Enzymes play an important role in the environment, they breakdown natural-occurring and anthropogenic molecules so that they can be transported into cells and utilized. Enzyme assays are routinely used in soil science and oceanography to measure the activities of specific processes and to serve as general indicators of microbial activity. Conventional enzyme assays are conducted as batch incubation of sediment and water samples. During these assays the concentration of product is measured and enzyme activity is then determined as the rate of product formation. Few studies have measured enzyme activities of groundwater. This work investigates the use of β-glucosidase and phosphatase assays for quantifying in situ enzyme activities in groundwater. Improvements to conventional enzyme assays using p-nitrophenyl substituted compounds were made by developing a high performance liquid chromatography method to improve quantitation limits of the product and to quantify concentrations of both the substrate and the product. An in situ single-well push pull test was then conducted to measure β-glucosidase activity in situ and to estimate the Michaelis constant (K[subscript m]) and the maximum reaction velocity (V[subscript max]) in petroleum-contaminated groundwater at a field site near Newberg, Oregon. An important feature of the single-well push pull test is the nonlinear drop in pore water velocity that the test solution experiences as it moves out from the injection point. The nonlinear drop in pore water velocity is of particular interest because enzyme-mediated reactions are very fast and changes in the hydraulic properties during the test may give rise to mass-transport limitations. Fast reactions lead to the simultaneous depletion of substrate and accumulation of product at the site of the reaction so substrate and product concentrations near the enzyme can be different then the concentrations in bulk solution. And the rates obtained from a single-well push pull tests may be a combination of the rates at which substrate diffuses to the microorganism and at which the reaction occurs. Laboratory experiments with sediment-packed columns were conducted with a range of pore water velocities typically achieved in the subsurface during as push-pull test as a means for examining the potential effects of inhibition and diffusion on phosphatase enzyme kinetics. In this set of column experiments rates of phosphatase-mediated reactions were investigated instead of β-glucosidase, which is an inducible enzyme. Numerical investigations were then conducted to examine the importance of diffusion limitations for describing the influence of transport processes on the observed rates of reaction. The theoretical investigation was conducted by formally upscaling the proposed sub-pore-scale processes to develop a macroscale (or Darcy scale) description of the transport of the substrate. These results indicate that mass-transfer limitations due to the diffusion of the substrate to the enzyme cause an increase in the apparent K[subscript m] but have no effect on V[subscript max]. In this study an analytical method was developed to measure rates of enzyme-mediated reaction in situ so that the measured rates reflected actual rates of microorganism in their natural environment. More carefully controlled laboratory experiments demonstrated that rates of enzyme-mediated reactions measured at low substrate concentrations depended on the flow properties of the test solution. / Graduation date: 2006

Thesis (M.S. - Geology)--University of Arizona. / Includes bibliographical references.

Thesis (M.S. - Agricultural Chemistry and Soils)--University of Arizona. / Includes bibliographical references (leaves 48-50).

Understanding the spatial and temporal dynamics of catchment systems is crucial to understanding how hydrological behaviour may change over time and how this impacts on crucial aspects of catchment management such as flood generation, water resource management and the sources and fluxes of sediment, nutrients and contaminants. A combined statistical analysis was undertaken to assess whether similar catchment groupings respond to changing climatic drivers in the same way. A k-means cluster and PCA analysis grouped catchments in four clusters, which were differentiated by their topographical differences between lowland and upland catchments. Lowland catchments exhibited similar behaviours to changing trends of key hydroclimatic variables whereas more upland catchments showed diverse responses. We assessed the behaviour of 8 mesoscale catchments with increasing lowland areas for spatial and temporal runoff dynamics via the application of environmental tracers (stable isotopes and Gran alkalinity). Mean transit times were estimated using a lumped convolution integral model and lowland catchments with greater coverage of sedimentary bedrock exhibited longer turnover for water and solute fluxes. Tracer data was then implemented into a conceptual rainfall-runoff model to develop a model, which could represent both spatial and temporal dynamics rather than simply recreating the observed stream hydrograph. Finally, we assessed the role of dominant landscape characteristics (urban environments and grazing pastures) on the sources and fluxes of microbial contaminant risk to water quality. Catchments which had larger urban coverage and higher portion of grazing pastures yielded higher concentration fluxes of faecal coliforms which provided a first-order approximation of water quality risk at the catchment scale.

