Rasmussen, Craig; Yamafuji, Kazumasa; Rasmussen, Craig; Artiola, Janick; Rasmussen, Craig; Walworth, James (The University of Arizona., 2014)
      Biochar (BC), produced through pyrolysis of organic residues, is increasingly being used as a beneficial soil amendment. We studied the effects of BC and animal manure additions on carbon dioxide (CO2) release and nitrogen (N) dynamics in three semi-arid climate soils. The objective of this study was to understand how BC application modifies soil nitrogen dynamics and moderate the effects of manure application in semi-arid agricultural systems on different textured soils: the loamy sand (LS) soil, the silty loam (SL) soil, and the clay loam (CL) soil. We found the positive interaction of BC and manure with BC suppressing CO2 emissions in manure amended soils. BC increased nitrogen mineralization in manure-amended soils towards the end of the incubation period 28 days. No significant N immobilization was observed in unamended soils. BC and manure soil additions reduced N deficiencies in all three soils. A second study focused on measuring carbon dioxide emissions from biochar-amended alkaline semi-arid soil. The objective of this study was to test if acidified and non-acidified BC released the same amount of CO2. The results showed that the soil samples amended with acidified BC released more CO2 than those amended with untreated BC with high alkalinity. It is postulated that untreated BC could absorb CO2; whereas, acidified BC with no alkalinity could not. The LS soil amended with BC released less CO2 than LS soil control perhaps due to the soil microbial activity inhibitory effects of the BC’s residual water soluble polynuclear aromatic hydrocarbons. Thus, the interaction with BC and steer manure application could suppress the release of CO2.
    • Soil Modulation of Ecosystem Response to Climate Forcing and Change Across the US Desert Southwest

      Rasmussen, Craig; Shepard, Christopher; Crimmins, Michael; Schaap, Marcel (The University of Arizona., 2014)
      The dryland ecosystems of the US Desert Southwest (SW) are dependent on soil moisture for aboveground productivity; the generation of soil moisture in the SW is dependent on both soil physical properties and climate forcing. This study is one of the first regional point-scale analyses that explores the role of soil physical properties in modulating aboveground vegetation dynamics in response to climate forcing in the SW. Soil texture accounted for significant differences in average aboveground primary productivity across the SW. However, soil texture could not account for differences in inter-annual aboveground productivity variation across the SW. Subsurface soil texture was tightly coupled with precipitation seasonality in accounting for differences in long-term average seasonal aboveground productivity in the Mojave and Sonoran Deserts. The results of this study indicate that the subsurface is a significant factor in modulating aboveground primary productivity, and needs to be included in future modeling exercises of dryland ecosystem response to climate forcing and change.
    • Batch and Column Transport Studies of Environmental Fate of 3-nitro-1,2,4-triazol-5-one (NTO) in Soils

      Dontsova, Katerina; Brusseau, Mark; Mark, Noah William; Dontsova, Katerina; Brusseau, Mark; Curry, Joan (The University of Arizona., 2014)
      NTO (3-nitro-1,2,4-triazol-5-one) is one of the new explosive compounds used in insensitive munitions (IM) and developed to replace traditional explosives, TNT and RDX. Data on NTO fate and transport is needed to determine its environmental behavior and potential for groundwater contamination. In this study, we measured how NTO in solution interacts with different types of soils and related soil properties to transport and fate behavior. We conducted a series of kinetic and equilibrium batch soil sorption experiments and saturated column transport studies under steady-state and transient conditions. NTO adsorbed very weakly to the studied soils. Adsorption coefficients (Kds) measured for NTO in a range of soils in batch experiments were less than 1 cm³ g⁻¹. There was a highly significant negative relationship between measured NTO adsorption coefficients and soil pH (P = 0.00011). In kinetic experiments, first order transformation rate estimates ranged between 0.0004 h⁻¹ and 0.0221 h⁻¹. There was a general agreement between batch and column-determined fate and transport parameters. However, transport studies showed an increase in the NTO transformation rate as a function of time, possibly indicating microbial growth.
    • Linking shorebird and marsh bird habitat use to water management in anthropogenic and natural wetlands in the Colorado River Delta

