To protect public health, detection and treatment technologies have been improved to monitor and inactivate pathogens in drinking water. The goal of this dissertation is to evaluate and utilize multiple online sensors and advanced oxidation processes to document both the detection as well as destruction of microbial contaminants in real-time. Reviews of rapid detection technologies for real-time monitoring of pathogens in drinking water and advanced technologies to inactivate pathogens in water are shown in Appendices A and B. The study in Appendix C evaluated the efficacy of real-time sensors for the detection of microbial contaminants. Bacillus thuringiensis was used in this research as a surrogate for Bacillus anthracis to determine each sensor response and detection capability. The minimum threshold responses of sensors were determined by injecting B.thuringiensis into deionized (DI), raw (unfiltered) tap water, or filtered tap water over a concentration range of 10² - 10⁵ spores/ml. The BioSentry sensor responded to increases in concentration over the range of 10² - 10⁵ spores/ml. Below this range, sensors provided signals undistinguishable from background noise. The select sensors can detect microbial water quality changes, and these advanced technologies can be integrated to monitor intrusion events in water distribution systems. The study in Appendix D evaluated the efficiency of the UV reactor for inactivation of MS2 coliphage. The virus MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a standard for UV reactor validation in the United States. In addition, MS2 is used as a surrogate for enteric viruses due to its similar size and morphology. Following UV radiation at a flow rate of 2gpm, infective MS2 showed a reduction of 5.3- log₁₀ when quantified with cultural plaque counts, whereas corresponding quantitative polymerase chain reaction (qPCR) data showed only a 1.7- log₁₀ reduction in viral RNA copy number. In contrast, plaque assay revealed a 5.8- log₁₀ inactivation; a slight increase in infective MS2 coliphage reduction at 1 gal per min but qPCR results indicate a 2.8- log₁₀ reduction in viral RNA copy number; a one log more inactivation compared to 2 gpm. When H₂O₂ was added at either 2.5 or 5 mg/l with UV at either flow rate, enhanced MS2 inactivation occurred with a greater than 7 log₁₀ reduction observed via plaque counts, indicating that all added MS2 had been inactivated, since no plaques were formed after incubation at 37°C for 24 hours. Correspondingly, qPCR data only showed a 3-4 log₁₀ reduction in viral RNA copy number. The study in Appendix E utilized online sensor to document the destruction of E.coli and Bacillus thuringiensis spores by UV/H₂O₂ treatment. In this study, Escherichia coli was tested for potential UV/H2O2 treatment in DI water and online sensors were also integrated to monitor the destruction in real-time. Pilot-scale experiments were performed using a Trojan UVSwift SC reactor (Trojan Technologies, London, ON, Canada) at a flow rate of 1 gal./min (gpm). UV radiation and UV/H₂O₂ combination in E.coli cell suspensions resulted in a >6 log₁₀ reduction of the viable counts. Similar exposure to B.thuringiensis spores resulted in a 3 log₁₀ reduction in viable counts. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E.coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following UV/H₂O₂ exposure, the BioSentry sensor showed an increase in the unknown, rod and spores counts, and did not correspond well when compared to viable counts assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B.thuringiensis spores which were more resistant to UV/H₂O₂.

