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Ozone Treatment to Prevent Microbial Growth in Air Conditioning Systems


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OZONE TREATMENT FOR PREVENTION OF MICROBIAL GROWTH IN AIR CONDITIONING SYSTEMS By AAKASH KHURANA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ENGINEERING UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Aakash Khurana

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To my Family and Friends

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ACKNOWLEDGMENTS This thesis would not have been completed without the help of several people whom I wish to thank. I would like to express my great appreciation to Dr. David Chynoweth, my committee chair and sponsoring professor, for his generosity and support, and for giving me this opportunity. He was always there when I needed any advice or to solve any problem. I would also like to give my special thanks to Dr. Arthur Teixeira for his support, interest, knowledge, problem solving and advice. I am grateful to Dr. Teixeira for recommending me to Dr. Chynoweth and suggesting that I contact him; without him, I would never have known about this opportunity. I am indebted to Dr. Jean Andino, who was there from the beginning to assist me with technical issues and allowed me to borrow her ozone analyzer instrument for this research work. Thanks to all my supervisory committee, whose comments and edits contributed substantially to my research and to this document. In addition, I would like to thank Mr. Bob Tonkinson for his help with my many assorted questions. Special thanks go to my friends, Luis Nogueira and Dr. Indaue Mello, who had a great influence on my personal and professional life. Finally, I would like to thank three very special people in my life; without them, this would have never been possible my Mother, Father and sister, to whom I dedicate this. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Problem.........................................................................................................................2 Objective.......................................................................................................................3 2 LITERATURE REVIEW.............................................................................................5 Indoor Air Quality........................................................................................................5 Biofilm Formation........................................................................................................6 Available Methods for Air Treatment..........................................................................9 Ozone as an Aerial Disinfectant.................................................................................11 Properties of Ozone....................................................................................................12 Ozone Generation.......................................................................................................17 Corona Discharge................................................................................................17 Ultraviolet Radiation...........................................................................................18 Electrolysis..........................................................................................................19 Stabilization and Storage of Ozone.....................................................................20 Ozone Measurement...................................................................................................22 Previous Work at UF..................................................................................................24 3 MATERIALS AND METHODS...............................................................................29 Scope of Work............................................................................................................29 Materials.....................................................................................................................29 Preliminary Studies.............................................................................................34 Preparation of Nutrient Media and Inoculum......................................................34 Ozone Generation and Measurement..................................................................36 Experimental Design..................................................................................................39 v

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4 RESULTS AND DISCUSSIONS...............................................................................42 Findings from Preliminary Studies.............................................................................42 Ozone Measurement and Generation..................................................................42 Nutrient Media and Preparation of Inoculum......................................................47 Exposure Treatments..................................................................................................49 5 CONCLUSIONS AND SUGGESTIONS..................................................................53 Conclusions.................................................................................................................53 Recommendations.......................................................................................................54 Suggestions for Future Work......................................................................................54 APPENDIX A INHIBITION DESIGN EXPERIMENT PICTURES................................................55 15 Minutes Ozone Treatment Duration......................................................................56 30 Minutes Ozone Treatment Duration......................................................................59 60 Minutes Ozone Treatment Duration......................................................................62 B PROCESS CONTROL PROGRAMS........................................................................66 C PETRI PLATE PICTURES TO SELECT NUTRIENT MEDIA AND INOCULATION DURATION...................................................................................74 LIST OF REFERENCES...................................................................................................78 BIOGRAPHICAL SKETCH.............................................................................................83 vi

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LIST OF TABLES Table page 2-1 Ozonation of bacteria and viruses in air...................................................................14 2-2 Summary of previous trials run on Serratia and Aspergillus inoculum...................26 2-3 Summary of previous trials run on natural airborne organisms...............................26 3-1 RGF-TURBOHYDROZONE ozone generator specifications.................................36 3-2 Experimental design matrix showing different combinations of treatment duration, frequency and ozone concentration carried out in study...........................40 4-1 Ozone concentration vs. flow rate achieved by small RGF generator using compressed air as feed gas.......................................................................................43 4-2 Ozone concentration vs. flow rate achieved by small RGF generator using pure oxygen as feed gas....................................................................................................44 4-3 Ozone concentration vs. flow rate achieved by large RGF generator using compressed air as feed gas.......................................................................................44 4-4 Ozone concentration vs. flow rate achieved by large RGF generator using pure oxygen as feed gas....................................................................................................44 4-5 Ozone concentration vs. flow rate achieved by small RGF generator using compressed air as feed gas and diluting exposure chamber with compressed air....45 4-6 Ozone concentration over time in an exposure chamber.........................................46 4-7 Results of experimental trials on Petri dishes containing different combinations of nutrient media strength and inoculation duration................................................48 4-8 Inhibition design matrix based on different combinations of treatment duration, frequency and ozone concentration..........................................................................50 vii

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LIST OF FIGURES Figure page 2-1 Death curves for A) Escherichia coli and B) Staphylococcus aureus in ozonated air. ............................................................................................................15 2-2 Biofilm formation in air conditioning systems........................................................27 2-3 Test apparatus to prevent microbial growth in air conditioning system..................27 2-4 Original apparatus attached to air conditioning system...........................................27 3-1 Lab scale experimental setup of ozone-treated air handling system........................31 3-2 Process flow diagram of ozone-treated air handling system....................................32 3-3 RGF-TURBOHYDROZONE ozone generators......................................................32 3-4 Mechanical damper in the inlet airline.....................................................................32 3-5 Biological containment hood...................................................................................33 3-6 Process automation using CR 10 as a datalogger and process controller................33 3-7 Sequence of steps taken to prepare humidity inside simulated air duct (A) Wet sponges inside exposure chamber (B) Petri dishes in an exposure chamber before starting experimental trial.............................................................................33 3-8 Sequence of steps taken to prepare nutrient media (A) Nutrient media prepared in a conical flask (B) Sterilization of nutrient media in a autoclave (C) Solidification of nutrient media in Petri plate under bacteria free environmental hood (D) Storage of Petri dishes in a refrigerator............................35 3-9 Petri dishes inoculated from a natural indoor home flora........................................36 3-10 Iodometric method test set-up for ozone concentration measurement....................38 3-11.The Dragger pump.....................................................................................................39 3-12 Comparison of ozone treated plate (left) with untreated plate (right)......................41 4-1 Ozone concentration levels achieved under different feed gas flow rate conditions.................................................................................................................43 viii

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4-2 Ozone concentration vs. time in an exposure chamber...........................................46 4-3 Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 15 minutes of treatment duration.....................................................................................................................51 4-4 Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 30 minutes of treatment duration.....................................................................................................................52 4-5 Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 60 minutes of treatment duration.....................................................................................................................52 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Engineering OZONE TREATMENT FOR PREVENTION OF MICROBIAL GROWTH IN AIR CONDITIONING SYSTEMS By Aakash Khurana December 2003 Chair: David P. Chynoweth Cochair: Arthur A. Teixeira Major Department: Agricultural and Biological Engineering Microbial contamination of indoor air by air conditioning systems represents a major public health concern. Biofilm formation around condensation coils and drip pans takes place due to the combination of collection of particulate organic matter and condensation of moisture in an air conditioning unit, which not only reduces the efficiency of heat transfer in these systems but also leads to a high level of fungi and bacteria that become airborne and transported throughout the buildings via air circulation. The success of ozone as a disinfectant in water systems suggested its potential for aerial applications to prevent microbial growth in air conditioning systems. Preliminary work suggested that ozone prevented accumulation of microbial mass in an actual home air conditioning system, and thus a method and apparatus to control the microbial growth in air handling systems were developed. x

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The goal of this study was to confirm and document the efficacy of ozone as a disinfectant to inhibit microbial growth in air conditioning systems and to determine practical ozone treatment conditions consisting of ozone concentration and treatment time and frequency for engineering applications of this invention. Findings from this study reveal that at 9 ppm and lower levels of ozone concentration frequent treatments are effective to inactivate microbial growth but as ozone concentration levels are increased from 11ppm and higher treatment duration plays a major role to inhibit microbial growth. Also, combinations of 30 minutes or longer treatment duration with concentration levels as low as 12 ppm achieve similar inhibition results. The results also show that one 15-minute treatment per day of exposure for ozone at a concentration of 45 ppm is effective to achieve microbial inactivation. Thus, the results presented in this research demonstrate that practical treatment conditions based on different combinations of ozone concentration, treatment duration and frequency can be applied to disinfect our air handling systems. xi

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CHAPTER 1 INTRODUCTION Background Transmission of respiratory infections in indoor environments represents a public health concern for which engineering alternatives are limited. Indoor air quality is affected by a variety of factors like accumulation of microorganisms in air conditioning systems, entrance of outdoor microorganisms into buildings, or contamination of building materials. The combination of collection of particulate organic matter and condensation of moisture provides an ideal environment for the growth of biofilms around drip pans, condensation coils, and ductwork of the air conditioning units and the recycling air from contaminated units leads to the transportation of airborne contaminants in the indoor environment. Biofilms not only lead to high levels of fungi and bacteria that become airborne and transported throughout the buildings via air recirculation, but also reduce the efficiency of heat transfer in these systems. Periodic cleaning of air conditioning system and ductwork with biocides does not eliminate all microorganisms which can cause variety of health problems including hyper-sensitivity, pneumonitis, sick building syndrome, legionnaires disease, asthma and allergies in humans. Common technologies, such as high efficiency particulate air (HEPA) filters and ultraviolet germicidal irradiation (UVGI) for air treatment, are proven less effective in real-world installations than in laboratory studies. The success of ozone to inactivate bacteria and fungi in water treatment systems (Blogoslawski and Rice, 1975) suggests its potential for aerial applications to prevent 1

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2 microbial growth in air conditioning systems. Gaseous ozone can treat areas, which are inaccessible to UV light and other cleaning methods. It is a strongest oxidant and disinfectant widely used for the treatment of water and wastewater. The high oxidationpotential of ozone results in a biocidal effect on the microorganisms. These attributes, plus the fact that ozone decomposes to form the innocuous byproduct oxygen, can make ozone ideal for air treatment. Problem The problem of contamination of air handling systems such as those used in commercial buildings, private dwellings and closed vehicles is particularly acute in warm climate areas that require lengthy air conditioner operation without the opportunity to permit the moisture producing coils and drip pans to dry. Most of the drip pans are not designed to drain completely and thus harbor microbial growth in air-conditioning units. It is recommended that the condensation producing apparatus of air-cooling equipment be regularly cleaned and disinfected. However, such routine maintenance is often overlooked, and the use of conventional disinfecting techniques creates problems because of the dissipation of disinfectants into the air flowing through the system exposes many occupants of buildings and vehicles to allergies and illnesses difficult to trace and diagnose. Microbial contamination of indoor air is a major cause of illness among individuals within office buildings and the like having sealed windows wherein air circulation is only through central cooling and heating units (Building Related Sickness, 1993). Chynoweth (Unpublished) observed and collected large quantities of microbial mass from the drip pan and condensation coils of his home air conditioning unit. He

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3 reasoned that when the blower transmits air across the evaporator coil, the microorganisms present in the biofilm on the coil and drip pan become air-borne particulates with the potential to cause health problems as allergies, rashes, colds, viruses, legionnaire disease and some other illnesses known. He installed a hot tub ozone unit to one of his two air conditioning units after cleaning them thoroughly during summer. The air conditioning unit with ozone unit installed did not show any sign of biofilm formation but the other unit had a significant biofilm formation across evaporator coil and drip pan after a few summer months. Thus, based on this observation Fannin and Chynoweth (1994) wrote a patent application for developing a method and apparatus to control the microbial growth in air handling systems by treating the unit with gaseous disinfectant. Initial tests were conducted at the home of an inventor and couple of years later a lab duct was built to do preliminary treatments. Preliminary work suggested that ozone prevented accumulation of microbial mass over a period in one summer. Follow up testing of this treatment system was conducted to measure the ozone output and test treatment regimes. The research report here developed was a detailed database for ozone treatment based on ozone concentration and the treatment time and frequency for engineering applications of this invention. Objective The goal of this study was to reconfirm the efficacy of ozone as a disinfectant to inhibit microbial growth in air conditioning systems and to determine practical treatment conditions. Specific objectives to the study were the following: 1. Determine the effectiveness of intermittent ozone treatment for prevention of bacterial and fungal growth in air conditioning systems 2. Evaluate the effect of various combinations of ozone concentration, exposure duration and treatment frequency on microbial growth.

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4 3. Recommend practical air conditioning unit treatment conditions based on the ozone concentration, treatment duration and frequency.

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CHAPTER 2 LITERATURE REVIEW Indoor Air Quality Poor indoor air quality is widely regarded as a significant health, environmental and economic problem. Microorganisms such as bacteria and fungi are ubiquitous in our indoor and outside environment. Excessive proliferation or distribution of microbial growth inside buildings has been known to cause respiratory related ailments as well as skin rashes. Evidence for the spread of respiratory diseases inside buildings, and specifically by ventilation systems exists in early epidemiological data (Hartman, 1925). Microbial contamination in buildings is often a function of moisture incursion from sources such as stagnant water in heating, ventilation and air conditioning (HVAC) air distribution systems and cooling towers. Such contamination of indoor air has been widely documented and is a cause of illness among individuals within office buildings and the like having sealed windows wherein air circulation is only through central cooling and heating units (Building Related Sickness, 1993). Health effects related to residency and/or work in closed buildings, such as residential blocks, offices, schools and nurseries, have become a growing public health concern. These problems are often called the sick building syndrome (SBS) and was originally defined by the World Health Organization (WHO, 1983) as the occurrence of an increased prevalence of non-specific symptoms among populations in certain buildings. Symptoms of SBS are believed to arise from multiple causes which, while not clearly understood, are associated mainly with air-conditioned office buildings. 5

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6 Most people are aware that outdoor air pollution can damage their health but many do not know that indoor air pollution can also have significant negative health effects. Environmental Protection Agency (EPA) studies of human exposure to air pollutants indicate that indoor levels of pollutants may be 2-5 times, and occasionally more than 100 times, higher than outdoor levels. These levels of indoor air pollutants may be of particular concern because most people spend about 90% of their time indoors (US EPA, 1995). Biofilm Formation A biofilm is a community of microbes embedded in an organic polymer matrix, adhering to a surface. The matrix of the biofilm consists largely of water and the remainder is an assortment of extracellular polymers (polysaccharides, glyco-protiens) which are referred to as extracellular polymeric substances (EPS) (Geesey, 1982). Bacteria gain a number of advantages from living in biofilms. In a biofilm, for example, the microbial cells are protected from possible attack from the outside environment, e.g., from ultraviolet (UV) radiation. They are also resistant to anti-bacterial agents like heat (Frank and Koffi, 1990) and bacteriophages (Hicks and Rowbury, 1987). Biofilms thus constitute a reservoir of many different species able to resist environmental fluctuations. Biofilm formation is a multi step process whereby bacteria adhere to equipment or surfaces surround themselves with a protective layer of polysaccharides and grow into a network of micro colonies and water channels. These channels serve to supply nutrients and remove bacterial waste products. According to Characklis (1981), the formation of a biofilm in a clean and inert surface containing organic and mineral molecules and microorganisms goes through four stages:

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7 1. Transport: First, molecules are carried towards the surface by diffusion or, in some cases, by turbulent flow. The larger particles, including the microorganisms, are slowed down by the laminar boundary flow; other mechanisms also intervene to explain how the microorganisms are carried right to the surface (gravity, thermophoresis and chemotaxis, fluid dynamic forces). 2. Adsorption of molecules: Molecules are almost instantly adsorbed at the surface and play a part in the events that follow. This conditioning of the surface alters its physico-chemical properties (surface free energies, electrostatic charge). The accumulation of organic and inorganic matters at the solid-liquid interface (explained by Gibbs law, and often referred to as the conditioning film) contains a notably higher concentration of molecules of nutrients than is found in the mass of the fluid. Nutrient transfer by diffusion is thus more rapid within the film. 3. Adhesion of nutrient microorganisms: Adhesion is due to various types of forces (Van der Waals attraction forces or electrostatic forces) that operate between the inert surface and the living cells (Oliveira, 1992). Unlike the previous stage, the adhesion of bacteria at this stage takes sometime, though this varies according to the species of microbe concerned. Marshall (1992) described a first stage in which adhesion is reversible and the bacteria still show Brownian motion and are easily removed (e.g., merely by rinsing); and a second phase, in which adhesion can be called irreversible, requiring much stronger forces to remove the bacteria (e.g., scraping, scrubbing). He reported on research involving Pseudonomas spp. took 4 hours for 73% of the adhering bacteria to become irreversibly attached.

