The high incidence of pathogens is one of the main causes for impaired surface water quality designations in the United States. Pathogen presence in fresh water is monitored through the detection of indicator bacteria. Indicator bacteria concentrations, spatial and temporal variability, and microbial risks were evaluated in two rural watersheds, the Bosque and Leon Rivers, and one predominantly urban watershed, the San Jacinto River, all in Texas. Human health risk was predicted from contaminated waters as indicated by ingestion of Escherichia coli found in surface water for contact recreation scenarios. The watersheds were chosen because many segments were previously placed on the 303 (d) list (published by the TCEQ) for failing the indicator bacteria standards. Predominantly urban areas of the San Jacinto River and rural portions of the Bosque and Leon Rivers, where Concentrated Animal Feeding Operations (CAFOs) are numerous, were compared to relatively pristine rural watersheds. Spatial analysis of the watersheds with E.coli concentrations exceeding the single sample (394 MPN/100mL) and the geometric mean standards (126 MPN/100mL) indicated that land use is a significant factor influencing the incidence of bacterial concentrations. Non-agricultural rural areas of the watersheds, such as forests and rangelands, had significantly lower E.coli concentrations compared to the agricultural areas and urban land uses. Human health risk due to ingestion of E.coli as an indicator organism indicated a similar pattern to that of their concentrations in that urban and agricultural areas had a greater risk compared to the other rural areas of the watersheds. The risk estimate for urban and agricultural areas exceeded the acceptable limit of one in ten thousand (10^-4), indicating a potential for adverse health effects to humans. Temporal variability in the watersheds as a function of streamflow, rainfall, and temperature indicated a positive correlation between bacterial concentration and high streamflow, rainfall and temperature. The positive correlation for these effects was greater in the rural areas compared to urban areas, indicating the presence of multiple factors responsible for E.coli concentrations in urban areas. Thus, land use was confirmed to be a major factor contributing to the presence of indicator bacteria in surface waters.

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used. The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum). Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces. In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

Anaerobic digestion process which has long been known to successfully reduce the organic content of sludge is one of the most common alternatives to meet pathogen reduction requirements for particular classes of biosolids. However, it has recently been reported that, significantly higher densities of indicator bacteria have been measured in dewatered cake samples compared to samples collected right after anaerobic digestion. In addition, this increase in bacterial population has been commonly observed after centrifugation but not after belt filter dewatering. Even though several theories have emerged to explain this occurrence, with the use of molecular tools such as Quantitative Polymerase Chain Reaction (Q

Water quality worldwide is assessed by enumeration of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, and enterococci) intended to act as surrogates for human enteric pathogens. In environmental waters, this predictive relationship is confounded by many possible sources of FIB with varying implications for human health. Many physico-chemical and biological factors influence the fate of enteric pathogens and FIB in aquatic habitats, but are poorly understood, thus limiting our understanding of the usefulness of FIB as fecal pollution indicators. These studies explored the field application of a “toolbox” approach to microbial source tracking (MST) intended to discriminate between human and non-human fecal pollution: a) in a Florida estuary used for shellfishing and recreational activities and b) at public beaches before and after remediation of wastewater infrastructure. Lastly, the effects of environmental factors (sediments, protozoa, sunlight) on survival of culturable E. coli were investigated in freshwater and seawater mesocosms simulating environmental conditions. Detection of a human- associated MST marker (the esp gene of Enterococcus faecium) at sites with suspected sewage contamination indicated that human fecal pollution is impacting water quality in Wakulla County, while Lagrangian drifters designed to follow current and tidal movement suggested that local hydrology plays an important role in bacterial transport and deposition pathways. Elevated FIB concentrations and frequent detection of human-associated MST markers (esp and human polyomaviruses) identified human sewage pollution at a public beach, facilitating remediation efforts (sewage main repair, removal of portable/abandoned restrooms), followed by significant decreases in FIB concentrations and MST marker detection. These studies show that comprehensive microbial water quality assessment can reliably identify contamination sources, thereby improving pollution mitigation and restoring recreational water quality. Protozoan predation, freshwater vs. seawater habitat and sediment vs. water column location affected the concentration of culturable E. coli in outdoor mesocosms. Sediments offered a refuge from predation where freshwater vs. seawater habitat was amore important determinant of survival. These findings provide important insight into the ecology of E. coli and their natural predators in aquatic habitats and underscore the inherent effect different habitats play in their survival.