Escherichia coli is a bacterial species that lives in the gut of all warm-blooded animals, fish, birds as well as reptiles and is commonly used as an indicator of fecal contamination in water. This project assessed currently used culture based media for the detection of E. coli in irrigation waters used in Arizona and California, and will present recommendations towards the most reliable media for the evaluation of irrigation waters used for produce. Currently, no microbial indicator standards exist for irrigation waters used for produce production in the United States. The produce industry suggests that the recreational water standard guideline (126 E. coli/100 ml) established by the United States Environmental Protection Agency (USEPA) be used. There is concern that the false positive rate of E. coli detection may be high in these waters giving false indications of the level of risk from enteric pathogens. This project evaluated three commercially available media for E. coli detection to test irrigation waters from three agricultural areas (Yuma and Maricopa, AZ and Imperial Valley, CA) and then assessed false positive rates by utilizing Polymerase Chain Reaction (PCR) and DNA sequencing of the bacterial isolates. The media that were chosen for evaluation were (1) MI Agar, (2) IDEXX Colilert Quanti-Tray® and (3) m-ColiBlue24® broth, all evaluation media accepted by the USEPA and widely used in the monitoring of irrigation water quality by the produce industry. Four hundred and fifty 1-L irrigation water grab samples were collected between March 2012 and November 2012. The samples were analyzed for both cultural counts and water quality parameters including temperature, salinity and pH. Isolates positive and negative for E. coli were then selected and assessed utilizing PCR and DNA sequencing. The false positive rate of each method was found to be high, with MI Agar, m-ColiBlue24® broth and the IDEXX Colilert Quanti-Tray® at an accuracy of 67%, 72%, and 51% respectively. A false positive result is reported when presumptive E. coli sub cultured from the media is found to be non-E. coli through molecular analysis. Overall the IDEXX Colilert Quanti-Tray® performed at a greater rate of accuracy than the other two media evaluated, however, high false positive rates may lead to inaccurate assessment of water quality.

The quantification and prediction of soil properties is fundamental to further understanding the Critical Zone (CZ). In this study we aim to quantify and predict soil properties within a forested catchment, Marshall Gulch, AZ. Input layers of soil depth (modeled), slope, Saga wetness index, remotely sensed normalized difference vegetation index (NDVI) and national agriculture imagery program (NAIP) bands 3/2 were determined to account for 95% of landscape variance and used as model predictors. Target variables including soil depth (cm), carbon (kg/m²), clay (%), Na flux (kg/m²), pH, and strain are predicted using multivariate linear step-wise regression models. Our results show strong correlations of soil properties with the drainage systems in the MG catchment. We observe deeper soils, higher clay content, higher carbon content, and more Na loss within the drainages of the catchment in contrast to the adjacent slopes and ridgelines.

Interfacial partitioning tracer tests (IPTT) are one method available for measuring air-water interfacial area (A(ia)).This study used the standard approach comprising tracer injection under steady unsaturated-flow conditions with a uniform water-saturation distribution within the column. Sodium dodecylbezene sulfonate (SDBS) and pentafluorobenzoic acid (PFBA) were used as the partitioning and nonreactive tracers, respectively. Three types of porous media were used for the study: a sandy soil, a well-sorted sand, and glass beads. Initial water saturations, S(w), were approximately 80%, 80%, and 26 % for the soil, sand, and glass beads, respectively. Water saturation was monitored gravimetrically during the experiments. The maximum interfacial areas (A(ia)/(1-S(w))) calculated from the results of the experiments are compared among the three porous media used in this work, and compared to previous air-water interfacial area studies.

Antibiotic resistance (AR) is reducing options for effective treatment of bacterial infections for clinically ill patients throughout the world. Our investigation examined the impact solids retention time (SRT) may have on the degradation of antibiotic compounds, proliferation of AR bacteria, and the persistence of antibiotic resistance genes (ARGs) during the wastewater treatment process. Results indicate the presence of ARGs related to clinically relevant antibiotics. Data analysis showed that antibiotic classes exhibit different removal efficiencies in the biological treatment processes (i.e. SRT); therefore, operating conditions at each WWTP can be optimized for highest efficiency removal. SRT of 19 days and higher indicated a high removal efficiency for all six ARGs of concern. Ultimately, identifying a critical value to optimize SRTs, where antibiotic degradation is maximized and AR is minimized, will provide information to WWTP operators, allowing treatment optimization to decrease harmful loading of Trace Organic Contaminants (TOrCs) in the environment.