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8 The extracellular polymeric substances (EPS) of cells in suspension condition the surface properties of the microorganisms and hence enhance their degree of adhesion to surfaces (Shea, 1991). Polysaccharide conformation varies in this respect according to environmental conditions of temperature and composition of solvent. 4. Colonization: Lastly, once they have attached, the microorganisms that produce EPS reproduce and multiply within the matrix so formed. This colonization results in the formation of a biofilm. It is a fairly slow process: depending on the culture conditions, it takes anything from a few hours to several months to reach a state of equilibrium. Thickness of biofilm depends on factors that impact on the lives and survival of microorganisms (e.g., pH, water activity (a w ), temperature and composition of the medium). When nutrient concentration in the medium increases, the thickness of the biofilm increases, as do the thickness thresholds beyond which diffusion of one nutrient element or other ceases (Trulear and Characklis, 1982). When the flow rate of the fluid in contact with the biofilm is increased until it becomes turbulent, the transport of molecules and microorganisms is improved and the rate of detachment of cells from the film increases. Research done in Hong Kong (Law et al., 2001) to determine the temporal concentration profile of bioaerosol inside office environments during office hours recorded highest bacteria concentrations at the early morning hours during the starting-up period of HVAC systems. The highest fungi concentrations were recorded during the weekend mornings. Elevated concentrations of bacteria and fungi occurred just after the morning HVAC system startup, and decreased within 1 2 h afterwards. Major variations in bioaerosol concentration occurred during the first few hours around the system startup

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9 time, and the bioaerosol concentration diminished gradually during office hours. This provided the insight that more treatment is needed within the first 3 h around the air-conditioning morning startup times than other times of the day. With these findings, it can be expected that a fairly high concentration of airborne fungi occur during the morning if the HVAC system is down for more than 8 hours. This burst of emission of bacteria and fungi was probably due to the incubation of bacteria and fungi during the nighttime HVAC shutdown hours. The study suggested that amplification of biological agents in HVAC reservoirs occurred due to the favorable conditions provided during the HVAC system shutdown hours. Available Methods for Air Treatment Cleaning and disinfection are the two methods used to eliminate biofilms from surfaces. Cleaning is intended to eliminate slime and disinfection to destroy any remaining microorganisms. Carpentier and Cerf (1992) mentioned that there is no method available to accurately count microorganisms on the surface due to two reasons. First, the usual techniques (scrapers, agar contact methods, sponges etc) remove only the top of a film containing only a fraction of the population. Secondly, because of the nutrient gradients found within thick biofilms (Characklis, 1981), the irregular inputs of nutrients and /or the stress produced by desiccation, cleaning and disinfection (Singh et al., 1986; Sall am and Donelly, 1992) some microorganisms remain undetected (fail to colonize) with usual laboratory culture media. Also, no media are suitable for growth of all organisms. Cleaning is an important treatment for minimizing microbial colonization on the surface. It seems to be of vital importance to eliminate as many microorganisms as possible before applying a disinfectant. Microorganisms are far more sensitive to

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10 disinfectants once they have been detached from surfaces. Mechanical actions (brush, medium and high pressure jets) are recognized as the most effective methods for eliminating biofilms (Exner, 1987; Holah, 1990 b). The extent to which biofilms are removed depends on the microbial strain (Holah, 1989). However, it is difficult to achieve 100% rate of bacterial removal by cleaning. Disinfection is considered to be the primary mechanism for the effective inactivation/destruction of organisms to prevent the spread of diseases. There is no perfect disinfectant available. A number of studies mentioned by Cerf and Carpentier (1992) indicated that resistance to disinfection increases with the biofilm age (Le Chevallier, 1988; Anwar, 1989; Frank and Koffi, 1990; Shin and Frank, 1991; Anwar, 1992). Further, Holah (1990a) showed that those disinfectants most effective against bacteria in suspension are not those that are most effective against those in biofilms. However, there are certain characteristics to look for when choosing a suitable disinfectant for a treatment .They could be 1. Ability to penetrate and destroy infectious agents under normal operating conditions; 2. Lack of characteristics that could be hazardous to people and the environment before or during disinfection; 3. Absence of toxic residuals and mutagenic or carcinogenic compounds after disinfection; 4. Affordable capital and operation and maintenance (O&M) costs. The two most common methods of disinfection are chlorination and ozonation. Chlorine, the most widely used disinfectant for municipal wastewater, destroys target organisms by oxidation of cellular material. It may be applied as chlorine gas, hypochlorite solutions, and other chlorine compounds in solid or liquid form. Like chlorine, ozone is a strong oxidizing agent but it is more effective in destroying viruses

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11 and bacteria. It is an unstable gas that is generated by an electrical discharge or UV radiation through dry air or pure oxygen. The ozonation process utilizes a short contact time (approximately 10 to 30 minutes) and there are no harmful residuals that are to be removed after ozonation because it decomposes rapidly to oxygen. Ozone as an Aerial Disinfectant Inactivating spore-forming bacteria in water with ozone is one thing, but in air it is extremely different matter, primarily because of the variations in relative humidities (Rice, 2002). Ozone has been previously investigated as an aerial disinfectant but the results were inconclusive (Elfred, 1942). Masoka (1982) showed promising results for disinfection of entire room with ozone but the data on ozone disinfection of air remain limited. Despite the growing research, there still remains an absence of data on the optimal concentration and exposure times to prevent the biofilm accumulations in the air conditioning ducts in warm climate areas. Ishizaki (1986) studied the inactivation of six strains of Bacillus spp; B .subtilis;B. cereus;B.licheniformis and B.megeterium with gas phase ozone (230-1400 ppm) at varying relative humdities and found a lag phase during the initial exposure followed by an exponential decrease in the number of survivors with time. No inactivation was observed at relative humidities below 50%. Increasing relative humidity influenced the lag phase, but not the inactivation rate. Whistler and Sheldon (1989) evaluated ozone as an alternative disinfectant in air to replace formaldehyde. Culture of Staphylococcus, Streptococcus, and Bacillus species previously isolated from poultry hatcheries and selected cultures of Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium, and Aspergillus fumigatus were spread plated on open Petri plates and independently fumigated with ozone or

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12 formaldehyde in a prototype laboratory poultry setter. Ozone (greater than 9000 ppm) reduced levels by >4 to 7 log 10 and fungal levels by >4 log 10 Naito (1989) studied the microbiocidal effects of ozone on Bacillus, Micrococcus and yeast present in a candy factory and found that the total microbial counts drastically reduced after gas phase ozone treatment. A most recent study by Currier et al. (2001) of gas phase ozone to inactivate both clumped and dirty spores of Bacillus globigii var. niger as a surrogate for Bacillus anthracis showed ozone inactivation at 9,000 ppm (~1% ozone in air) ozone during 15 hours of exposure. Properties of Ozone In gaseous form, ozone is colorless, whereas liquid ozone is almost opaqueand bluish-black and its crystals are violet-blue. It has a characteristic odor, reminiscent of phosphorous and sulphur dioxide. Olfactory organs are extremely sensitive to ozone; it can be detected by humans at a dilution of 1 parts per million (ppm) of air. Ozone freezes to bluish-black crystals at the boiling point of hydrogen. Ozone half-life in air is typically about 15 minutes in open areas (can be hours in enclosed areas) and increases with lower temperature and lower humidity. Ozone is about 50% heavier than air and has a low vapor pressure, so it tends to sink to the floor and does not disperse if there is no air circulation. Maximum ozone concentration in water varies directly with concentration of the gas in the sparging air and inversely with temperature: for example ozone at 1.5% sparging gas (by weight) will have a maximum concentration of about 11 ppm in water at 5 o C and 6.4 ppm at 20 o C. Doubling the concentrations of ozone in the feed gas will double the concentration in water at any given temperature.

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13 Antimicrobial Properties. Ozone is a strong, broad-spectrum antimicrobial agent that is active against bacteria, fungi, viruses, protozoa, and bacterial and fungal spores (Kim et al., 2000, Kim et al., 1999, Yousuf, 2001). Inactivation of bacteria by ozone occurs by lysing (i.e., oxidative rupture) of the cell wall. The anti microbial activity of ozone is based essentially on its powerful oxidizing effect, which causes irreversible damage to the fatty acids in the cell membrane and to cellular macromolecules, such as proteins, and DNA (Hoffman 1971, Fetner and Ingols, 1959, Naitoh, 1994). This action is particularly effective in air at high relative humidity level. Gram-positive bacteria are more sensitive to ozone than gram-negative. Disinfection rate depends on the type of organism and is affected by ozone concentration, treatment time (Kim and Yousuf, 2000, Kim et al., 1999), temperature, relative humidity, pH, turbidity, the presence of ozone-oxidizable materials, the tendency (or not) for the microorganisms to form clumps, and the type of ozone contactor. Ewell (1938) stated that depending on cleanliness, minimum continuous concentrations of 0.6 to 1.5 ppm ozone (in air) were necessary to prevent mold growth on eggs kept at 90% relative humidity (RH), whereas 2.5 to 3.0 ppm ozone were required to control molds on beef stored under similar conditions. Kim (2002) conducted a detailed study of the effects of relative humidity on the ozone inactivation of Bacillus subtilis. He concluded that high relative humidity is needed for the inactivation of microorganisms by ozone in gas environment. It is believed that hydration of dry microorganisms in humid atmospheres makes them susceptible to ozone. The optimum relative humidity found by Kim (2000) for microbial inactivation by ozone in air is 90-95%. Elford and Ende (1942) used low ozone concentrations and long exposures at variable relative humidity to disinfect airborne microorganisms. At RH

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14 <45%, the germicidal power of ozone was negligible. Ewell (1946) also demonstrated that microorganisms were killed more readily by ozone in an atmosphere having a high rather than low RH. Holah (1995) constructed a special aerobiology cabinet to study the destruction of airborne microorganisms by disinfectant fogging, by ozone and by ultraviolet light. Exposure to ozone above 4 ppm in air (5 and 10 min exposure) significantly reduced the numbers of airborne P. aeroginosa. Kowalski et. al. (1998) studied the bactericidal effects of airborne ozone against E. coli and Staphylococcus aureus by placing Petri dishes containing the microorganisms in the chamber and exposing for 10 to 480 seconds to ozone concentrations between 300 and 1500 ppm. Levels of population reduction in excess of 99.99% (4 log 10 cycles) were achieved for both species. However, practical applications of such higher ozone concentrations for air treatment are quite questionable. Thus, concentrations lower than 300 ppm could also be equally effective to achieve similar reduction in population levels. Table 2-1 lists data on ozonation of airborne bacteria and viruses reported by previous investigators and compiled by Kowalski et al. (1998). Data taken in Kowalski et. al. (1998) experimental trials were combined to form the death curves presented in Figure 2-1-a and 2-1-b. Table 2-1. Ozonation of bacteria and viruses in air (Kowalski et al., 1998). Test Organism Ozone (ppm) Time(sec) % Survival Reference S. salivarius 0.6 600 2 Elford & van de Eude,1942 S. epidermis 0.6 240 0.6 Heindel et al., 1993 pX174 (virus) 0.4 480 0.1 de Mik, 1977

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15 A B Figure 2-1. Death curves for (A) Escherichia coli and (B) Staphylococcus aureus in ozonated air. Ozone concentrations during this series of experiments varied from 300 to 1500 ppm.

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16 Physiological Effects of Ozone on Humans Ozone inhalation causes a wide range of symptoms, from the unpleasant smell to pulmonary edema or lethal poisoning. Irritation by ozone primarily affects the lungs. Reaction of the mucous membranes in the upper respiratory tracts manifests itself in dryness of the mouth, nose and throat. Irritating symptoms are also exhibited in the eye; visual acuity diminishes, followed by the appearance of tears and swelling. Coughing, headache, dizziness, dribbling and sweating are also experienced. After prolonged exposure, symptoms become more serious, exhaustion sets in along with an increase in the pulse rate. All these symptoms can be associated with nausea and an intensification of symptoms can lead to unconsciousness, pulmonary congestion, hemorrhage of the nose, and eventually to death. Actual and expected physiological effects of ozone on humans were investigated in a detailed manner by Clamann and Bancroft (1959).The adverse health affects of ozone exposure are also well documented by Weinstein (1991).The individual physiological symptoms manifest themselves depending on the concentration and the exposure time. OSHA has established a time-weighted average permissible exposure level for workers for an eight-hour day of 0.10 ppm and a short-term exposure limit of 0.30 ppm for an exposure less than 15 minutes (OSHA, 1975). The symptoms experienced on exposure to 0.1-1 ppm ozone are headache, throat dryness, irritation of the respiratory passages, and burning of the eyes caused by the formation of aldehydes and peroxyacyl nitrates. Exposure to 1-100 ppm ozone can cause asthma-like symptoms such as tiredness and lack of appetite. Short-term exposure to higher concentrations can cause throat irritations, hemorrhaging, and pulmonary edema.

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17 Ozone Generation Ozone is formed from oxygen molecules as a result of electrical discharge or ultraviolet (UV) radiation. Oxygen atoms are formed by the splitting of diatomic oxygen molecules into two atoms, which then recombine with other oxygen molecules to produce ozone molecules. The exothermic reaction leading mostly to ozone formation is O 2 + O O 3 Commercially, ozone can be generated on-site by three following wayscorona discharge, UV radiation and electrolysis. Corona Discharge The corona discharge technique is a commercially significant method (the so-called silent electrical discharge procedure) to manufacture ozone. In a corona discharge ozone generator, the feed gas (dried air, oxygen, or mixtures thereof), passes between two closely spaced electrodes (one of which is coated with a dielectric material) under a nominal applied voltage potential of ~10 kV. A silent or barrier discharge occurs when the gas becomes partially ionized, resulting in a characteristic violet glow when air is the feed gas (with high purity oxygen the violet coloration is seldom observed). Efficiency of ozone production by corona discharge depends, for the most part, on the strength of micro discharges (which is influenced by a number of factors such as the gap width, gas pressure, properties of the dielectric and metal electrodes, power supply, and the presence of moisture). In weak discharges, a significant fraction of the energy is consumed by ions, whereas in stronger discharges, almost all of the discharge energy is transferred to electrons responsible for the formation of ozone. The optimum is a compromise that

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18 avoids energy losses to ions but at the same time obtains a reasonable conversion efficiency of oxygen atoms to ozone. If compressor air is used as the feed gas, it must be scrupulously dried and be free of traces of oils and greases (oxidized by ozone). Moist air gives rise to nitrogen oxides in the ozone generator which can form nitric acid and corrode the generator, requiring frequent maintenance and down time. Ozone is quite readily available in nature, most often as a result of lightning strikes that occur during thunderstorms. The "fresh, clean, spring-like rain" smell that we notice after a storm most often results from nature's creation of ozone. Natural ozone is produced by the process of electrically rupturing oxygen atoms to produce oxygen ions or atoms that combine with more oxygen to produce ozone and liberating considerable amount of heat. Corona discharge method also works on the same principle and the heat generated during this process is removed from the generator to avoid the reverse reaction (ozone reverting to oxygen) from taking over and decreasing the efficiency of ozone generation. Thus, cooling of the corona discharge ozone generator becomes a critical component in generator design. Corona discharge ozone generators commercially available these days are capable of producing ozone in gas phase concentrations of 1-5% by weight in air and up to 14% by weight in high purity oxygen. Ultraviolet Radiation The mechanism of photochemical production of ozone is similar to that which occurs in the stratosphere, that is, oxygen atoms formed by the photo-dissociation of oxygen by short wavelength UV radiation (240 nm) react with oxygen molecules to

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19 form ozone. Although the theoretical quantum yield of ozone by this technique is ~2, in practice the actual yield is more on the order of ~ 0.5, because the low pressure mercury lamps produce not only the 185 nm radiation responsible for the production of ozone, but also the 254 nm radiation that destroys ozone. Medium pressure UV that produces higher levels of 185 nm radiation produces more ozone. An advantage of generating ozone by UV radiation is that ambient air can be used efficiently as a feed gas. On the other hand, quantities of ozone generated per 40W UV bulb are low at maximum ozone concentration of 2400 ppm. However, these maximum ozone yields and concentrations cannot be attained simultaneously by the UV method. The low concentrations of ozone available from UV generators limit their applicability for water treatment to special applications. However, their use to generate ozone for air treatment can be effective where very high ozone concentrations are not required. Electrolysis High current density electrolysis of aqueous phosphate solutions at room temperature produces ozone and oxygen in the anodic gas. Electrolysis of sulfuric acid can produce very high ozone concentration in oxygen when a well-cooled cell is used. Although electrolysis of water can produce high concentrations of ozone, the output is low, and the cost is several times more than that of the corona discharge or UV radiation process. Ozone produced by the corona discharge method has a higher ozone concentration but requires extra energy and effort to compress, dry, produce and then dilute the produced ozone in comparison to ozone produced by the ultraviolet light method. Air treatment usually requires large volumes of low ozone concentration. Ozone produced by UV, in comparison, generally needs no air pretreatment and is produced at

