The need for rapid, specific and sensitive assays that provide a detection of bacterial indicators are important for monitoring water quality. Rapid detection using biosensor is a novel approach for microbiological testing applications. Besides, validation of rapid methods is an obstacle in adoption of such new bio-sensing technologies. In this study, the strategy developed is based on using the compound 4-methylumbelliferyl glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-D-glucuronidase (GUD) enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells present in water samples. The detection time required for the biosensor response ranged from 30 to 120 minutes, depending on the number of bacteria. The specificity of the MUG based biosensor platform assay for the detection of E. coli was examined by pure cultures of non-target bacterial genera and also non-target substrates. GUD activity was found to be specific for E. coli and no such enzymatic activity was detected in other species. Moreover, the sensitivity of rapid enzymatic assays was investigated and repeatedly determined to be less than 10 E. coli cells per reaction vial concentrated from 100 mL of water samples. The applicability of the method was tested by performing fluorescence assays under pure and mixed bacterial flora in environmental samples. In addition, the procedural QA/QC for routine monitoring of drinking water samples have been validated by comparing the performance of the biosensor platform for the detection of E. coli and culture-based standard techniques such as Membrane Filtration (MF). The results of this study indicated that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. The procedural QA/QC of the biosensor will provide both industry and regulatory authorities a useful tool for near real-time monitoring of E. coli in drinking water samples. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.

Bacteria of the Legionella genus are a water-borne pathogen of increasing concern due to being responsible for more annual drinking water related disease outbreaks in the United States than all other microbes combined. Unfortunately, the development of public health policies concerning Legionella has impeded by several key factors, including a paucity of data on their interactions and growth requirements in water distribution networks, a poor understanding of potential transmission sources for legionellosis, and limitations in current methodology for the characterization of these pathogens. To address these issues, a variety of research approaches were taken. By measuring Legionella survival in tap water, association in pipe material biofilms, population dynamics in a model distribution system, and occurrence in drinking water distribution system biofilms, key aspects of Legionella ecology in drinking water systems were revealed. Through a series of experiments qualitatively and quantitatively examining the growth of Legionella via nutrients obtained from several water sources, environmental nutritional requirements and capability for growth in the absence of host organisms were demonstrated. An examination of automobile windshield washer fluid as a possible source of legionellosis transmission revealed Legionella survival in certain windshield washer fluids, growth within washer fluid reservoirs, high levels and frequency of contamination in washer fluid reservoirs, and the presence of viable cells in washer fluid spray, suggesting the potential for exposure to Legionella from this novel source. After performing a systematic and quantitative analysis of methodology optimization for the analysis of Legionella cells via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, several strains of this microbe isolated from separated and varied environmental water sampling sites were distinctly typed, demonstrating a potential application of this technology for the characterization of Legionella. The results from this study provide novel insight and methodology relevant to the development of programs for the monitoring and treatment of Legionella in drinking water systems.

Research in microbial biofuels has dramatically increased over the last decade. The bulk of this research has focused on increasing the production yields of cyanobacteria and algal cells and improving extraction processes. However, there has been little to no research on the potential impact of viruses on the yields of these phototrophic microbes for biofuel production. Viruses have the potential to significantly reduce microbial populations and limit their growth rates. It is therefore important to understand how viruses affect phototrophic microbes and the prevalence of these viruses in the environment. For this study, phototrophic microbes were grown in glass bioreactors, under continuous light and aeration. Detection and quantification of viruses of both environmental and laboratory microbial strains were measured through the use of a plaque assay. Plates were incubated at 25º C under continuous direct florescent light. Several environmental samples were taken from Tempe Town Lake (Tempe, AZ) and all the samples tested positive for viruses. Virus free phototrophic microbes were obtained from plaque assay plates by using a sterile loop to scoop up a virus free portion of the microbial lawn and transferred into a new bioreactor. Isolated cells were confirmed virus free through subsequent plaque assays. Viruses were detected from the bench scale bioreactors of Cyanobacteria Synechocystis PCC 6803 and the environmental samples. Viruses were consistently present through subsequent passage in fresh cultures; demonstrating viral contamination can be a chronic problem. In addition TEM was performed to examine presence or viral attachment to cyanobacterial cells and to characterize viral particles morphology. Electron micrographs obtained confirmed viral attachment and that the viruses detected were all of a similar size and shape. Particle sizes were measured to be approximately 50-60 nm. Cell reduction was observed as a decrease in optical density, with a transition from a dark green to a yellow green color for the cultures. Phototrophic microbial viruses were demonstrated to persist in the natural environment and to cause a reduction in algal populations in the bioreactors. Therefore it is likely that viruses could have a significant impact on microbial biofuel production by limiting the yields of production ponds.