Siesta Key Beach, located on the Gulf Coast of Florida, is frequently mentioned among the top ten beaches in the US. In summer 2004, high levels of indicator bacteria caused health warnings to be posted, and a storm drainage system was implicated as a possible source of microbial contamination. A study was initiated to determine whether indicator bacteria that persisted in the stormwater system could contribute to high microbial loads in receiving waters. Two sampling events, one within 48 hours of a rain event and the other during dry conditions, were conducted. Water and sediment samples were taken at various sites from the storm drainage system to the beach. Fecal coliforms and Enterococcus spp. were enumerated, and genotypic fingerprints of E. coli and Enterococcus spp. were generated by BOX-PCR. Diversity of E. coli and Enterococcus populations was calculated with the Shannon-Weiner diversity index. Similarity of E. coli and Enterococcus populations was calculated with the population similarity coefficient. After the rain event, levels of fecal coliforms and Enterococcus spp. were high in sediments and exceeded the regulatory standard for all water samples. In dry conditions, levels were lower in water samples, but still high in sediment samples. Significantly greater population diversity was observed in the rain event compared to the dry event for both E. coli and Enterococcus populations, and greater population similarity was vi observed in dry conditions. Enterococcus population diversity was significantly higher in untreated sewage and the Siesta Key rain event when compared to dry conditions, and to a site on the Myakka River (no known human input or urban stormwater runoff). Siesta Key populations in dry conditions were most similar to Myakka, and sewage was the least similar to all other populations. Increased population similarity for E. coli and Enterococcus spp. during dry conditions suggests that a portion of the population is composed of “survivor” isolates. Persistence of survivor isolates in the storm drainage system, where urban runoff can sit for days, suggests a reservoir for indicator bacteria that can be flushed through the system to the Gulf, causing high levels of indicator bacteria in receiving waters.