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20 the ideal concentrations required to destroy airborne bioaerosols and volatile organic compounds. Reduced air/oxygen flow rate in the UV radiation-based ozone generator gives higher ozone concentration due to longer retention time of oxygen molecules in the presence of ultraviolet light. The factors that can influence the production yield of ozone are chemical composition ( percentage of oxygen) and physical properties and process conditions like pressure, temperature, impurities, moisture of the feed gas, dielectric material, thickness and discharge gap of the generator, type of the UV bulbs (wavelength, frequency, energy consumption) used in the generator (Masschelein, 1997). The most obvious source for producing ozone gas is atmospheric air, but it does not possess high purity oxygen. High purity oxygen does not require further drying; it gives much higher ozone concentration at low energy consumption, reduces the size of the generator and eliminates the by production of nitrous oxides from the nitrogen contained in the air. Therefore, for large-scale ozone production, oxygen is a more favorable option (Masschelein, 1997). However, for lower ozone concentration, air as a feed gas in more practical and economical. Stabilization and Storage of Ozone The bulk of ozone consumed is produced in situ and introduced to the process or to the place of its application directly, without intermediate storage or transport. The rate of spontaneous thermal decomposition of ozone depends primarily on the temperature according to the general laws of reaction kinetics. By decreasing the temperature, the rate of decomposition strongly diminishes, for example a half-life of 3 days at 293 K, 8 days at 248 K and 3 months at 223 K is specified generally (Rompps Chemie-Lexicon, 1974). According to Streng and Grosse (1961), the rate of homogeneous decomposition of pure

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21 ozone gas at atmospheric pressure and a temperature of 195 K is less than 10 -5 per cent per day. At temperatures above room temperature the rate of decomposition can rise with increasing temperature to an extent that the slow decomposition is transformed into a thermal explosion. Pure or concentrated ozone is inclined to explode, both in liquid and in gaseous state, due to its instability, independent of its rate of spontaneous (slow) thermal decomposition, which is essentially dependent only on temperature and the presence of catalysts. The primary requirement for safe handling pure, or highly concentrated ozone, either in the liquid or gaseous state, is the total absence of trace substances catalyzing decomposition of, or generally reacting with, ozone. This requirement has to be satisfied at three different levels in the following way: 1. Feedstock oxygen for ozone generation should be freed from various contaminants, mainly organic, before entering into the ozone generator. 2. Special care should be exercised in cleaning the internal surface of vessels, apparatus and piping in contact with ozone. In the case of glass apparatus, this can be achieved by handling the internal surfaces with sulphuric acid-potassium dichromate solution followed by rinsing first with distilled water and subsequently with a pure, dry oxygen stream. 3. Finally, materials reluctant to enter into any reaction with, and having no catalytic effect on, the effect of ozone should be selected as structural materials for equipment parts in contact with ozone. For high concentrations of ozone only glass, stainless steel and fluorinated polymers can be considered for use as construction materials.

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22 Pure gaseous ozone and gaseous mixtures of high ozone content cannot be stored in gas bottles used normally for the storage of other gases, due to their high reactivity and tendency to explosion with ozone. Although Grosse and Streng (1958) stated that ozone-oxygen mixtures containing less than 16 per cent ozone could be safely stored in standard gas bottles at atmospheric pressure and low temperatures, this is, however, of no practical use. Ozone Measurement Ozone concentrations are measured in the air and liquid by various methods. Commonly used ozone measurement methods are standard iodometric method, dragger pumps and ozone analyzer. The amount of ozone circulating through the test apparatus is critical in analyzing the experimental results. Chemical determination of ozone concentration by the Standard Iodometric method is based on the oxidation of the iodide ion in an aqueous solution to iodine and its subsequent reduction with sodium thiosulfate (Rakness, K. et. al., 1996).The reactions of ozone with iodide are as follows: O3 + I O2 + IO IO+ H 2 O HIO + OHHIO + 2 I I 3 + OH 3 HIO + 3 OH IO 3 + 2I + 3H 2 O Upon acidification the overall process formula becomes: O3 + I + H 2 O I 3 + O2 + 2OH Iodometric determination of ozone is the oldest process in ozone analysis which cannot be totally replaced by more recent methods, even today, despite its apparent limitations. Although the reaction is not specific and can lead to unwanted deviations

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23 from stoichiometry due to possibility of side reactions, it still has the great advantage of being an independent method that requires no separate calibration and can be used equally well for the determination of low or high concentrations of ozone both in gas phase and solution. Dragger pump, a military device, is another ozone measurement apparatus. This device consists of a hand-held pump and a gas sample tube, which receives the airflow and contains a color indicator that changes from greenish-blue indicating layer to yellow in the presence of ozone. A direct measurement of the ozone concentration is seen on a scale (operating range is 20-300 ppm ozone concentration) on the gas sample tube. Lack of accuracy often limits practical application of this method. Ozone Analyzers can also be used to measure ozone concentration. It has a fast response time and balanced flow system. Ultraviolet (UV) photometric ambient ozone analyzer (Model 49) is a time-shared dual cell instrument capable of measuring ambient level ozone concentration on a continuous real time basis. The UV photometer in ozone analyzer determines the ozone concentration by measuring the attenuation of light due to ozone in the absorption cell; at a wavelength of 254 nm .The ozone concentration is directly proportional to the magnitude of the attenuation. Also, ozone analyzer requires zero air as a reference gas for the calculation of ozone concentration. Reference (Zero air) passes into the absorption cell to establish Zero light intensity reading (Io). The solenoid then switches and the sample pass through the absorption cell to establish sample light intensity (I). It works on Beer Lamberts law, which says that transmittance is directly proportional to the ozone concentration, absorption coefficient and the length of the absorption cell in the analyzer. Mathematically,

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24 I/Io = eklc where, I/Io = Transmittance or measure of light absorbed by the ozone. K = absorption coefficient = 308 cm -1 at 0 o C & 1atm. l = length of the cell (approx. 37.4cm) c = ozone concentration in ppm. The major advantage of ozone analyzers is that they have a very fast response time in comparison to conventional chemical method of ozone determination. However, the absorption cross-section of ozone at 254 nm is by orders of magnitude larger than those of most other gases, therefore the interference of other air pollutants (nitrogen oxides, carbon monoxide, hydrocarbons and water vapor) can be neglected. Dust and water vapor condensation in the measuring cell must be avoided for reliable measurements. High cost and narrow range of ozone concentration measurement are some other limitations associated with ozone analyzers. Previous Work at UF Chynoweth observed that air handling systems employing heat exchanging coils, water pans, or the like, exposed to moisture and subject to microbial contamination (Figure 2-2). He installed a hot tub ozone unit to one of his two thoroughly cleaned home air conditioning units and found no sign of biofilm formation while the other unit had a significant biofilm formation across evaporator coil and drip pan during summer. Thus, based on the initial tests conducted at the home of an inventor Fannin and Chynoweth (1994) patented a method and apparatus to prevent buildup of microbial growth in an air-conditioning unit which does not allow gaseous disinfectant to come in contact with indoor air (Figure 2-3 and Figure 2-4). Condensation apparatus located within a chamber

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25 defined in an air handling system was disinfected by temporarily interrupting normal air flow, sealing the chamber with respect to the air handling system, introducing an anti-microbial growth gaseous disinfectant into the chamber, removing the anti-microbial gas from the chamber after disinfecting the condensation apparatus and then restoring normal air flow through the chamber. The preferred anti-microbial gas was produced by using ozone generator in conjunction with an auxiliary air flow system. Based on their methodology, experiments were conducted on prototype cells of bacterium Serratia marcescens and fungus Aspergillus niger. Their results supported the qualitative observations that ozonated air prevented microbial growth in air conditioning systems (Chynoweth, 1997). They found that ozone concentration of 2.2 ppm for a treatment duration of 30 minutes and frequency of two times per day prevented visible accumulation of microbial biofilm growth in an air conditioning unit over a period of six months and a treatment duration of 120 minutes at same ozone concentration and treatment frequency completely inhibited Serratia marcescens and partial inhibition of Aspergillus niger growth (Table 2-2). An ozone concentration of 9 ppm for a duration of 30 minutes with two times treatment frequency per day resulted in a total inhibition of natural airborne organisms (Table 2-3). Their results showed that higher ozone concentration at shorter treatment duration and frequency per day was effective to inhibit microbial growth. However, the desired combination for complete inhibition was not found (Table 2-2).

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26 Table 2-2. Summary of previous trials run on Serratia and Aspergillus inoculum by Chynoweth (Unpublished Data). Trial# Ozone Concentration (ppm) Treatment Duration (min.) Treatment Frequency (per day) Serratia Growth Aspergillus Growth 1 2.2 20 2 Limited Limited 2 2.2 20 12 Limited Limited 3 2.2 120 2 None Limited 4 2.2 60 2 Limited Limited 5 13.6 60 2 None Limited 6 9.0 15 24 None None 7 9.0 60 2 Limited Limited 8 9.0 60 2 Limited Limited Table 2-3. Summary of previous trials run on natural airborne organisms by Chynoweth (Unpublished Data). Trial# Ozone Concentration (ppm) Treatment Duration (min.) Treatment Frequency (per day) Bacteria Growth Fungal Growth 1 9.0 30 4 None Limited 2 9.0 30 6 None None 3 9.0 30 12 None None 4 No Ozone 30 2 Heavy Heavy 5 9.0 30 2 None Limited 6 9.0 30 2 None Limited 7 9.0 30 2 None None

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27 Figure 2-2. Biofilm formation in air conditioning systems (Fannin and Chynoweth, 1994). Figure 2-3. Test apparatus to prevent microbial growth in air conditioning system (Fannin and Chynoweth, 1994). Figure 2-4. Original apparatus attached to air conditioning system (Fannin and Chynoweth, 1994).

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28 Therefore, the objectives of this research work were to determine the effectiveness of intermittent ozone treatment for prevention of bacterial and fungal growth in air conditioning systems; evaluate the effect of various combinations of ozone concentration, exposure duration and treatment frequency on microbial growth; and to recommend practical air conditioning unit treatment conditions based on the ozone concentration, treatment duration and frequency.

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CHAPTER 3 MATERIALS AND METHODS Scope of Work The scope of work for this research was divided into three phases: Preliminary studies to choose the best method for ozone generation, ozone measurement, preparation of nutrient media and inoculation of plates, Development and execution of design of experiments to evaluate effects of various combinations of ozone concentration, treatment duration and frequency on microbial inactivation Development of recommendations for practical air conditioning unit treatment conditions. Microbial cultures of natural air flora were inoculated on nutrient media by placing Petri dishes in an indoor environment which were then ozonated in an experimental chamber. Various trials were run following an experimental design matrix to test the efficacy of various combinations of ozone concentration, treatment frequency and duration. Each experimental trial was three days long and after completion of each trial, microbial growth on plates was observed and compared to that found on untreated plates. Depending on the effectiveness of a combination of treatment conditions in one trial, the conditions were changed accordingly in subsequent experiments in search of optimum treatment conditions. Materials Test Apparatus. A method and apparatus that was developed by Fannin and Chynoweth (1994) for controlling microbial growth in air handling systems was used to carry out this lab scale research work (Figures 3-1 and 3-2). The exposure chamber was 29

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30 intended to simulate a home air duct, and was fabricated from a section of galvanized steel ventilation duct of 0.6 by 0.15 meter in size. A constant speed fan provided air circulation in the chamber, but as soon as ozone flow was started the normal airflow was interrupted by temporarily redirecting the primary air-mover. The hinged opening of the exposure chamber was securely sealed with an aluminum tape to prevent ozone leakage to the ambient environment. Ozone produced from the ozone generator (Figure 3-3) flowed through the exposure chamber when required to carry out each experiment. Accidental back flow of ozone in the airline during experiments was prevented by closing a damper in the airline (Figure 3-4). Ozone was removed by purging air through the chamber, after which the system was returned to the normal airflow operation. Ozone exhaust gas was removed from the system by connecting the exhaust to a biological containment hood (Figure 3-5). Copper tubings and fittings were used throughout the system because ozone has caustic effects on rubber materials. A data logger/process controller (CR-10) for this set up was connected to a laptop computer equipped with PC208W software to carry out process control and datalogging tasks. The programme written in PC208W-based software, depending upon preset conditions of ozone treatment duration and frequency, was transferred to the data logger (Appendix B). Once the connection between the two was established, the datalogger/controller automatically controlled the complete process by continually monitoring process conditions and transmitting control signals. Thus, the operation of the method and apparatus was automatically controlled without requiring much attention. Automation of the process had eliminated the need to be physically present to control the

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31 ozone generator, fan, mechanical dampers and air switches. It also provided additional flexibility by implementing a wider range of treatment duration times, which were not available with used the time relays used earlier (Figure 3-6). Wet towels or sponges were kept inside the chamber to maintain a humidity level of 85-95 % which simulated actual air conditioning humidity environment and prevented drying of inoculated samples during the experiment (Figure 3-7-A and Figure 3-7-B). Relative humidity was measured by using Dicksons temperature and humidity handheld device during each experimental trial to confirm that sufficient humidity levels were maintained in the exposure chamber. Figure 3-1. Lab scale experimental setup of ozone-treated air handling system.

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32 Figure 3-2. Process flow diagram of ozone-treated air handling system. Figure 3-3. RGF-T URBOHYDROZONE ozone generators. Figure 3-4. Mechanical damper in the inlet airline.

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33 Figure 3-5. Biological containment hood. Figure 3-6. Process automation using CR 10 as a datalogger and process controller. Figure 3-7. Sequence of steps taken to prepare humidity inside simulated air duct (A) Wet sponges inside exposure chamber (B) Petri dishes in an exposure chamber before starting experimental trial.

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34 Preliminary Studies Preliminary work for the purpose of method development had been divided into three sections; preparation of inoculum, ozone generation and ozone measurement. Preparation of Nutrient Media and Inoculum Nutrient Media A general-purpose tryptic soy agar was used to prepare one-tenth and full strength nutrient media for the cultivation of microorganisms. One-tenth strength nutrient medium was prepared by mixing 4 grams of tryptic soy broth (Bacto Tryptic Soy Broth without Dextrose manufactured by DIFCO laboratories) with 15 grams of general agar (A-360 Purified Grade manufactured by Fisher Scientific) in one liter distilled water (Figure 3-8-A) and full strength medium was prepared by mixing 40 grams broth in one liter distilled water. The prepared medium was sterilized in an autoclave at 121 o C for 15 minutes (Figure 3-8-B) and was then allowed to cool down at room temperature before pouring nutrient media into 100mm x 115 mm Petri dishes under a bacteria-free hood environment (Figure 3-8-C) .Inverted plates were kept in plastic bags before storing them in a refrigerator (Figure 3-8-D). Preparation of Inoculum Petri plates containing nutrient media were inoculated with natural airborne microorganisms to simulate actual air conditioning environment. Ambient building air of old Rogers Hall was used to inoculate plates in previous work by Chynoweth. During this work, the inoculation of Petri plates was first attempted using ambient air in Rogers Hall, the new Agricultural and Biological Engineering Department building but with no success. This building had been recently renovated with a new air handling system and air ducts, which contained no appreciable air-borne microbial contamination. Thus, open

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35 plates of both nutrient media strength were placed in a private home environment with an older established air-handling system where natural micro flora could inoculate the Petri dishes for 6, 8,12 hours with natural microorganisms found in an indoor setting (Figure 3-9). Two replicates were carried out for each combination of nutrient strength and inoculation duration. Plates were then closed and one replicate of each combination of nutrient media strength and inoculation duration was kept for 72 hours of incubation while another replicate was placed in an exposure chamber for ozone treatment. Plates were treated with varying ozone concentrations and microbial growth on both nutrient media strength plates was analyzed to decide inoculation duration and nutrient media strength of plates to be used during experiments. D C B A Figure 3-8. Sequence of steps taken to prepare nutrient media (A) Nutrient media prepared in a conical flask (B) Sterilization of nutrient media in a autoclave (C) Solidification of nutrient media in Petri plate under bacteria free environmental hood (D) Storage of Petri dishes in a refrigerator.

