A thesis presented to. the faculty of. In partial fulfillment. of the requirements for the degree. Master of Science. Joseph P. Morris.

Transcription

1 Disinfection of Bacillus Subtilis Spores Using Ultraviolet Light Emitting Diodes A thesis presented to the faculty of the Russ College of Engineering and Technology of Ohio University In partial fulfillment of the requirements for the degree Master of Science Joseph P. Morris June Joseph P. Morris. All Rights Reserved.

2 2 This thesis titled Disinfection of Bacillus Subtilis Spores Using Ultraviolet Light Emitting Diodes by JOSEPH P. MORRIS has been approved for the School of Electrical Engineering and Computer Science and the Russ College of Engineering and Technology by Wojciech M. Jadwisienczak Associate Professor of Electrical Engineering and Computer Science Dennis Irwin Dean, Russ College of Engineering and Technology

3 3 ABSTRACT MORRIS, JOSEPH P., M.S., June 2012, Electrical Engineering Disinfection of Bacillus Subtilis Spores Using Ultraviolet Light Emitting Diodes Director of Thesis: Wojciech M. Jadwisienczak Water disinfection by ultraviolet (UV) radiation is a technique that neither leaves residual chemicals nor causes any aesthetically displeasing results. UV light generated from UV light emitting diodes (LEDs) has the potential to begin a new wave of mercuryfree, light-weight, completely solid-state, cost-effective, and low-power-consuming water treatment systems for developing residential and commercial locations. This thesis gives a report on the effectiveness tests of commercially available UVLEDs used for laboratory water disinfection. Bacillus subtilis spores were harvested in water from B. subtilis colonies grown using a standard culture method. The water containing these spores was irradiated by UVLEDs operating at 250 nm and 270 nm. It was found that 3-log inactivation occurred at approximately 29 minutes and 24 minutes, respectively, thus confirming the plausibility of using UVLEDs in a water disinfection system. Additionally, this thesis also explores the plausibility of generating UV radiation from the impact excitation of BN phosphors. Approved: Wojciech M. Jadwisienczak Associate Professor of Electrical Engineering and Computer Science

4 4 ACKNOWLEGMENTS Above all else, I am most grateful to our savior Jesus Christ for the sacrifice He made to give all life purpose and meaning. Without that sacrifice all of this work would be a vain attempt on my part to give myself meaning and purpose. He brought me to Ohio University and has guided me through all my experiences here, all the while teaching me emulate and display the love and passion He has for all the people on Earth. I will move forward offering my work and all that I am as a living sacrifice because He is worthy of all that I can give. I would like to bestow thanks upon Dr. Wojciech Jadwisienczak, who has served as my graduate advisor. He has provided me with all the resources necessary to complete my work as well as teaching me how pursue additional opportunities in grants and fellowships. He has provided me with many challenges to overcome and has always inspired me to put my best work into everything that I do. I would like to thank the remaining members of my advisory committee, Dr. Ralph Whaley, Dr. Savas Kaya, and Dr. Kelley Johnson. Their technical knowledge and expertise have aided me in many ways during the pursuit of my master s degree. They have challenged me to learn difficult concepts to broaden my understanding of applied electric engineering. To Dr. Johnson I extend special thanks for jumping aboard last minute to fill a crucial role in the defense of this thesis. I would like to thank Dr. Guy Riefler and Alex Anderson for their assistance in lab and for teaching me to cultivate and propagate the bacteria.

5 5 Lastly, I would like to recognize and thank my parents, Dennis and Katherine Morris, my sisters, brothers-in-law, and sister-in-law, Jeff and Bethany Buckner, Randy and April Massie, Linda Morris, and Jessica Heim, my father- and mother-in-law, Walter and Beverly Heim, and my friends Tucker and Brittnee Barlow, for their continued support and prayer. I would especially like to thank my beautiful wife, Rachel Morris, for her continual love, support, and inspiration throughout all the areas of my life.

6 6 TABLE OF CONTENTS Page Abstract... 3 Acknowlegments... 4 List of Tables... 8 List of Figures Introduction Motivation Disinfection of Water UV Light Generation High Cost of Current Disinfection Systems Objectives and Foci Impact Background/Theory A Brief History of Water Treatment UV Induced DNA Damage Bacteria Dark Reactivation and Photoreactivation Considerations Advantages of UV Water Disinfection UV Light and UV Light Generation Development of UVLEDs Experiment Development of Experimental Setup Design Criteria Design and Construction Additional Instrumentation UV Light Source Analysis UVLED Research Standard Spectrum Analysis of Commercial UVLEDs Power Density Analysis of Commercial UVLEDs Power Supply and Circuit Analysis... 51

7 7 3.3 Bacteria Preparation and Growth Rationale for Bacillus subtilis Bacillus subtilis Preparation Bacteria Exposure Experiments Testing Procedure Percent Inactivation Calculation Results Bacillus subtilis disinfection results for 250 nm and 270 nm UVLEDs The Exclusion of the 295 nm UVLED Discussion EPA Standards and Experimental Log Inactivation Dose Comparison UV Wavelengths Inactivation Effectiveness Comparison Large Scale Disinfection System Analysis Conclusion Future Work The Next Step Excitation of BN by Impact Excitation Initial Cathodoluminescence Experiments Plan for Impact Excitation of BN Experimentation References Appendix A: Partial SETi UVTOP Catalogue Appendix B: Partial ATCC 6633 B. subtilis Data Sheet... 98

8 8 LIST OF TABLES Page Table 3.1: UVLED characteristics at their maximum rated output during water disinfection trials. Power density measured at 40 mm from source Table 3.2: Log inactivation dose for specified bacteria or virus as stated in referenced publications Table 3.3: Ingredients for Columbian agar Table 3.4: Ingredients for sporulation media Table 4.1: Inactivation percentages for the three trials of 250 nm UVLED exposures Table 4.2: Inactivation percentages for the three trials of 270 nm UVLED exposures Table 5.1: B. subtilis UV dosage required and demonstrated by experiments in order to reach 2-log and 3-log inactivation Table 5.2: Cost and power out characteristics for SETi UVLEDs used in this thesis

9 9 LIST OF FIGURES Page Figure 2.1: The images show the chemical structure interactions between DNA and RNA bases where A designates adenine, T designates thymine, G designates guanine, C designates cytosine, and U designates uracil which is nearly identical to thymine but lacking the addition of the methyl group. Additionally within the image the R is not an element or compound like the other unbolded letters, rather R is the location where the base binds to the ribose or deoxyribose sugar Figure 2.2: The DNA absorbance spectra of (A) Decoated spores, (B) B. subtilis spores observed by Chen, and (C) DNA isolated from spores [8] Figure 2.3: When DNA is exposed to UV light one of the most common forms of damage to occur is a thymine dimer. When exposed two adjacent thymine bases bond together creating a bulge in the double helix that prevents replication and the original bonds between the thymine and adenine degrade Figure 2.4: Generic setup for a sand filtration system in which gravity is utilized to remove visible impurities from water Figure 2.5: Absorption coefficients in the clearest natural waters as stated by Smith and Baker for the wavelengths between 200 nm and 400 nm [21] Figure 2.6: UV light has an emission wavelength range from 100nm to 400nm. Within this band of wavelengths there are four primary sub-bands known as UV-A, UV-B, UV-C, and Vacuum UV [13] Figure 2.7: Each band of UV light has its own set of applications. This figure shows several examples of applications as a function of wavelength [23] Figure 2.8: UV low pressure and medium pressure mercury vapor germicidal lamp spectrum Figure 2.9: AlN/AlGaN based superlattice structure used in 2006 produced a UVLED with an emission wavelength of 210 nm which is very close to the minimum possible wavelength using AlN and ternary compounds Figure 2.10: Idealized band diagram of the device depicted in Figure 2.9 shows the basic operation of an LED with an AlN active region Figure 2.11: (A) GaN is deposited on an AlN substrate, which creates tension in the AlN and compression of the GaN. (B) AlN is deposited on a GaN substrate, which

10 causes tension in the AlN and compression in the GaN. (C, D) The results of each deposit in which the circles highlight the lattice dislocations produced as a result of the stress produced by lattice mismatch Figure 3.1: Basic design of the experimental setup taking into account the LED emission pattern and making use of a magnetic stir plate to encourage uniform UV distribution. (A) A side view of the test apparatus. (B) Atop down view of the test apparatus Figure 3.2: Photograph of the experimental setup including all essential equipment. Included are the (A) Power supply and multimeters, (B) UVLED array, (C) UV spectrometer, (D) Computer interface, (E) Plexiglass testing apparatus, and (F) Newport Power Meter Figure 3.3: Spectral analysis results of SETi 250 nm, 270 nm, and 295 nm UVLEDs Figure 3.4: Normalized spectral analysis results of SETi 250 nm, 270 nm, and 295 nm UVLEDs Figure 3.5: Displayed are the results of the power density analysis. The 250 nm UVLED peaked at about 15 μw/cm 2, the 270 nm UVLED peaked at about 50 μw/cm 2, and the 295 nm UVLED peaked at about 39 uw. The reference point is a 254 nm low end mercury vapor lamp which has a power density of about 57 μw/cm 2. * Indicates the power density of a 254 nm mercury vapor lamp Figure 3.6: This flowchart details the disinfection testing process for the 250 nm UVLED. This process was repeated three times per wavelength Figure 4.1: B. subtilis spore inactivation percentage as a function of exposure time to 250 nm UV radiation Figure 4.2: B. subtilis spore inactivation percentage as a function of exposure time to 270 nm UV radiation Figure 4.3: B. subtilis spore inactivation percentage of the averaged values of 250 nm and 270 nm exposure trials as a function of exposure time. The miniature plot zooms in on the 3-log inactivation area Figure 7.1: BN impact excitation theory. (A) This simple schematic of the structure of a device that would utilize the impact excitation of BN to generat UV light. (B) This very basic illistration demonstrates an electron colliding with a BN atom and producing a UV photon

11 Figure 7.2: Room temperature CL spectrum of h-bn material excited by electron accelerated with 6 kev. Inset shows comparison between normalized CL spectra of h-bn material measured at (a) 12 K and (b) 300 K. Vertical arrows indicate deep UV excitonic recombination peaks and a deep level related emission band centered at 270 nm, respectively

12 12 1 INTRODUCTION 1.1 Motivation Water contamination with bacterial and viral agents is an issue as old as the human race. It is a problem that mankind has been trying to solve for millennia. Ancient water treatment methods date back as far as 4000 B.C. These attempts, however, because of the lack of understanding of bacteria and microscopic organisms, only amounted to improving the aesthetic quality of water. Making water clearer and better tasting were the primary goals in early water treatment until ancient Greek civilizations began the earliest forms of water disinfection, boiling and filtering water [1]. Today, disinfection systems can be purchased that are capable of inactivating bacteria and viruses to a 6-log ( %) level. However, despite the numerous and exhaustive efforts of the human race, each year 1.8 million children worldwide die because of contaminated drinking water. In fact, the number of deaths from cholera and typhoid stemming from waterborne bacteria are higher than those from AIDS. Approximately 1 billion people around the world do not have access to safe drinking water [2]. The simple, devastating truth is that although disinfection systems are available, they are far too expensive for the places that most desperately need clean water, and sickness and death are consequences for these people who cannot afford to disinfect their water.