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:01:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Vanessa_Klug.pdf: 783938 bytes, checksum: 79ed032707f8126d95d822fe1b7883a5 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:16:19Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Vanessa_Klug.pdf: 783938 bytes, checksum: 79ed032707f8126d95d822fe1b7883a5 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:16:28Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Vanessa_Klug.pdf: 783938 bytes, checksum: 79ed032707f8126d95d822fe1b7883a5 (MD5) / Made available in DSpace on 2018-05-21T20:16:28Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Vanessa_Klug.pdf: 783938 bytes, checksum: 79ed032707f8126d95d822fe1b7883a5 (MD5) Previous issue date: 2016-09-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A chia é uma planta mexicana, originária de países subtropicais e tropicais, é da família das labiadas, herbácea, anual, possui talos quadrangulares, acanelados, com vilosidades; folhas opostas, pecioladas, serrilhadas e flores reunidas em espigas auxiliares ou terminais, cada fruto leva quatro sementes bem pequenas de forma oval, lisas, brilhantes, de cor cinzenta com manchas avermelhadas. Essa semente tem sido muito consumida, pois possui uma boa qualidade nutricional em sua composição, rica em fibras, minerais, proteínas e ácidos graxos ômega-3 e ômega-6, os quais quando presentes na dieta de indivíduos promove uma redução na incidência de doenças cardiovasculares. Por ser um grão sem glúten, este pseudocereal se torna uma boa alternativa na substituição do glúten. O glúten é formado pelas proteínas gliadina e glutenina, a doença celíaca é uma intolerância à ingestão de glúten, contido em cereais como cevada, centeio, trigo e malte, em indivíduos geneticamente predispostos, caracterizada por um processo inflamatório que envolve a mucosa do intestino delgado, levando a atrofia das vilosidades intestinais, má absorção e uma variedade de manifestações clínicas por portadores de doença celíaca. O objetivo desse estudo foi avaliar a composição química dos grãos de chia, elaborar biscoitos com grãos de chia e farinha de arroz em diferentes formulações, afim de atender os consumidores com intolerância ao glúten, uma vez que o produto elaborado é isento do mesmo; e realizar análises de composição química nos mesmos. Foram realizadas análises de proteínas, fibras, umidade, cinzas, gorduras, proteínas solúvel e insolúvel, antioxidantes e fenóis totais nos e grãos e biscoitos elaborados. Foi avaliado o índice de aceitação e intenção de compra dos biscoitos através de testes sensoriais. Foi avaliada atividade antimicrobiana de um extrato elaborado com chia, frente as bactérias indicadoras, Slmonella, Listeria, E. coli e S. aureus. Os grãos de chia apresentaram valores de composição química semelhantes aos já estudados por outros autores, e pode ser considerado uma boa fonte nutricional. Os biscoitos obtiveram valores variados na sua composição química, os mesmos tiveram boa aceitabilidade e intenção de compra. O extrato obtido dos grãos de chia, inativaram as bactérias Listeria e Salmonella por um tempo determinado. / The chia is a Mexican plant, native to subtropical and tropical countries, is the family of Labiatae, herbaceous, annual, has quadrangular stems, acanelados with villi; opposite leaves, petiolate, knurled and flowers gathered in spikes or auxiliary terminals, each fruit takes four very small seeds oval, smooth, shiny, gray in color with reddish spots. This seed has been very consumed because it has a good nutritional quality in its composition, rich in fiber, minerals, protein and omega-3 fatty acids and omega-6, which when present in the diet of individuals promotes a reduction in the incidence of diseases cardiovascular. Being a grain gluten, this pseudocereal becomes a good alternative to replace the gluten. Gluten is formed by gliadin protein and glutenin, celiac disease is an intolerance to gluten ingestion, contained in cereals such as barley, rye, wheat and malt, in genetically predisposed individuals, characterized by an inflammatory process involving the mucosa of the small intestine, leading to atrophy of the intestinal villi, malabsorption and a range of clinical manifestations in patients with celiac disease. The aim of this study was to evaluate the chemical composition of chia grains, prepare cookies with chia grains and rice flour in different formulations in order to meet consumers intolerant to gluten, as the manufactured product is free of it; and perform chemical composition analysis on them. protein analyzes were performed, fiber, moisture, ash, fat, soluble and insoluble proteins, antioxidants and phenolic compounds in grains and prepared and biscuits. It evaluated the acceptance rate and purchase intent of cookies through sensory tests. We evaluated the antimicrobial activity of a prepared statement with chia, front indicator bacteria, Slmonella, Listeria, E. coli and S. aureus. The chia grains showed chemical composition values similar to those already studied by other authors, and can be considered a good source of nutrition. The biscuits obtained different values in their chemical composition, they had good acceptability and purchase intent. The extract obtained from chia grains, inactivate the bacteria Listeria and Salmonella for a time.