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36 Figure 3-9. Petri dishes iflora. noculated from a natural indoor home Ozone Generation and Measurement Ozone Generation Ultravoilet (UV) radiationbased ozone generators were selected to produce on-site ozone because they generally do not need air pretreatment and delivered concentrations required to destroy airborne microbes for this research work. Two different size large swimming pool RGF-TURBOHYDROZONE ozone generators were used to attain varying levels of ozone concentration based on feed gas flow rate. Specifications of generators are presented in Table 3-1. Table 3-1. RGF-TURBOHYDROZONE ozone generator specifications. MODEL 120 MODEL 60 20 scfm 5 cfm 20 scfm 5 cfm 1 cfm 1 cfm mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm 1200 550 1700 383 3940 158 242 225 400 127 1500 63 Based on the ozone generator specifications, feed gas flow rate was varied from 1 cubic foot per minute to 5 cubic feet per minute in the generator. Flow meters connected at the inlet and outlet of the ozone generator measured the flow rate of incoming feed gas inside the generator and ozonated gas leaving the generator. Both compressed air and pure oxygen were tested as a feed gas to the generator. Experiments were carried out to measure the maximum and minimum levels of concentration that could be achieved by

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37 using compressed air and pure oxygen with both ozone generators. A minimum possibleozone concentration in the exposure chamber was obtained by diluting the ozonated air inthe chamber with a fixed flow rate of compressed air to the chamber. The purpose of reaching lowest possible concentration in the chamber was to develop a microbial inhibition design based on broader range of ozone concentration data. Experiments were also conducted to measure the time required t o stabilize ozone concewere employed to measure ozone concentration in the exposure cham other n e feed as allowed to come in contact with 400 mL o ntration in the exposure chamber by collecting periodic samples of chamber atmosphere every three minutes and measuring ozone concentration. Ozone Measurement Various methods ber. Initial ozone concentration measurements were done with a UV photometric ambient ozone analyzer (Model 49), but its narrow range of ozone concentration measurement (Full scale range 0 to 1 ppm), insensitivity towards interference fromgaseous constituents and high cost precluded its application for this research work. The Standard Iodometric method (Chemical Method) and Dragger pump method were used ithe study side by side to measure ozone concentration and later compared with one another. Feed gas flow rates were varied in the range of 2.5 cfm to 5 cfm to meet manufacturers specifications and ozone concentration was measured on respectivgas flow rate using the Chemical and Dragger pump methods. Procedure for Standard Iodometric Method Ozonated air from the exposure chamber w f 2% potassium iodide (KI) solution placed in two conical flasks (400 mL in each), as shown in Figure 3-3. An open gas bubbling device (diffuser) was attached to the end of tubing which brought the ozone in contact with potassium iodide solution for uniform

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38 absorption of ozone in the KI solution. Both KI flasks were connected in series so that unabsorbed ozone from the first flask could be transferred and absorbed in the second flask. The sampling line was connected to the absorber and the gas containing ozone wbubbled to the KI solution at a flow rate of 1 cubic foot per minute (cfm) for 3 minutes by a vacuum pump. Unabsorbed gas was sucked from the vacuum pump and vented out to the biological hood. The iodine formed in the solutions of KI, immediately after acidification with 10 mL of 1N sulphuric acid was titrated with a freshly standard0.2N sodium thiosulfate solution. This was followed by addition of 2 mL of the starch indicator solution to the titrant just before the end point (recommended, but can be optional, depending on skill and experience of the operators). The amount of sodiumthiosulfate needed to convert iodide ions of KI solution to iodine was measured, and thnecessary calculations were performed to determine ozone concentration. The assembly for a Standard Iodometric method for ozone product-ga as ized e s measu rement is shown in Figure 3-10 below. Figure 3-10. Iodometric method test set-up for ozone concentration measurement. ation of ozone(C) in mg/L equals: f thiosulfate/ Vc x 28.316 Calculations Concentr 24 x volume of thiosulfate in L x Normality o

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39 where Vc= corrected sample volume = V x (PPw)/ 760 x298/(273+T) V = sam p le volume in cubic feet P = Total Pressure (barometric pres sure) temperature T air = 467 ppm ozone in air mp and a gas sample tube, which receivf tion of 10-15 Pw= water vapor pressure corresponding to T = Temperature 1 mg/L of ozone in The Dragger Pump consisted of a hand-held pu ed airflow from the exposure chamber and changed the color of indicator from greenish-blue to yellow in the presence of ozone (Figure 3-11). The operating range othe gas sample tube (dragger tube) was 20-300 ppm. As per manufacturers specifications, the ozone concentration measured by this method had a devia%(+/-) in comparison to the Standard Iodometric method. Figure 3-11.The Drag ger pump. Experimental Design Each experimental trial conlated Petri dishes to a given ozone out at each ozone concentration; as shown in Table 3-2. sisted of exposing inocu concentration in the chamber for a specified period of time (duration) and at a frequency of either 1, 2, or 3 times within a 24-hour period. Experiments were carriedat 9 different ozone concentrations, and for time durations of either 15, 30, or 60 minutes

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40 Table 3-2. Experimental design matrix showing different combinations of treatment 15 Minutes Treatment 30 Minutes T zone duration, frequency and ozone concentration carried out in study. reatment 60 Minutes Treatment Day OConc. (p1 4 1 4 1 4 2 Times per Day Times per Day Times per pm) 2 2 2 5.7 9 11 17 30 35 45 At the coment, the exposed Petri dishes were incubated to romote outgrowth of surviving microorganisms for visual observation of colony forming units pletion of each experim 3.5 p and compared with untreated plates (Figure 3-12). Results were reported as either growth (+) or no growth (-) for each treatment combination tested. Each experiment was replicated a minimum of three times.

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41 igure 3-12. Compted plate (right). arison of ozone treated plate (left) with untrea F

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CHAPTER 4 RESULTS AND DISCUSSIONS Findings from Preliminary Studies Preliminary work for the purpose of method development had been divided into three sections; ozone generation, ozone measurement, preparation of nutrient media and inoculum. Ozone Measurement and Generation Ozone Generation Selection of ozone generator and feed gas flow rate was based on the ozone concentration levels needed to achieve microbial inhibition and cost of feed gas respectively. Compressed air was selected as a feed gas for this study. Ozone concentration data obtained with oxygen-fed generators were also collected to verify the maximum concentration level that could be attained by both ultraviolet radiation based ozone generators. Reducing air/oxygen flow rate through the ozone generators gave higher ozone concentrations due to longer retention time of oxygen molecules in the presence of ultraviolet light. Results ozone concentrations achieved under different flow rate conditions are shown graphically in Figure 4-1. Ozone Measurement Ozone measurement was done by using the Standard Iodometric method because the percentage difference of ozone concentration measured by the Dragger method in comparison to the Chemical method was much higher than the deviations mentioned by the manufacturer. Also, the Chemical Method is a standard method and thus has greater 42

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43 accuracy in comparison to other available methods. Although, ozone concentration data were collected by using easy-to-use but less reliable Dragger method to compare ozone concentrations obtained by two different measurement methods but only results obtained by the Chemical method are shown. The results of ozone concentration levels are shown in Table 4-1, 4-2, 4-3 and 4-4. Ozone Concentration vs Feed Gas Flowrate0501001502002503003502.63.63.94.55.3Feed Gas Flowrate(CFM)Ozone Concentration(PPM) Large Generator(Oxygenfeed gas) Small Generator(Oxygenfeed gas) Large Generator(Air feedgas) Small Generator(Air feedgas) Figure 4-1. Ozone concentration levels achieved under different feed gas flow rate conditions. Table 4-1. Ozone concentration vs. flow rate achieved by small RGF generator using compressed air as feed gas. Air Flow rate (cfm) Ozone Concentration (ppm) 2.5 3.2 3.7 4.3 30 23 20 17

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44 Table 4-2. Ozone concentration vs. flow rate achieved by small RGF generator using pure oxygen as feed gas. Oxygen Flow rate (cfm) Ozone Concentration (ppm) 2.7 3.8 4.4 4.9 320 210 180 165 Table 4-3. Ozone concentration vs. flow rate achieved by large RGF generator using compressed air as feed gas. Oxygen Flow rate (cfm) Ozone Concentration (ppm) 2.6 3.6 3.9 4.5 5.3 110 90 88 80 68 Table 4-4. Ozone concentration vs. flow rate achieved by large RGF generator using pure oxygen as feed gas. Air Flow rate (cfm) Ozone Concentration (ppm) 2.6 3.7 4.2 4.5 4.9 50 45 40 35 32

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45 The results of minimum possible ozone concentration level attained by diluting exposure chamber with compressed air are shown in Table 4-5. Table 4-5. Ozone concentration vs. flow rate achieved by small RGF generator using compressed air as feed gas and diluting exposure chamber with compressed air to achieve lower levels of ozone concentration for fixed air flow rate of 4 cfm to generator. Air Pressure to Chamber (psi) Ozone Concentration(ppm) 3.5 4.5 6.5 7.5 9.0 11 09 5.7 3.5 2.0 The time required to stabilize ozone concentration in the exposure chamber was determined by collecting periodic samples of chamber atmosphere and measuring ozone concentration with the Standard Iodometric method. A potassium iodide (KI)-ozone sample was collected every three minutes (3-minutes interval) and subsequently titrated with sodium thiosulfate solution to measure ozone concentration. These results show that approximately 15 minutes were needed to stabilize ozone concentration in the chamber. Based on these result, treatment durations of 15 minutes, 30 minutes and 60 minutes were selected to prepare a treatment regime for the design of experiments. The results of ozone concentration based on quantity of sodium thiosulfate consumed to titrate ozone-KI solution are presented in Table 4-6.

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46 Table 4-6. Ozone concentration over time in an exposure chamber. Time based Samples (minutes) Sodium Thiosulfate consumed (mL) Ozone Concentration (ppm) 3 0.7 17 6 0.9 24 9 1.0 26.7 12 1.1 29.4 15 1.1 29.4 18 1.1 29.4 21 1.1 29.4 24 1.1 29.4 Ozone Concentration vs. Time05101520253035051015202530Time (minutes)Ozone Concentration(PPM) Figure 4-2. Ozone concentration vs. time in an exposure chamber.

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47 Nutrient Media and Preparation of Inoculum Visual observation of different nutrient media strength Petri dishes inoculated for 6, 8 and 12 hours when treated with ozone revealed that higher strengths of nutrient media induced very fast population growth of microorganisms on plates that was unrealistic and non representative of expected levels of contamination(Appendix C). Presence of such high strength nutrients in an air handling unit via air flow under normal conditions was also not possible. Thus, the appropriate nutrient media strength chosen to inoculate Petri dishes with natural airborne microorganisms captured in an actual home setting was found to be 10%. Petri plates were inoculated for no longer than six hours because longer duration caused undesirable rapid growth of microbes on plates. Overly frequent ozone treatments (greater than 4 times per day) were also avoided because shut down of the air handling unit too frequently would be inconvenient for any home or office setting. The results from experiments on different nutrient media strength plates inoculated for 6, 8 and 12 hours are shown in Table 4-7.

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48 Table 4-7. Results of experimental trials on Petri dishes containing different combinations of nutrient media strength and inoculation duration. Ozone Concentration (ppm) Dragger Method Chemical Method Inoculation Duration (hrs) Treatment Duration (min.) Treatment Frequency (per day) Results for media strength 10% 100% 240 210 12 1 30 225 180 12 1 30 + + 225 180 12 2 30 + + 225 180 12 4 30 85 60 08 1 30 + + 85 60 08 2 30 + + 85 60 08 4 30 85 60 06 1 30 85 60 06 2 30 85 60 06 4 30 30 20 06 1 30 30 20 06 2 30 30 20 06 4 30 25 17 06 1 30 + All data points are from a minimum of 3 replicates. + Represents Growth and -Represents No Growth.

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49 Exposure Treatments Preliminary experimental data obtained prior to this study demonstrated that partial inhibition of Serratia and Aspergillus inoculum was achieved using lower levels of ozone concentration in a simulated air conditioning environment. However, higher ozone concentration levels were not tested and no fixed treatment duration time and frequency were maintained to prepare a database upon which a defined treatment regime could be specified. This study involved a design of experiments with several trials covering a wide range of combinations of ozone concentration, predetermined treatment duration and frequency. Results of this work are summarized in Table 4-8, which is divided into two parts namely treatment zone of effective treatment and treatment zone of ineffective treatment. The effective treatment zone shows combinations of ozone concentration, treatment duration and frequency required to achieve complete microbial inactivation while only partial microbial inhibition was achieved in treatment zone of ineffective conditions. These results reveal that treatment duration during ozone exposure is more important than frequency in affecting the rate of microbial inactivation. The results reveal that at 9 ppm and lower levels of ozone concentration frequent treatments are effective to inactivate microbial growth but as ozone concentration levels are increased from 11ppm and higher treatment duration play a major role to inhibit microbial growth. Combinations of 30 minutes or longer treatment duration with concentration levels as low as 12 ppm achieve similar inhibition results. The results also show that one 15-minute treatment per day of exposure for ozone at a concentration of 45 ppm is effective to achieve microbial inactivation.

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50 Table 4-8. Inhibition design matrix based on different combinations of treatment duration, frequency and ozone concentration. 15 Minutes Treatment 30 Minutes Treatment 60 Minutes Treatment Times Per Day Times Per Day Times Per Day Ozone Concentration (ppm) 1 2 4 1 2 4 1 2 4 02 + + + + + + + + + 3.5 + + + + + + + + + 5.7 + + + + + + + + + 09 + + + + + 11 + + + + 17 + + + 20 + + + 23 + + + 30 + + 35 45 Treatment Zone of Ineffective Conditions Treatment Zone of Effective Conditions All data points are from a minimum of 3 replicates. Pictures in Appendix A + represents Growth -represents No Growth An intermittent ozone treatment regime limited biofilm regrowth potential because it provided only partial residual protection against recontamination. Under suitable environmental conditions after ozone treatment, regrowth of bacteria and fungi on the Petri dishes took place but at a much slower rate. Petri plate treated with 45ppm concentration for 15 minutes at a treatment frequency of one time per day did not show

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51 any growth but when the same plate was kept in the indoor environment for the next 48 hours initiation of microbial growth was quite visible. Therefore, regular ozone treatment of air-handling systems at least once per day is necessary to achieve complete microbial inhibition. The experimental results of intermittent application of ozone have indicated the importance of adopting an effective ozone dose that is capable of inhibiting biofilm formation in an air handling system. Figure 4-3, 4-4 and 4-5 show that effective microbial inactivation of airstreams with airborne microbial contamination can be achieved with ozone treatment. The results presented here suggest that an ozonation system capable of disinfecting condensation coil surfaces and duct pans of an air conditioning system can be developed based upon the treatment conditions given in the effective region of Table 4-8. 15 Minutes Treatment Duration05101520253035404550124Treatment Frequency(per day)Ozone Concentration(PPM) Growth No Growth Figure 4-3. Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 15 minutes of treatment duration.

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52 30 Minutes Treatment Duration05101520253035124Treatment Frequency(per day)Ozone Concentration(PPM) Growth No Growth Figure 4-4. Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 30 minutes of treatment duration. 60 Minutes Treatment Duration051015202530124Treatment Frequency (per day)Ozone Concentration (ppm) Growth No Growth Figure 4-5. Microbial inactivation in a simulated air conditioning duct based on varying ozone concentration, treatment frequency and 60 minutes of treatment duration.