13 Disinfection of Water There are several different ways to disinfect water from harmful and deadly bacteria such as Escherichia coli, which is known to cause severe food poisoning, gastroenteritis, and meningitis, and Salmonella which is known to cause a variety of diseases including Enteric and Typhoid Fever, as well as from viruses that cause life threatening illnesses. Unfortunately, many of these disinfection techniques introduce harmful, chemical contaminants during the disinfection process that are difficult to remove and are often overlooked due to the lack of other options. Chlorine is one such chemical that is often introduced to kill bacteria and viruses, via destructive oxidation reactions. However, after chlorine addition, it is then either left in the water and consumed, or removed through a decontamination process which requires the introduction of more foreign chemicals to the water in order to remove the chlorine. There is currently only one form of water disinfection that has been shown to leave no residual chemicals, ultraviolet (UV) radiation. UV radiation has been used as a water disinfection method in the United States since 1916 and does not introduce any environmentally harmful chemicals or materials. Additionally, if the UV is generated by solid state devices there is no involvement of mercury either, thus furthering the characteristics that make it environmental friendly and safe for humans. It is very safe, assuming the light is contained inside the disinfection system, and it has been shown to be effective enough to produce up to 6-log inactivation of many viruses and bacteria [3].

14 UV Light Generation The generation of light has been explored for more than 150 years beginning with Joseph W. Swan in Great strides have been taken since then and especially when Thomas Edison invented the electric light bulb in Edison s invention remained relatively unchanged and dominated the lighting market for over a century, until the rise of compact fluorescent lights because of their energy efficiency. Now, with the energy crisis at hand, researchers are searching for new answers to the efficiency problem, among which light emitting diodes (LEDs) are at the forefront. The first LED was invented in 1927 however it wasn t until the recent development of microelectronic processing techniques that their full potential has been understood. The generation of light in the UV spectrum (100 nm 400 nm) has sparked interest among biological researchers as they consider the disinfection potential. As a result, UV light generation has been a growing area of research among physics, optics, and electronics experts in recent years in order to cater to the new demand for UV disinfection systems. The most widely used form of UV generation is through mercuryvapor lamps, which generate a wide spectrum of light. Low-pressure mercury-vapor lamps use a phosphor to limit their output wavelength to 254 nm, which is deep within the UV spectrum. Despite their wide use and long standing implementation, these lamps are inefficient, requiring high input power to generate a sufficient amount of UV light for disinfection. Moreover, as their name suggests, they require the utilization the element

15 15 Mercury (Hg), which has been proven to be extremely hazardous not only to the environment but human life as well [4]. Recently, researchers have turned to LEDs for UV light generation. The first UVLED was produced in the late 1990 s. Since they are still early in their developmental stages, these UVLEDs are not yet as efficient as the most common LEDs emitting light in the visible spectrum. Nonetheless, as with their visible spectrum counterparts, they have the potential to take lighting technology far beyond what is currently possible and into the solid state technology realm. Although there is much ground to cover, LEDs are a promising technology for a safer, more efficient form of UV light generation. 1.4 High Cost of Current Disinfection Systems The high cost of current disinfection systems is one of the driving forces in the current push to develop additional water disinfection systems. An expensive system simply is not practical when the vast majority of the 1 billion people without clean drinking water do not have the money or resources to purchase or build one of the current disinfection systems. Burch and Thomas [5] provide a detailed analysis of costs of water disinfection systems and assess the feasibility of each for large scale disinfection in developing countries. The striking fact in this research is that the village grade disinfection systems cost more than $2000 initially and have high capacity costs of above $90/m 3 /day. This cost analysis shows that the current disinfection systems are unsuitable

16 for citizens in developing countries because of their high initial, daily usage, and maintenance costs Objectives and Foci In order to design a water disinfection system for a developing country, there are two fundamental research issues that must be addressed: the necessity for disinfection of water contaminated with high microbial loads and the lack of highly efficient forms of UV light generation. This research focuses on the design and testing of a UV germicidal light engine for a water disinfection system. There are three major foci in the consideration of this research. First is the analysis of the properties and characteristics of nine commercially available UVLEDs; three 250 nm UVLEDs, three 270 nm UVLEDs, and three 295 nm UVLEDs purchased from Sensor Electronic Technology, Inc (SETi). Next the effectiveness of these UVLEDs in bacteria inactivation is examined. The third and final focus is an assessment of the UVLEDs feasibility as a component in large scale disinfection systems for developing countries. 1.6 Impact This research has great potential far beyond Ohio University. Ohio University s Engineers Without Boarders (EWB) chapter is partnered with a small village in Maase- Offinso, Ghana and EWB has been working to get water into the village for some time.

17 17 They have recently constructed a pumping and storage system that brings an adequate supply of water to the village. However, this water is still contaminated with deadly bacteria and viruses as the well that the water is being pumped from has been polluted with human waste for centuries. This research has the potential to not only help this village directly through Ohio University and EWB but thousands of other villages just like it. The implementation of UVLEDs in large scale disinfection systems that is capable of disinfecting and providing clean water to a village the size of Maase-Offinso, Ghana (over 1000 people) could begin a revolution in the drinking water disinfection market. In addition an examination of other sources of UV radiation will open up doors to new and possibly more efficient and powerful forms of UV generation. One such future source that will be outlined in this research is the use of Boron Nitride (BN) phosphors as a building block of a large scale hybrid low voltage UV-C light source, utilizing electroluminescence techniques. This BN UV light would allow for extensive future research in bacteria disinfection with UV radiation from other sources than UV mercury vapor lamps and even UVLEDs. Additionally, it will be a major step in the development of affordable and efficient forms of UV light generation for water disinfection, which has the potential to save millions of lives.

18 18 2 BACKGROUND/THEORY 2.1 A Brief History of Water Treatment Since the invention of the microscope by Hans and Zacharias Janssen in 1590 [6] humans started to understand the microbial world and the necessity of water disinfection. For the first time, rather than merely observing their effects on the human race, researchers were able to observe bacteria. While the ability to visibly see individual bacterium and viruses was still a few years off, the microscope started the exploration. As the understanding of the microbial world developed, water purification moved from simply increasing its aesthetic appeal towards disinfection. Filtration has been a major form of water disinfection since the 1700 s [1]. Early in the 1800 s European cultures had already began wide scale use of slow sand filtration systems. These types of systems are still in use today and were the early versions of some septic systems. These systems while typically inexpensive, are, unfortunately, not effective at completely disinfecting water, but rather they focused on filtering out visible contaminants. While filtration does aid in disinfection it is not nearly as thorough as modern disinfection systems for which the sole purpose is the disinfection of water. Several types of modern disinfection systems include chemical, ozone, and UV radiation disinfection. Common chemicals used include chlorine gas as well as several chlorine containing molecules including sodium hypochlorite, chlorine dioxide, and chloramine. Pure chlorine works effectively in water disinfection due to the irreparable

19 19 levels of oxidative damage it causes in microbes; however, it has the same effects on human cells and the health risk associated with chlorine consumption has compelled researchers to study variations of chlorine like those stated above. The major difficulty with the primary variations of chlorine is that they are less efficient in water disinfection, especially for viruses. Chlorine dioxide is the only form that provides sufficient disinfection from all contaminants that chlorine, however, it also provides a sufficient number of disadvantages in that it requires on-site power generation, transportation and storage of flammable chemicals, and also leaves a distinct odor and taste when it comes in contact with various organic materials [7]. The use of ozone (O 3 ) as a water disinfectant has been used for several decades. Due to its unstable molecular structure, ozone is a powerful oxidizing agent and powerful disinfectant, but, like chlorine, ozone oxidize human cells as well as microbes and must be removed from drinking water to prevent harmful human consumption. Additionally, upon reacting with some organic compounds, ozone disintegrates and becomes a nutrient for microorganisms, thus facilitating water infection [7]. UV radiation is currently the most explored form of disinfection mainly because of the rise of solid state lighting and their potential for producing efficient, low power forms of water disinfection. However, UV radiation systems, while being very effective and not leaving any residual chemicals in water, have been very expensive in both the initial capital and operating costs [7]. Reaching, essentially, a stalemate in disinfection technology, the opportunity to produce inexpensive and efficient forms of UV light has not gone unnoticed; it has seen a great deal of interest in recent years, culminating in the production of UVLEDs.