      Glenn, Edward P.; Gómez Sapiens, Martha Marina; Flessa, Karl W.; Fitzsimmons, Kevin; Glenn, Edward P. (The University of Arizona., 2014)
      I estimated patterns of shorebird abundance and species diversity in the Colorado River Delta and Upper Gulf of California wetlands in order to determine the relative contribution of intertidal wetlands and non-tidal anthropogenic wetlands to support shorebird habitat use. Species richness varied from 15 to 26 species among sites and 29 species were detected across sites. Density during the peak migration month was higher at the anthropogenic wetland Cienega de Santa Clara (mean = 168 ind/ha, 95% C.I. 29-367), and the intertidal Golfo de Santa Clara (mean = 153 ind/ha, 95% C.I. 17-323). Anthropogenic wetlands (playa and lagoons) supported high abundance of shorebirds along with intertidal wetlands in the Colorado River Delta (mudflats). In contrast, intertidal wetlands farther south on the Sonoran Coast presented lower abundance but higher diversity of shorebird, likely as a result of the higher diversity of habitats (rocky shore, sandy beach, estuary). I modeled water management scenarios for the Cienega in order to determine the response of the dominant vegetation (southern cattail, Typha domingensis Pers.) and the area of the outflow pool below the marsh to different scenarios of water management. The model indicates that if the inflow rate is reduced below the current 4-5 m³s⁻¹ the vegetated area of the Cienega that supports habitat for marsh birds would decrease in proportion, as would the area of the outflow pool in the Santa Clara Slough identified previously as shorebird habitat. Increases in salinity will also reduce the vegetated area due to the low salt tolerance of T. domingensis. In winter about 90% of inflow water exits the Cienega into the Santa Clara Slough due to low evapotranspiration contributing to inundate areas that are used by wintering and migrating shorebirds. Lastly, I explored the feasibility of using Vegetation Indices (NDVI and EVI) to model Yuma Clapper Rail detections in the Cienega de Santa Clara as well as the effects of adding other habitat variables and the presence of fire events in the performance of linear models based on NDVI. Both NDVI and EVI were positively related to the Yuma Clapper Rail detections. The relationship was weak to moderate, but significant (P<0.001), which suggests other factors besides the vegetation condition play an important role in the bird distribution pattern. A model including all the variability among years was a better predictor of the rails detected per transect, than models for fire and non-fire years. We did not find a significant effect from adding habitat features (water % or vegetation %), and we recommend to include variables at both microhabitat level and landscape level, relevant before and during the breeding season in order to increase the explanatory power of models.
    • A Comprehensive Approach in Identifying Sources of Contamination, Understanding Water Quality Perception, and Translating Information through Community Outreach in the Upper Gila Watershed in Clifton, Arizona

      Rock, Channah; Rivera, Berenise; Rock, Channah; Maier, Raina; Gerba, Charles P. (The University of Arizona., 2014)
      As of 2010, there are approximately twenty one surface water locations classified as impaired for Escherichia coli (E. coli) contamination in the State of Arizona. Of note is the San Francisco River (SFR) which is currently listed on the US EPA 303d list of impaired waters due to E. coli bacteria present at higher concentrations than the US EPA standards for partial- and full-body contact. In 2010-2011 surface water samples were collected at sites within the impaired region to monitor E. coli and areas known for heavy recreational uses. Of 70 samples collected over 1 year, 81% were positive for universal Bacteroides marker (Allbac). Of the 57 Allbac-positive samples, 68% show contributions of the human-specific marker and 60% were positive for bovine-specific marker. While 28% of the total samples assayed showed elevated levels of E. coli (>235 MPN/100mL), there were minimal significant correlations between Bacteroides and generic E. coli across all samples. While this information is significant, past research has suggested that successfully distinguishing the sources of fecal contamination will not alone reduce or eliminate disease associated with contaminated water unless these investigations are coupled with public outreach and education. With this in mind a survey was developed to gather information about water quality perceptions, water use, peoples' attitudes, knowledge, and behaviors related to the water resources in Clifton, AZ. Survey questions consisted of multiple choice and Likert scales questions and were provided in both English and Spanish and were conducted during the summer of 2012 and winter of 2013. A total of 150 surveys were deployed with 38 surveys completed for a response rate of 25%. Our study findings indicate mixed attitudes on water quality with 80% reporting the SFR has poor water quality for drinking and 39% agree the SFR has poor water quality for swimming. Yet, 84% consider the river safe enough for picnics and activities near the water. Also, it was interesting to note participants' opinions regarding consequences of poor water quality with 66% of respondents indicating that they are concerned with poor water quality and their health. Clifton is a very tight knit community so it was not unexpected that the majority of the respondents (61%) get water quality information by having conversations with other people and 68% from newspapers, factsheets and brochures. Based on the survey responses, our team worked to develop two peer reviewed Extension publications entitled; Microbial Source Tracking: Watershed Characterization and Source Identification (Arizona Cooperative Extension, #AZ1547) and Water Quality, E. coli, and Your Health (#AZ1624). Publications have been developed in both English and Spanish and will be part of future outreach to this and other Arizona communities. It is our goal that these survey findings can be used to better tailor outputs appropriate for the targeted audience, namely the local Hispanic population. These results are important because they add to understanding perceptions of water quality and health risks in this rural community; and can lend towards enhanced outreach practices in other similar communities.
    • Water Quality Assessment of the Santa Cruz River in Southern Arizona