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CHAPTER 5 CONCLUSIONS AND SUGGESTIONS Conclusions The results presented in this research demonstrate that airborne ozone is an effective disinfectant to achieve microbial inhibition with promising applications to treat air handling systems. This research reconfirmed the results obtained by previous work of Chynoweth that an ozone concentration of at least 9 ppm for duration of 30 minutes with two times treatment frequency per day can result in a total inhibition of natural airborne microorganisms. The results reveal that one 15-minute treatment per day of exposure for ozone at a concentration of 45 ppm is effective to achieve total microbial inactivation. The results also show that at 11 ppm and higher ozone concentration treatment duration is more important than frequency in affecting the rate of microbial inactivation while at lower levels of ozone treatment frequency has more impact on microbial inhibition. Definitive inhibition design is developed to optimize conditions for applying gas-phase ozone to inhibit microbial contamination, to obtain maximum decontamination by applying a correlation between ozone concentration, exposure time and treatment frequency and to suggest practical treatment conditions to be applied to disinfect air conditioning systems. The treatment regime suggested in this research to disinfect air handling systems is not a replacement to the general cleaning of the system. A clean and air leak proof system are prerequisite to obtain best disinfection results and to avoid any ozone leakage in the indoor air environment respectively. Ozone treatment in night time or early morning may 53

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54 be effective to achieve microbial inhibition and convenient to the building residents as their routine work will not be affected by the shutdown of air handling system. Recommendations Based on the results of testing different combinations of ozone concentration, treatment frequency and treatment duration, the most practical conditions suggested by inhibition design matrix to achieve total microbial inhibition in an air conditioning systems treatment are One 15-minute treatment per day of ozone exposure at a concentration of 45 ppm, Alternatively, combination of 30-minutes treatment with concentration levels as low as 25 ppm one time per day. Suggestions for Future Work The studies here open some areas for expansion of this research. Further research on studying the effect of ozone on specific microorganisms like Bacillus anthracis and plotting death curves to determine the exact number of log cycle reduction of microbes achieved by airborne ozone in the air conditioning systems can be done. Further modification in the process can be done to treat exhaust ozone gas from air conditioning system by reducing the ozone concentration to an acceptable maximum level/ or decomposing ozone into environment friendly oxygen for discharge to the local environment. This can be accomplished thermally and/or by catalytic means, or by ozone passage through wet granular activated carbon beds. Cost economics study can be further done to check the commercial feasibility of this research project. The cost of ozone disinfection system will primarily be dependent on the area of the air conditioning system and duct work to be disinfected.

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APPENDIX A INHIBITION DESIGN EXPERIMENT PICTURES

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15 Minutes Ozone Treatment Duration 1 Time / Day 2 Times / Day 4 Times / Day 11 ppm 17 ppm 56 Growth Growth Growth Growth Growth Growth

PAGE 68

57 23 ppm 30 ppm 1 Time / Day 2 Times / Day 4 Times / Day Growth Growth Growth Growth Growth Growth

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58 35 ppm 45 ppm 1 Time / Day 2 Times / Day 4 Times / Day No Growth No Growth No Growth No Growth No Growth No Growth

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30 Minutes Ozone Treatment Duration 1 Time / Day 2 Times / Day 4 Times / Day 2 ppm 5.7 ppm Growth Growth Growth Growth 59 Growth Growth Growth

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60 11 ppm 1 Time / Day 2 Times / Day 4 Times / Day Growth Growth Growth 17 ppm Growth Growth Growth

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61 23 ppm 1 Time / Day 2 Times / Day 4 Times / Day No Growth No Growth No Growth 35 ppm No Growth No Growth No Growth

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60 Minutes Ozone Treatment Duration 1 Time / Day 2 Times / Day 4 Times / Day 2 ppm 5.7 ppm Growth Growth Growth 62 Growth Growth Growth

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1 Time / Day 2 Times / Day 4 Times / Day 9 ppm No Growth No Growth Growth 11 ppm 63 No Growth No Growth No Growth

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1 Time / Day 2 Times / Day 4 Times / Day 17 ppm 23 ppm No Growth No Growth No Growth 64 No Growth No Growth No Growth

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65 35 ppm 1 Time / Day 2 Times / Day 4 Times / Day No Growth No Growth No Growth

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APPENDIX B PROCESS CONTROL PROGRAMS 15 Minutes Ozone Treatment One Time Treatment per Day *Table 1 Program 01: 60 Execution Interval (seconds) 1: If time is (P92) 1: 900 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 2: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0110 C4..C1 = low/high/high/low 3: End (P95) 4: Volt (Diff) (P2) 1: 1 Reps 2: 3 25 mV Slow Range 3: 2 DIFF Channel 4: 1 Loc [ _________ ] 5: 1.0 Mult 6: 0.0 Offset 5: If time is (P92) 1: 915 Minutes (Seconds --) into a 66

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67 2: 1440 Interval (same units as above) 3: 30 Then Do 6: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 7: End (P95) 8: Do (P86) 1: 10 Set Output Flag High 9: Real Time (P77) 1: 221 Day,Hour/Minute,Seconds (midnight = 2400) 10: Sample (P70) 1: 1 Reps 2: 1 Loc [ _________ ] *Table 2 Program 02: 0.0000 Execution Interval (seconds) *Table 3 Subroutines End Program Two Times Treatment per Day Table 1 Program 01: 60 Execution Interval (seconds) 1: If time is (P92) 1: 625 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do

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68 2: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0110 C4..C1 = low/high/high/low 3: End (P95) 4: If time is (P92) 1: 640 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 5: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 6: End (P95) 7: If time is (P92) 1: 1345 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 8: Set Port(s) (P20) 1: 0000 C8..C5 = low/low/low/low 2: 0110 C4..C1 = low/high/high/low 9: End (P95) 10: If time is (P92) 1: 1360 Minutes (Seconds --) into a 2: 1440 Interval (same units as above)

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69 3: 30 Then Do 11: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 12: End (P95) 13: Do (P86) 1: 10 Set Output Flag High 14: Real Time (P77) 1: 221 Day,Hour/Minute,Seconds (midnight = 2400) 15: Sample (P70) 1: 1 Reps 2: 1 Loc [ _________ ] *Table 2 Program 02: 0.0000 Execution Interval (seconds) *Table 3 Subroutines End Program Four Times Treatment per Day *Table 1 Program 01: 60 Execution Interval (seconds) 1: If time is (P92) 1: 1015 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 2: Set Port(s) (P20)

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70 1: 0000 C8,C7,C6,C5 Options 2: 0110 C4..C1 = low/high/high/low 3: End (P95) 4: If time is (P92) 1: 1030 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 5: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 6: End (P95) 7: If time is (P92) 1: 1375 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 8: Set Port(s) (P20) 1: 0000 C8..C5 = low/low/low/low 2: 0110 C4..C1 = low/high/high/low 9: End (P95) 10: If time is (P92) 1: 1390 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do

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71 11: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 12: End (P95) 13: If time is (P92) 1: 295 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 14: Set Port(s) (P20) 1: 0000 C8, C7, C6, C5 Options 2: 0110 C4..C1 = low/high/high/low 15: End (P95) 16: If time is (P92) 1: 310 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 17: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 18: End (P95) 19: If time is (P92) 1: 655 Minutes (Seconds --) into a 2: 1440 Interval (same units as above)

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72 3: 30 Then Do 20: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0110 C4..C1 = low/high/high/low 21: End (P95) 22: If time is (P92) 1: 660 Minutes (Seconds --) into a 2: 1440 Interval (same units as above) 3: 30 Then Do 23: Set Port(s) (P20) 1: 0000 C8,C7,C6,C5 Options 2: 0001 C4..C1 = low/low/low/high 24: End (P95) 25: Do (P86) 1: 10 Set Output Flag High 26: Real Time (P77) 1: 221 Day,Hour/Minute,Seconds (midnight = 2400) 27: Sample (P70) 1: 1 Reps 2: 1 Loc [ _________ ] *Table 2 Program 02: 0.0000 Execution Interval (seconds)

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73 *Table 3 Subroutines End Program

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APPENDIX C PETRI PLATE PICTURES TO SELECT NUTRIENT MEDIA AND INOCULATION DURATION Comparison of 10% (right) with 100 %( left) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 4 hours and incubated for 2 days. Comparison of 10% (left) with 100 %( right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 6 hours and incubated for 2 days. 74

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75 Comparison of 10% (left) with 100% (right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 8 hours and incubated for 2 days. Comparison of 10% (left) with 100% (right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 12 hours and incubated for 2 days.

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76 Comparison of 10% (left) with 100% (right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 4 hours and incubated for 3 days. Comparison of 10% (right) with 100% (left) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 6 hours and incubated for 3 days.

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77 Comparison of 10% (left) with 100% (right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 8 hours and incubated for 3 days. Comparison of 10% (left) with 100% (right) nutrient media strength plates when both the plates were inoculated in natural home micro flora for 12 hours and incubated for 3 days.

PAGE 89

LIST OF REFERENCES Anwar H, 1989, Interaction of Biofilm Bacteria with Antibiotics in a Novel in vitro Chemostat System. Antimicrobial Agents and Chemotherapy 33:1824-1826. Anwar H and Costerton J, 1990, Effective Use of Antibiotics in the Treatment of Biofilm-Associated Infections. American Society of Microbiology News 58:665-668. Building Related Sickness, Building Services, March Ed.: 21-31, 1993. Blogoslawski W and Rice R, 1975, Aquatic Applications of Ozone. International Ozone Institute Inc., Syracuse, New York, Ed. 1975, pp. 1-226. Carpentier B and Cerf O, 1993, Biofilms and their Consequences, with Particular Reference to Hygiene in the Food Industry. Journal of Applied Bacteriology 75:499511. CharacklisW, 1981, Fouling Biofilm Development: A Process Analysis. Biotechnological Bioengineering 23:1923-1960. Chynoweth D, May 1997, Influence of Ozone on Growth and accumulation of Microorganisms in Air Conditioning Systems. Proposal for The Canadian Institute for Advanced Research (CIAR) May 30, 1997. Clamann H and Bancroft R, 1959, Properties and Analytical Properties of Ozone. Ozone. Academic Press, London, Ed. 1985 pp. 58-63, Chapter 1. Currier R, Toracco D, and Cross J, 2001, Deactivation of Clumped and Dirty Spores of Bacillus globigii. Ozone Science & Engineering 23(4):285-294. Dales R, 1991, Respiratory Health Effects of Home Dampness and Molds among Children. American Journal of Epidemiology 134:196-203. Elford W and Eude J, 1942, An Investigation of the Merits of Ozone as an Aerial Disinfectant. Journal of Hygiene 42:240-265. Ewell A, 1938, Present Use and Future Prospects of Ozone in Food Storage. Food Resources 3: 101-108. Ewell A, 1946, Recent Ozone Investigation. Journal of Applied Physics 17:908-911. 78

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79 Exner M, 1987, Influence of Biofilms by Chemical Disinfectants and Mechanical Cleaning. Zentralblatt fur Bacteriologie und Hygiene B183:549-563. Fannin K and Chynoweth D, 1994, Method and Apparatus for Controlling Microbial Growth on Condensation Coils. US Patent no. 5,286,447. Fetner R and Ingols R, 1959, Bactericidal Activity of Ozone and Chlorine against Escherichia coli at 1 o C. Ozone Chemistry and Technology, Advances in Chemistry Series, No. 21 (Washington, DC: Am. Chem. Soc.), pp. 370-374. Frank J and Koffi R, 1990, Surface Adherent Growth of Listeria monocytogenes is Associated with Increased Resistance to Surfactant Sanitizers and Heat. Journal of Food Protection 53: 550-554. Geesey G, 1982, Microbial Exopolymers: Ecological and Economic Considerations. American Society of Microbiology News 48, 9-14. Geesey G, 1992, New Technologies Focus on Biofilm Heterogeneity. American Society of Microbiology News 58:546-547. Grosse A and Streng A, 1958, Storage of explosive gases.U.S.Patent 2928529, 1960. Hartman F, 1925, Journal of American Society of Heating and Ventilation Engineers 31:33. Hicks S and Rowbury R, 1987, Bacteriophage Resistance of Attached Organisms as a Factor in the Survival of Plasmid Bearing Strains of Escherichia coli. Letters in Applied Microbiology 4:129-132. Hoffman R, 1971, Inhibition and Destruction of the Microbial Cell. Ozone. Academic Press, London, Ed. 1985 pp. 251-253, Chapter 4. Holah J, 1989, The Use of Epifluorescence Microscopy to Determine Surface Hygiene. International Biodeterioration 25:147-153. Holah J, 1990 a, A Conductance based Surface Disinfection Test for Hygiene. Letters in Applied Microbiology 11: 255-259. Holah J, 1990 b, The Spread of Listeria by Cleaning Systems. Technical memorandum no.590. Chipping Campden: The Campden Food and Drink Research Association. Holah J, 1995, The Evaluation of Air Disinfection Systems. R&D report, Campden & Chorleywood, Food Resource Association 13: 40. Horn E, 1994, Criteria for the Selection of the Feed Gas for Ozone Generation. Ozone Science and Engineering18: 57-71.

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80 Husman T, 1993, Respiratory Symptoms and Infections among Residents in Dwellings with Moisture Problems or Mold Growth. Proceedings of Indoor Air l4:171-174. Ishizaki K and Shinriki N, 1986, Inactivation of Bacillus Spores by Gaseous Ozone. Journal of Applied Bacteriology 60(1):67-72. Kaur K, 1992, Effects of Ozone As a Biocide in an Experimental Cooling Water System. Ozone Science and Engineering14:.517-530. Kim J and Yousef A, 2000, Inactivation Kinetics of Foodborne Spoilage and Pathogenic Bacteria by Ozone. Journal of Food Science 65(3): 521-528. Kim J and Yousef A, Dave S, 1999, Application of Ozone for Enhancing the Microbiological Safety and Quality of Foods: A Review. Journal of Food Protection 62(9): 1071-1087. Kim J, Yousuf A, and Khadre A, 2002, Ozone and its Current and Future Application in the Food Industry. Advances in Food and Nutrition Research. Kowalski, Bahnfleth, and Whitam, 1998, Bactericidal Effects of High Airborne Ozone Concentrations on Escherichia coli and Staphylococcus aureus. Ozone Science and Engineering 20:205-221. Lavoie, Jacques, and Comtois P, 1993, Microbial Decontamination of Ventilation Systems. Indoor Environment 2(5-6):291-300. Law A, Chau C, and Chan G, 2001, Characterstics of bioaerosol profile in Office Buildings in Hong Kong. Building and Environment 36:527-541. LeChevallier M, 1988, Factors Proving Survival of Bacteria in Chlorinated Water Supplies. Applied and Environmental Microbiology 54:649-654. Marshall K, 1992, Biofilms: An Overview of Bacterial Adhesion, Activity, and Control at Surfaces. American Society of Microbiology News 8: 202-207. Masoka T and Yositsugu K, 1982, Ozone Decontamination of Bioclean Rooms. Applied and Environmental Microbiology 43(3) 509-513. Masschelein W, 1997, Ozone Generation: Use of Air, Oxygen or Air Simphonized with Oxygen. Ozone Science and Engineering 20:191-203. Naitoh S, 1994, Inhibition of Food Spoilage Fungi by Application of Ozone. Journal of Food Microbiology 11:11-17. Occupational Safety & Health Administration (OSHA), 1975, Federal Register 40:47261. Oliveira D, 1992, Physico-Chemical Aspect of Adhesion. Biofilms-Science and Technology ed. Dordrecht: Kluwer Academic Press, pp.45-58(1992).

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81 Rakness K, Gordon G, and Masschelein W, 1996, Guideline for Measurement of Ozone Concentration in the Process Gas from an Ozone Generator. Ozone Science and Engineering18:209-229. Rice R, 2002, Ozone and AnthraxKnowns and Unknowns. Ozone Science and Engineering 24:151-158. Rompps Chemie-Lexicon and Neumuller O, 1974, Stabilization and Storage of Ozone. Ozone. Academic Press, London, Ed.1985 pp 232238, Chapter 4. Sallam S and Donnelly C, 1992, Destruction, Injury and Repair of Listeria Species Exposed to Sanitizing Compounds. Journal of Food Protection 55:771-776. Shea C, 1991, Comparison of the Adhesion Properties of Deleya Marina and the Exopolysaccharide-defective Mutant Strain DMR. Applied and Environmental Microbiology 57:3107-3113. Shin H and Frank J, 1991, Inactivation of Surface Adherent Listeria monocytogenes. Hypochlorite and Heat. Journal of Food Protection 54:4-6. Shun Cheng Lee, Wai-Ming Li, and Chio-Hang Ao, 2001, Investigation of indoor air quality at residential homes in Hong Kongcase study. Atmospheric Environment 36(2):225-237. Singh A, Yeager R, and McFeters G, 1986, Assessment of invivo Revival, Growth, and Pathogenicity of Escherichia coli strains after Copper and Chlorine-induced Injury. Applied and Environmental Microbiology 52:832-837. Spendlove J and Fannin K, 1983, Significance and Control of Indoor Microbial Aerosols: Human Health Aspects. U.S.Public Health Reports17:229-244. Streng A and Grosse A, 1961, Stabilization and Storage of Ozone. Ozone. Academic Press, London, Ed.1985 pp 231-256, Chapter 4. Trulear M and Characklis W, 1982, Dynamics of Biofilm Process. Journal of Water Pollution Control Federation 54:1288-1301. United States Environmental Protection Agency and the United States Consumer Product Safety Commission Office of Radiation and Indoor Air (6604J), April 1995. The Inside Story: A Guide to Indoor Air Quality .EPA 402-K-93-007. Wells W, 1935, Air-borne Infection and Sanitary Air Control. Journal of Industrial Hygiene and Toxicology17:253-257. Weinstein R, 1991, Epidemiology and Control of Nosocomial Infections in Adult Intensive Care Units. American Journal of Medicine 91:179S-184S.