20 UV Induced DNA Damage While there are many ways to disinfect water, a common difficulty that all methods face is killing bacterial endospores. An endospore (spore) is a bacterial protection mechanism in unfavorable environments such as in the presence of high temperatures or lack of nutrients. These spores protect the vital macromolecules inside the cell that are essential for life, mainly the DNA containing the genome. The primary structural units in the genome are nucleic acids, which are composed of a sugar, ribose or deoxyribose, a phosphate group which catalyzes polymerization, and a variable base. While they can exist and function as independent monomers within cells, nucleic acids polymerize into two main forms: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). DNA is responsible for housing the information with which the bacteria produces proteins, which facilitate most cellular functions, while RNA is responsible for the process of converting the DNA code into proteins. The bases of DNA and RNA are divided into two groups based on their chemical ring structures. Purines, guanine (G) and adenine (A), have a double ring structure, while pyrimidines, cytosine (C), thymine (T), and uracil (U), contain a single ring structure. Inter-base pairing occurs through hydrogen bonding. G always binds to C and vice versa with three hydrogen bonds, and A always binds to T in DNA and U in RNA and vice versa with two hydrogen bonds. DNA exists as a double stranded alpha helix in which one strand contains the coding information and binds via complementary base pairing to the other strand. RNA,

21 which contains an additional hydroxyl group on the ribose sugar, is not as stable as DNA and generally exists in a single strand that can interact and base pair within itself. 21 Figure 2.1: The images show the chemical structure interactions between DNA and RNA bases where A designates adenine, T designates thymine, G designates guanine, C designates cytosine, and U designates uracil which is nearly identical to thymine but lacking the addition of the methyl group. Additionally within the image the R is not an element or compound like the other unbolded letters, rather R is the location where the base binds to the ribose or deoxyribose sugar. Messenger RNA (mrna) is transcribed from the coding DNA strand via complementary base pairing. It is then translated to a sequence of amino acids called a polypeptide that fold three dimensionally to form a protein. Translation of mrna is mediated by large protein complexes called ribosomes, which contain catalytic ribosomal RNA (rrna). The actual translation of bases to amino acids is accomplished by transfer RNA (trna). Within the mrna, bases are read in threes, and each set of three bases is

22 22 called a codon. Each codon in the mrna binds to a complementary three base anticodon in a precise region of the trna. After the binding of the codon and anticodon, with the help of the catalytitc rrna, the ribosome removes the amino acid bound to the trna, adds it to the growing polypeptide, and releases the used trna, freeing space for the binding of the next trna with the correct anticodon. At the end of the mrna sequence, a special codon called a STOP codon signals the end of the transcript, causing the release of the polypeptide for folding into a protein. Since DNA plays a crucial role in all organisms and cells, the integrity of the code must be maintained and when a cell or organism divides, a complete copy of the entire genome must be placed in each daughter cell/organism. Thus organisms and cells have developed elaborate pathways to maintain genomic integrity and to duplicate their genome for each subsequent generation. Without the DNA replicating properly, the single celled organisms like bacteria cannot replicate. Bacterial infection is dependent on replication rates exceeding death rates due to the infected organism s immune system. Without the ability to replicate, the bacterium is unable to infect a person through drinking water. Bacillus subtillus is a bacterium that regularly forms a protective spore making it one of the most difficult organisms to kill when disinfecting water [1]. For this reason, Bacillus subtillus spores were propagated and used in showing that UV-light exposure can be an effective way to disinfect water. During exposure, DNA absorbs UV-light. The amount of absorption is dependent upon the wavelength of light the DNA is being exposed to. This is shown in detail for B. subtilis in Figure 2.2 [8].

23 23 Figure 2.2: The DNA absorbance spectra of (A) Decoated spores, (B) B. subtilis spores observed by Chen, and (C) DNA isolated from spores [8]. Upon absorption of UV radiation becomes compromised in one of three important ways, all involving structural changes in the nucleic acid sequence [1] [9]. The first and most common way that UV radiation causes damage is the creation of a pyrimidine dimer [9]. A pyrimidine dimer results from extra radiation causing a covalent linkage between two adjacent pyrimidine bases, usually two Ts, on the same strand of DNA [9]. This causes a bulge in the strand, inhibiting accurate RNA synthesis, and, more importantly, preventing replication as the unidirectional cellular replication machinery is halted by this structural change. As a result, the bacterium dies without replicating. Figure 2.3 gives a depiction of the most common type of pyrimidine dimer, usually called a thymine dimer because when UV light hits the DNA strand two adjacent

24 thymine bases bond together creating the aforementioned bulge and the bonds between the thymine and adenine degrade. 24 Figure 2.3: When DNA is exposed to UV light one of the most common forms of damage to occur is a thymine dimer. When exposed two adjacent thymine bases bond together creating a bulge in the double helix that prevents replication and the original bonds between the thymine and adenine degrade.

25 25 The second form of DNA damage is the formation of a pyrimidine (6-4) pyrimidone photoproduct, which forms in a similar way as a pyrimidine dimers, but results in a different structure [9]. The final effect is protein-dna cross-linkage. The UV-radiation can cause an amino acid of a protein to bind with a base of the DNA strand, which also blocks replication [9]. Since UV-radiation and other DNA damaging agents are experienced on a daily basis, organisms are equipped with methods and pathways that identify and correct this damage through continuous genome scanning. However, cases of extreme damage cannot be remedied, and hence UV light is extremely effective disinfectant. 2.3 Bacteria Dark Reactivation and Photoreactivation Considerations UV radiation causes DNA alterations that are almost exclusively detrimental to all organisms. However, nearly all organisms are exposed to UV radiation in their natural environment (predominantly UV-A and some levels of UV-B) because of the UV light permitted to pass to the Earth s surface by the atmosphere. Thus, life is equipped with coping mechanisms. One such mechanism is the nucleotide excision repair or dark repair [10] [11]. By this mechanism, when DNA damage is recognized, the damage and the surrounding DNA is excised, leaving a section of single stranded DNA on the normally double stranded macromolecule. This gap is then filled by normal cellular replication machinery by re-synthesizing the section of DNA that was excised using the other strand

26 26 as a template. Most organisms, especially higher level organisms like humans deal with UV-induced DNA damage by this mechanism [11]. Simpler organisms that do not have their DNA protected in a membrane-enclosed nucleus and that divide rapidly contain an additional UV coping mechanism to specifically repairs pyrimidine dimers called photoreactivation or light repair. This mechanism does not require the removal of DNA, but rather utilizes near UV-light to catalyze the reverse reaction of UV-induced dimerization. Photons between 320 and 370 nm activate the enzyme photolyase, which then splits pyrimidine dimers. This reverses the DNA damage, thereby preventing mutation and allowing replication to proceed [11]. Both previously described pathways are repair mechanisms of normal levels of DNA damage experienced by organisms in their environment and are not sufficient to repair high levels of damage. The highest levels of UV-induced DNA damage are caused by UV-C, which is not a part of the natural environment as it is entirely absorbed by Earth s atmosphere. Thus, organisms are not equipped to repair damage of the magnitude induced by UV-C, and despite repair pathways, high levels of UV-C exposure is fatal [11]. 2.4 Advantages of UV Water Disinfection In 1903, Niels Ryberg Finsen was awarded the Nobel Prize in Physiology or Medicine primarily for his contribution to treating diseases including tuberculosis with concentrated UV light radiation [12]. The first UV disinfection system was developed in

27 27 France in 1910 [13] following the invention of the mercury vapor lamp in 1901 and the use of quartz in 1906 as a transmission medium [13]. However, because of the low cost of chlorine as well as issues in the operation of these early UV disinfection systems, their use was minimal in comparison to their chlorine counterparts. The comeback of the UV disinfection system began with the first reliable disinfection system, which was used for the disinfection of municipal drinking water in Switzerland and Austria in The number of UV disinfection systems in these countries continued to increase until in 1985 where approximately 500 small scale systems were installed in Switzerland and approximately 600 small scale systems in Austria. The primary motivation for alternatives to chemical disinfection systems stems from a desire to reduce the production of carcinogenic disinfection byproducts that form from chlorine-based disinfectants and to reduce the threat to local populations posed by storing and delivering large quantities of chlorine gas. Soon after the discovery of the carcinogenic disinfection byproducts of chlorine several other countries began to adopt the search for alternative water disinfection technique [13]. It was at this time that UV light came out of the shadows and began to emerge as a primary candidate to become the replacement for chlorine-based disinfection. UV disinfection was initially used for the disinfection of wastewater [14]. In wastewater disinfection, the lack of residual chemicals is the primary advantage to UV treatment over chemical disinfection. However by the year 2000, there were over 400 UV drinking water disinfections systems worldwide usually treating flows of less than 1 million gallons per day. In Seattle, Washington a UV drinking water disinfection system currently produces 180 million

28 28 gallons of drinking water per day [13]. An additional benefit of UV drinking water disinfection systems that has further increased its popularity is that UV radiation is more effective than chemicals in inactivating protozoa, another type kind of disease-causing microorganism that infects water [15] [16] Other advantages to UV light can be seen in considering the unique opportunity to use UVLEDs instead of mercury vapor lamps. UVLEDs, despite being a immature technology, have been shown to operate for more than 10,000 hours [17]. Their visible LED counterparts can last in many cases 100,000 hours or more showing that even though they are showing a respectable lifetime currently there is quite a bit of room for improvement for these UVLEDs. Despite leading the market of UV light generation for a long time mercury vapor lamps have an average operating lifetime of only 8,800 hours [18]. Operating at full power a mercury vapor lamp consumes 61% more energy than a UVLED for the same output power [19]. UVLEDs are also very durable in that they are completely solid state. In comparison mercury vapor lamps are very fragile because of the quartz casing. The double negative in this case is that on top of being fragile they contain mercury which needs to be contained to be safe. On the other hand there are some obstacles that can be a hindrance in UV water disinfection. Two primary difficulties in UV water disinfection are the necessity for clear water and the absorption of UV light in water. It is absolutely imperative that water is visibly clear in order for UV radiation disinfection to occur as it is supposed to. In dealing with light reflection and absorption are the two most important factors in UV disinfection. Reflection can become a problem when anything is in the water in addition

29 29 to the bacteria. Light cannot pass through all objects and therefore, for example, if there is a piece of sand or dirt in the water it can cover up the bacteria and thus the UV light will not be able to alter the DNA of that bacterium. Thus keeping water clear is the first necessity in UV disinfection. On such technique that works well and has been implemented since the 1800 s is slow sand filtration. Figure 2.4 shows the typical operation of a slow sand filtration system [20]. Figure 2.4: Generic setup for a sand filtration system in which gravity is utilized to remove visible impurities from water. This system is very simple because it allows gravity to do the majority of the work as the water interacts with the sand in the filter bed and then the gravel. The water enters the system through the water in nozzle and, because of gravity, is pulled down

30 30 through the filter bed, typically sand which acts as a sieve, effectively trapping the visible contaminants. The water then passes through the support gravel and is released through the controlled water out valve. This process is one very simple way to keep water clear before implementing the disinfection through the use of UV light. The other primary difficulty in water disinfection with UV light is UV light absorption in water. As the wavelength of light decreases through the UV spectrum the absorption coefficient increases dramatically and therefore is absorbed quickly in water. Figure 2.5 shows the absorption coefficients of light in water as a function of wavelength from 200 nm to 400 nm created using data collected by Smith and Baker [21]. 3.5 Absorption Coefficient in Water α (m -1 ) λ (nm) Figure 2.5: Absorption coefficients in the clearest natural waters as stated by Smith and Baker for the wavelengths between 200 nm and 400 nm [21].