      Rock, Channah M.; McOmber, Todd Christian; Rock, Channah M.; McLain, Jean; Curry, Joan E. (The University of Arizona., 2014)
      Utilization of areas adjacent to rivers for agricultural and industrial purposes can have detrimental effects on water quality and can potentially impact human and ecosystem health downstream. In this study we tested water quality along a stretch of the effluent-dependent Santa Cruz River near Nogales, AZ. This stretch of river has historically been impaired, but prior to upgrades to the Nogales International Wastewater Treatment Plant (NIWTP) in 2009. Our work endeavored to assess water quality according to the Arizona Department of Environmental Quality (ADEQ) standards, and identify sources of pollution entering the river system. Heavy metals were analyzed via ICP. Three IDEXX quantification systems were used to detect E. coli, Enterococcus, and P. aeruginosa as fecal indicators or potential threats to public health. Potential fecal sources were analyzed using quantitative PCR targeting Bacteroides DNA markers for total, human, and bovine feces (Allbac, HF183, and CowM2, respectively). The NIWTP effectively removed chemical and biological contaminants. The lowest E. coli counts occurred at the site located at the NIWTP outfall (mean = 5 MPN/100ml) while the highest counts (mean = 348 MPN/100 ml) came from Nogales Wash, a tributary receiving untreated flow from Nogales, Mexico. The Allbac marker was detected in all samples, while approximately 97% of samples tested positive for HF183 and 33% tested positive for the CowM2 marker. Continued monitoring of effluent effects on river quality and evaluation of water quality degradation will lead to improvements in the management of Arizona's riparian areas and will ultimately contribute to healthy water bodies.
    • The Occurrence and Control of Pathogens on Fomites

      Gerba, Charles P.; Sexton, Jonathan D.; Gerba, Charles P.; Reynolds, Kelly A.; Bright, Kelly R.; Pepper, Ian L. (The University of Arizona., 2013)
      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.
    • Does Increasing Solids Retention Time During the Wastewater Treatment Process Affect the Persistence of Antibiotic Resistance Genes?

      Rock, Channah; Walston, Stefan Eugene; McLain, Jean; Abrell, Leif; Gerrity, Daniel (The University of Arizona., 2013)
      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.
    • Analysis of the Phylogenetic and Functional Dynamics of Microbial Communities in Metalliferous, Acid-Generating Mine Tailings Subject to a Phytostabilization Treatment

      Maier, Raina M.; Valentín-Vargas, Alexis; Maier, Raina M.; Chorover, Jonathan D.; Rich, Virginia I.; Vedantam, Gayatri (The University of Arizona., 2013)
      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.
    • Monitoring Microbial Water Quality via Online Sensors