Recent advancements in molecular biology such as next generation sequencing and more sensitive and rapid molecular detection methods like qPCR, have historically been developed for clinical applications in human genetics and for health care diagnostic purposes. The high demand for faster and more accurate molecular assays in the health care field has driven rapid development of inexpensive molecular techniques that when applied to the science of environmental microbiology, provides an unprecedented level of understanding of the microbial world around us. The goal of this dissertation is to begin to apply more advanced molecular technologies to problems in applied environmental microbiology. Appendix A is a brief literature review of next generation sequencing technologies for applications in environmental microbiology. Appendix B focuses on the development of a more robust virus nucleic extraction kit for the detection of viral genomes from environmental samples found to contain high concentrations of qPCR inhibitors, such as humic acids or heavy metals. Appendix C summarizes one of the largest virus surveys done in the US, using state of the art qPCR technologies in both wastewater influent and effluent from two wastewater treatment plants in the Southwest. Data suggests that traditional virus indicators may not be a viable tool to evaluate fecally impacted source water or virus removal during water treatment. The third study summarized in Appendix D, provides one of the first insights into the microbial ecology of biofilms utilized as biological treatment media using Roche 454 amplicon sequencing of the 16S rRNA gene.

Extensive research conducted over the last decade has demonstrated the great potential of phytostabilization for the reclamation of abandoned mine tailing piles. The right combination of plant species and soil amendments can facilitate the growth of a permanent vegetative cover on the tailings that will help minimize the mobilization of metal-bearing particles by means of wind dispersion and water erosion. Despite previous research efforts, the diversity and potential role of microbial populations inhabiting the root zone of the plants on the stabilization of the metal(loid) contaminants remains mostly unresolved. The study presented in this dissertation represents one of the first comprehensive efforts aimed to understand the ecology and dynamics of microbial communities colonizing both bulk and rhizosphere tailings during phytostabilization as an initial step towards elucidating the role of microbes in the stabilization of metal(loid) contaminants during the remediation treatment. This study was divided into two main projects: (1) the first aimed to monitor the temporal variations in functional and taxonomic diversity of prokaryotic populations in acid-generating metalliferous mine tailings during phytostabilization to determine how they respond to and/or influence changes in environmental parameters and to identify key patterns in their composition that may serve as bioindicators of soil health and the success of the remediation treatment; and (2) the second aimed to expand our understanding of the dynamics of root-associated bacterial, fungal and archaeal communities during mine tailing phytostabilization and how the dynamic behavior of the communities correspond to the growth of plants, the addition of soil amendments, and fluctuations in environmental conditions. The results presented here demonstrate that different microbial groups respond differently to changes in environmental conditions during phytostabilization, suggesting that by monitoring the behavior of specific microbial groups in the systems (as bioindicators) we may be able to assess the effectiveness of the remediation treatment. Furthermore, the results from the taxonomic and functional analysis of the microbial communities served as the basis for the development of a model that explains the ecology and distribution of dominant microbial groups in the tailings that may significantly contribute to the oxidation of iron-sulfides, the production of acid mine drainage, and to facilitate plant establishment and survival during phytostabilization.

The increasing food demand as well as the need to predict the impact of warming climate on vegetation makes it critical to find the best tools to assess crop production and carbon dioxide (CO₂) exchange between the land and atmosphere. Photosynthesis is a good indicator of crop production and CO₂ exchange. Chlorophyll fluorescence (ChF) is directly related to photosynthesis. ChF can be measured at leaf-scale using active techniques and at field-scales using passive techniques. The measurement principles of both techniques are different. In this study, three overarching questions about ChF were addressed: Q1) How water, nutrient and ambient light conditions determine the relationships between photosynthesis and ChF? Which is the optimum irradiance level for detecting water and nutrient deficit conditions with ChF?; Q2) which are the limits within which active and passive techniques are comparable?; and Q3) What is the seasonal relationship between photosynthesis and ChF when nitrogen is the limiting factor? To address these questions, two main experiments were conducted: Exp1) Concurrent photosynthesis and ChF light-response curves were measured in camelina and wheat plants growing under (i) intermediate-light and (ii) high-light conditions respectively. Plant stress was induced by (i) withdrawing water, and (ii) applying different nitrogen levels; and Exp2) coincident active and passive ChF measurements were made in a wheat field under different nitrogen treatments. The results indicated ChF has a direct relationship with photosynthesis when water or nitrogen drives the relationship. This study demonstrates that the light level at which plants were grown was optimum for detecting water and nutrient deficit with ChF. Also, the results showed that for leaf-average-values, active measurements can be used to better understand the daily and seasonal behavior of passive ChF. Further, the seasonal relation between photosynthesis and ChF with nitrogen stress was not a simple linear function. Our study showed that at times in the season when nitrogen was sufficient and photosynthesis was highest, ChF decreased because these two processes compete for available energy. These results demonstrated that ChF is a reliable indicator of crop stress and has great potential for better understand the CO₂ exchange between the land and atmosphere.