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82 Whistler P and Sheldon B, 1989, Biocidal Activity of Ozone versus Formaldehyde Against Poultry Pathogens Inoculated in a Prototype Setter. Poultry Science 68:1068-1073. Williams R, 1960, Intramural Spread of Bacteria and Viruses in Human Populations. Ann. Rev. Microbiology, 14:43-64. World Health Organization (WHO), 1983, Indoor Air Pollutants: Exposure and Health Effects. EURO Reports and Studies No. 78 Copenhagen (1983). Yousuf A and Kim J, 2001, Microbiological Aspects of Ozone Applications in Food: A Review. Journal of Food Science 66:1242-1252. Zetterberg J, 1973, A Review of Respiratory Virology and the Spread of Virulent and Possibly Antigenic Viruses via Air Conditioning Systems. Annals of Allergy 31:228-299.

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BIOGRAPHICAL SKETCH Aakash Khurana was born in Sitapur city of Uttar Pradesh state in India. He was awarded a Bachelor of Science degree in chemical engineering by Dr. Babasaheb Ambedkar Marathwada University, India, in 1998. He worked as a Chemical Process Engineer in Vam Organic Chemical Limited, India for two and a half years before coming to United States for higher studies. He expects to be awarded a Master of Engineering degree in agricultural and biological engineering from the University of Florida in August 2003 and plans to pursue a career in the food or pharmaceutical industry. 83


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Title: Ozone Treatment to Prevent Microbial Growth in Air Conditioning Systems
Physical Description: Mixed Material
Copyright Date: 2008

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OZONE TREATMENT FOR PREVENTION OF MICROBIAL GROWTH IN AIR
CONDITIONING SYSTEMS

















By

AAKASH KHURANA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Aakash Khurana




























To my Family and Friends















ACKNOWLEDGMENTS

This thesis would not have been completed without the help of several people

whom I wish to thank. I would like to express my great appreciation to Dr. David

Chynoweth, my committee chair and sponsoring professor, for his generosity and

support, and for giving me this opportunity. He was always there when I needed any

advice or to solve any problem. I would also like to give my special thanks to Dr. Arthur

Teixeira for his support, interest, knowledge, problem solving and advice. I am grateful

to Dr. Teixeira for recommending me to Dr. Chynoweth and suggesting that I contact

him; without him, I would never have known about this opportunity. I am indebted to Dr.

Jean Andino, who was there from the beginning to assist me with technical issues and

allowed me to borrow her ozone analyzer instrument for this research work. Thanks to all

my supervisory committee, whose comments and edits contributed substantially to my

research and to this document.

In addition, I would like to thank Mr. Bob Tonkinson for his help with my many

assorted questions. Special thanks go to my friends, Luis Nogueira and Dr. Indaue Mello,

who had a great influence on my personal and professional life. Finally, I would like to

thank three very special people in my life; without them, this would have never been

possible my Mother, Father and sister, to whom I dedicate this.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ......... ..................... ....... ........... ....... ............ .. vii

A B ST R A C T ................. .......................................................................................... x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

B a c k g ro u n d ............................................................................................. 1
P problem ..................................................2
O bj ectiv e ...................................................................................... . 3

2 LITER A TU R E R EV IEW .............................................................. ....................... 5

Indoor A ir Q quality .......................................................5
B iofilm F orm ation ............... ... ...................................... ................. ....................6
Available M ethods for Air Treatm ent ........................................ ...................... 9
O zone as an A erial D isinfectant ......................................................... ... ............ 11
Properties of O zone ..... .... .......................................... .... ..... .. ............ 12
O zone G generation ..................................... ..................... .. .......... 17
Corona D ischarge .................. ............................. ......... .. .......... 17
U ultraviolet R radiation ..................... .. ...................... .. .. ...... ........... 18
E lectrolysis ..................................... ........................... .. ... .......... 19
Stabilization and Storage of O zone ........................................ .....................20
O zone M easurem ent .................................................... .... .. ............ 22
Previous W ork at U F ........................ ................ .... ....... .... ....... 24

3 M ATERIALS AND M ETHODS ........................................ ......................... 29

Scope of W ork ...................................... ............................... .......... 29
M a te ria ls ........................................................................... 2 9
Prelim inary Studies ............. ............................................ ....... ... 34
Preparation of Nutrient Media and Inoculum........................................................34
Ozone Generation and M easurement ...................................... ............... 36
E xperim mental D design .......................................... .. .. .... .... ... .... 39




v









4 RESULTS AND DISCU SSION S......................................... .......................... 42

Findings from Prelim inary Studies................................... ............................. ..... 42
Ozone M easurem ent and Generation .............................................. ............42
Nutrient Media and Preparation of Inoculum................................... ................47
Exposure Treatm ents ........................ ................ ... .... ........ ......... 49

5 CONCLUSIONS AND SUGGESTIONS ...................................... ............... 53

C o n c lu sio n s ........................................................................................................... 5 3
R ecom m endations................... ............................................. ........ 54
Suggestions for Future W ork................................... ....................... ............... 54

APPENDIX

A INHIBITION DESIGN EXPERIMENT PICTURES ............................................55

15 Minutes Ozone Treatment Duration........... ...................................56
30 Minutes Ozone Treatment Duration.............................................................59
60 Minutes Ozone Treatment Duration.............................................................62

B PROCESS CON TROL PROGRAM S ............................................. .....................66

C PETRI PLATE PICTURES TO SELECT NUTRIENT MEDIA AND
IN OCULA TION DURA TION .............................................................. ............... 74

L IST O F R E FE R E N C E S ....................................................................... ... ...................78

BIOGRAPH ICAL SKETCH ..................................................... 83















LIST OF TABLES


Table page

2-1 Ozonation of bacteria and viruses in air .... ........... ....................................... 14

2-2 Summary of previous trials run on Serratia and Aspergillus inoculum...................26

2-3 Summary of previous trials run on natural airborne organisms.............................26

3-1 RGF-TURBOHYDROZONE ozone generator specifications.............................. 36

3-2 Experimental design matrix showing different combinations of treatment
duration, frequency and ozone concentration carried out in study.........................40

4-1 Ozone concentration vs. flow rate achieved by small RGF generator using
com pressed air as feed gas. ........................................................... .....................43

4-2 Ozone concentration vs. flow rate achieved by small RGF generator using pure
oxygen as feed gas.................... .... ..................... .......... 44

4-3 Ozone concentration vs. flow rate achieved by large RGF generator using
com pressed air as feed gas. ........................................................... .....................44

4-4 Ozone concentration vs. flow rate achieved by large RGF generator using pure
oxygen as feed gas.................... .... ..................... .......... 44

4-5 Ozone concentration vs. flow rate achieved by small RGF generator using
compressed air as feed gas and diluting exposure chamber with compressed air....45

4-6 Ozone concentration over time in an exposure chamber. ......................................46

4-7 Results of experimental trials on Petri dishes containing different combinations
of nutrient media strength and inoculation duration. ................. ............... 48

4-8 Inhibition design matrix based on different combinations of treatment duration,
frequency and ozone concentration ............................. .............. ............... 50















LIST OF FIGURES
Figure pge

2-1 Death curves for A) Escherichia coli and B) Staphylococcus aureus in
ozon ated air. ..................................................... ................. 15

2-2 Biofilm formation in air conditioning systems ................................ ............... 27

2-3 Test apparatus to prevent microbial growth in air conditioning system ................27

2-4 Original apparatus attached to air conditioning system. ........................................27

3-1 Lab scale experimental setup of ozone-treated air handling system......................31

3-2 Process flow diagram of ozone-treated air handling system...............................32

3-3 RGF-TURBOHYDROZONE ozone generators. .............................................. 32

3-4 M mechanical damper in the inlet airline. ........................................ ............... 32

3-5 Biological contain ent hood. .............................................................................33

3-6 Process automation using CR 10 as a datalogger and process controller ...............33

3-7 Sequence of steps taken to prepare humidity inside simulated air duct (A) Wet
sponges inside exposure chamber (B) Petri dishes in an exposure chamber
before starting experim ental trial. ........................................ ........................ 33

3-8 Sequence of steps taken to prepare nutrient media (A) Nutrient media prepared
in a conical flask (B) Sterilization of nutrient media in a autoclave
(C) Solidification of nutrient media in Petri plate under bacteria free
environmental hood (D) Storage of Petri dishes in a refrigerator..........................35

3-9 Petri dishes inoculated from a natural indoor home flora. .....................................36

3-10 lodometric method test set-up for ozone concentration measurement....................38

3-11 .The D ragger pum p ........... ....... .......... .. ...... ........................ 39

3-12 Comparison of ozone treated plate (left) with untreated plate (right)....................41

4-1 Ozone concentration levels achieved under different feed gas flow rate
conditions. ...........................................................................43









4-2 Ozone concentration vs. time in an exposure chamber.................... ...............46

4-3 Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 15 minutes of treatment
d u ratio n ...................... .. .. ......... .. .. .................................................. 5 1

4-4 Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 30 minutes of treatment
d u ratio n ...................... .. .. ......... .. .. .................................................. 5 2

4-5 Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 60 minutes of treatment
d u ratio n ...................... .. .. ......... .. .. .................................................. 5 2















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Engineering


OZONE TREATMENT FOR PREVENTION OF MICROBIAL GROWTH IN AIR
CONDITIONING SYSTEMS

By

Aakash Khurana

December 2003

Chair: David P. Chynoweth
Cochair: Arthur A. Teixeira
Major Department: Agricultural and Biological Engineering

Microbial contamination of indoor air by air conditioning systems represents a

major public health concern. Biofilm formation around condensation coils and drip pans

takes place due to the combination of collection of particulate organic matter and

condensation of moisture in an air conditioning unit, which not only reduces the

efficiency of heat transfer in these systems but also leads to a high level of fungi and

bacteria that become airborne and transported throughout the buildings via air circulation.

The success of ozone as a disinfectant in water systems suggested its potential for

aerial applications to prevent microbial growth in air conditioning systems. Preliminary

work suggested that ozone prevented accumulation of microbial mass in an actual home

air conditioning system, and thus a method and apparatus to control the microbial growth

in air handling systems were developed.









The goal of this study was to confirm and document the efficacy of ozone as a

disinfectant to inhibit microbial growth in air conditioning systems and to determine

practical ozone treatment conditions consisting of ozone concentration and treatment time

and frequency for engineering applications of this invention.

Findings from this study reveal that at 9 ppm and lower levels of ozone

concentration frequent treatments are effective to inactivate microbial growth but as

ozone concentration levels are increased from 1 Ippm and higher treatment duration plays

a major role to inhibit microbial growth. Also, combinations of 30 minutes or longer

treatment duration with concentration levels as low as 12 ppm achieve similar inhibition

results. The results also show that one 15-minute treatment per day of exposure for ozone

at a concentration of 45 ppm is effective to achieve microbial inactivation. Thus, the

results presented in this research demonstrate that practical treatment conditions based on

different combinations of ozone concentration, treatment duration and frequency can be

applied to disinfect our air handling systems.














CHAPTER 1
INTRODUCTION

Background

Transmission of respiratory infections in indoor environments represents a public

health concern for which engineering alternatives are limited. Indoor air quality is

affected by a variety of factors like accumulation of microorganisms in air conditioning

systems, entrance of outdoor microorganisms into buildings, or contamination of building

materials. The combination of collection of particulate organic matter and condensation

of moisture provides an ideal environment for the growth of biofilms around drip pans,

condensation coils, and ductwork of the air conditioning units and the recycling air from

contaminated units leads to the transportation of airborne contaminants in the indoor

environment. Biofilms not only lead to high levels of fungi and bacteria that become

airborne and transported throughout the buildings via air recirculation, but also reduce the

efficiency of heat transfer in these systems.

Periodic cleaning of air conditioning system and ductwork with biocides does not

eliminate all microorganisms which can cause variety of health problems including

hyper-sensitivity, pneumonitis, sick building syndrome, legionnaire's disease, asthma and

allergies in humans. Common technologies, such as high efficiency particulate air

(HEPA) filters and ultraviolet germicidal irradiation (UVGI) for air treatment, are proven

less effective in real-world installations than in laboratory studies.

The success of ozone to inactivate bacteria and fungi in water treatment systems

(Blogoslawski and Rice, 1975) suggests its potential for aerial applications to prevent









microbial growth in air conditioning systems. Gaseous ozone can treat areas, which are

inaccessible to UV light and other cleaning methods. It is a strongest oxidant and

disinfectant widely used for the treatment of water and wastewater. The high oxidation-

potential of ozone results in a biocidal effect on the microorganisms. These attributes,

plus the fact that ozone decomposes to form the innocuous byproduct oxygen, can make

ozone ideal for air treatment.

Problem

The problem of contamination of air handling systems such as those used in

commercial buildings, private dwellings and closed vehicles is particularly acute in warm

climate areas that require lengthy air conditioner operation without the opportunity to

permit the moisture producing coils and drip pans to dry. Most of the drip pans are not

designed to drain completely and thus harbor microbial growth in air-conditioning units.

It is recommended that the condensation producing apparatus of air-cooling equipment be

regularly cleaned and disinfected. However, such routine maintenance is often

overlooked, and the use of conventional disinfecting techniques creates problems because

of the dissipation of disinfectants into the air flowing through the system exposes many

occupants of buildings and vehicles to allergies and illnesses difficult to trace and

diagnose. Microbial contamination of indoor air is a major cause of illness among

individuals within office buildings and the like having sealed windows wherein air

circulation is only through central cooling and heating units (Building Related Sickness,

1993).

Chynoweth (Unpublished) observed and collected large quantities of microbial

mass from the drip pan and condensation coils of his home air conditioning unit. He









reasoned that when the blower transmits air across the evaporator coil, the

microorganisms present in the biofilm on the coil and drip pan become air-borne

particulates with the potential to cause health problems as allergies, rashes, colds, viruses,

legionnaire disease and some other illnesses known. He installed a hot tub ozone unit to

one of his two air conditioning units after cleaning them thoroughly during summer. The

air conditioning unit with ozone unit installed did not show any sign of biofilm formation

but the other unit had a significant biofilm formation across evaporator coil and drip pan

after a few summer months. Thus, based on this observation Fannin and Chynoweth

(1994) wrote a patent application for developing a method and apparatus to control the

microbial growth in air handling systems by treating the unit with gaseous disinfectant.

Initial tests were conducted at the home of an inventor and couple of years later a lab duct

was built to do preliminary treatments. Preliminary work suggested that ozone prevented

accumulation of microbial mass over a period in one summer. Follow up testing of this

treatment system was conducted to measure the ozone output and test treatment regimes.

The research report here developed was a detailed database for ozone treatment based on

ozone concentration and the treatment time and frequency for engineering applications of

this invention.

Objective

The goal of this study was to reconfirm the efficacy of ozone as a disinfectant to

inhibit microbial growth in air conditioning systems and to determine practical treatment

conditions. Specific objectives to the study were the following:

1. Determine the effectiveness of intermittent ozone treatment for prevention of
bacterial and fungal growth in air conditioning systems
2. Evaluate the effect of various combinations of ozone concentration, exposure
duration and treatment frequency on microbial growth.






4


3. Recommend practical air conditioning unit treatment conditions based on the
ozone concentration, treatment duration and frequency.














CHAPTER 2
LITERATURE REVIEW

Indoor Air Quality

Poor indoor air quality is widely regarded as a significant health, environmental

and economic problem. Microorganisms such as bacteria and fungi are ubiquitous in our

indoor and outside environment. Excessive proliferation or distribution of microbial

growth inside buildings has been known to cause respiratory related ailments as well as

skin rashes. Evidence for the spread of respiratory diseases inside buildings, and

specifically by ventilation systems exists in early epidemiological data (Hartman, 1925).

Microbial contamination in buildings is often a function of moisture incursion from

sources such as stagnant water in heating, ventilation and air conditioning (HVAC) air

distribution systems and cooling towers. Such contamination of indoor air has been

widely documented and is a cause of illness among individuals within office buildings

and the like having sealed windows wherein air circulation is only through central

cooling and heating units (Building Related Sickness, 1993).