Thesis (M.S. - Hydrology and Water Resources)--University of Arizona, 1982. / Includes bibliographical references (leaves 100-103).

Thesis (Ph. D. - Microbiology)--University of Arizona, 1982. / Includes bibliographical references (leaves 95-101).

The primary objective of this study was to investigate the use of hydrogen as a structural substitute or as a reducing equivalent in the anaerobic biodegradation of methanol, methyl tert-butyl ether (MTBE), toluene, phenol, and 2,4- dichlorophenol. In addition, biodegradation rates of these compounds at various initial concentrations with and without inhibition of sulfate reducing bacteria were determined along with anaerobic biodegradation rate constants for each of the compounds studied. Rates of methanol biodegradation were only slightly altered in molybdate amended microcosms indicating that methanol is a noncompetitive substrate in Blacksburg soil. MTBE biodegradation was slow and followed first order kinetics with respect to initial concentration. Molybdate had no affect on MTBE biodegradation alone, but increased the biodegradation rate in MTBE microcosms which were amended with ethanol. Toluene, phenol, and 2,4-dichlorophenol biodegradation proceeded at two different rate versus initial concentration relationships for lower and upper concentration ranges. Phenol biodegradation followed first order kinetics. The 2,4-dichlorophenol biodegradation rate order varied from 0.78 to 1.75. Monod kinetics were followed by methanol, toluene, and phenol, but not by MTBE, ethanol amended MTBE, or 2,4-dichlorophenol. Addition of molybdate to inhibit sulfate reduction increased the degradation rates more for compounds which may require hydrogen in a structural position (2,4-dichlorophenol, MTBE) than those which require hydrogen for proton reduction (methanol). Biodegradation or recalcitrant compounds may be stimulated by the addition of organics (such as ethanol) which produce hydrogen upon biodegradation. / Master of Science

The bacterial flora of groundwater obtained from wells and distribution sites was investigated from both an ecological and a public health perspective. A majority of the isolates were oxidase positive, non-fermentative, gramnegative bacilli. Extensive heterogeneity of groundwater microflora, as determined by biochemical characterization and antibiograms, was observed both within and among welldistribution (N-D) systems. Of the unique isolates (sorts) found in the W-D systems, 75% were resistant to 2 or more antibiotics at clinically significant concentrations. Community diversity within W-D systems was evaluated by rarefaction which failed to reveal a general trend. Standard plate counts in 2 of 3 wells were significantly higher on Standard Methods Agar diluted ten-fold than on the same medium at the standard concentration. Pseudomonas aeruginosa, Flavobacterium sp., Aeromonas hydrophila and Yersinia enterocolitica were detected in 21, 18, 7 and 3% respectively of water samples analyzed over a 12 month period. A selective medium was developed for the isolation of Flavobacterium sp. and was effective in suppressing 98% of the background flora when used in a membrane filtration (MF) procedure. Yersinia Selective Agar was employed in an MF procedure for the isolation of Y. enterocolitica. The use of anaerobic incubation in this procedure effectively suppressed background growth both in the presence and absence of an antimicrobic supplement. Anaerobic incubation of m-endo LES Agar (AN-MF) was effective in suppressing non-coliform growth in the total coliform MF test and markedly reduced the frequency of overgrown plates. The AN-MF appeared to obviate the need for selective chemical agents and thus has potential value in the isolation of stressed coliforms.

The objective of this study was to determine the potential for biodegradation in subsurface soils and groundwater from sites in Williamsport, PA, Wayland, NY, and Dumfries, VA. These subsurface systems were characterized both physically, chemically and biologically. Bacterial populations were substantial in all systems and ranged from 10³ to 10⁸ colony forming units per gram. Soil sampling was done in a quality-controlled aseptic manner using conventional drilling end sampling equipment. A matrix of test-tube microcosms was used to determine biodegradation rates of methanol and t-butyl alcohol at concentrations ranging from 1 to 1000 mg/L. Methanol degraded readily at all sites ranging from 0.8 mg/L/day to 20.4 mg/L/day and rates were generally greater in the saturated zone. TBA biodegraded at all sites, but was refractory in nature. Biodegradation rates for TBA in anaerobic subsurface systems were found to increase directly with initial concentration from 10⁻⁴ mg/L/day for 1 mg/L to 10⁻¹ mg/L/day for 80 mg/L. TBA biodegradation in the aerobic system was essentially constant over all concentrations. Biokinetic coefficients were determined for methanol and TBA at each site based on plots of utilization rates versus substrate concentration and reciprocal plots of these values. The K values found suggest that aerobic subsurface systems can utilize alcohols at a greater rate than anoxic subsurface systems and can be used for comparative purposes. The K_s of anoxic subsurface systems were found to be large due to the low temperature (10°C) found in aquifers. The results indicate that methanol contamination in groundwater has much less associated risk to drinking water supplies due to the ease of biodegradation. However, TBA poses a much greater risk due to the very slow removal rates at low concentrations, which could result in a residual level for over a decade in some cases. / Ph. D.