      Pepper, Ian L.; Sherchan, Samendra Prasad; Pepper, Ian L.; Gerba, Charles P.; Snyder, Shane A. (The University of Arizona., 2013)
      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.
    • Coupled Transport, Fractionation and Stabilization of Dissolved Organic Matter and Rare Earth Elements in the Critical Zone

      Chorover, Jon; Vázquez-Ortega, Angélica; Rasmussen, Craig; McIntosh, Jennifer; Perdrial, Julia; Chorover, Jon (The University of Arizona., 2013)
      It is important to understand the processes that influence the critical zone (CZ) evolution to ensure its sustainability. This thesis reports on laboratory and field experiments designed to measure the behavior of biogenic and lithogenic chemical species and their interaction in the CZ from column to pedon to catchment scales. We postulated that interactions between organic matter and rock-derived metals drive coupled processes of carbon stabilization and chemical weathering and denudation in the Jemez River Basin Critical Zone Observatory (JRB-CZO). First, we observed that secondary mineral coatings (Al and Fe (oxy)hydroxides) on primary silicate surfaces play a major role in sequestering aromatic and "humified" dissolved organic matter (DOM) into sorbate form, significantly retarding their subsurface transport. Further, reinfusion to OM-reacted-porous-media of a different DOM source resulted in exchange reactions consistent with a zonal model of OM adsorption at mineral surfaces. This dissertation also aimed to examine the influence of water and DOM fluxes on the CZ weathering processes. Rare earth elements (REE) were selected because of their coherent trends in reactivity toward organic ligands common to soils. Specifically, trends in REE fractionation were explored for their utility to inform on biogeochemical weathering processes in forested terrain in the JRB-CZO. Mineral weathering mechanisms are expected to differentially influence REE release, fractionation, and transport and the relative importance of such processes should be reflected in REE signatures of bulk soil, pore and surface waters. Our studies showed: (1) REE depletion trends with depth in bulk soils are correlated with topographically-induced variation in water and dissolved organic carbon (DOC) flux (reflected in negative correlations between total water and C fluxes) and solid phase REE concentrations measured at the same depths; (2) REE and DOC concentrations in stream waters were strongly correlated during snowmelt periods of high discharge, consistent with REE complexation and mobilization in association with organic ligands during shallow subsurface flow; (3) preferential sequestration of Eu occurs during formation of secondary Mn(IV)-oxides, explaining patterns of Eu enrichment in bulk soils; and (4) the incremental increase in positive Ce-anomalies with depth in bulk soils are apparently controlled by adsorption/co-precipitation with secondary Fe-(oxy)hydroxide minerals.
    • Quantifying catchment scale soil variability in Marshall Gulch, Santa Catalina Mountains Critical Zone Observatory

      Rasmussen, Craig; Holleran, Molly E.; Chorover, Jon; Guertin, David P. (The University of Arizona., 2013)
      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.
    • Determination, Characterization, and Control Measures of the Agent Causing Early Mortality Syndrome (EMS) also known as Acute Hepatopancreatic Necrosis Syndrome (AHPNS) in Farmed Penaeid Shrimp

      Fitzsimmons, Kevin M.; Lightner, Donald V.; Tran, Loc Huu; Fitzsimmons, Kevin M.; Lightner, Donald V.; Glenn, Edward P.; Gerba, Charles P.; Pantoja, Carlos R. (The University of Arizona., 2013)
      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.
    • Application of Advanced Molecular Techniques in Applied Environmental Microbiology

      Pepper, Ian L.; Gerba, Chuck P.; Iker, Brandon Charles; Rock, Channah; Bright, Kelly; Pepper, Ian L.; Gerba, Chuck P. (The University of Arizona., 2013)
      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.
    • The Assessment of Escherichia coli as an Indicator of Microbial Quality of Irrigation Waters used for Produce

      Rock, Channah; Brassill, Natalie A.; McLain, Jean E.; Gerba, Charles; Nolte, Kurt (The University of Arizona., 2013)
      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.
    • Drinking Water Quality Monitoring

