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Picoeukaryotes are protist 3 µm belonging to a wide diversity of taxonomic groups, and they are an important constituent of the ocean microbiota, performing essential ecological roles in marine trophic chains and in nutrient and carbon budgets. Despite this, the true extent of their diversity is currently unknown, and in the last decade molecular surveys have uncovered a substantial number of previously unknown groups from all taxonomic levels. No studies on this group have been done so far on the Red Sea, a unique marine environment characterized by oligotrophic conditions and high irradiance, salinity and water temperature. We sampled the surface waters of a site near the northern Red Sea coast, and analyzed the picoeukaryotic diversity through the construction of PCR clone libraries using the 18S ribosomal gene. The community captured by our library is dominated by three main groups, the alveolates (32%), chlorophytes (32%) and Stramenopiles (20.55%). Members of Radiolaria, Cercozoans and Haptophyta were also found, although in low abundances. Photosynthetic organisms are especially diverse and abundant in the sample, with heterotrophic organism mostly composed by the mostly parasitic novel alveolates and bacterivorous stramenopiles. Novel clades were detected among the Novel Alveolates- II and the photosynthetic stramenopiles taxa, which suggests that they may be part of a number of groups unique to the basin and adapted to the high salinity and temperature conditions. This is the first study done on the Red Sea focusing on the diversity of the complete picoeukaryotic fraction, and provides a stepping stone in the characterization of the picoeukaryotic component of the microbial diversity of the basin.

Reclaimed roads on the Little Missouri National Grasslands of southwestern North Dakota have not returned to pre-disturbance conditions. Phospholipid fatty acid analysis was performed on soil samples collected from reclaimed roads and adjacent prairie to assess reclamation effects on the microbial community. Additionally, nutrient cycling capacity was measured by four enzyme assays. Ordination analysis of PLFA data identified a distance gradient indicating microbial communities of reclaimed roads were different from the prairie. Specifically, Gram-negative bacteria and arbuscular mycorrhizal fungi are associated with roads; soil organic matter was associated with prairie sites. Soil enzyme activities associated with prairie sites indicate greater nutrient cycling. The soils of reclaimed roads have not accumulated sufficient organic matter to sustain both plant and microbial communities characteristic of the surrounding prairie.

Microbes exist in unfathomably diverse, dynamic and intricately structured ecosystems. However, we lack the tools to fully capture the complexity of these microbiomes, which in turn limits our ability to understand their ecology and function. Here, I address these shortcomings by developing new high-throughput measurement tools to characterize microbiomes across functionally distinct axes. First, from a synthetic biology perspective, I leverage the bacterial CRISPR-Cas immune system to enable a new class of population-wide passive recording devices in cells for capture temporally varying signals and horizontally transferred DNA sequences. Second, in the microbiome arena, I develop a new suite of tools (experimental and theoretical) to capture and analyze the spatiotemporal dynamics of microbiomes at macroscopic and microscopic length scales. Taken together, these measurements provide deep insights into the ecology of complex microbiomes, and constitute a suite of powerful new tools to study microbes in their native context.

Traditionally, the analysis of soil health has overlooked the biological component of soil due to poor understanding of connections between the microbiome and empirically measured soil health indicators. The purpose of this study was to assess the effects of environmental and anthropogenic stressors on the soil microbiome, with the aim of identifying measurable soil biological indicators. Chosen soils were examined under distinct conditions to evaluate the effect of selected environmental and anthropogenic stressors on the microbiome. Soil biological responses were analyzed via enzymatic response, microbial functional genes, and microbial community. Environmental factors such as soil moisture and organic matter showed significant influence on the microbiome with each selected biological indicator showing importance. Anthropogenic factors provided various responses dependent largely on the nature of the soil amendment. This study demonstrates that in addition to traditional soil health indicators, soil biological indicators should be included in the process of determining healthy soils.

