Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

Quantification of natural organic matter (NOM) in water is limited by the complex and varied nature of compounds found in natural waters. Current characterization techniques, which identify and quantify fractions of NOM, are often expensive and time consuming suggesting the need for rapid and accurate characterization methods. In this work, principal component analysis of fluorescence excitation-emission matrices (FEEM-PCA) was investigated as a NOM characterization technique. Through the use of jar tests and disinfection by-product formation tests, FEEM-PCA was shown to be a good surrogate for disinfection by-product precursors. FEEM-PCA was also applied in order to characterize differences in humic-like, protein-like, and Rayleigh scattering between multiple source waters and due to differing treatment processes. A decrease in Rayleigh scattering influence was observed for a deep lake intake, and multiple processes were found to significantly affect humic-like substances, protein-like, and Rayleigh scattering fractions.

Quantification of natural organic matter (NOM) in water is limited by the complex and varied nature of compounds found in natural waters. Current characterization techniques, which identify and quantify fractions of NOM, are often expensive and time consuming suggesting the need for rapid and accurate characterization methods. In this work, principal component analysis of fluorescence excitation-emission matrices (FEEM-PCA) was investigated as a NOM characterization technique. Through the use of jar tests and disinfection by-product formation tests, FEEM-PCA was shown to be a good surrogate for disinfection by-product precursors. FEEM-PCA was also applied in order to characterize differences in humic-like, protein-like, and Rayleigh scattering between multiple source waters and due to differing treatment processes. A decrease in Rayleigh scattering influence was observed for a deep lake intake, and multiple processes were found to significantly affect humic-like substances, protein-like, and Rayleigh scattering fractions.

Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

abstract: Disinfection byproducts are the result of reactions between natural organic matter (NOM) and a disinfectant. The formation and speciation of DBP formation is largely dependent on the disinfectant used and the natural organic matter (NOM) concentration and composition. This study examined the use of photocatalysis with titanium dioxide for the oxidation and removal of DBP precursors (NOM) and the inhibition of DBP formation. Water sources were collected from various points in the treatment process, treated with photocatalysis, and chlorinated to analyze the implications on total trihalomethane (TTHM) and the five haloacetic acids (HAA5) formations. The three sub-objectives for this study included: the comparison of enhanced and standard coagulation to photocatalysis for the removal of DBP precursors; the analysis of photocatalysis and characterization of organic matter using size exclusion chromatography and fluorescence spectroscopy and excitation-emission matrices; and the analysis of photocatalysis before GAC filtration. There were consistencies in the trends for each objective including reduced DBP precursors, measured as dissolved organic carbon DOC concentration and UV absorbance at 254 nm. Both of these parameters decreased with increased photocatalytic treatment and could be due in part to the adsorption to as well as the oxidation of NOM on the TiO2 surface. This resulted in lower THM and HAA concentrations at Medium and High photocatalytic treatment levels. However, at No UV exposure and Low photocatalytic treatment levels where oxidation reactions were inherently incomplete, there was an increase in THM and HAA formation potential, in most cases being significantly greater than those found in the raw water or Control samples. The size exclusion chromatography (SEC) results suggest that photocatalysis preferentially degrades the higher molecular mass fraction of NOM releasing lower molecular mass (LMM) compounds that have not been completely oxidized. The molecular weight distributions could explain the THM and HAA formation potentials that decreased at the No UV exposure samples but increased at Low photocatalytic treatment levels. The use of photocatalysis before GAC adsorption appears to increase bed life of the contactors; however, higher photocatalytic treatment levels have been shown to completely mineralize NOM and would therefore not require additional GAC adsorption after photocatalysis. / Dissertation/Thesis / M.S. Civil and Environmental Engineering 2011

This research uses Bayesian analysis of fluorescence spectroscopy results to determine if wastewater from the Heber Valley Special Service District (HVSSD) lagoons in Midway, UT has seeped into the adjacent Provo River. This flow cannot be directly measured, but it is possible to use fluorescence spectroscopy to determine if there is seepage into the river.Fluorescence spectroscopy results of water samples obtained from HVSSD lagoons and from upstream and downstream in the Provo River were used to conduct this statistical analysis. The fluorescence 'fingerprints' for the upstream and lagoon samples were used to deconvolute the two sources in a downstream sample in a manner similar to the tools and methods discussed in the literature and used for source apportionment of air pollutants. The Bayesian statistical method employed presents a novel way of conducting source apportionment and identifying the existence of pollution.This research demonstrates that coupling fluorescence spectroscopy with Bayesian statistical methods allows researchers to determine the degree to which a water source has been contaminated by a pollution source. This research has applications in determining the affect sanitary wastewater lagoons and other lagoons have on an adjacent river due to groundwater seepage. The method used can be applied in scenarios where direct collection of hydrogeologic data is not possible. This research demonstrates that the Bayesian chemical mass balance model presented is a viable method of performing source apportionment.