      Gerba, Charles P.; Kilungo, Aminata Peter; Gerba, Charles P.; Pepper, Ian; Bright, Kelly (The University of Arizona., 2013)
      This dissertation involves two different studies. The first concerns the real-time detection of microbial contamination in drinking water using intrinsic fluorescence of the microorganisms. The prototype, “Blinky”, uses LEDs that emit light at 365nm, 590nm, and 635nm for ultraviolet, amber, and red light, respectively. At 365 nm, the cellular components excited include reduced pyridine nucleotides (RPNs), flavins, and cytochromes to distinguish viable bacteria; at 590 nm, the cellular components excited include cytochromes for non-viable bacteria; at 635 nm, the cellular components excited include calcium dipicolinic acid (DPA) for spores. By using these three different wavelengths, the prototype can differentiate between viable and non-viable organisms and also has the potential to detect spores. The aim of this study was to improve the detection limit by modifying the design of the instrument and to establish the detection limit of viable and non-viable bacteria and spores. The instrument was modified by replacing existing LEDs with LEDs that had 50% more intensity. Two additional LEDs were added for amber and red light, bringing the total to four LEDs for each. The LEDs were also positioned closer to the photomultiplier tube so as to increase sensitivity. For UV, only two LEDs were used as previous. The detection limit of the viable bacteria was ~50 live bacteria/L. No change in the intrinsic fluorescence below the concentration of ~10⁸ dead bacteria/L was observed. The results for spore measurements suggested that most of the spores had germinated before or during the measurements and could not be detected. The instrument was successful in detection of viable bacteria and also differentiating viable and non-viable bacteria. The instrument was not successful in detection of spores. The second study was designed to assess the water quality of well construction in southeastern Tanzania. Three designs were tested: Msabi rope pump (lined borehole and covered), an open well converted into a closed well (uncovered well into a covered and lined well), and an open well (uncovered and may or may not be lined). The study looked at the microbial and chemical water quality, as well as turbidity. The survey included 97 water collection points, 94 wells and three rivers. For microbial analysis, heterotrophic plate count (HPC), total coliforms and E. coli tests were performed. Fifteen of these wells were further analyzed for microflora and diversity for wells comparison purposes, using culture methods, followed by polymerase chain reaction (PCR) and genome sequencing. Ten wells out of the fifteen were analyzed for calcium (water hardiness), potassium, nitrates, nitrites, chloride, fluoride, bromide, sulfate, iron, and arsenic. Two water collection points were also selected for organic compound analysis (gasoline components). All samples tested positive for coliforms. Two samples tested positive for Escherichia coli for the lined borehole (Msabi rope pump) and four samples from closed wells. All open wells tested positive for E. coli. There was more microbial diversity in open wells than the closed wells and Msabi rope pumps. Potential bacterial pathogens were detected in seven wells out of the fifteen examined. The wells that tested positive were one Msabi rope pump, one closed well; the rest were from open water sources. Open wells had high turbidity followed by closed wells. Msabi rope pumps had low turbidity comparing to the two wells designs. No traces of gasoline components were detected in any of the water sources. One well out of ten had high amounts of nitrates-nitrogen (> 10 mg/L). The results of this study showed that the Msabi rope pumps performed comparably to the closed wells in terms of microbial quality but performed better with regard to turbidity. The open wells performed poorly in terms of microbial water quality as well and turbidity. There was a statistical difference in HPC, total coliforms, E.coli numbers and turbidity between open wells, closed wells and the Msabi rope pumps. However, there was no statistical difference in HPC, total coliforms and E.coli numbers between the closed wells and Msabi rope pumps. Msabi rope pumps performed better in turbidity
    • Chlorophyll Fluorescence Response to Water and Nitrogen Deficit

      Moran, Mary Susan; Papuga, Shirley Anne; Cendrero Mateo, Maria del Pilar; Moran, Mary Susan; Papuga, Shirley Anne; van Leeuwen, Willem; Monson, Russell K.; Thorp, Kelly; Moreno, Jose (The University of Arizona., 2013)
      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.
    • Measuring Air-Water Interfacial Area in Unsaturated Porous Media Using the Interfacial Partitioning Tracer Test Method

      Brusseau, Mark L.; El Ouni, Asma; Curry, Joan E.; Zhong, Hua (The University of Arizona., 2013)
      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.
    • Transfer of Microorganisms from Fomites to Hands and Risk Assessment of Contaminated and Disinfected Surfaces

