The Aim of this study is to investigate human viral contamination in urban rivers and its impact on coastal waters of southern California. During the first year study, three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR at eleven rivers and creeks along southern California coast. Fecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both fecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the coastal water quality than following storm events. During the second year of study, a detailed seasonal assessment of microbial pollution was conducted in two major Southern California urban rivers, San Gabriel and Los Angeles. A total of 114 river samples from five different sample locations along these two rivers were collected over a one-year period and analyzed for fecal indicator bacteria, including total coliform, fecal coliform, and enterococcus and indicator viruses, including F-specific and somatic coliphage, and human entero and adenoviruses. Based on the California recreational water quality standards, 52.5% of the samples exceeded at least one fecal indicator bacteria standard. Both somatic and F-specific coliphage were detected in higher concentrations (up to 5x102 PFU/100ml) during storm events. Enterovirus was detected in ~13% of the samples by reverse-transcription PCR. Adenovirus was detected by real-time PCR in approximately 50% of the samples, with concentrations ranging from 10 to >106 genomes per liter. However, plaque assay using two human tissue culture cell lines, HEK-293A and A549, yielded negative results suggesting adenoviruses detected by real-time PCR may not be infectious. Correlations between human adenoviruses and F-specific (p=0.13) and somatic coliphages (p=0.06) were found. However, these correlations were not statistically significant at 95% level. No significant correlation between human adenoviruses and fecal indicator bacteria (total coliform, fecal coliform, and enterococcus) was observed. This study presents the first quantitative measure of human adenoviruses in urban rivers and their statistical relationship to fecal indicator bacteria and coliphages. This study also provides the first direct evidence that human viruses are prevalent in southern California urban rivers.

The effects of climate change, population growth, and future hydrologic uncertainties necessitate increased water conservation, new water resources, and a shift towards sustainable urban water supply portfolios. Diversifying water portfolios with non-traditional water sources can play a key role. Rooftop harvested rainwater, stormwater, recycled wastewater and greywater, desalinated seawater and brackish water, and atmospheric and condensate harvesting are all currently utilized and rapidly emerging non-traditional water sources. Given the unique challenges and various pros and cons of each individual source, quantitative models and process analyses highlight the strength of comparative assessments across scenarios and water supply options to ultimately aid in decision making efforts. Larger frameworks including technoeconomic assessment (TEA) and quantitative microbial risk assessment (QMRA) paired with new, scenario specific efforts can quantify priority metrics to highlight areas of both improvement and concern. Through this research, three different non-traditional water sources were approached individually to demonstrate three aims: 1) assessment of processes, layouts, and local factors for specific facilities to identify opportunities for capital cost reduction and conditions required to adopt seawater reverse osmosis desalination as a water source; 2) locally conducted surveys and water sample collection used to both quantify health risks and local perception of water use and quality for harvested rainwater after major tropical hurricanes; and 3) implementation of a dose-response model accounting for antibiotic resistance to produce a new outcome of quantified health risks to augment our understanding of risk-based regulations for non-potable reclaimed wastewater. The outcomes of this research will provide better understanding of cost origins, health risks, and where critical efforts are needed to improve design and use of these water sources.

Rapid and sensitive monitoring of water quality can effectively prevent accidental human exposure to waterborne pathogens. In this project, a rapid quantification method was developed for Enterococcus faecalis, a fecal indicator bacterium (FIB), used as a surrogate for water quality assurance. The loop-mediated isothermal amplification (LAMP) assay was combined with a microfluidic technology to produce thousands of individual reactions that convert the qualitative LAMP assay to quantitative results based on Poisson distribution. This “Lab on a Chip” (LOC) assay is considerably more rapid than the conventional culture-based and the polymerase chain reaction (PCR) based methods (i.e. qPCR and droplet digital PCR). The isothermal amplification removes the need of thermal cycling, which makes it suitable for field development. In this assay, the water sample is mixed with LAMP reagents in the presence of fluorescent dye. The mixture is then dispersed into thou- sands of droplets encapsulated within the oil phase using a microfluidic droplet generator chip, where each droplet acts as an individual LAMP reaction. By counting the number of positive droplets among total droplets in the viewing field, the most probable number of the E. faecalis can be quantified statistically. Several types and compositions of oil phases were tested for the compatibility with sample/LAMP reagent mixture for droplet generation and stability. The optimized oil phase can generate monodisperse droplets of 55 μm in diameter, which were stable during LAMP reaction at 65◦C for 30 mins. The droplets that stained with dsDNA fluorescent dye could be clearly visualized using fluorescence microscope and counted by image J software. The limit of detection of droplet LAMP assay is 4 cfu/reaction in pure culture.