31 31 As shown in Figure 2.5 the absorption coefficient as the wavelength approaches 200 nm increases significantly. In fact, as the wavelength approaches shorter wavelengths, for example 100 nm in the V-UV the absorption coefficient increases to approximately 1000 m -1 [22]. Such a dramatic increase in absorption makes water disinfection difficult because at short wavelengths UV light can be absorbed by the water before it even has the opportunity to alter the DNA of any bacteria. Despite these possible issues with UV disinfection the potential that UVLEDs have to decrease power consuption, increase efficiency, and increase the lifetime of UV disinfection systems is clear. Additionally, these issues are easily avoidable one with the implimentation of some type of filter like a slow sand filtration system and being mindful to optimize UV light output to provide the optimal amount of UV radiation without entering too far into the spectrum of UV light that is quickly absorbed in water. 2.5 UV Light and UV Light Generation The invention of the mercury vapor lamp in 1901 [13] opened the door to new possibilities of water, air, and food disinfection. UV light comes in a range of specifications. Figure 2.6 illustrates the location of UV on the electromagnetic spectrum and the specific bands within the UV spectrum [13]. Figure 2.7 further indicates several applications within each band [23]. UV light lies between x-rays and visible light with a wavelength ranging between 100 nm and 400 nm. It is most often broken down into three major bands of emission which are most typically labeled as UV-A, UV-B, and

32 UV-C, although the range of wavelengths between 100 nm and 200 nm, although considered within the UV-C band is sometimes referred to as Vacuum UV band. 32 Figure 2.6: UV light has an emission wavelength range from 100nm to 400nm. Within this band of wavelengths there are four primary sub-bands known as UV-A, UV-B, UV- C, and Vacuum UV [13]. Figure 2.7: Each band of UV light has its own set of applications. This figure shows several examples of applications as a function of wavelength [23].

33 33 The range of wavelengths from 315 nm to 400 nm is specified as long wave UV or UV-A [13]. UV-A also sometimes referred to as black light has a number of applications included UV curing of polymers and printer inks, light therapy in medicine, medical imaging of cells, and bug zappers as flies and other insects are drawn to light emitting at or around 365 nm. Solar UV-A radiation composes 95% of all the UV light that reaches the Earth s surface. The range of wavelengths from 280 nm 315 nm is specified as medium wave or UV-B [13]. UV-B applications include protein analysis, DNA sequencing, and drug discovery as well as some medical imaging of cells. UV-B composes the other 5% of solar UV-radiation at the earth s surface and is attributed with the DNA damage that leads to skin cancer. The wavelengths between 100 nm and 280 nm are specified as short wave or UV-C [13]. Applications for UV-C range from forensic analysis to optical sensors. A fourth category of UV light emerges from within the UV-C band and this is typically referred to as vacuum UV or V-UV. V-UV contains the wavelengths from 100 nm 200 nm [13]. It is referred to as V-UV because emission at these wavelengths, in the vast majority of media including air and in our case water, is absorbed so quickly that it cannot be used for any applications. Thus the only environment where it is not absorbed immediately is in a vacuum. The specifications and categories of UV light are subject to interpretation however these four categories are the most widely accepted. Contained within the UV-C band are also the most widely used wavelengths for disinfection purposes (265 nm to 280 nm). Chen et. al. shows the absorption tendencies of DNA based on wavelength, shown in Figure

34 [8], which demonstrates that the absorption of UV radiation by DNA is reaches a local maximum at around 270 nm, within the UV-C range. An important fact to notice from Figure 2.2 is that although there is a local minimum in DNA absorption of UV radiation around 240 nm as the wavelength continues to decrease DNA sbsorptions increases dramatically and is about three times the absorption at 254 nm by the time the wavelength reaches 220 nm. Conventional UV treatment relies on low and medium pressure lamps that typically emit UV light at 254 nm. Some special low pressure lamps have been designed to emit UV light between 185 nm and 254 nm. Additionally medium pressure UV lamps are capable of emission at a wide range of UV wavelengths [24]. The typical emission spectrum of these lamps is shown in Figure 2.8..

35 35 Figure 2.8: UV low pressure and medium pressure mercury vapor germicidal lamp spectrum The aforementioned photoreactivation comes into question here as there is a peak from the medium pressure mercury vapor lamp within the band of wavelengths that can repair some of the damage done to DNA by shorter UV-C wavelengths. With the rise in research for new UV light engines the number of ways in which to produce UV light has increased with a primary contender in the realm of solid state UVLEDs. By using ternary compounds of III-nitride semiconductors like Aluminum Gallium Nitride (AlGaN) and some quaternary compounds such as Aluminum Indium Gallium Nitride (AlInGaN) scientists can now create semiconductor LEDs capable of emitting at a wide range of wavelengths in the UV spectrum. The use of AlN and AlGaN has produced resulting

36 36 UVLEDs with emission at wavelengths as low as 210 nm [25]. Additionally, and more recently the use of AlInGaN, AlGaN, and AlN have been used to create superlattices and multi-quantum wells within the active layer and the cladding of UVLEDs to increase their efficiency by lowering the operating voltage and encouraging electron-hole recombination at certain wavelengths. 2.6 Development of UVLEDs Publications about the development of UVLEDs started to increase in the late 1980 s. In 1988 Mishima developed one of the first UVLEDs [26]. It was constructed from a simple cubic BN pn junction and had an emission spectrum centered at around 340 nm-360 nm and extending from 215 nm to the visible red. Other experimentations continued through the 1990 s in which UVLED light was generated from various materials as the ability to mix multiple semiconductors into ternary and quaternary compounds increased the quality of the LEDs which further increased the ability to finetune the devices. The most common ternary and quaternary compounds used in the development of UVLEDs are AlGaN and AlInGaN which are typically deposited with an undoped AlN sample to produce UV light at wavelengths varying as a function of the fine-tuning between the bandgap of AlN and the ternary and quaternary superlattices surrounding it. Each AlN based LED has a theoretical minimum just below 6.1 ev or just above 205 nm because the AlN component has a bandgap of ev [27] to 6.2 ev [28] at 300 K whereas the GaN is between 3.2 ev (for Zinc Blende) and 3.39 ev (for

37 37 Wutzite) [28] at 300 K and InN is about 1.9 ev to 2.05 ev at 300 K [27] [28], respectively. In 2006 an AlN LED was produced with an emission of 210 nm using AlN/AlGaN superlattices [25]. The structure of this AlN/AlGaN UVLED is shown in Figure 2.9. Figure 2.10 shows an idealized band diagram for this AlN/AlGaN UVLED. The superlattice represented by the regions marked as p-type and n-type AlN/AlGaN superlattice acts to decrease operating voltage [25]. As depicted in Figure 2.10 when a voltage is applied electrons begin to pass through the active region and when a hole with the same energy is matched to the electron, the electron recombines with the hole and during that recombination process a photon can be emitted at the same energy as the recombination energy. Figure 2.9: AlN/AlGaN based superlattice structure used in 2006 produced a UVLED with an emission wavelength of 210 nm which is very close to the minimum possible wavelength using AlN and ternary compounds.

38 38 Figure 2.10: Idealized band diagram of the device depicted in Figure 2.9 shows the basic operation of an LED with an AlN active region. In more modern cases the implementation of multi-quantum wells [29] is used to encourage recombination at a specific wavelength. This encouragement takes place by implementing quantum wells in the active layer of the LED. The electrons then become trapped at an energy state within the quantum well. Being trapped in the active region the electron is forced to recombine from the conduction band to the hole in the valence band and emit light at the wavelength specified by the band gap of the quantum well.

39 39 The implementation of these quantum wells not only increases the chance of correct recombination but the efficiency of the device as well [30]. Quantum wells are created in ternary and quaternary compounds by depositing a layer of the compound with a certain concentration (e.g. Al.4 Ga.6 N where the material is 40% AlN and 60% GaN) and then a layer of the same (different in some cases) compound with a different concentration (e.g. Al.5 Ga.5 N where the compound is 50% AlN and 50% GaN). This layer combination would be repeated many times. This type of engineering of material bandgaps is called bandgap engineering [29]. The principles behind bandgap engineering are relatively simple since there is a semi-linear relationship between the bandgap of the ternary compound (e.g. AlGaN) and the bandgaps of the two separated compounds (e.g AlN and GaN). There is however, a bending, or bowing, in this linearity when combining two or more elements from group III on the Periodic Table [31]. This effect is referred to as Vegard s Law and is further accentuated when Indium is involved. The bowing is directly related to the concentration of the group III elements within the structure. Equation 2.1 displays this relationship for a ternary compound of Al x Ga 1-x N compound. In equation 2.1, E g represents the bandgap of the specified material, x refers to the concentration of the specified group III element, and b represents the bowing parameter [31]. The primary reason behind this bowing in the linearity is due to the crystal lattice

40 defects which arise from the fact that each of these semiconductors (AlN, GaN, and InN) 40 has a different lattice constant. When materials of different lattice constants are combined, there is an associated stress or strain introduced in an attempt to equalize the lattice structure. This stress or strain in this material creates lattice defects which hinder the material s ability to perform as expected. Specifically with regards to radiative recombination, a necessary action in LED operation, dislocations like these sometimes manifest themselves in energy levels within the bandgap. These incidental energy levels can encourage non-radiative or radiative recombination at an undesired wavelength by providing an intermediate energy step between the conduction band and the valence band. The whole purpose behind LEDs is to create light, thus these dislocations and defects can become a very real issue in LED development. Figure 2.11 further illustrates this. The increase in tension and/or compression placed on the lattice by combining two materials with different lattice constants directly relates to the increase in crystal lattice defects and thus an increase in non-radiative recombination decreasing the efficiency of the UVLED.