This study was devised to elucidate key information concerning the potential risk posed by Legionella in reclaimed water. A series of biological experiments and a recharge basin soil column study were conducted to examine the survival, growth, and transport of L. pneumophila through engineered reclaimed water systems. A pilot-scale, column study was set up to measure Legionella transport in the columns under Arizona recharge basin conditions. Two columns, A and B, were packed to a depth of 122 cm with a loamy sand media collected from a recharge basin in Mesa, Arizona. The grain size distribution of Column A differed from that of Column B by the removal of fines passing the #200 sieve. The different soil profiles represented by column A and B allowed for further investigation of soil attributes which influence the microbial transport mechanism. Both clear PVC columns stand at a height of 1.83 m with an inner diameter of 6.35 cm. Sampling ports were drilled into the column at the soil depths 15, 30, 60, 92, 122 cm. Both columns were acclimated with tertiary treated waste water and set to a flow rate of approximately 1.5 m/d. The columns were used to assess the transport of a bacterial indicator, E. coli, in addition to assessing the study's primary pathogen of concern, Legionella. Approximately, 〖10〗^7 to 〖10〗^9 E. coli cells or 〖10〗^6 to 〖10〗^7Legionella cells were spiked into the columns' head waters for each experiment. Periodically, samples were collected from each column's sampling ports, until a minimum of three pore volume passed through the columns.

The pilot-scale, column study produced novel results which demonstrated the mechanism for Legionella to be transported through recharge basin soil. E. coli was transported, through 122 cm of the media in under 6 hours, whereas, Legionella was transported, through the same distance, in under 30 hours. Legionella has been shown to survive in low nutrient conditions for over a year. Given the novel results of this proof of concept study, a claim can be made for the transport of Legionella into groundwater aquifers through engineering recharge basin conditions, in Central Arizona.

Granular activated carbon (GAC) filters are final polishing step in the drinking water treatment systems for removal of dissolved organic carbon fractions. Generally filters are colonized by bacterial communities and their activity reduces biodegradable solutes allowing partial regeneration of GAC's adsorptive capacity. When the bacteria pass into the filtrate due to increased growth, microbiological quality of drinking water is compromised and regrowth in the distribution system occurs. Bacteria attached to carbon particles as biofilms or in conjugation with other bacteria were observed to be highly resistant to post filtration microbial mitigation techniques. Some of these bacteria were identified as pathogenic.

This study focuses on one such pathogen Legionella pneumophila which is resistant to environmental stressors and treatment conditions. It is also responsible for Legionnaires' disease outbreak through drinking water thus attracting attention of regulatory agencies. The work assessed the attachment and colonization of Legionella and heterotrophic bacteria in lab scale GAC media column filters. Quantification of Legionella and HPC in the influent, effluent, column's biofilms and on the GAC particles was performed over time using fluorescent microscopy and culture based techniques.