The primary concern associated with water reuse practices is the public health risk caused by the potential exposure to microbial contaminants, such as pathogenic protozoa, bacteria and viruses. The detection and removal of microbial pathogens is therefore of great importance to ensure the reclaimed water is “safe” for the intended end uses. This research studied the microbial pathogen removal in three different water reuse schemes. In the first study, a rapid direct virus detection method based on Accuri C6 flow cytometry (FCM) was developed to quantify the virus removal rate in a water reclamation plant using microfiltration-reverse osmosis (MF-RO) process for indirect potable reuse water production. This new method, in combination with online total organic carbon and nanoparticle analysis, has shown to be a viable way for online monitoring of high-pressure RO membrane integrity and the potential breakthrough of viral particles. In the second study, the effectiveness of microbial pathogen removal by constructed stormwater wetlands (CSW) in the U.S. and Australia was investigated using digital droplet PCR (ddPCR) and 454-pyrosequcing techniques. The results showed that the two US CSW and one of the three Australian CSW had good performance in terms of indicator bacteria removal during dry weather flow. The treated stormwater can meet the recreational water quality criteria/guidelines. No Cryptosporidium was detected in any of the CSW, while Adenovirus were present at all sites. Human specific HF183 Bacteroides were only found in Australian sites indicating the potential contamination from sewage ingression. The microbial community analysis showed a clear increase of Cyanobacteria in the outflow of CSW with better performance. The water residence time was determined as a critical factor affecting the efficacy of microbial pathogen removal or inactivation. In the last study, pathogen removal efficiency was investigated in a solar-powered mobile toilet system for decentralized wastewater treatment. The wastewater electrolysis cell (WEC) uses solar energy to generate oxidants via electrochemical (EC) reaction for of microbial pathogen disinfection. The results showed 5 log10 reductions of bacteria (E. coli and Enterococcus) and viruses (coliphage MS2 and adenovirus) were achieved within 1 h reaction at applied cell voltage of +4V. The dominating role of free reactive chlorine generated in situ during EC disinfection process was verified using laboratory model waters. The formation of organic disinfection byproducts trihalomethanes (THMs) and haloacetic acids (HAA5) during EC treatment were found to increase with the rise of applied cell voltage. The EC treated toilet wastewater is suitable for many non-potable reuse applications (e.g., toilet flushing and irrigation) with significantly reduced microbial infection risk. As there is no need for supporting chemicals, the WEC system can be developed into commercial viable, self-sufficient, solar-powered mobile toilets for decentralized wastewater treatment.

Coastal seawater desalination using reverse osmosis (RO) membranes has the potential to alleviate water stress in arid regions. However, membrane biofouling, caused by bacterial biofilm formation, is a significant challenge for seawater desalination plants. Biofilm formation is regulated by quorum sensing (QS) pathways where bacteria secrete auto-inducer molecules to communicate with neighboring bacteria to activate biofilm formation. This research investigated the role of the QS system and the effect of QS inhibiting (QSIs) compounds on marine biofilm production and membrane biofouling. This study revealed that four different marine bacteria isolated from fouled RO membranes in a desalination plant produced two low molecular weight auto-inducer 1 (AI-1) QS molecules. Vanillin and cinnamaldehyde were then identified as the most effective QSI compounds with reduction of marine biofilm formed by RO membrane biofouling isolates and native uncultured seawater bacterial communities by more than 79% and 70%, respectively in a microtiter plate assay. Further investigation into the anti-biofouling capabilities of vanillin and cinnamaldehyde in a cross-flow membrane bio-monitoring system indicated that vanillin in the bulk fluid (1200 mg/L) significantly reduced extracellular polysaccharides (>40%) and dead cells (>20%) on the RO membrane surface. In order to improve the membrane in-situ anti-biofouling potential, vanillin and cinnamaldehyde were physically adsorbed onto various RO membrane surfaces. The addition of the QSI layer on the RO membrane surface significantly altered the membrane surface contact angle along with a less than 16% reduction in pure water permeability, but there was no significant change in salt rejection compared to unmodified membranes. Under biofouling conditions consisting of four mixed marine bacterial species in a high pressure RO system, QSI modified membranes experienced a minimal loss in permeate flux compared to unmodified membranes. Extracellular polysaccharide production, live cells, and dead cells were significantly suppressed on vanillin and cinnamaldehyde modified membrane surfaces by more than 15%, 58%, and 61%, respectively. These findings indicate that QSIs have the potential to suppress marine biofilm formation and membrane biofouling for seawater desalination.

Aims: To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA. Methods and Results: Three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR from 11 rivers and creeks. Faecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both faecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the quality of coastal water than following storm events. Conclusions: This study provides the first direct evidence that human viruses are prevalent in southern California urban rivers. Urban run-off impacts coastal water quality most significantly during the storm season. Significance and Impact of the Study: To protect human health during water recreational activities, it is necessary to develop effective strategies to manage urban run-off during storm events.