41 Figure 2.11: (A) GaN is deposited on an AlN substrate, which creates tension in the AlN and compression of the GaN. (B) AlN is deposited on a GaN substrate, which causes tension in the AlN and compression in the GaN. (C, D) The results of each deposit in which the circles highlight the lattice dislocations produced as a result of the stress produced by lattice mismatch. 41

42 42 3 EXPERIMENT 3.1 Development of Experimental Setup Design Criteria In developing a testing apparatus and experimental setup for a UVLED based water disinfection system safety, among other things, must be considered. The system had to be a closed system which would contain all UV radiation. Depending on the method of disinfection there were a couple of options. The two major types of disinfection systems are flowing and static. A flowing system disinfects the water while it is in motion, and optimal flow rate is determined by the strength of the UV radiation power density. A static system disinfects a certain volume of water, drains the disinfected water, refills with untreated water, and then restarts the process. In a flowing system the UV light is usually contained within a cylindrical metal tube that has an inlet and an outlet for water to flow however, in implementing UVLEDs this system was rejected due to the difficulty of assembly. A static system allowed more freedom with the design. The design agreed upon for this research and testing was a static system that incorporated a magnetic stir plate in order to ensure a more evenly distributed amount of UV radiation throughout the water. This design while not ideal for a large scale disinfection system is very useful in a laboratory setup for a study of inactivation effectiveness. Because of the nature of the UVLED test sample (producing a maximum

43 power density with the 270 nm UVLED of 50 µw/cm 2 ) only 20 ml of infected water was placed in a beaker to be disinfected and this beaker was surrounded with an acrylic 43 plexiglass box with a thickness sufficient enough to dissipate stray UV rays. An additional consideration in this experimental process is the incident application of ambient light. As previously discussed there is the possibility of photoreactivation and dark reactivation during the disinfection process, however unlikely. If there is light being permitted to enter the testing apparatus that is between 320 nm and 370 nm then photoreactivation could be in effect [11]. Before conducting these experiments the ambient light in the room was measured and there was no trace of UV light between 320 nm and 370 nm. Therefore it was safe to assume that photoreactivation would not be a problem Design and Construction Figure 3.1 is a diagram that displays the design for the testing apparatus and experimental setup.

44 44 Figure 3.1: Basic design of the experimental setup taking into account the LED emission pattern and making use of a magnetic stir plate to encourage uniform UV distribution. (A) A side view of the test apparatus. (B) Atop down view of the test apparatus. UVLEDs with a design similar to SETi s, [32] while considered to be a point source, are not a perfect point source. Instead these UVLED follow a lambertian emission pattern shown in the SETi data sheets in Appendix A. Taking into account the fact that a UVLED is not a perfect point source the intensity of UV radiation at the edges of the beaker was not anticipated to be the same as in the middle. In order to encourage a more uniform distribution of UV radiation the magnetic stirrer was used to create mini vortex so that bacteria in the water would spend equal time at the edge of the beaker as they would in the center. Additionally a structural base was added to the testing apparatus to allow for spectral and output power measurements of the UV radiation. Other testing solutions were considered including a system developed by Sandia labs for UV disinfection testing [33]. The system implemented by Crawford et. al. implemented a UV lamp whose light was guided through a collimator and diffuser to ensure uniform exposure. Because of the limited resources and time in the experiments conducted for

45 this thesis the approach described earlier was implemented. However for future testing to avoid the need of a magnetic stirring plate this technique could be implemented Additional Instrumentation In addition to the testing apparatus a couple of other instruments were utilized. For the purpose of spectral analysis an Ocean Optics USB UV/Vis Spectrometer was used. In order to calculate the UV dose received a Newport model 1815-C power meter was used to measure power density at a distance equal to the distance between the water and the UVLEDs. The power required to operate the LEDs was generated by a Tektronix PS503A dual power supply. In order to monitor the operating characteristics of the UVLEDs three Tektronix DM502A auto-ranging digital multimeters were used. These instruments were required to ensure that the UVLEDs were operating at the correct and optimal parameters. Data sheets for the UVLEDs used in this thesis are included in Appendix A. The final experimental setup, including all equipment used, is shown in Figure 3.2.

46 46 Figure 3.2: Photograph of the experimental setup including all essential equipment. Included are the (A) Power supply and multimeters, (B) UVLED array, (C) UV spectrometer, (D) Computer interface, (E) Plexiglass testing apparatus, and (F) Newport Power Meter. 3.2 UV Light Source Analysis UVLED Research Standard The UVLEDs set as the standard for this research and analysis were purchased from SETi [34]. The typical wavelength standard for most mercury vapor UV disinfection systems is 254 nm. Additionally the vast majority of the Concentration- Time (CT) tables for UV radiation, which provide necessary doses for disinfection of particular bacteria and viruses, are also based on UV radiation at 254 nm. However given the goal of producing a low cost system and examining the pricing differences in

47 47 the deep UVLEDs offered by SETi the decision was made to explore several different wavelengths of UV radiation and examine the effectiveness of each wavelength in the disinfection process. Three LED wavelengths were chosen for this research primarily in order to explore the inactivation effectiveness as a function of wavelength as well as to provide a range of disinfection results. The three original wavelengths that this research was based upon were 250 nm, 270 nm, and 295 nm (The 295 nm UVLEDs were later discarded from this research because of their extreme inefficiency against the control bacteria This will be discussed in further detail in the Discussion Section of this thesis) Spectrum Analysis of Commercial UVLEDs The spectral analysis for the SETi UVLEDs was performed with an Ocean Optics USB UV/Vis Spectrometer. The intensity of light was measured in counts as a function of wavelength. Figure 3.3 shows the spectral results of each UVLED and Figure 3.4 show the normalized spectral results.

48 48 Figure 3.3: Spectral analysis results of SETi 250 nm, 270 nm, and 295 nm UVLEDs. Figure 3.4: Normalized spectral analysis results of SETi 250 nm, 270 nm, and 295 nm UVLEDs.

49 49 The spectral analysis of the SETi commercial UVLEDs revealed a variance in specified details, which is important to note in this research. The spectral peaks for these UVLEDs while specified at 250 nm, 270 nm, and 295 nm were actually measured at nm, nm, and nm, respectively. The data sheets for the UVLEDs specify that the specified wavelength is the minimum wavelength and that an error of ±9 nm Power Density Analysis of Commercial UVLEDs UV optical lower density analysis was performed with a Newport 1815-C power meter. The power density was measured at a distance of 40 mm from the source when directly in front to the UVLED with the sensor perpendicular to the UV light. A measurement was taken every 2.5 mm laterally on the plane perpendicular the UV light. Therefore the actual distance from the UVLED light source to the sensor increased based upon Pythagorean s theorem displayed in Equation 3.1 below, where is the minimum distance from the UVLED to the perpendicular plane. The results from the power density analysis are displayed in Figure 3.5.

50 UVLED Power Distribution 60.0 Power Density (µw/cm 2 ) nm 254nm* 270nm 295nm Lateral (Angled) Distance from LED (mm) Figure 3.5: Displayed are the results of the power density analysis. The 250 nm UVLED peaked at about 15 μw/cm 2, the 270 nm UVLED peaked at about 50 μw/cm 2, and the 295 nm UVLED peaked at about 39 uw. The reference point is a 254 nm low end mercury vapor lamp which has a power density of about 57 μw/cm 2. * Indicates the power density of a 254 nm mercury vapor lamp. The SETi 250 nm, 270 nm, 295 nm UVLEDs had a peak power density at 15 µw/cm 2, 50 µw/cm 2, and 39 µw/cm 2, respectively, at an absolute distance of 40 mm from the source. As expected when moving laterally from the away from the perpendicular UV rays the power density diminishes by about 1.5 times when 20 mm laterally from normal. The additional UV light source specified in Figure 3.5, shown in black and asterisked in the legend, is not a measured statistic but rather it is the rated output power of a low end mercury vapor lamp developed by Trojan UV emitting at a

51 51 wavelength of 254 nm. This reference is placed in Figure 3.5 to allow comparison of power density output of these UVLEDs in comparison to the commercially available UV mercury vapor lamps. The 270 nm SETi UVLED is closely comparable in terms of peak power density to the low end mercury vapor lamp. This observation substantiates previous statements that these UVLEDs, although they are early in the developmental stages, are in some cases very comparable to, in terms of output power, low pressure mercury vapor lamps Power Supply and Circuit Analysis To increase power output and decrease necessary exposure time three of each UVLED were arrayed in a triangle pattern 1cm apart from each other and were connected in series. Using a resistor decade box and the Tectronix dual power supply, a consistent current of 19.9 ma was propagated through the circuit. Table 3.1 shows the values of the decade box, power supply, voltmeter, and ammeter for each UVLED array varying only by wavelength.

52 Table 3.1: UVLED characteristics at their maximum rated output during water disinfection trials. Power density measured at 40 mm from source. UVLED Characteristics Max rated output Serial # UVLED R (Ω) I (ma) V s (V) V d (V) Power Density at normal (90º) and 40 mm (µw/cm 2 ) 0Y Z53 250nm ON N N8 270nm D IM IM52 295nm IC Bacteria Preparation and Growth Rationale for Bacillus subtilis In order for a water disinfection system to be approved by the Environmental Protection Agency (EPA) Cryptosporidium and Giardia must be shown to achieve 3-log inactivation. Log inactivation is an alternative measurement for percentage where 2-log is equivalent to 99%, 3-log is equivalent to 99.9%, and so forth. Because of the difficulty in the propagation and maintenance of Cryptosporidium and Giardia, Bacillus subtilis spores were used. Results with B. subtilis were able to validate the trial and receive treatment credit for both Cryptosporidium and Giardia because B. subtilis UV resistance

53 53 is considerably higher than both Cryptosporidium and Giardia. In addition the EPA suggested the inactivation of the viral MS2 Phage propagated in Escherichia Coli. However, this research focused on the propagation and inactivation of B. subtilis spores because of their extremely high resistance to UV disinfection and similarity in inactivation dose to the MS2 Phage. Table 3.2 below shows a comparison of bacteria and virus considered and their respective dosages needed in order to achieve various log inactivations. Table 3.2: Log inactivation dose for specified bacteria or virus as stated in referenced publications. Delivered UV Dose (mj/cm 2 ) to Achieve Indicated Log inactivation Microorganism 1-Log 2-Log 3-Log 4-Log Reference Giardia < 2 < 2 < 4 [35] Cryptosporidium [36] Salmonella typhi [37] B. subtilis spores [38] E. coli [39] MS2 Phage [40] Poliovirus [41] Rotavirus SA [42] Bacillus subtilis Preparation The B. subtilis spores were propagated using Schaeffer s medium [43] [44] [45]. The exact procedure used was an adaptation of the procedure in [45] as suggested by the

54 EPA in [13]. The first step was the preparation of the Columbian agar. For each liter produced the materials and amounts needed of each are listed in Table 3.3 below. 54 Table 3.3: Ingredients for Columbian agar. Columbian Agar Ingredient Amount (g) Special Peptone Starch 1.00 NaCl 5.00 Agar After measuring and mixing the materials deionized water was added to make a solution with a total volume of 1 L and then the solution was buffered until the measured ph was 7 using a phosphate buffer. The solution was then autoclaved at 120 ºC for 20 minutes. Following the autoclave procedure the solution was poured into disposable petri dishes and allowed to cool to form the agar medium that would be most suitable for B. subtilis growth. Following the rehydration of B. subtilis (Data Sheet for ATCC 6633 is included in Appendix B) the bacteria were spread on several petri dishes containing the Columbian agar and were allowed to grow by incubation at 37 ºC for hours. After the initial propagation of B. subtilis, several colonies were placed in a media optimized for long term bacterial storage and placed in a refrigerator at 4 ºC to minimize bacterial replication. A T-streak was performed every two weeks for the B. subtilis as a backup for the long term storage in the event that the bacteria in long term storage failed.