The results indicated gradual increase in the colonization of the GAC particles with HPC bacteria. Initially high number of Legionella cells were detected in the column effluent and were not detected on GAC suggesting low attachment of the cells to the particles potentially due to lack of any previous biofilms. With the initial colonization of the filter media by other bacteria the number of Legionella cells on the GAC particles and biofilms also increased. Presence of Legionella was confirmed in all the samples collected from the columns spiked with Legionella. Significant increase in the Legionella was observed in column's inner surface biofilm (0.25 logs up to 0.52 logs) and on GAC particles (0.42 logs up to 0.63 logs) after 2 months. Legionella and HPC attached to column's biofilm were higher than that on GAC particles indicating the strong association with biofilms. The bacterial concentration slowly increased in the effluent. This may be due to column's wall effect decreasing filter efficiency, possible exhaustion of GAC capacity over time and potential bacterial growth.

Nanotechnology is a scientific field that has recently expanded due to its applications in pharmaceutical and personal care products, industry and agriculture. As result of this unprecedented growth, nanoparticles (NPs) have become a significant environmental contaminant, with potential to impact various forms of life in environment. Metal nanoparticles (mNPs) exhibit unique properties such as increased chemical reactivity due to high specific surface area to volume ratios. Bacteria play a major role in many natural and engineered biogeochemical reactions in wastewater treatment plants and other environmental compartments. I have evaluated the laboratory isolates of E. coli, Bacillus, Alcaligenes, Pseudomonas; wastewater isolates of E. coli and Bacillus; and pathogenic isolate of E. coli for their response to 50 & 100 nm sized Cu nanoparticles (CuNPs). Bactericidal tests, scanning electron microscopy (SEM) analyses, and probable toxicity pathways assays were performed. The results indicate that under continuous mixing conditions, CuNPs are effective in inactivation of the selected bacterial isolates. In general, exposure to CuNPs resulted in 4 to >6 log reduction in bacterial population within 2 hours. Based on the GR, LDH and MTT assays, bacterial cells showed different toxicity elicitation pathways after exposure to CuNPs. Therefore, it can be concluded that the laboratory isolates are good candidates for predicting the behavior of environmental isolates exposed to CuNPs. Also, high inactivation values recorded in this study suggest that the presence of CuNPs in different environmental compartments may have an impact on pollutants attenuation and wastewater biological treatment processes. These results point towards the need for an in depth investigation of the impact of NPs on the biological processes; and long-term effect of high load of NPs on the stability of aquatic and terrestrial ecologies.

Bacteroides have been suggested as alternative indicators of fecal pollution since they are highly abundant in feces and are thought to have limited potential to grow in environment. However, recent literature suggests that Bacteroides can potentially survive within water distribution systems. The first objective of this study was therefore to investigate the validity of Bacteroides as a fecal indicator for drinking water through laboratory experiments and field studies. Experiments were performed using a laboratory scale PVC model water distribution system that was spiked with 109 Bacteroides. Samples were collected over the following four and analyzed by culture and molecular-based techniques. Second, field studies were performed by collecting water meters from two large chlorinated water distribution systems in central Arizona. Upon removal for repair by city personnel, meters were collected and biofilms samples were gathered within two hours. The biofilms were then analyzed using culture and molecular-based assays. The results from these studies support the hypothesis that Bacteroides DNA may be found in water distribution systems despite the difficulty of cultivating these bacterial cells. These experiments present the importance of considering biofilm interactions with fecal indicator bacteria when performing molecular assays on environmental samples, as biofilms may provide protection from high oxygen concentrations and grazing protozoa in bulk water that limit the persistence Bacteroides in the environment. Although the significance of biofilm interactions with surface or recreational waters may be small, they are likely important when considering drinking water delivered through distribution systems. The second objective of this study was to investigate alternative detection methodologies for the fecal indicator Bacteroides. In particular, this study focused on using a simplified protocol of Nucleic Acid Sequence Based Amplification (NASBA) and Thermophilic Helicase-Dependent Amplification (tHDA) to amplify the highly conserved 16s rRNA gene in the genomic DNA of fecal indicator Bacteroides. The results of this study show that the simplified NASBA procedure was not able to amplify the target, while continuous problems with tHDA exposed the methods lack of reliability. These results suggest higher reliability in the isothermal amplification methods needs to be achieved before application to environmental samples.