Mais de 50% da dose de radiação efetiva anual recebida por um ser humano está relacionada com o Radônio e seus produtos. Os principasis mecanismos que levam o Radônio no interior das residências são a emanação do solo e a liberação do Radônio na água. Esse trabalho apresenta uma proposta de mitigação, método para redução de Radônio-222 em níveis de água de poço, utilizando o processo de aeração da água e medidas da concentração de Radônio -222 nas águas e solo. As amostras foram coletadas em poço e solo na região do Pinheirinho em Curitiba, Brasil. As medidas foram realizadas durante o período de Fevereiro a Junho de 2011, sendo analisadas em média dez amostras de cada coleta de água, entre elas oito sob o processo de aeração. As medidas foram obtidas com a câmara de ionização Radon Professional Monitor (AlfaGUARD), aparelho que verifica a concentração de Radônio na faixa de 2 - 2x106 Bq/L, ajustado em um fluxo de 0,5L/min, em um tempo aproximado de 60 minutos, no Laboratório de Radiações Ionizantes da Universidade Tecnológica Federal do Paraná (UTFPR). Os níveis de concentração de Radônio-222 foram medidos através do software AlfaEXPERT, e formulações adequadas caracterizaram a concentração desse gás em água corrigindo o tempo de decaimento devido ao atraso da coleta das amostras em relação às medições. O método de mitigação utilizado foi o de aeração, chamado Aeração Difusora, na qual ocorre a gaseificação da água em um processo de injeção de ar por um aerador conectado em uma placa porosa durante um período de 24 horas em 4 dias, já que a meia-vida do Radônio é cerca de 3,82 dias, para a diminuição da concentração de Radônio-222 nas amostras. Inicialmente as amostras apresentaram concentrações de Radônio-222 de aproximadamente 20 Bq/L, nível acima de 11,11 Bq/L recomendado pela USEPA (Agência de Proteção Ambiental dos Estados Unidos). Após aplicar a medida de mitigação nessas amostras de água, as concentrações de Radônio-222 diminuíram satisfatoriamente e ficaram abaixo do valor esperado pelo decaimento natural do gás. / More than 50% of the effective annual radiation dose received by a human being is related to the Radon and its progeny. Among main mechanisms that bring Radon inside the dwelling are the soil emanation and radon release from the water. This present work represents a proposal of mitigation, method to reduce Radon-222 levels in well water, using the process of water aeration and measurements of Radon-222 in the water and soil. Samples were collected in a pit and soil in the region of "Pinheirinho" in the city of "Curitiba" Brazil. The Measurements were taken during the period of February till June of 2011, being analyzed by an average of ten samples of each water collected, among them eight were under the process of aeration. The measurements were obtained with ionization chamber Randon Professional Monitor (AlfaGUARD), a device that checks concentrations of Radon in the range of 2-2x106 Bq/L, adjusted in a flow of 0.5L/min, with approximate time of 60 minutes, in the laboratory for Ionizing Radiation of the Univeristy " Federal do Parana" (UTFPR). The levels of concentration of Radon-222 were measured by the software called AlfaEXPERT, and suitable formulations characterized the concentration of this gas in the water by correcting the time decay due to the delay in the collection of samples for measurements. The method of mitigation used was aeration, called Diffusing Aeration, in which there is the gasification of water in a process that injects air by an aerator connected in a porous plate during a period of 24 hours in 4 days, since the half life of Radon is approximately 3,82 days, for the reduction of the concentration of Radon-222 on samples. Initially samples had concentrations of Radon-222 aproximately from 20 Bq/L, a level above 11.11 Bq/L recomended by USEPA (United States Enviromental Protection Agency). After Applying the measurements to mitigate these samples of water, the concentration of Radono-222 decreased satisfactorily and remained below the expected value by the natural decaying of the water.