      Gerba, Charles P.; Lopez, Gerardo Urquijo; Reynolds, Kelly A.; Pepper, Ian L.; Bright, Kelly R.; Gerba, Charles P. (The University of Arizona., 2013)
      It is now widely accepted that surface contamination plays an important role in the transmission of both respiratory and gastrointestinal infections in the domestic environment and community setting. The efficiency of transfer of a pathogen to the hand from a fomite is important in modeling transmission in microbial risk assessment models. The objective of this study was to use published literature to assess the role of fomites and hands in disease transmission, and to conduct fomite-to-finger transfer studies from various porous and nonporous fomites under different relative humidity condition using non-pathogenic strains of Escherichia coli, Staphylococcus aureus, MS2 coliphage, Bacillus thuringiensis spores, and poliovirus 1; to evaluate the persistence of bacteria and viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to conduct a foodborne quantitative microbial risk assessment using Campylobacter jejuni from the data obtained in these studies. It was found that numerous factors influence the transfer efficiency of microorganisms, with moisture being the most important, with greater transfer under humid conditions. Other factors influencing transfer include drying time, contact time, pressure, friction, type of material, and porosity of the fomite. Percent transfer was greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer under high relative humidity (40 - 65%) compared to low relative humidity (15 - 32%). Relative humidity and fomite type influenced the survival of all studied organisms; survival was greater on nonporous surfaces than those for porous surfaces. Test organisms were reduced up to 99.997% on the fomites after the surfaces were wiped with a disinfectant wipe. Microbial fomite-to-finger transfer from disinfectant wipe-treated surfaces were, lower than from non-treated surfaces. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%.
    • Bacterial Diversity of the Atacama Desert, Chile: The Challenges of Characterizing the Community Dynamics of Extreme Oligotrophic Ecosystems

      Maier, Raina M.; Neilson, Julia Worsley; Pepper, Ian L.; Rasmussen, Craig; Arnold, Anne Elizabeth; Maier, Raina M. (The University of Arizona., 2012)
      This dissertation examines the bacterial diversity of hyperarid and arid regions of the Atacama Desert, Chile, as a first step towards understanding the global biogeochemical significance of arid-land microbial communities. The specific objectives were to characterize bacterial diversity and infer the possible metabolic potential of these bacterial communities, and to evaluate the influence of moisture exposure on community structure. In addition, the strengths and limitations of available tools for probing microbial diversity and activity in terrestrial ecosystems were characterized for their application to extreme oligotrophic communities. Preliminary PCR-DGGE analysis of a west-east elevational transect from the Pacific Ocean near Antofagasta to the western slopes of the central Andes indicated that bacterial communities along this transect belonged to two distinct community types: 1) hyperarid (700 - 2000 m) and 2) arid (2500 - 4500 m) communities that included both vegetated and unvegetated regions. Subsequent diversity analysis of these two regions revealed novel but distinct communities in both regions. A greater diversity was observed in the unvegetated arid regions than in the unvegetated hyperarid areas. The unvegetated arid sites were characterized by a bacterial community harboring a combination of radiotolerant and halotolerant heterotrophs as wells as diverse phylotypes closely related to chemolithoautotrophs. These rare phylotypes may be uniquely adapted to arid ecosystems. Molecular tools evaluated for community diversity analysis included PCR-DGGE, Sanger-clone and 454-pyrosequencing analysis of 16S rRNA gene libraries, and the use of reverse transcriptase quantitative PCR (RT-qPCR) for quantifying the impact of environmental variables on the metabolic activity of a specific organism. These techniques were evaluated using the ecosystems of the Atacama Desert as well as model ecosystems designed to address specific questions. Molecular tools are invaluable to the study of microbial ecology because they facilitate the study of fastidious organisms that are difficult or impossible to culture, but the analysis presented in this dissertation demonstrates that each of these methods has limitations and biases which must be acknowledged to avoid inaccurate conclusions from skewed results. The most complete picture of the taxonomic and functional profile of a microbial community is obtained by employing a combination of molecular techniques.