55 55 The next step in the procedure was the preparation of the sporulation medium. For every 1 L of sporulation medium the materials and amounts needed of each are listed in Table 3.4 below. Table 3.4: Ingredients for sporulation media. Sporulation Media Ingredient Amount (g) MgSO 4 H 2 O KCl FeSO 4 7H 2 O Nutrient broth After mixing the materials deionized water was added to make a solution with a total volume of 1 L and then the solution was buffered to ph 7 using a phosphate buffer. The solution was then autoclaved at 120 ºC for 20 minutes. After the solution cooled to room temperature it was divided into three separate 500 ml Erlenmeyer flasks each with a volume of about 330 ml of sporulation media in each flask. These solutions were inoculated with 3-5 colonies of B. subtilis from the cultures propagated after rehydrating the bacteria and then placed in a shaker operating at 2 Hz and incubated at a temperature of 37 ºC for 72 hours. Following the incubation period the solutions were recombined and sonicated for 10 minutes at 50,000 Hz and at a temperature of 10 ºC. After the sonication procedure was completed the spores were harvested from the solution by using a centrifuge operating at 5000G and a temperature of 10 ºC for 10 minutes. The

56 56 harvested spores were washed by re-suspending them in 20 ml of distilled water and recentrifuged. This process of washing and re-centrifuging was repeated for a total of three times. Following the washings, the spores were re-suspended in 100 ml of M phosphate-saline buffer (PBS). The solution was then heated to 80 ºC for 10 minutes in order to inactivate vegetative B. subtilis. After sonicating again at 50,000 Hz and 10 ºC for 10 minutes to shed the outer membrane of each bacterium the spores were removed from the inactivated cells. This resulting solution was stored at 4 ºC to ensure that the spores remain in the spore state and did not begin to form active, replicating bacteria. This resulting spore-containing solution consistently produced between colony forming units (CFU) at a dilution of 10 4 for the first batch and between CFU at a 10 4 dilution for the second batch. This was sufficient to test for a 3-Log inactivation and thus the experimentation continued to water disinfection testing. 3.4 Bacteria Exposure Experiments Testing Procedure The testing procedure for the disinfection portion of these experiments made use of redundancy to ensure correct readings for each test result. Three separate disinfection tests were performed for each UVLED wavelength. Each of these three tests was performed at five exposure times. After each exposure the resulting solution was spread

57 onto the Columbian agar in three separate petri dishes. The flowchart in Figure 3.6 helps to grant a more detailed image of the testing process. 57 Figure 3.6: This flowchart details the disinfection testing process for the 250 nm UVLED. This process was repeated three times per wavelength.

58 58 As Figure 3.6 shows, each exposure test began with a single 20 ml solution of B. subtilis spore infected water at a dilution of From there, 100 µl was removed and diluted down to 10 4 and three 100 µl samples of the 10 4 dilution was spread onto three separate plates to serve as the reference. Additionally, from this sample 100 μl was diluted by another factor of 10 to 10 5 and plated in the same manner as the 10 4 dilution. This repetition at different dilutions ensured an accurate plate count and thus and accurate calculation of the exact number of CFUs in the water prior to disinfection. The original sample was then exposed to UV light for a certain time period, either 10, 15, 20, 25, or 30 minutes. After the exposure, 500 µl of the sample was removed. Of the 500 μl, 200 μl was stored at 4 C and the remaining 300 µl was spread onto three separate plates in 100 µl volumes without any dilution. Thus the UV exposed water contained a 10 2 dilution of B. subtilis spores. After the exposure tests were complete the plates were incubated for 24 hours at 37 ºC and then the CFU were counted. The process described above was repeated for each exposure test each exposure starting with 20 ml of B. subtilis infected water Percent Inactivation Calculation Diluting down to 10 5 before spreading the spores on the reference plates and plating the exposed water at a dilution of 10 2 was necessary in order to accurately assess the degree of inactivation and verify that it was sufficient to satisfy the EPAs standards

59 for drinking water disinfection of B. subtilis spores. The concentration of B. subtilis on a cultured plate is shown by Equation 3.2, 59 where, is the dilution factor, is the number of CFU, and is the volume of the solution plated. Adaptation of this summation to calculate a percentage as shown in Equation 3.3 reveals the inactivation percentage. In Equation 3.3, is the reference sample and refers to the exposed sample. Thus, if the dilution of the reference was 10 5 (the dilution was selected between 10 5 and 10 6 to ensure that there was between 30 and 300 CFU on a reference plate for accuracy) and there were the same number of CFU on the exposed sample which was at dilution of 10 2, then the inactivation percentage would be 99.9% or 3-log, exactly what is necessary to satisfy the EPA standards. Diluting the original sample down to 10 5 allowed the possibility of achieving accurate inactivation results greater than 99.9% or 3- log.

60 60 4 RESULTS 4.1 Bacillus subtilis disinfection results for 250 nm and 270 nm UVLEDs The results from the disinfection experiments were recorded and the inactivation percentages shown in Table 4.1 and Table 4.2 the graphs shown in Figure 4.1, Figure 4.2, and Figure 4.3 were produced. Table 4.1: Inactivation percentages for the three trials of 250 nm UVLED exposures 250 nm UVLED Disinfection Results Exposure Time (min) Trial 1 Trial 2 Trial 3 Average Std. Dev % 84.76% 83.15% 83.43% 1.22% % 93.40% 92.20% 92.87% 0.61% % 99.28% 98.72% 98.82% 0.42% % 99.89% 99.73% 99.74% 0.15% % 99.96% 99.93% 99.93% 0.02% Table 4.2: Inactivation percentages for the three trials of 270 nm UVLED exposures. 270 nm UVLED Disinfection Results Exposure Time (min) Trial 1 Trial 2 Trial 3 Average Std. Dev % 99.72% 99.67% 99.68% 0.03% % 99.80% 99.77% 99.77% 0.03% % 99.87% 99.85% 99.85% 0.03% % 99.91% 99.90% 99.90% 0.01% % 99.94% 99.93% 99.94% 0.01% Table 4.1 displays the inactivation percentage results of the 250 nm UVLED experimental from 10 minutes to 30 minutes exposure time in 5 minute increments. The

61 61 results of the 250 nm UVLED exposures shows that 3-log in activation was achieved between 25 minutes and 30 minutes, most likely closer to 30 minutes based on an interpolation of the data in Figure 4.3. Table 4.2 displays similar results from the 270 nm UVLED experiments. The required time to achieve 3-log inactivation of the B. subtilis spores exposed to 270 nm UVLED radiation was 25 minutes, which was shorter than that of the 250 nm UVLEDs. This would appear, on the surface, to suggest that 270 nm UV light is more effective in disinfection of bacteria and organisms. However after using the power density and exposure time to calculate the required dose to reach 3-Log inactivation it was obvious that it is actually higher at 270 nm than 250 nm which is discussed in more detail in the discussion section.

62 Figure 4.1: B. subtilis spore inactivation percentage as a function of exposure time to 250 nm UV radiation. 62

63 63 Figure 4.2: B. subtilis spore inactivation percentage as a function of exposure time to 270 nm UV radiation. Figure 4.1 and Figure 4.2 provide a good indication of how the disinfection process works based on time and constant power density. It could be implied that the disinfection process follows a semi-logarithmic pattern. This implication is supported by the majority of articles focusing on UV B. subtilis disinfection including Sommer [38] and Nicholson [46]. While this is an expected result in practice as it is nearly impossible to achieve a complete disinfection, it also helps analyze the bacteria s resistivity to UV disinfection treatment and gives better expectations for future testing. Other potential increases in the resistance of some bacteria to disinfection involved the aforementioned

64 64 processes of photoreactivation and dark reactivation. However, it is unlikely that these effects made any significant impact in the tests results described in this thesis. The variation in the y-axis of Figure 4.1and Figure 4.2 was necessary to indicate a change in disinfection percentage for the 270 nm UVLED. It became apparent immediately that the 270 nm UVLED was more effective at disinfection than the 250 nm UVLED, wheret after only 10 minutes, a 2-log inactivation had already occurred in all three trials. Reasons for this seemingly contradictory fact (one would expect that the higher energy UV radiation would be more effective against microorganisms) are explored in more detail in the discussion section. Figure 4.3 provides a comparison between the plots of the 250 nm exposure trials and the 270 nm exposure trials.

65 65 Figure 4.3: B. subtilis spore inactivation percentage of the averaged values of 250 nm and 270 nm exposure trials as a function of exposure time. The miniature plot zooms in on the 3-log inactivation area. Figure 4.3 shows more plainly the difference in effectiveness initially in favor of the 270 nm UVLED. However, even though the 270 nm UVLED reached 2-Log inactivation much quicker than the 250 nm UVLED, the additional time required to reach 3-log from 2-log for the 270 nm UVLED in comparison to the 250 nm UVLED was over 6 minutes. After reaching 2-log inactivation the 270 nm UVLED took approximately 15 additional minutes to reach 3-log inactivation whereas the 250 nm UVLED only needed an additional 7-8 minutes to reach 3-log inactivation after achieving 2-log inactivation. Reasoning behind this interesting phenomenon is provided in the discussion section.

66 The Exclusion of the 295 nm UVLED As previously alluded to the 295 nm UVLED was removed from analysis after the first couple rounds of testing. The primary reason for the removal was the extreme ineffectiveness in disinfecting B. subtilis spores. This can be explain by an number of things. First and foremost Figure 2.2 shows that at 295 nm DNA absorption of UV radiation is at approximately 10% as compared to the absorption at 254 nm. That fact in combination with the 15 µw/cm 2 lower power density of the 295 nm UVLEDs as compared to the 270 nm UVLEDs as well as the consideration that the longer wavelength corresponds to less energy, is a likely reason that disinfection at this wavelength was not effective. Disinfection at this wavelength, while entirely plausible, would take a longer time for the 295 nm UVLED than that either of the 270 nm and 250 nm UVLEDs. If exposed to significantly longer doses, it could potentially disinfect the water just as the other two wavelengths, however for the purpose of this thesis and the development of an efficient water disinfection system the theoretical time requirements to achieve inactivation of B. subtilis spores was simply too long to pursue. Thus, because of the statistical insignificance and irrelevance of the 295 nm UVLED testing the research and data was removed from this thesis and discarded as a research possibility.