Health effects related to residency and/or work in closed buildings, such as

residential blocks, offices, schools and nurseries, have become a growing public health

concern. These problems are often called the sick building syndrome (SBS) and was

originally defined by the World Health Organization (WHO, 1983) as the occurrence of

an increased prevalence of non-specific symptoms among populations in certain

buildings. Symptoms of SBS are believed to arise from multiple causes which, while not

clearly understood, are associated mainly with air-conditioned office buildings.









Most people are aware that outdoor air pollution can damage their health but many

do not know that indoor air pollution can also have significant negative health effects.

Environmental Protection Agency (EPA) studies of human exposure to air pollutants

indicate that indoor levels of pollutants may be 2-5 times, and occasionally more than

100 times, higher than outdoor levels. These levels of indoor air pollutants may be of

particular concern because most people spend about 90% of their time indoors (US EPA,

1995).

Biofilm Formation

A biofilm is a community of microbes embedded in an organic polymer matrix,

adhering to a surface. The matrix of the biofilm consists largely of water and the

remainder is an assortment of extracellular polymers (polysaccharides, glyco-protiens)

which are referred to as extracellular polymeric substances (EPS) (Geesey, 1982).

Bacteria gain a number of advantages from living in biofilms. In a biofilm, for

example, the microbial cells are protected from possible attack from the outside

environment, e.g., from ultraviolet (UV) radiation. They are also resistant to anti-bacterial

agents like heat (Frank and Koffi, 1990) and bacteriophages (Hicks and Rowbury, 1987).

Biofilms thus constitute a reservoir of many different species able to resist environmental

fluctuations. Biofilm formation is a multi step process whereby bacteria adhere to

equipment or surfaces surround themselves with a protective layer of polysaccharides and

grow into a network of micro colonies and water channels. These channels serve to

supply nutrients and remove bacterial waste products.

According to Characklis (1981), the formation of a biofilm in a clean and inert

surface containing organic and mineral molecules and microorganisms goes through four

stages:









1. Transport: First, molecules are carried towards the surface by diffusion or, in

some cases, by turbulent flow. The larger particles, including the microorganisms, are

slowed down by the laminar boundary flow; other mechanisms also intervene to explain

how the microorganisms are carried right to the surface (gravity, thermophoresis and

chemotaxis, fluid dynamic forces).

2. Adsorption of molecules: Molecules are almost instantly adsorbed at the surface

and play a part in the events that follow. This conditioning" of the surface alters its

physico-chemical properties (surface free energies, electrostatic charge). The

accumulation of organic and inorganic matters at the solid-liquid interface (explained by

Gibbs law, and often referred to as the 'conditioning film') contains a notably higher

concentration of molecules of nutrients than is found in the mass of the fluid. Nutrient

transfer by diffusion is thus more rapid within the film.

3. Adhesion of nutrient microorganisms: Adhesion is due to various types of forces

(Van der Waals attraction forces or electrostatic forces) that operate between the inert

surface and the living cells (Oliveira, 1992). Unlike the previous stage, the adhesion of

bacteria at this stage takes sometime, though this varies according to the species of

microbe concerned. Marshall (1992) described a first stage in which adhesion is

reversible and the bacteria still show Brownian motion and are easily removed (e.g.,

merely by rinsing); and a second phase, in which adhesion can be called irreversible,

requiring much stronger forces to remove the bacteria (e.g., scraping, scrubbing). He

reported on research involving Pseudonomas spp. took 4 hours for 73% of the adhering

bacteria to become irreversibly attached.









The extracellular polymeric substances (EPS) of cells in suspension condition the

surface properties of the microorganisms and hence enhance their degree of adhesion to

surfaces (Shea, 1991). Polysaccharide conformation varies in this respect according to

environmental conditions of temperature and composition of solvent.

4. Colonization: Lastly, once they have attached, the microorganisms that produce

EPS reproduce and multiply within the matrix so formed. This colonization results in the

formation of a biofilm. It is a fairly slow process: depending on the culture conditions, it

takes anything from a few hours to several months to reach a state of equilibrium.

Thickness of biofilm depends on factors that impact on the lives and survival of

microorganisms (e.g., pH, water activity (aw), temperature and composition of the

medium). When nutrient concentration in the medium increases, the thickness of the

biofilm increases, as do the thickness thresholds beyond which diffusion of one nutrient

element or other ceases (Trulear and Characklis, 1982). When the flow rate of the fluid in

contact with the biofilm is increased until it becomes turbulent, the transport of molecules

and microorganisms is improved and the rate of detachment of cells from the film

increases.

Research done in Hong Kong (Law et al., 2001) to determine the temporal

concentration profile of bioaerosol inside office environments during office hours

recorded highest bacteria concentrations at the early morning hours during the starting-up

period of HVAC systems. The highest fungi concentrations were recorded during the

weekend mornings. Elevated concentrations of bacteria and fungi occurred just after the

morning HVAC system startup, and decreased within 1-2 h afterwards. Major variations

in bioaerosol concentration occurred during the first few hours around the system startup









time, and the bioaerosol concentration diminished gradually during office hours. This

provided the insight that more treatment is needed within the first 3 h around the air-

conditioning morning startup times than other times of the day. With these findings, it

can be expected that a fairly high concentration of airborne fungi occur during the

morning if the HVAC system is down for more than 8 hours. This burst of emission of

bacteria and fungi was probably due to the incubation of bacteria and fungi during the

nighttime HVAC shutdown hours. The study suggested that amplification of biological

agents in HVAC reservoirs occurred due to the favorable conditions provided during the

HVAC system shutdown hours.

Available Methods for Air Treatment

Cleaning and disinfection are the two methods used to eliminate biofilms from

surfaces. Cleaning is intended to eliminate slime and disinfection to destroy any

remaining microorganisms. Carpentier and Cerf (1992) mentioned that there is no method

available to accurately count microorganisms on the surface due to two reasons. First, the

usual techniques (scrapers, agar contact methods, sponges etc) remove only the top of a

film containing only a fraction of the population. Secondly, because of the nutrient

gradients found within thick biofilms (Characklis, 1981), the irregular inputs of nutrients

and /or the stress produced by desiccation, cleaning and disinfection (Singh et al., 1986;

Sall am and Donelly, 1992) some microorganisms remain undetected (fail to colonize)

with usual laboratory culture media. Also, no media are suitable for growth of all

organisms.

Cleaning is an important treatment for minimizing microbial colonization on the

surface. It seems to be of vital importance to eliminate as many microorganisms as

possible before applying a disinfectant. Microorganisms are far more sensitive to









disinfectants once they have been detached from surfaces. Mechanical actions (brush,

medium and high pressure jets) are recognized as the most effective methods for

eliminating biofilms (Exner, 1987; Holah, 1990 b). The extent to which biofilms are

removed depends on the microbial strain (Holah, 1989). However, it is difficult to

achieve 100% rate of bacterial removal by cleaning.

Disinfection is considered to be the primary mechanism for the effective

inactivation/destruction of organisms to prevent the spread of diseases. There is no

perfect disinfectant available. A number of studies mentioned by Cerf and Carpentier

(1992) indicated that resistance to disinfection increases with the biofilm age (Le

Chevallier, 1988; Anwar, 1989; Frank and Koffi, 1990; Shin and Frank, 1991; Anwar,

1992). Further, Holah (1990a) showed that those disinfectants most effective against

bacteria in suspension are not those that are most effective against those in biofilms.

However, there are certain characteristics to look for when choosing a suitable

disinfectant for a treatment .They could be

1. Ability to penetrate and destroy infectious agents under normal operating
conditions;
2. Lack of characteristics that could be hazardous to people and the environment
before or during disinfection;
3. Absence of toxic residuals and mutagenic or carcinogenic compounds after
disinfection;
4. Affordable capital and operation and maintenance (O&M) costs.
The two most common methods of disinfection are chlorination and ozonation.

Chlorine, the most widely used disinfectant for municipal wastewater, destroys target

organisms by oxidation of cellular material. It may be applied as chlorine gas,

hypochlorite solutions, and other chlorine compounds in solid or liquid form. Like

chlorine, ozone is a strong oxidizing agent but it is more effective in destroying viruses









and bacteria. It is an unstable gas that is generated by an electrical discharge or UV

radiation through dry air or pure oxygen. The ozonation process utilizes a short contact

time (approximately 10 to 30 minutes) and there are no harmful residuals that are to be

removed after ozonation because it decomposes rapidly to oxygen.

Ozone as an Aerial Disinfectant

Inactivating spore-forming bacteria in water with ozone is one thing, but in air it is

extremely different matter, primarily because of the variations in relative humidities

(Rice, 2002). Ozone has been previously investigated as an aerial disinfectant but the

results were inconclusive (Elfred, 1942). Masoka (1982) showed promising results for

disinfection of entire room with ozone but the data on ozone disinfection of air remain

limited. Despite the growing research, there still remains an absence of data on the

optimal concentration and exposure times to prevent the biofilm accumulations in the air

conditioning ducts in warm climate areas.

Ishizaki (1986) studied the inactivation of six strains of Bacillus spp; B .subtilis;B.

cereus;B.licheniformis and B.megeterium with gas phase ozone (230-1400 ppm) at

varying relative humdities and found a lag phase during the initial exposure followed by

an exponential decrease in the number of survivors with time. No inactivation was

observed at relative humidities below 50%. Increasing relative humidity influenced the

lag phase, but not the inactivation rate.

Whistler and Sheldon (1989) evaluated ozone as an alternative disinfectant in air to

replace formaldehyde. Culture of Staphylococcus, Streptococcus, and Bacillus species

previously isolated from poultry hatcheries and selected cultures of Escherichia coli,

Pseudomonasfluorescens, Salmonella typhimurium, and Aspergillusfumigatus were

spread plated on open Petri plates and independently fumigated with ozone or









formaldehyde in a prototype laboratory poultry setter. Ozone (greater than 9000 ppm)

reduced levels by >4 to 7 logo and fungal levels by >4 logio. Naito (1989) studied the

microbiocidal effects of ozone on Bacillus, Micrococcus and yeast present in a candy

factory and found that the total microbial counts drastically reduced after gas phase ozone

treatment.

A most recent study by Currier et al. (2001) of gas phase ozone to inactivate both

clumped and dirty spores of Bacillus globigii var. niger as a surrogate for Bacillus

a/ul/1La i showed ozone inactivation at 9,000 ppm (-1% ozone in air) ozone during 15

hours of exposure.

Properties of Ozone

In gaseous form, ozone is colorless, whereas liquid ozone is almost 'opaque'and

bluish-black and its crystals are violet-blue. It has a characteristic odor, reminiscent of

phosphorous and sulphur dioxide. Olfactory organs are extremely sensitive to ozone; it

can be detected by humans at a dilution of 1 parts per million (ppm) of air. Ozone freezes

to bluish-black crystals at the boiling point of hydrogen. Ozone half-life in air is typically

about 15 minutes in open areas (can be hours in enclosed areas) and increases with lower

temperature and lower humidity. Ozone is about 50% heavier than air and has a low

vapor pressure, so it tends to sink to the floor and does not disperse if there is no air

circulation. Maximum ozone concentration in water varies directly with concentration of

the gas in the sparging air and inversely with temperature: for example ozone at 1.5%

sparging gas (by weight) will have a maximum concentration of about 11 ppm in water at

5C and 6.4 ppm at 200C. Doubling the concentrations of ozone in the feed gas will

double the concentration in water at any given temperature.









Antimicrobial Properties. Ozone is a strong, broad-spectrum antimicrobial agent

that is active against bacteria, fungi, viruses, protozoa, and bacterial and fungal spores

(Kim et al., 2000, Kim et al., 1999, Yousuf, 2001). Inactivation of bacteria by ozone

occurs by lysing (i.e., oxidative rupture) of the cell wall. The anti microbial activity of

ozone is based essentially on its powerful oxidizing effect, which causes irreversible

damage to the fatty acids in the cell membrane and to cellular macromolecules, such as

proteins, and DNA (Hoffman, 1971, Fetner and Ingols, 1959, Naitoh, 1994). This action

is particularly effective in air at high relative humidity level. Gram-positive bacteria are

more sensitive to ozone than gram-negative. Disinfection rate depends on the type of

organism and is affected by ozone concentration, treatment time (Kim and Yousuf, 2000,

Kim et al., 1999), temperature, relative humidity, pH, turbidity, the presence of ozone-

oxidizable materials, the tendency (or not) for the microorganisms to form clumps, and

the type of ozone contactor. Ewell (1938) stated that depending on cleanliness, minimum

continuous concentrations of 0.6 to 1.5 ppm ozone (in air) were necessary to prevent

mold growth on eggs kept at 90% relative humidity (RH), whereas 2.5 to 3.0 ppm ozone

were required to control molds on beef stored under similar conditions.

Kim (2002) conducted a detailed study of the effects of relative humidity on the

ozone inactivation of Bacillus subtilis. He concluded that high relative humidity is

needed for the inactivation of microorganisms by ozone in gas environment. It is believed

that hydration of dry microorganisms in humid atmospheres makes them susceptible to

ozone. The optimum relative humidity found by Kim (2000) for microbial inactivation by

ozone in air is 90-95%. Elford and Ende (1942) used low ozone concentrations and long

exposures at variable relative humidity to disinfect airborne microorganisms. At RH









<45%, the germicidal power of ozone was negligible. Ewell (1946) also demonstrated

that microorganisms were killed more readily by ozone in an atmosphere having a high

rather than low RH. Holah (1995) constructed a special aerobiology cabinet to study the

destruction of airborne microorganisms by disinfectant fogging, by ozone and by

ultraviolet light. Exposure to ozone above 4 ppm in air (5 and 10 min exposure)

significantly reduced the numbers of airborne P. aeroginosa. Kowalski et. al. (1998)

studied the bactericidal effects of airborne ozone against E. coli and Staphylococcus

aureus by placing Petri dishes containing the microorganisms in the chamber and

exposing for 10 to 480 seconds to ozone concentrations between 300 and 1500 ppm.

Levels of population reduction in excess of 99.99% (4 logo cycles) were achieved for

both species. However, practical applications of such higher ozone concentrations for air

treatment are quite questionable. Thus, concentrations lower than 300 ppm could also be

equally effective to achieve similar reduction in population levels. Table 2-1 lists data on

ozonation of airborne bacteria and viruses reported by previous investigators and

compiled by Kowalski et al. (1998). Data taken in Kowalski et. al. (1998) experimental

trials were combined to form the death curves presented in Figure 2-1-a and 2-1-b.

Table 2-1. Ozonation of bacteria and viruses in air (Kowalski et al., 1998).
Test Organism Ozone (ppm) Time(sec) % Survival Reference

S. salivarius 0.6 600 2 Elford & van de Eude,1942
S. epidermis 0.6 240 0.6 Heindel et al., 1993
pX174 (virus) 0.4 480 0.1 de Mik, 1977











A 1
Death Curve for Escherichia coli in airborne ozone
0.1

0
0.01- 88
S8- o
0
2 0.001 0 Fitted Data Curve
P 0.001
00


0.0001

Theoretical ultrasonicated curve
0.00001
0 100 200 300 400 500

Time, seconds



B 1

Death Curve for Staphylococcus aureus in airborne ozone
0.1


0
*| 0.01


Z Fitted data curve
2: 0.001 o
0 0
Io o o
0.0001


p--- Theoretical ultrasonicated curve 0
0.00001
0 100 200 300 400 500
Time, seconds


Figure 2-1. Death curves for (A) Escherichia coli and (B) Staphylococcus aureus in
ozonated air. Ozone concentrations during this series of experiments varied
from 300 to 1500 ppm.









Physiological Effects of Ozone on Humans

Ozone inhalation causes a wide range of symptoms, from the unpleasant smell to

pulmonary edema or lethal poisoning. Irritation by ozone primarily affects the lungs.

Reaction of the mucous membranes in the upper respiratory tracts manifests itself in

dryness of the mouth, nose and throat. Irritating symptoms are also exhibited in the eye;

visual acuity diminishes, followed by the appearance of tears and swelling. Coughing,

headache, dizziness, dribbling and sweating are also experienced. After prolonged

exposure, symptoms become more serious, exhaustion sets in along with an increase in

the pulse rate. All these symptoms can be associated with nausea and an intensification of

symptoms can lead to unconsciousness, pulmonary congestion, hemorrhage of the nose,

and eventually to death.