No / Organic matter (OM) causes many problems in drinking water treatment. It is difficult to monitor OM concentrations and character during treatment processes due to its complexity. Fluorescence spectroscopy is a promising tool for online monitoring. In this study, a unique dataset of fluorescence excitation emission matrixes (EEMs) (n = 867) was collected from all treatment stages of five drinking water treatment plants (WTPs) situated in diverse locations from subtropical to temperate climate. The WTPs incorporated various water sources, treatment processes and OM removal efficiencies (DOC removal 0%–68%). Despite these differences, four common fluorescence PARAFAC components were identified for characterisation of OM concentration and treatability. Moreover, fluorescence component ratios showed site-specific statistically significant correlations with OM removal, which contrasted with correlations between specific UV absorbance at 254 nm (SUVA) and OM removal that were not statistically significant. This indicates that use of fluorescence spectroscopy may be a more robust alternative for predicting DOC removal than UV spectroscopy. Based on the identified fluorescence components, four optical locations were selected in order to move towards single wavelength online OM monitoring.

Les activités anthropiques ont apporté des changements majeurs à notre système global. Par ailleurs, la matière organique dissoute(MOD) du littoral a une grande influence sur le cycle global du carbone et donc sur le changement climatique. L'apport côtier enMOD représente la matière organique terrestre. Les rivières urbanisées sont fortement impactées par la MOD anthropiqueprovenant des usines de traitement des eaux usées. La MOD chromophorique est un sous-groupe de la MOD qui peut absorber lalumière. La MOD fluorescente est à son tour un sous-groupe de la MOD chromophore. Le signal de fluorescence de la MODanthropogénique dans la zone côtière n'est pas bien caractérisé et évalué dans la littérature. Les dégradations induites parphotochimie et les changements au niveau moléculaire sont peuvent de plus influencer la MOD. Dans la présente étude, plusieursexpériences d'irradiation solaire ont été menées avec plusieurs modes de filtration de mélange d’eau de rivière, d’eau de mer et d'uneffluent de station de traitement des eaux usées dans le but de trouver un signal spécifique de fluorescence comme un traceur de laMOD anthropique en utilisant les matrices d'émission d'excitation de la spectroscopie de fluorescence (EEMs) couplées à latechnique statistique chimiométrique de l'analyse factorielle parallèle CP/PARAFAC. Un modèle de régression multilinéaire a étédéveloppé entre la contribution des composantes CP/PARAFAC et la composition du mélange. La cinétique des paramètres derégression multilinéaire a également été étudiée. Des suivis géographiques de l'évolution du signal de fluorescence dans la rivièreGapeau jusqu'à la mer ont été menées ainsi qu’une étude temporelle du signal de fluorescence. Le modèle de régressionmultilinéaire développé a été appliqué pour modéliser les résultats des expériences de champs géographiques et temporelles. Lesrésultats ont montré que le modèle de régression multilinéaire est excellent. Par contre la recherche d'un signal ou d'une signaturede fluorescence spécifique pour l'eau de rivière, les stations d'épuration des eaux usées ou l'eau de mer n'a pas pu être réalisée dansce travail. Dans la zone côtière affectée par l'homme, les matières organiques fluorescentes résiduelles proviennent principalementsinon uniquement de l'usine de traitement des eaux usées, et aucun signal spécifique provenant de l'eau de mer n'a pu être détectéprès de la côte. / Anthropogenic activities have done major changes to our global system. The coastal dissolved organic matter has great influenceon the global carbon cycle and hence climate change. The riverine input of dissolved organic matter represents the terrestrialorganic matter. Urbanized rivers is greatly impacted by the anthropogenic dissolved organic matter coming from wastewatertreatment plants . Chromophoric dissolved organic matter is a subgroup of the dissolved organic matter which can absorb light.Fluorescent dissolved organic matter in turn is a subgroup of the chromophoric dissolved organic matter .The fluorescence signalof the anthropogenic dissolved organic matter in the coastal zone is not well characterized and evaluated in the literature.Photochemically induced degradations and changes at the molecular level is considered to be a great process which could influencethe dissolved organic matter . In the present study, Laboratory mixing experiments several sunlight irradiation experiments wereconducted with several modes of filtration of three endmember mixing components ( River water, Sea water , wastewater treatmentplant effluent discharge) with the objective of finding a specific signal of fluorescence which could be a tracer of the anthropogenicdissolved organic matter through using the fluorescence spectroscopy excitation emission matrices (EEMs) coupled with thechemometric statistical technique of Parallel Factor analysis CP/PARAFAC. Moreover, multilinear regression model between thecontribution of CP/PARAFAC components and two content fraction of River water and Seawater endmember was developed. Inaddition the kinetics of the multilinear regression parameters were investigated. On top of that , geographical investigations of theevolution of fluorescence signal in the Gapeau river till the sea were conducted. Furthermore, Temporal investigation of thefluorescence signal for four water points in the pathway of Gapeau river were done. The multilinear regression model developedwas applied to model the results of the geographical and temporal field experiments. Results have shown that Multilinearregression model for contribution of CP/PARAFAC components is excellent and could be done for the three endmembers. Inaddition the search for specific fluorescence signal or signature for river water, wastewater treatment plants and sea water couldn’tbe done in this work. In human impacted coastal zone, residual fluorescent organic matter come from wastewater treatment plant,and no specific signal from sea water could be detected near the coast.