Pathogen contamination, typically quantified by elevated levels of fecal indicator bacteria (FIB), remains the leading cause of surface water-quality impairments in the United States. Continuous watershed-scale models are typically employed to facilitate Total Maximum Daily Load (TMDL) restoration efforts. Due to limited understanding of microbial fate and transport, predictions of FIB concentrations are associated with considerable uncertainty relative to other water-quality contaminants. By focusing on a data-rich instrumented urban watershed, this study aims to improve understanding of microbial fate and transport processes. Weekly FIB concentrations in both the water column and streambed sediments were monitored for one year, and statistical correlations with hydrometeorological and physicochemical variables were identified. An intensive six storm intra-sampling campaign quantified and contrasted loading trends of both traditional regulatory FIB and emerging Microbial Source Tracking (MST) markers. Together, these intensive monitoring efforts facilitated evaluation of the impacts of bacteria-sediment interactions on the predictions of daily FIB concentrations in Hydrological Simulation Program-Fortran (HSPF) over multiple years. While superior overall model performance was demonstrated as compared to earlier efforts, the inclusion of bacteria-sediment interactions did not improve performance. Large wet-weather microbial loading appears to have dwarfed the effects of FIB release and resuspension from sediment. Although wet-weather loading is generally considered as a primary source of waterbody microbial loads, dry-weather periods are more directly associated with public health concern, which may be a more suitable area for future model-refinement efforts. Site evaluation is critical to determine whether the added model complexity and effort associated with partitioning phases of FIB can be sufficiently offset by gains in predictive capacity. Finally, a stochastic framework to translate simulated daily FIB concentrations into estimates of human illness risks is presented that can be can be readily integrated into existing TMDLs. As even small concentrations of FIB from human sources are associated with great risk, and monitoring efforts indicated moderate/high levels of human-associated MST marker in this watershed, remediation efforts to protect public health would be best directed toward infrastructure improvements. Uncertainty analysis indicates more site-specific knowledge of pathogen presence and densities would best improve the estimation of illness risks. / Ph. D.

E. coli and Enterococcus were used to determine the fate and survival of fecal indicator bacteria (FIB) in sand and sediments. The microbial source tracking (MST) methods antibiotic resistance analysis (ARA), Bacteroides human-specific primer test, and fluorometry were compared against the FIBs to determine how reliable each method was in detecting the presence of human fecal contamination. Two phases (Summer 2009 and 2010) were evaluated based on the type of contamination event. A combined sewage overflow (CSO) event was simulated in Phase I, where large amounts of influent were added to sand and bay water columns over 1 to 4 days. In 2010, a low volume sewage leak was simulated in which smaller doses of influent were added to sand and bay water columns over a period of 5 to 15 days. Within each of the phases, both non- and re-circulated columns were also evaluated. Evaluation of FIB survival indicated that Enterococcus was able to stabilize and re-grow in the water and at the sediment/water interface within the Phase I non-circulated columns. E. coli was unable to re-grow and/or stabilize within any environment. Comparisons between the ARA and the FIBs revealed a large majority of isolates identified as coming from either bird or wildlife sources. Human sources were identified but at much lower concentrations than expected. Bacteroides results indicated strong relationships between the increase of FIB concentrations and the presence of the human-specific Bacteroides. Fluorometry results did not indicate any relationship with the FIBs. Unexpectedly, fluorometry readings increased as time progressed indicating that another compound was present that fluoresced at the same wavelength as optical brighteners (OBs). This project was one of the first to study the differences related to two different pollution events (CSO vs. sewage leak) while also evaluating what happens to pollution as it settles into the sediment. It was also unique because it compared bacterial (ARA), molecular (Bacteroides), and chemical (fluorometry) MST methods. / Master of Science

