem gradient. Surprisingly, coupling of dinoflagellate biology with reservoir physics and chemistry varied along this continuum; recruiting cells were sensitive to reservoir physics (e.g., flow rate, solar radiation) in the upstream riverine zone, while recruitment was related to reservoir chemistry (e.g., dissolved oxygen, nutrients) in the downstream lacustrine zone. This study indicates that upstream habitats should be monitored when studying reservoir phytoplankton dynamics. Next, I investigated the environmental drivers of the vertical distribution and biomass of a hypolimnetic cyanobacterial bloom over two consecutive summers. I collected high-resolution in situ phytoplankton data, and measured environmental variables throughout the water column. Across both years, the vertical distribution of this population was determined by light availability, while the cyanobacterial biomass was predicted by both light and nutrients. These two studies demonstrate that changing physics and chemistry across environmental gradients can regulate phytoplankton dynamics in reservoirs, and phytoplankton monitoring should include more spatially comprehensive sampling approaches. / Master of Science

Three strains of cyanobacteria (Anabaena PCC7120, A. variabilis and Nostoc commune), all belonging to the family Nostocaceae, were found to be capable of modulating the production and chemical composition of extracellular polysaccharides (EPS) in response to carbon and nitrogen availability as well as pH. While the carbohydrate compositions of the glycans produced by the different organisms were indicative of their recent evolutionary divergence, there were measurable differences that were dependent upon growth conditions. The EPS resulting from biofilm growth conditions was reduced in glucuronic acid levels in both Anabaena variabilis ATCC 29413 and Anabaena PCC 7120. Under planktonic conditions, the glycan from A. variabilis contained glucuronic acid when grown in nitrate-free BG-11₀ medium whereas A. PCC 7120 produced similar levels in standard BG-11 medium. This suggests that phylogeneticallyrelated cyanobacteria respond very differently to changes in their local environment. The pH of BG-11 cultures increased to 9-10 for all three strains of cyanobacteria. The increase resulted in an increase in the amount of dissolved inorganic carbon available in the medium, creating an imbalance in the carbon-nitrogen ratio, with the complete consumption of 17.65 mmol L⁻¹ nitrates raising the pH to near 10 in BG-11 medium. While increased carbon availability has been shown to induce capsulated morphologies in strains of cyanobacteria, only Nostoc commune DRH-1 exhibited this behavior, and only when grown in BG-11 medium. Carbon and nitrogen availability as well as pH modulate the monosaccharide composition of the glycan generated by cyanobacteria investigated. The different characteristics of the glycans produced can affect the survivability of the organisms and the community structure of cyanobacterial biofilms and microbial mats found in nature. As cyanobacteria are ubiquitous organism both now and in the past, they play a pivotal role in the biological and geological processes of the Earth, controlling the availability and cycling of carbon and nitrogen both actively and passively. / Master of Science in Life Sciences

The molecular biology of desiccation tolerance was investigated in the cyanobacteria with emphasis on Nostoc commune. Analysis of DNA from 41 samples of desiccated Nostoc spp. of varied age and global distribution led to the amplification of 43 independent tRNALEU(UAA) group 1 intron sequences. Phylogenetic analysis of the entire data set made it possible to define the form species Nostoc commune. The synthase (spsA) and phosphatase (sppA) genes required for the synthesis of sucrose were isolated from cyanobacterium Synechocystis sp. strain PCC 6803 and overexpressed in E. coli in two different vector constructions. Transformants had a marked increased capacity for desiccation tolerance. Sucrose synthesis was confirmed through thin layer chromatography (TLC) analysis of cell extracts from transformants. Long-term stability of DNA in desiccated Nostoc samples was demonstrated by the ability to amplify selected gene loci from samples stored dry for decades. Successful amplification in some samples was possible only after treatment with phenacylthiazolium bromide, a reagent that disrupts covalent cross-links; indicating that the DNA was modified by cross-links that occurred between reducing sugars and the primary amines on the DNA. Abundant superoxide dismutase was released following rehydration of desiccated field material N. commune CHEN after 13 years in the dry state. sodF mRNA was present in the dry material but was turned over within 15 min of rehydration. mRNA levels then rose and appeared to reach steady state levels after 3 hours and remained abundant after 24 hours of rehydration. / Master of Science