Assessing a Fluorescence Spectroscopy Method for In-Situ Microbial Drinking Water Quality

Abstract

Waterborne disease is a significant contributor to the global burden of disease, in particular among high-risk populations in developing nations. State-of-the-art methods for the enumeration of microbial pathogens in drinking water sources have important limitations, including high initial cost, 24-48 hour delays in results, high staffing and facility requirements, and training requirements which all become especially problematic in the developing nation context.A number of alternative approaches to microbial water quality testing have been proposed, with the goal of decreasing the required testing time, decreasing overall costs, leveraging appropriate technology approaches, or improving sensitivity or specificity of the water quality testing method. One approach that may offer solutions to some of these limitations involves the deployment of sensor networks using fluorescent spectroscopy to detect intrinsic protein fluorescence in water samples as a proxy for microbial activity. In recent years, a number of researchers have found significant and meaningful correlations between indicator bacteria species and the protein fluorescence of drinking water samples. Additionally, advances in the semiconductor industry could be used to drive down the cost of such sensors. This technology may also be extensible to other water quality parameters, including dissolved organic matter or the presence of fluorescent pollutants.In this thesis, a literature review describes the fundamentals of fluorescence spectroscopy, historical and recent work regarding the fluorescence of the amino acid tryptophan and associated bacterial fluorescence, possible mechanisms for this ii association, and potential applications of this technology for drinking water quality monitoring and waste water process control. Extensibility of the technology is also discussed.Next, experimental methodology in reproduction of similar results is described.Samples were taken from seven (7) surface water sources and tested using membrane filtration and an off-the-shelf fluorescence spectrometer to help examine the association between the presence of indicator bacteria and the tryptophan fluorescence of the water sample. The results, showing an association of R 2 = 0.560, are compared to the results of recent similar experiments.Finally, two prototypes are described, including their design requirements and data from prototype testing. The results of the testing are briefly discussed, and next steps are outlined with the goal of developing a low-cost, in-situ microbial water quality sensor using fluorescence spectroscopy principles.iii Dedication Dedicated to my parents, who taught me to start with the written word in my search for understanding; and who taught me to see even the biggest problems as surmountable and critical. And to my brothers and sisters, who have shown me a halfdozen fascinating ways of interacting with and impacting the world. I'm proud of every one of you.iv

Monitoring Approaches for Faecal Indicator Bacteria in Water: Visioning a Remote Real-Time Sensor for E. coli and Enterococci

“…Absorption spectroscopy measurements [8,29,[62][63][64][65][66][67][68]72,130,137,138] Absorption spectroscopy measures the absorption of light by an unknown substance or sample generally performed on molecules dissolved in transparent solvents or aqueous buffers. Samples are illuminated with electromagnetic rays of determinant wavelength depending on the choice; these can include visible, ultraviolet, or the lower infrared spectrum and then the absorbance of the sample is used to calculate the concentration.…”

“…Spectral Signature of E. coli and Enterococci Fluorescent spectrum signatures exist for bacterial species including E. coli (and likely enterococci), but these signatures might in turn be dependent on the cellular or extracellular accumulation of the fluorescent material, as suggested by Sharpe [8]. However, several reports indicate the correctness of the general agreement between excitation and emission wavelengths for E. coli; enterococci require further investigation.…”

“…In contrast with standard water-testing procedures, the fluorescence measurement enables fast or near-instantaneous results, and it can be used with or without reagents, thus reducing costs; however, currently, for E. coli and enterococci, results can be delayed by sample collection, preprocessing, and fluorescence measurement in laboratories [8,38,74]. Furthermore, most typical fluorescence sensors are bulky and expensive and are commonly affected by optical inferences; findings have shown that miniaturisation, filters, and compensation algorithms could overcome such limitations, but more experimentation is required [62,72,73].…”

“…Improved result accuracy requires minimising or eliminating false positives and false negatives [36]. Promisingly, Sharpe [8] found that both sensitivity and specificity for a PFS device increase with improved or additional optical components or a converging lens. They made this a suggested measure to be incorporated into proposed device developments.…”

“…coli are rod-shaped, Gram-negative facultative anaerobic bacilli found in the intestines of warm-blooded animals [5,6]. The presence of E. coli indicates the likely occurrence of pathogens linked to human faecal contamination and is a preferred indicator of faecal contamination in various water types [7][8][9]. Although many E. coli strains have low infectious doses, virulent strains (E. coli 0157:H7) can result in diarrheal infections, neonatal meningitis, septicaemia, and urinary tract infections [6,10], and there have been increasing reports of certain E. coli strains blooming in environments without evidence of faecal contamination [11].…”