Between 1970 and 2010 almost one-third of drinking water related waterborne disease outbreaks reported to the US Centers for Disease Control and Prevention were associated with systems dependent on untreated groundwater (i.e., most commonly, household wells). This is unsurprising, given that numerous past efforts to monitor household well water quality have indicated a high prevalence of fecal coliforms and/or E. coli at the point of use. Non-point sources of pollution, including septic tank leakages and poorly constructed drain fields, have been identified as the leading risk factors associated with outbreaks in households dependent on groundwater. Ideally, the integration of emerging source tracking (ST) analyses in well monitoring programs could be used to identify whether the presence of fecal indicator bacteria (FIB) is associated with human or non-human sources in order to inform remediation strategies. However, the application of ST to groundwater has been limited, and the interpretation of data is consequently difficult. This research compares the fate and transport of FIB (E. coli and enterococci) with a chemical (optical brighteners, OB) and a molecular (Bacteroides HF183) ST target in order to evaluate their potential use as indicators of water quality issues in private drinking water systems. Eighteen PVC soil columns were constructed in an outdoor soil column facility to represent small-scale septic drainfield models; they received synchronized doses of primary-treated wastewater twice daily and were monitored bi-weekly over a 7-month period. Columns were subject to variable influent loading rates of wastewater effluent, and differing degrees of soil compromisation (i.e. synthetic solution channels). Results show that while column effluent volume and constituent levels were related to dosage, they were not always related to soil compromisation (ANOVA, p < 0.05). E. coli and enterococci concentrations were associated with effluent volume and OB levels (Spearman's rank, p < 0.05). The presence of Bacteroides HF183 was not strongly associated with the other measured ST target levels (Point-biserial correlation, p < 0.05). Findings from this study suggest surface water ST methodologies may have a role in groundwater quality monitoring efforts. Quantifying the relative recovery of ST targets and FIB from controlled groundwater simulations will assist in the development of strategies to identify non-point sources of human wastewater pollution efficiently and effectively to inform remediation. / Master of Science

The increase of animal agriculture coupled with excess manure production, and the reduced availability of land has led to the over application of animal manure on agricultural fields. The excessive application of manure is responsible for nutrient and bacterial pollution of downstream waterbodies. Manure application based on the crop phosphorus (P) requirements has been recommended as a viable method to reduce nutrient pollution. A plot scale study was conducted to measure the loss of nutrients and bacterial transport in runoff from cropland treated with poultry litter, dairy manure and inorganic fertilizer according to the P requirements of the crop. Three simulated rainfall events were conducted 1, 2 and 35 days after planting of corn. Highest P and N concentrations were observed in the runoff from plots treated with poultry litter, followed by dairy manure and inorganic fertilizer. The poultry litter treated plots exhibited highest concentrations of bioavailable P in the runoff, compared to all other treatments. The P from poultry litter treated plots was also mostly in the soluble form, which underscores the need to control the runoff from cropland in order to decrease the P losses from the poultry litter treated fields. The edge of the field nutrient concentrations observed in this study were high enough to cause severe to moderate eutrophication problems in downstream waterbodies unless they are diluted. In general, nutrient concentrations were lower during the second simulated event, compared with those from the first event. A significant reduction in the nutrient concentrations of runoff was observed from the second to the third simulated event for all the treatments. This reduction was attributed to the loss of nutrients by natural rainfall-runoff events during the time period between the second and the third simulated rainfall event, plant uptake of nutrients, sorption and leaching processes. The indicator bacteria analyzed in the present study were fecal Coliform (FC), Escherichia Coli (E.Coli) and Enterococcus (ENT). The bacterial concentrations reported in the runoff for the first and second simulated events were 104 to 105 times higher than the federal and state limits for primary contact recreation waters. No significant effect of treatments was observed on the bacterial concentrations in runoff. The highest concentrations were observed for FC, followed by ENT and EC in the runoff. The ratio of bacteria removed in runoff to the bacteria applied also followed the above trend. The concentrations of bacteria generally increased from the first to second simulated event; unlike the nutrients. However, the bacterial concentrations dropped significantly from second to the third simulated rainfall event to the levels lower than those designated for primary contact recreation water limits. This reduction was attributed to the washing away of bacteria by the heavy rainfall-runoff events in the period between second and third simulated rainfall events and the die-off of bacteria. The results reported from this study suggest that the manure application based on crop P requirements can also be a significant source of nutrient pollution and should be coupled with other best management practices (BMPs) also to reduce nutrient pollution. The results also suggest that the manure treated cropland can be a source for significant indicator bacterial pollution and appropriate BMPs are required to mitigate their effect. / Master of Science