67 67 5 DISCUSSION 5.1 EPA Standards and Experimental Log Inactivation Dose Comparison According to Sommer [38] along with most sources for B. subtilis disinfection the log inactivation dose is reiterated by Table 5.1 along with calculated doses for the 250 nm UVLED from this research. Table 5.1: B. subtilis UV dosage required and demonstrated by experiments in order to reach 2-log and 3-log inactivation. Delivered UV Dose (mj/cm 2 ) to Achieve Indicated Log Inactivation of B. subtilis spores 1-Log 2-Log 3-Log 4-Log Reference [38] UVLED Test Table 5.1 demonstrates that the 2-log and 3-log inactivation doses required from this research testing the UVLEDs was 40.8 mj/cm 2 and 59.2 mj/cm 2, respectively. Since the majority of UV disinfection tables are from low pressure 254 nm UV mercury vapor lamps only the 250 nm UVLED was included in Table 5.1. Table 5.1 also demonstrates that this testing is consistent with previous publications as the 2-log inactivation was within 5% of the doses measured by Sommer [38]. In addition the 3-log inactivation was within 2% of the previously stated dose. It is important to note that variations in required doses are common and usually expected in different trials and can be attributed to

68 variations in the cleanliness and therefore the absorption coefficient of the media infected with the bacteria which was discussed earlier UV Wavelengths Inactivation Effectiveness Comparison One striking observation from this research comes from the comparison of the inactivation efficiency and effectiveness of the 270 nm and 250 nm UVLEDs. The 270 nm UVLED was clearly shown to inactivate the B. subtilis spores at a faster rate than the 250 nm UVLED, but the 250 nm UVLED showed a lower dose requirement to achieve various log inactivation steps and was therefore more effective against the bacterial spores in question. While the 270 nm UVLED reached 3-log inactivation quicker than the 250 nm UVLED it is also important to note that the 270 nm UVLED displayed an output power that was over 3 times greater than that of the 250 nm UVLED. If they had the same dose requirement, it would be expected that because of the power difference, the 270 nm UVLED would reach 3-log inactivation much sooner (approximately 3 times) than the 250 nm UVLED instead of only slightly before. Instead the 270 nm UVLED displayed much higher required doses to reach 2-log and 3-log inactivation. This was initially a slightly puzzling result considering that the DNA UV absorption plot shown by Chen et. al. [8] in Figure 2.2 suggests that absorption is reaches a local maximum at approximately 270 nm while at 250 nm the absorption is approaching a local minimum. The primary explanation that can be for this result is due to the fact that 250 nm UV radiation carries more energy per photon than 270 nm UV radiation. Even though more

69 69 UV light is being absorbed at 270 nm, it does not do as much damage to the DNA as its 250 nm counterpart. Therefore, even though the 250 nm UV radiation is not absorbed as frequently, when it is absorbed, it is more effective. 5.3 Large Scale Disinfection System Analysis The final necessary evaluation is the plausibility of these UVLEDs being implemented into a large scale disinfection system. It has been shown in this thesis that these UVLEDs are capable or disinfecting water from B. subtilis spores and therefore most likely all other bacterial and viral contaminants. Table 5.2 shows several characteristics of SETi UVLEDs used in this thesis along with pricing. Table 5.2: Cost and power out characteristics for SETi UVLEDs used in this thesis. Catalog Number Purchase Date Wavelength (nm) Current (ma) P out (µw) Min Typical Cost (USD) UVTOP255TO18FW Oct $ UVTOP270TO18FW Oct $ UVTOP295TO18FW Oct $ The costs of these UVLEDs per unit are expensive as shown in Table 5.2. The young age of this technology currently limits the opportunity for affordability.

70 70 6 CONCLUSION It has been shown that the selected UVLEDs are as capable as other UV sources in water disinfection. This research has shown UVLEDs can inactivate B. subtilis spores to EPA standards of 3-log inactivation and reflected a similar inactivation dosage requirement as previous publications of B. subtilis UV radiation inactivation. Although a significant amount of time was required to disinfect a small volume of water, it is entirely plausible that a by increasing the number of UVLEDs, this project could be modified to create a quick, large-scale water disinfection system capable of providing clean water daily to villages like Maase-Offinso with over 1000 people. This is primarily because disinfection time requirements are directly proportional to the UV output power. Simply adding more UVLEDs to the system will increase the output power and UV intensity, and thereby decrease the time required to disinfect. However, because of the current cost of individual UVLEDs, considering the original goal of our project, to deliver a large scale, efficient and low cost disinfection system for developing countries, it unlikely that anyone would be able to produce an inexpensive and efficient large scale disinfection system without the UVLEDs cost coming down. Additionally, the cleanliness of the water to be disinfected is a primary factor in the efficiency of UV disinfection systems to allow for full penetration of the light. In order to have an effective UV disinfection system a suitable filtration system must be implemented first. Despite these economic considerations the concept behind UV disinfection systems is sound and it appears that the only major issue in the way of UVLED disinfection systems is the cost. Beyond this

71 71 fact UVLEDs have been shown to be superior in many ways in comparison to the mercury vapor lamp alternatives, including life expectancy, durability, and energy consumption. The potential issue of dark reactivation still remains but photoreactivation can be easily avoided by engineering of the LEDs.

72 72 7 FUTURE WORK 7.1 The Next Step It has been shown to a reasonable degree that producing an efficient and affordable large scale disinfection system out of commercially available UVLEDs would be difficult at this point in time. Despite the benefits and other positive attributes of UVLED operation described in this thesis, until the cost per unit is decreased significantly it would be difficult to provide a disinfection system capable of providing clean water for a village the size of Maase-Offinso, Ghana on the budget of a developing country. Therefore in light of the original goal it was necessary to begin looking for answers in other directions. The focus of continued research will shift to the generation of UV light from other potentially more efficient devices capable of providing more output power than the UVLEDs examined. A quick glance around the semiconductor world and it is apparent that the direction to move towards is exploring more UV light generation within wide bandgap semiconductors. Commonly used semiconductors like Silicon (Si) or Gallium Arsenide (GaAs) have bandgaps of 1.12 ev and 1.42 ev, respectively. Semiconductors are typically referred as wide bandgap semiconductors when their bandgap exceeds 1.7 ev. However, in order to emit in the UV range the bandgap must be greater than 3.1 ev using the conversion shown by Equation 7.1,

73 73 where is the bandgap, is plank s constant, c is the speed of light, and is the wavelength. Therefore the initial examination will focused on wide bandgap semiconductors. Within the family of wide bandgap semiconductors, only a few have bandgaps wide enough to fit the needs of generating deep UV radiation. After preliminary research into several of these candidates, Boron Nitride (BN) stood out because it has yet to be thoroughly researched and it has been used in the past and recently to produce UV light through several different techniques. The exploration of BN will be a primary focus in the works following this thesis. 7.2 Excitation of BN by Impact Excitation There are several techniques that can be implemented to allow BN to emit light. One technique described by Mishima [26] in 1988 is to simply implement BN into a supperlattice to create an LED. However, more modern techniques aided by newer technologies can grant similar or in some cases better results with a different configuration, such as electron field emission through carbon nanotubes [47]. An adaptation of this technique could be used to generate a larger area of UV light emitter thus increasing the uniformity of exposure if implemented into a BN base structure. Rather than a point source device, a device similar to this could be made into a large area of material that could then be irradiated, in this case, by energetic electrons. Another design allows similar results and more flexibility in design, with respect to adapting

74 74 future devices to fit current disinfection systems, is the adaptation of impact excitation. The theory behind impact excitation was described in great detail by Moiseiwitsch [48] in Despite the rich history of impact excitation, the technology to put this theory into practice for BN base devices has not had the necessary precision until recently. The basic concept behind the impact excitation of BN is displayed in Figure 7.1. Figure 7.1: BN impact excitation theory. (A) This simple schematic of the structure of a device that would utilize the impact excitation of BN to generat UV light. (B) This very basic illistration demonstrates an electron colliding with a BN atom and producing a UV photon. Figure 7.1 (A) depicts a simple schematic of a theoretical circuit for a BN impact excitation device. A certain voltage is applied to a conducting material that is separated from the BN powder by a thin insulator. The insulator serves to provide a barrier to make it difficult for any electrons to pass from the conducting material to the BN. However when enough voltage is applied the electrons gain enough energy to pass through the

75 75 insulator. In transit the electrons will acceleration through the material and collide with the BN on the other side. Then, as Figure 7.1(B) shows, upon impact part the energy of the collision will be converted into energy producing a photon at an energy unique to the material of impact which in the case of BN can be as large as about 5.8 ev. Using Equation 7.1 the wavelength corresponding to this energy is 214 nm. 7.3 Initial Cathodoluminescence Experiments In the initial experiments with BN a cathodoluminescence spectrum was measured to confirm the low wavelength emission. The synthesized h-bn material was characterized using cathodoluminescence (CL) spectroscopy revealing several sharp excitonic peaks between 210 nm and 240 nm at 12 K when excited with energetic electrons. The room temperature CL is dominated by a single band at 233 nm due to intrinsic impurity bound exciton with negligible deep levels emission at longer wavelength. An example of the room temperature CL spectrum observed for studied h-bn samples is shown in Figure 7.2. The CL spectrum is dominated by an intense ultraviolet luminescence band peaking at nm with a shoulder on the lower energy wing at ~233 nm and the featureless low intensity emission background at longer wavelengths. The comparison between normalized CL spectra measured with increased spectral resolution from 200 nm to 290 nm at 12 K and 300 K, respectively, is shown in the inset of Figure 7.2. It is seen that the CL spectrum measured at 12 K shows several peaks