In Taiwan, more than 25% of all water uses comes from groundwater, and thus groundwater is a very valuable water resource for both domestic and industrial uses. However, groundwater at many existing former industrial sites and disposal areas was contaminated by halogenated organic compounds that were released into the environment. The chlorinated solvent trichloroethene (TCE) is one of the most ubiquitous of these compounds. In this laboratory-scale feasibility study, a hybrid two-stage process combining fiber filtration (FF) and nanofiltration (NF) was applied to remove to suspended solids (SS) and TCE from contaminated groundwater for water purification. In this study, a man-made kaolin solution was used to simulate groundwater purification using FF system. Then, microfiltration (MF), ultrafiltration (UF), and NF systems were applied for TCE removal. The hybrid membrane process using FF and NF units was used to evaluate the feasibility on TCE removal. The scanning electron microscope (SEM) and energy dispersive spectroscope (EDS) were used to investigate membrane morphology and structure after use. A 3-D excitation emission fluorescence matrix (EEFM) was used to evaluate the potential of membrane organic fouling. Results show that the optimization filtration velocity of FF was 15.3 m/hr, and the observed TCE and SS removal efficiencies were 80% and 60%, respectively. Removal mechanisms for MF and UF were mainly sieving, and the removal mechanism for NF was mainly electrostatic repulsion. Results indicate that NF had the highest TCE removal efficiency (98.2%). When initial TCE concentration was 1 mg/L, NF membrane pore might shrink caused increased TCE removal (rejection). When TCE concentration was higher 1 mg/L, membrane damage and pore enlargement was observed with decreased TCE removal efficiency. The observed SS, sulfate, and hardness removal efficiencies were 99.8%, 98.7%, and 98.7% respectively, when FF and NF hybrid process was used. Higher TCE concentration might enlarge membrane pore, which caused decreased membrane separation and increased flux. Approximately 46% of flux drop was observed when NF was used alone compared to the hybrid membrane process using FF as the first treatment stage. Membrane analyses show that residual TCE was adsorbed on the membrane. Low zeta potential of groundwater was observed due to the compressed electric double layer, which caused aggregation of particle. High zeta potential of permeate was due to the particle dispersive through hybrid process. Results from SEM analysis show that membrane morphology was damaged by TCE, and heavy metal in groundwater deposited on membrane. Results of EEFM analysis indicate that groundwater contained humic acid (HA) and soluble microbial by-product (SMP). HA and SMP might be adsorbed on fiber filter, and extracellular polymeric substances (EPS) that attached on fiber filter might be washed out. The organic powders on the surface of the fiber filter might be washed out causing the increased in NPDOC concentrations. Humic acid could be removed through NF process, and SMP might be adsorbed in membrane pore caused organic fouling, and SMP might be washed out after treatment by the FF+NF hybrid process. Results indicate that FF as pre-treatment can maintain higher flux. Higher TCE concentration caused membrane destruction and decreased membrane separation. TCE contaminated groundwater can be affectively treated by the hybrid membrane system to meet the groundwater standard and reclaimed water standard. Reclaimed water could be used for industrial cooling water and irrigation purposes.

Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.