A series of studies were conducted on an emerging disease in farmed penaeid shrimp. This disease was first named as Early Mortality Syndrome (EMS) or more descriptively as Acute Hepatopancreatic Necrosis Syndrome (AHPNS). As part of the outcome of this research, the etiology of EMS/AHPNS was demonstrated. EMS was first classified as an idiopathic disease because no causative agent had been identified. Preliminary studies conducted in Vietnam in 2012 by the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) indicated that EMS is infectious (Tran et al., 2013). The agent was identified as a unique strain of Vibrio parahaemolyticus. Hence, EMS has a bacterial etiology confirmed by satisfying Koch's Postulates. Further studies focusing on the bacterial isolate causing EMS revealed that the agent could produce toxin(s), which is responsible for the primary pathology in affected shrimp. Since the causative agent has been identified, we propose a new name for EMS as Acute Hepatopancreatic Necrosis Disease (AHPND). Characterizations of the AHPND-causing Vibrio parahaemolyticus: Biochemical methods and molecular methods were used. Based on these results, various diagnostic methods were developed including polymerase chain reaction (PCR) test and biochemical tests. Other aspects of the AHPND causing V. parahaemolyticus were also run to determine such as antibiogram and the development of resistance mechanism of the bacteria exposed to farm conditions with antibiotic medications, pathogenicity, and infection dose of the bacteria, mode of infection, mechanisms governing the toxin production, and effects of environmental parameters on the invasion of the agent. Some proposed control measures for AHPND: Several antibiotic-free approaches were tested to determine viable control methods for AHPND. The principle proposed control method is to increase biosecurity. With the PCR method that has been developed, potential sources of the pathogen such as post-larvae and broodstock can be tested. As more and more insights of the pathogen were explored, the behavior of the pathogen was further elucidated. Based on this, control methods such as using polyculture with tilapia, probiotics, and bioflocs system were also tested. Several improvements in shrimp farming practices that may reduce the outbreak of the disease were also proposed.

Microbes survive for an extended period of time on fomites. With new strains of pathogens constantly emerging, it is important to understand their survival and spread and to evaluate the efficiency of new disinfection methods. The purpose of this study was to determine the occurrence of pathogens on fomites, and evaluate different disinfection methods (household bleach and steam vapor). Fomites were sampled in a variety of environments for the presence of pathogens including methicillin intermediate- and resistant- Staphylococcus aureus, Clostridium difficile, Penecillium brevi-compactum, Alternaria alternata and novel H1N1 influenza A. Samples were assayed using both cultural and genetic techniques to determine the microbial occurrence. In disinfection studies, samples were collected before and after disinfectant application. Steam vapor was effective at reducing microbial occurrence by>90% with a minimal contact time of 10-20 seconds on hospital fomites. The material and shape of the surface had an effect on the reduction rate, due to access of the disinfecting agent. In addition, low levels of initial contamination limited the reduction rate. Many surfaces had a starting contamination level of less than 3 log10 and after disinfection the contamination levels, in 69% of the samples, were at or near the limit of detection. This suggests a higher reduction is feasible. Household bleach reduced the presence of mold spores and mycelium by >99% on gypsum-wall board. Reduction rates were influenced by the growth stage of mold, with the mycelium requiring additional disinfectant application to achieve the 99% reduction rate. Novel H1N1 influenza A was not recovered on any surfaces in day care facilities and elementary schools. This is consistent with previous studies looking at H1N1 influenza occurrence in similar environments, though it is different from H3N2 influenza A studies. Survival differences among varying influenza strains are expected to have an impact on pathogen spread and human health risks. Differences can be quantitatively evaluated and used to develop more advanced risk assessment models. Steam vapor and household bleach are effective at reducing risks of pathogens in the environment and are critical interventions in an overall strategy to minimize exposure and prevent disease.