Since the work of John Snow, scientists and medical professionals have understood that individuals develop cholera by means of consuming contaminated water. Despite the knowledge of cholera's route of infection, many countries have experienced and still experience endemic cholera. Cholera is caused by the Vibrio cholerae (V. cholerae) bacterium and presents with acute diarrhea and vomiting. If untreated, infected individuals may die due to dehydration. Cholera is a disease that most commonly affects countries with poor infrastructure and water sanitation. Despite efforts to control cholera in such countries, the disease persists. One such example is Haiti which has been experiencing a cholera outbreak since 2010. While there has been much research in the field of microbiology to understand V. cholerae, there has been comparably less research in the field of mathematical biology to understand the dynamics of V. cholerae in the environment. A mathematical model of V. cholerae incorporating a phage population is coupled with a SIRS disease model to examine the impact of vibrio and phage interaction. It is shown that there might exist two endemic equilibria, besides the disease free equilibrium: one in which phage persist in the environment and one in which the phage fail to persist. Existence and stability of these equilibria are established. Disease control strategies based on vibrio and phage interactions are discussed.

Metagenomics is a culture-independent framework for deciphering the complexity of biological communities, often with a focus on microbial communities in a specific environment. The applicability of this approach is widespread due to the ubiquity and presence of unculturable microbes in many environments which can only be investigated using culture-independent methods. With advances in DNA sequencing and the introduction of high-throughput sequencing technologies, studying microbial life as communities has become more accessible. However, the breadth of data generated dictates that computational processing steps must be in place to analyze the data. Due to the large diversity in computational and bioinformatic steps possible for metagenomic data, differences in methods of analysis can lead to discordant interpretations of results. The performance of different metagenomics methods must therefore be assessed to establish the effect on the interpretation of results. Taxonomic classification is an integral step in metagenomic analysis and many tools exist for this purpose. To determine which tools are better suited for particular types of metagenomic data, a comparative analysis of performance was conducted for numerous tools. The findings suggest that hybrid programs may have the best performance and warrant further investigation. Programs such as CLARK, KRAKEN, and MEGAN also performed well and are suitable for metagenomic analysis. Utilizing these methods, investigation into the bacterial populations of four freshwater beaches was examined. Bacterial communities in beach waters and sands were more distinct in terms of taxonomic composition than communities in different lakes. Functional capacity was stable between beach habitats, although enrichment of anaerobic and stress related genes in the sand suggests that this is a relatively harsh environment. The detection of sequences belonging to pathogens in the sands of these beaches also has implications for public health and warrants changes in water quality monitoring procedures. / Thesis / Master of Science (MSc)

The microbial world is a rich tapestry of communities experienced at the micron scale, but often studied at the macro scale. The methods commonly used to study microbial communities are unable to capture this heterogeneity, limiting insight into the processes that create them. High resolution methods have been applied to the human gut, but not at sufficient scale to sufficiently characterize the spatial structure of the community. Here, we examine the methods we use to study microbial communities, their limitations, and present SAMPL-seq, a method that allows for micron-scale interrogation of human and murine gut communities. In this we discover novel and consistent colocalization of microbial taxa across people. Further, we use many of the methods discussed to explore spatial heterogeneity of the microbes of hospital rooms and fusobacterial strains in the gut, revealing before unseen natural diversity.

<p>Microbes are ubiquitous in the biosphere and play important roles in natural ecosystems. They are typically present as diverse, complex communities, and in humans these communities are present on all exposed surfaces and mucosal tissues. The human upper respiratory tract harbors a complex microbiome and the composition includes what are traditionally considered commensal organisms, including a significant proportion of anaerobic bacteria. It is generally assumed that most of the bacteria from any particular environment cannot be readily cultivated, including the human microbiome. Some<em> in vitro </em>microfluidic and <em>in vivo </em>models are available to study the airway microbial communities, however these methods are expensive, limited and are not practical for experiments manipulating the community. A robust culture-based approach that can propagate these polymicrobial communities has been developed in this study to investigate spatial-temporal changes in bacterial populations <em>in vitro</em>. Matrix embedded synthetic bacterial communities, comprised of aerobes and anaerobes, were cultivated in continuous flow cell systems. The structure of communities propagated in these systems was compared to those in static and shaken batch cultures. The data shows that reproducible stable bacterial communities can be propagated with these culture methods, however the community composition varies considerably with the approach used. Only matrix embedded communities, cultured under continuous flow conditions, could successfully retain obligate anaerobes when flow cell systems were operated in an aerobic environment. This optimized method was used for culturing complex and diverse natural communities from clinical samples (sputum). The majority of bacteria present in the original sample were recovered in flow cell cultures and the methodology was consistent. This study provides an experimental system that can be used for examining microbial community dynamics and community structure-function relationship.</p> / Master of Science (MSc)

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)