Real-time PCR was applied to quantify the abundance of human adenoviruses in two southern California urban rivers, the San Gabriel and Los Angeles. A total of 114 river samples from five different locations were collected over a 1-year period and analyzed for human adenoviruses, along with fecal indicator bacteria and coliphages. Adenoviruses were detected by real-time PCR in similar to 16% of the samples, with concentrations ranging from 10(2) to 10(4) genomes per liter. However, a plaque assay using two human tissue culture cell lines, HEK-293A and A549, yielded negative results, suggesting that adenoviruses detected by real-time PCR are likely noninfectious. Enterovirus genome was detected in similar to 7% of the samples by reverse transcription-PCR. Analysis by Spearman's rho rank order correlation showed significant correlations between fecal indicator bacteria and indicator virus (total coliform, fecal coliform, enterococcus, and coliphage values). However, no significant correlations were found between human adenoviruses quantified by real-time PCR and culturable coliphages or fecal indicator bacteria. Kruskall-Wallis chi-square analysis showed significant seasonal variability of all fecal indicator bacteria and coliphages, while no significant variability was observed for adenoviruses or enteroviruses. This study presents the first quantitative measurement of human adenovirus genomes in urban rivers and their statistical relationship to fecal indicator bacteria and coliphages. The uncoupling between high-number genome copies of adenoviruses detected by real-time PCR and the absence of infectivity detected by tissue culture suggests that genome-based detection methods are inadequate for direct assessment of human health risk.

Increases in frequency and intensity of freshwater cyanobacterial blooms (cyanoHABs) place significant challenges to drinking water plants for human health protection from exposure to algal toxins. Specifically, microcystins (MC), the hepatotoxin produced by cyanobacteria, is not effectively removed by the current water treatment processes. The goal of this study is to isolate and characterize bacteria that are capable of degrading MC in order to develop biofilters that can effectively remove the toxin. Water and sediment samples from a drinking water reservoir and a freshwater pond were collected and enriched for MC degrading bacteria. Among nearly 100 fast growing bacterial isolates were obtained from the enrichment culture and identified by gene sequencing, none were MC degraders due to the carryover of the trace amount of ethanol in the MC stock that were used by fast growing non-degraders. On the contrary, the bacterial consortia obtained from enrichment cultures can efficiently degrade MC. Although both consortia enriched in the presence or absence of ethanol can perform MC degradation, the degradation rates were improved for most of the consortia when the ethanol was removed from the enrichment culture in spite of lower cell growth rate. This indicates that the fast growing bacteria in the enrichment culture are not the key players in the degradation of MC. Bacterial community analysis also confirmed the higher diversity in consortia associated with ethanol. However, there is not a clear separation of bacterial diversity between the MC degrading and non-degrading consortia, indicating the complexity of consortia in MC degradation.

Antibiotic resistance has emerged as a critical global health threat in the 21st century, leading to untreatable infectious diseases and significantly increasing human morbidity and mortality. Rapid, portable, and user-friendly detection of antibiotics, microbial pathogens, and antibiotic-resistant organisms in aquatic environment plays a key role in controlling the spread of diseases and combating the emergence of antibiotic resistance. Unfortunately, current environmental monitoring practices are tedious, expensive and slow. These limitations impede timely detection and response to emerging threats, allowing the spread of diseases and antibiotic-resistant pathogens to human populations. This dissertation research aims to develop environmental monitoring strategies that streamline the current monitoring timeline and make it accessible to a wider range of users. This effort will contribute to more frequent and efficient monitoring of antibiotics, microbial pathogens, and antibiotic-resistant organisms in water matrices. Through this research, I first investigated the application of Surface Enhanced Raman Scattering (SERS) for label-free monitoring of antibiotics in wastewater. By utilizing carefully designed SERS substrates and artificial intelligence (AI) algorithms, I successfully quantified the target antibiotics even in the presence of other organic and inorganic molecules in wastewater. Next, I developed a portable and semi-automatic virus detection centrifugal microfluidic disc (CD) that integrates sample concentration, purification, amplification, and quantification steps for environmental water monitoring. The on-CD assay is completed in less than 1.5 hours, and its performance is comparable to that of the benchtop in-tube assay. Lastly, building upon the foundation of the virus detection CD system, I further designed a microfluidic CD that integrates the phenotypic bacterial culture with genotypic pathogen identification for monitoring of antibiotic resistant bacteria. This CD system successfully detected indigenous antibiotic-resistant bacteria in raw wastewater in under 3 hours. This dissertation serves as an important foundation for developing more advanced, field-applicable techniques and methodologies in environmental monitoring.