76 76 located at ~215 nm, 220 nm, nm, nm and 233 nm, respectively. The 215 nm band is assigned to the h-bn free exciton luminescence. The remaining CL peaks between 220 nm and 240 nm could be assigned as excitons bound by impurities or defects, or a phonon replica of free exciton luminescence bands [49]. The deep UV CL band shape changes with increased temperature, the observed spectral features became less resolved, due to inhomogeneous broadening; however the peaks positions do not change significantly. Furthermore, the room temperature CL spectrum shows a broad band emission tail between 260 nm to 280 nm probably attributable to vacancies or residual impurities [50]. It should be mentioned here that this low intensity deep level emission band indicates a low density of structural defects and intrinsic impurities in synthesized h-bn material. However, at this time we cannot completely rule out that the shape of observed deep UV CL spectrum is also affected by the different morphologies of BN nanomaterial. Recently Watanabe et al. investigated the low temperature CL of free standing h-bn single crystals grown by high pressure-high temperature method [51]. Interestingly enough they reported on the observation of the CL spectra change caused by plastic deformation of free standing h-bn crystal. The evolution of the CL spectra exhibited red-shift of the strongest excitonic peak position from 215 nm to 227 nm at 300 K. It was proposed that this effect is due to the exciton localized around glided planes luminescence energy lowering under applied pressure induced by the weak coupling between the h-bn sp 2 layers as compared to that between boron and nitrogen within a layer. Our samples were synthesized on foreign substrates and hence the h-bn is subjected to the strain developed at substrate-h-bn hetero-boundary. Thus, we believe,

77 that the observed CL spectrum maximum is red-shifted as compared to the CL spectrum 77 of a free standing h-bn. In general, synthesized h-bn shows promising excitonic spectral features in deep UV spectral range without visible emission. Further optical studies should reveal better inside in to origin of the impurity bound exciton peaks as well as the h-bn phase-purity. Typically for CL measurements samples were mounted on the cold finger of a closed-cycled helium refrigerator operating down to 6 K. The CL was generated by the Staib Instruments, Inc. electron gun EK-20-R system being in common vacuum (of 1x10-7 Torr) with the cryostat. The electron beam was incident upon the sample at a 45 angle from an electron gun. The CL depth of the excitation could be easily varied by varying the electron acceleration voltage between 500 ev up to 20 kev. Samples were excited with an electron acceleration voltage of 6 kev and current density of 1.2 µa/mm2 and resulting CL spectra were dispersed by a 0.3 m Acton spectrograph and analyzed by a Princeton Instrument PI-MAX CCD camera equipped with UV intensifier, operating in the spectral region nm.

78 78 Figure 7.2: Room temperature CL spectrum of h-bn material excited by electron accelerated with 6 kev. Inset shows comparison between normalized CL spectra of h-bn material measured at (a) 12 K and (b) 300 K. Vertical arrows indicate deep UV excitonic recombination peaks and a deep level related emission band centered at 270 nm, respectively. Thus far it has been established that wavelengths between 250 nm and 270 nm are the typical wavelengths for water disinfection and that 254 nm is the most common standard because it is produced by mercury vapor lamps. UV light disinfection at 214 nm, the wavelength of emission for BN, has not been well studied. Additionally, recalling Chen s observations of the absorbance tendencies of DNA [8], shown in Figure 2.2, it is clear in his absorption chart that there is a local maximum in absorbance of UV light between 260 nm and 280 nm. There is a local minimum at shorter wavelengths

79 79 between 240 nm and 250 nm. Despite this local minimum at about 245 nm, if the tendency is followed below 240 nm toward shorter wavelengths the DNA absorbance of UV radiation increases significantly. This increase suggests that UV radiation at 214 nm would be more readily absorbed by the DNA and do significantly more damage due to its higher energy. It was seen in the analysis of the UVLEDs in this thesis that the 250 nm UVLED during the disinfection process was much more effective that the 270 nm UVLED, requiring less of a UV dosage to reach inactivation of B. subtilis. While the 270 nm UVLED reached 3-log inactivation faster due to a higher output power than the 250 nm UVLED, much more UV light was being emitted by the 270 nm UVLED than the 250 nm UVLED. In the case of the BN at 214 nm, more UV radiation (at least 3 times the amount of 250 nm radiation) will be absorbed by the DNA than the 270 nm UVLED according to Chen [8] and it will do more damage, when absorbed, than the 250 nm UVLED. This double advantage suggests that a 214 nm UV radiation would be much more effective and efficient than either of the two UVLEDs. One potential drawback at this short wavelength is the problem of water absorption discussed earlier. At 214 nm the absorption coefficient of UV light in water is about 0.02 cm -1. This absorption coefficient is about four times larger than it is at 250 nm and over four time larger than at 275 nm. This could cause difficulty in disinfection however a carefully designed apparatus and disinfection system will likely relax this issue.

80 Plan for Impact Excitation of BN Experimentation The first step in beginning research into the development of BN impact excitation devices would be a thorough investigation into the plausibility of its success. A series of papers were published during the early 1990 s by E. Bringuier outlining the electroluminescence characteristics and anatomy of Zinc Sulfide (ZnS) devices [52] [53] [54] [55]. Through these papers he details impact excitation characteristics of ZnS films through a series of simulations and experiments. Thus, in moving forward with research and production of BN impact excitation devices, it would be beneficial to repeat these simulations replacing the static characteristics of ZnS with those of BN. Repeating these simulations will either bolster the assumptions that BN will be a great material for an impact excitation based device, or it will save valuable time, effort, and resources by revealing flaws that have not yet been considered. In either circumstance it is worthwhile to pursue a modeling simulation before actual experimentation. There are two models referred to by Bringuier [55]. The first of these models is referred to as the lucky-drift model presented by Ridley [56] which is most often coupled with the calculation of bandto-band impact ionization statistics. This model does not contain explicit references to the band structure of the material. The Monte Carlo computer simulations however incorporate a realistic band structure into statistics calculations. This modeling tool was introduced by Fawcett in 1970 [57] and revisited by Shichijo in 1981 [58]. Both models have their advantages and disadvantages but Bringuier identifies that for the most accurate modeling one must consider the results derived from both. Bringuier goes on to

81 81 suggest that modeling impact-ionization rates do not give the most accurate model but instead one should consider using impact excitation, because impact excitation is simpy the inelastic collision of an energetic carrier with an impurity, it is a one-body process which is easier to deal with that carrier-induced hole-electron pair production, a three body process [55]. Impact excitation rate, α e, can be defined by where, is drift velocity at saturation, is the number of electrons, is the effective isotropic cross section, is the group velocity, and ( ) is the energy distribution. Before simply calculating impact excitation an analysis of material characteristics is important. Bringuier focused on the statistics of ZnS in his calculations of high field transport and impact excitation. Among the other things that were taken into account were the electron populations as a function of energy, conduction current as a function of internal field, and average electron energy as a function of internal field. Outlining the behavior of the material before and during impact excitation will be an important step in considering the impact excitation of BN. Both the lucky-drift and Monte Carlo simulation models must be utilized to assess the feasibility of using BN as the primary material in an impact excitation device. Following the outline for the simulations given by Fawcett [57] and Shichijo [58] and using the adaptation presented

82 by Bringuier [52] [53] [54] [55] an accurate assessment of BN for impact excitation base UV emitting devices can be made. 82

83 83 REFERENCES [1] U.S. Environmental Protection Agency, "The History of Drinking Water Treatment," Washington D.C., [2] R. George, The Big Necessity: The Unmentionable World of Human Waste and Why It Matters.: Holt Paperbacks, [3] W. Hijnen, "Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: A review," Water Research, vol. 40, no. 1, pp. 3-22, [4] S. B. O'Reilly, "Mercury Exposure and Children's Health," Current Problems in Pediatric and adolescent Health Care, vol. 40, no. 8, pp , [5] J. D. Burch, "Water Disinfection for Developing Countries and Potential for Solar Thermal Pasteurization," Solar Energy, vol. 64, no. 1-3, pp , [6] (2011) Microscopes: Time Line. [Online]. [7] V. M. Bakhir. (2003) Disinfection of drinking water: problems and solutions. [Online]. [8] R. Z. Chen, S. A. Craik, and J. R. Bolton, "Comparison of the action spectra and relative DNA absorbance spectra of microorganisms: Information important for the determination of germicidal fluence (UV dose) in an ultraviolet disinfection of water," Water Research, vol. 43, no. 20, pp , [9] P. Setlow, "Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals," Journal of Applied Microbiology, vol. 101, no. 3, pp , [10] S. Leuko, B.A. Neilan, B.P. Burns, M.R. Walter, and L.J. Rothschild, "Molecular assessment of UVC radiation-induced DNA damage repair in the stromatolitic halophilic archaeon, Halococcus hamelinensis," Journal of Photochemistry and Photobiology B: Biology, vol. 102, no. 2, pp , [11] P.J. Russell, igenetics: A Molecular Approach, 3rd ed.: Benjamin Cummings, [12] The Nobel Prize in Physiology or Medicine [Online].

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87 87 [50] K. Watanabe, T. Taniguchi, T. Niiyama, K Miya, and M. Taniguchi, Nature Photonics, vol. 3, pp , [51] K. Watanabe and T. Taniguchi, International Journal of Applied Ceramic Technology, vol. 8, pp , [52] E. Bringuier, "Electron multiplication in ZnStype electroluminescent devices," Journal of Applied Physics, vol. 67, no. 11, pp , June [53] E. Bringuier, "Impact excitation in ZnStype electroluminescence," Journal of Applied Physics, vol. 70, no. 8, pp , October [54] E. Bringuier, "Tentative anatomy of ZnStype electroluminescence," Journal of Applied Physics, vol. 75, no. 9, pp , May [55] E. Bringuier, "High-field transport statistics and impact excitation in semiconductors," Physical Review B, vol. 49, no. 12, pp , March [56] B.K. Ridley, Journal of Physics C, vol. 16, p. 3373, [57] W. Fawcett, A.D. Boardman, and S. Swain, Journal of Physics and Chemical Solids, vol. 31, p. 1963, [58] H. Shichijo and K. Hess, Physical Review B, vol. 23, p. 4197, [59] Inc Sensor Electronic Technology. (2011) UVTOP Deep UV LED Technical Catalogue. [Online]. [60] ATCC. Product Information Sheet for ATCC [Online].

88 88 APPENDIX A: PARTIAL SETI UVTOP CATALOGUE [59]

89 89 [59]

90 90 [59]

91 91 [59]

92 92 [59]

93 93 [59]

94 94 [59]

95 95 [59]

96 96 [59]

97 97 [59]

98 98 APPENDIX B: PARTIAL ATCC 6633 B. SUBTILIS DATA SHEET [60]

99 99 [60]

100 100 [60]

101 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Thesis and Dissertation Services!