Actual and expected physiological effects of ozone on humans were investigated in

a detailed manner by Clamann and Bancroft (1959).The adverse health affects of ozone

exposure are also well documented by Weinstein (1991).The individual physiological

symptoms manifest themselves depending on the concentration and the exposure time.

OSHA has established a time-weighted average permissible exposure level for workers

for an eight-hour day of 0.10 ppm and a short-term exposure limit of 0.30 ppm for an

exposure less than 15 minutes (OSHA, 1975). The symptoms experienced on exposure to

0.1-1 ppm ozone are headache, throat dryness, irritation of the respiratory passages, and

burning of the eyes caused by the formation of aldehydes and peroxyacyl nitrates.

Exposure to 1-100 ppm ozone can cause asthma-like symptoms such as tiredness and

lack of appetite. Short-term exposure to higher concentrations can cause throat irritations,

hemorrhaging, and pulmonary edema.









Ozone Generation

Ozone is formed from oxygen molecules as a result of electrical discharge or

ultraviolet (UV) radiation. Oxygen atoms are formed by the splitting of diatomic oxygen

molecules into two atoms, which then recombine with other oxygen molecules to produce

ozone molecules. The exothermic reaction leading mostly to ozone formation is

02 + O 03

Commercially, ozone can be generated on-site by three following ways- corona

discharge, UV radiation and electrolysis.

Corona Discharge

The corona discharge technique is a commercially significant method (the so-called

"silent electrical discharge" procedure) to manufacture ozone. In a corona discharge

ozone generator, the feed gas (dried air, oxygen, or mixtures thereof), passes between two

closely spaced electrodes (one of which is coated with a dielectric material) under a

nominal applied voltage potential of -10 kV. A silent or barrier discharge occurs when

the gas becomes partially ionized, resulting in a characteristic violet glow when air is the

feed gas (with high purity oxygen the violet coloration is seldom observed). Efficiency of

ozone production by corona discharge depends, for the most part, on the strength of

micro discharges (which is influenced by a number of factors such as the gap width, gas

pressure, properties of the dielectric and metal electrodes, power supply, and the presence

of moisture). In weak discharges, a significant fraction of the energy is consumed by

ions, whereas in stronger discharges, almost all of the discharge energy is transferred to

electrons responsible for the formation of ozone. The optimum is a compromise that









avoids energy losses to ions but at the same time obtains a reasonable conversion

efficiency of oxygen atoms to ozone.

If compressor air is used as the feed gas, it must be scrupulously dried and be free

of traces of oils and greases (oxidized by ozone). Moist air gives rise to nitrogen oxides

in the ozone generator which can form nitric acid and corrode the generator, requiring

frequent maintenance and down time.

Ozone is quite readily available in nature, most often as a result of lightning strikes

that occur during thunderstorms. The "fresh, clean, spring-like rain" smell that we notice

after a storm most often results from nature's creation of ozone. Natural ozone is

produced by the process of electrically rupturing oxygen atoms to produce oxygen ions or

atoms that combine with more oxygen to produce ozone and liberating considerable

amount of heat. Corona discharge method also works on the same principle and the heat

generated during this process is removed from the generator to avoid the reverse reaction

(ozone reverting to oxygen) from taking over and decreasing the efficiency of ozone

generation. Thus, cooling of the corona discharge ozone generator becomes a critical

component in generator design.

Corona discharge ozone generators commercially available these days are capable

of producing ozone in gas phase concentrations of 1-5% by weight in air and up to 14%

by weight in high purity oxygen.

Ultraviolet Radiation

The mechanism of photochemical production of ozone is similar to that which

occurs in the stratosphere, that is, oxygen atoms formed by the photo-dissociation of

oxygen by short wavelength UV radiation (# 240 nm) react with oxygen molecules to









form ozone. Although the theoretical quantum yield of ozone by this technique is -2, in

practice the actual yield is more on the order of- 0.5, because the low pressure mercury

lamps produce not only the 185 nm radiation responsible for the production of ozone, but

also the 254 nm radiation that destroys ozone. Medium pressure UV that produces higher

levels of 185 nm radiation produces more ozone. An advantage of generating ozone by

UV radiation is that ambient air can be used efficiently as a feed gas. On the other hand,

quantities of ozone generated per 40W UV bulb are low at maximum ozone

concentration of 2400 ppm. However, these maximum ozone yields and concentrations

cannot be attained simultaneously by the UV method. The low concentrations of ozone

available from UV generators limit their applicability for water treatment to special

applications. However, their use to generate ozone for air treatment can be effective

where very high ozone concentrations are not required.

Electrolysis

High current density electrolysis of aqueous phosphate solutions at room

temperature produces ozone and oxygen in the anodic gas. Electrolysis of sulfuric acid

can produce very high ozone concentration in oxygen when a well-cooled cell is used.

Although electrolysis of water can produce high concentrations of ozone, the output is

low, and the cost is several times more than that of the corona discharge or UV radiation

process.

Ozone produced by the corona discharge method has a higher ozone concentration

but requires extra energy and effort to compress, dry, produce and then dilute the

produced ozone in comparison to ozone produced by the ultraviolet light method.

Air treatment usually requires large volumes of low ozone concentration. Ozone

produced by UV, in comparison, generally needs no air pretreatment and is produced at









the ideal concentrations required to destroy airborne bioaerosols and volatile organic

compounds. Reduced air/oxygen flow rate in the UV radiation-based ozone generator

gives higher ozone concentration due to longer retention time of oxygen molecules in the

presence of ultraviolet light.

The factors that can influence the production yield of ozone are chemical

composition ( percentage of oxygen) and physical properties and process conditions like

pressure, temperature, impurities, moisture of the feed gas, dielectric material, thickness

and discharge gap of the generator, type of the UV bulbs (wavelength, frequency, energy

consumption) used in the generator (Masschelein, 1997). The most obvious source for

producing ozone gas is atmospheric air, but it does not possess high purity oxygen. High

purity oxygen does not require further drying; it gives much higher ozone concentration

at low energy consumption, reduces the size of the generator and eliminates the by

production of nitrous oxides from the nitrogen contained in the air. Therefore, for large-

scale ozone production, oxygen is a more favorable option (Masschelein, 1997).

However, for lower ozone concentration, air as a feed gas in more practical and

economical.

Stabilization and Storage of Ozone

The bulk of ozone consumed is produced in situ and introduced to the process or to

the place of its application directly, without intermediate storage or transport. The rate of

spontaneous thermal decomposition of ozone depends primarily on the temperature

according to the general laws of reaction kinetics. By decreasing the temperature, the rate

of decomposition strongly diminishes, for example a half-life of 3 days at 293 K, 8 days

at 248 K and 3 months at 223 K is specified generally (Rompps Chemie-Lexicon, 1974).

According to Streng and Grosse (1961), the rate of homogeneous decomposition of pure









ozone gas at atmospheric pressure and a temperature of 195 K is less than 10-5 per cent

per day. At temperatures above room temperature the rate of decomposition can rise with

increasing temperature to an extent that the slow decomposition is transformed into a

thermal explosion.

Pure or concentrated ozone is inclined to explode, both in liquid and in gaseous

state, due to its instability, independent of its rate of spontaneous (slow) thermal

decomposition, which is essentially dependent only on temperature and the presence of

catalysts. The primary requirement for safe handling pure, or highly concentrated ozone,

either in the liquid or gaseous state, is the total absence of trace substances catalyzing

decomposition of, or generally reacting with, ozone. This requirement has to be satisfied

at three different levels in the following way:

1. Feedstock oxygen for ozone generation should be freed from various

contaminants, mainly organic, before entering into the ozone generator.

2. Special care should be exercised in cleaning the internal surface of vessels,

apparatus and piping in contact with ozone. In the case of glass apparatus, this can be

achieved by handling the internal surfaces with sulphuric acid-potassium dichromate

solution followed by rinsing first with distilled water and subsequently with a pure, dry

oxygen stream.

3. Finally, materials reluctant to enter into any reaction with, and having no

catalytic effect on, the effect of ozone should be selected as structural materials for

equipment parts in contact with ozone. For high concentrations of ozone only glass,

stainless steel and fluorinated polymers can be considered for use as construction

materials.









Pure gaseous ozone and gaseous mixtures of high ozone content cannot be stored in

gas bottles used normally for the storage of other gases, due to their high reactivity and

tendency to explosion with ozone. Although Grosse and Streng (1958) stated that ozone-

oxygen mixtures containing less than 16 per cent ozone could be safely stored in standard

gas bottles at atmospheric pressure and low temperatures, this is, however, of no practical

use.

Ozone Measurement

Ozone concentrations are measured in the air and liquid by various methods.

Commonly used ozone measurement methods are standard iodometric method, dragger

pumps and ozone analyzer. The amount of ozone circulating through the test apparatus is

critical in analyzing the experimental results.

Chemical determination of ozone concentration by the Standard Iodometric method

is based on the oxidation of the iodide ion in an aqueous solution to iodine and its

subsequent reduction with sodium thiosulfate (Rakness, K. et. al., 1996).The reactions of

ozone with iodide are as follows:

03 + I -F 02 + IO-

10- + H20 HIO + OH-

HIO + 2 I- I3- + OH-

3 HIO + 3 OH- 103- + 21- + 3H20

Upon acidification the overall process formula becomes:

03 + I + H20 "- 13 + 02 + 20H-

Iodometric determination of ozone is the oldest process in ozone analysis which

cannot be totally replaced by more recent methods, even today, despite its apparent

limitations. Although the reaction is not specific and can lead to unwanted deviations









from stoichiometry due to possibility of side reactions, it still has the great advantage of

being an independent method that requires no separate calibration and can be used

equally well for the determination of low or high concentrations of ozone both in gas

phase and solution.

Dragger pump, a military device, is another ozone measurement apparatus. This

device consists of a hand-held pump and a gas sample tube, which receives the airflow

and contains a color indicator that changes from greenish-blue indicating layer to yellow

in the presence of ozone. A direct measurement of the ozone concentration is seen on a

scale (operating range is 20-300 ppm ozone concentration) on the gas sample tube. Lack

of accuracy often limits practical application of this method.

Ozone Analyzers can also be used to measure ozone concentration. It has a fast

response time and balanced flow system. Ultraviolet (UV) photometric ambient ozone

analyzer (Model 49) is a time-shared dual cell instrument capable of measuring ambient

level ozone concentration on a continuous real time basis. The UV photometer in ozone

analyzer determines the ozone concentration by measuring the attenuation of light due to

ozone in the absorption cell; at a wavelength of 254 nm .The ozone concentration is

directly proportional to the magnitude of the attenuation. Also, ozone analyzer requires

zero air as a reference gas for the calculation of ozone concentration. Reference (Zero air)

passes into the absorption cell to establish 'Zero' light intensity reading (Io). The

solenoid then switches and the 'sample' pass through the absorption cell to establish

sample light intensity (I). It works on Beer Lamberts law, which says that transmittance is

directly proportional to the ozone concentration, absorption coefficient and the length of

the absorption cell in the analyzer. Mathematically,









I/Io = e-kl

where,

I/Io = Transmittance or measure of light absorbed by the ozone.

K = absorption coefficient = 308 cm-1 at 0C & latm.

1 = length of the cell (approx. 37.4cm)

c = ozone concentration in ppm.

The major advantage of ozone analyzers is that they have a very fast response time

in comparison to conventional chemical method of ozone determination. However, the

absorption cross-section of ozone at 254 nm is by orders of magnitude larger than those

of most other gases, therefore the interference of other air pollutants (nitrogen oxides,

carbon monoxide, hydrocarbons and water vapor) can be neglected. Dust and water vapor

condensation in the measuring cell must be avoided for reliable measurements. High cost

and narrow range of ozone concentration measurement are some other limitations

associated with ozone analyzers.

Previous Work at UF

Chynoweth observed that air handling systems employing heat exchanging coils,

water pans, or the like, exposed to moisture and subject to microbial contamination

(Figure 2-2). He installed a hot tub ozone unit to one of his two thoroughly cleaned home

air conditioning units and found no sign of biofilm formation while the other unit had a

significant biofilm formation across evaporator coil and drip pan during summer. Thus,

based on the initial tests conducted at the home of an inventor Fannin and Chynoweth

(1994) patented a method and apparatus to prevent buildup of microbial growth in an air-

conditioning unit which does not allow gaseous disinfectant to come in contact with

indoor air (Figure 2-3 and Figure 2-4). Condensation apparatus located within a chamber









defined in an air handling system was disinfected by temporarily interrupting normal air

flow, sealing the chamber with respect to the air handling system, introducing an anti-

microbial growth gaseous disinfectant into the chamber, removing the anti-microbial gas

from the chamber after disinfecting the condensation apparatus and then restoring normal

air flow through the chamber. The preferred anti-microbial gas was produced by using

ozone generator in conjunction with an auxiliary air flow system.

Based on their methodology, experiments were conducted on prototype cells of

bacterium Serratia marcescens and fungus Aspergillus niger. Their results supported the

qualitative observations that ozonated air prevented microbial growth in air conditioning

systems (Chynoweth, 1997). They found that ozone concentration of 2.2 ppm for a

treatment duration of 30 minutes and frequency of two times per day prevented visible

accumulation of microbial biofilm growth in an air conditioning unit over a period of six

months and a treatment duration of 120 minutes at same ozone concentration and

treatment frequency completely inhibited Serratia marcescens and partial inhibition of

Aspergillus niger growth (Table 2-2). An ozone concentration of 9 ppm for a duration of

30 minutes with two times treatment frequency per day resulted in a total inhibition of

natural airborne organisms (Table 2-3). Their results showed that higher ozone

concentration at shorter treatment duration and frequency per day was effective to inhibit

microbial growth. However, the desired combination for complete inhibition was not

found (Table 2-2).









Table 2-2. Summary of previous trials run on Serratia and Aspergillus inoculum by
Chynoweth (Unpublished Data).


Trial# Ozone Treatment Treatment Serratia Aspergillus
Concentration Duration Frequency Growth Growth
(ppm) (min.) (per day)

1 2.2 20 2 Limited Limited

2 2.2 20 12 Limited Limited

3 2.2 120 2 None Limited

4 2.2 60 2 Limited Limited

5 13.6 60 2 None Limited

6 9.0 15 24 None None

7 9.0 60 2 Limited Limited

8 9.0 60 2 Limited Limited


Table 2-3. Summary of previous trials run
(Unpublished Data).


on natural airborne organisms by Chynoweth


Trial# Ozone Treatment Treatment Bacteria Fungal
Concentration Duration Frequency Growth Growth
(ppm) (min.) (per day)

1 9.0 30 4 None Limited

2 9.0 30 6 None None

3 9.0 30 12 None None

4 No Ozone 30 2 Heavy Heavy

5 9.0 30 2 None Limited

6 9.0 30 2 None Limited

7 9.0 30 2 None None























Figure 2-2. Biofilm formation in air conditioning systems (Fannin and Chynoweth,
1994).


Figure 2-3. Test apparatus to prevent microbial growth in air conditioning system (Fannin
and Chynoweth, 1994).


Figure 2-4. Original apparatus attached to air conditioning system (Fannin and
Chynoweth, 1994).






28


Therefore, the objectives of this research work were to determine the

effectiveness of intermittent ozone treatment for prevention of bacterial and fungal

growth in air conditioning systems; evaluate the effect of various combinations of ozone

concentration, exposure duration and treatment frequency on microbial growth; and to

recommend practical air conditioning unit treatment conditions based on the ozone

concentration, treatment duration and frequency.














CHAPTER 3
MATERIALS AND METHODS

Scope of Work

The scope of work for this research was divided into three phases:

Preliminary studies to choose the best method for ozone generation,
ozone measurement, preparation of nutrient media and inoculation of
plates,
Development and execution of design of experiments to evaluate effects
of various combinations of ozone concentration, treatment duration and
frequency on microbial inactivation,
Development of recommendations for practical air conditioning unit
treatment conditions.
Microbial cultures of natural air flora were inoculated on nutrient media by placing

Petri dishes in an indoor environment which were then ozonated in an experimental

chamber. Various trials were run following an experimental design matrix to test the

efficacy of various combinations of ozone concentration, treatment frequency and

duration. Each experimental trial was three days long and after completion of each trial,

microbial growth on plates was observed and compared to that found on untreated plates.

Depending on the effectiveness of a combination of treatment conditions in one trial, the

conditions were changed accordingly in subsequent experiments in search of optimum

treatment conditions.

Materials

Test Apparatus. A method and apparatus that was developed by Fannin and

Chynoweth (1994) for controlling microbial growth in air handling systems was used to

carry out this lab scale research work (Figures 3-1 and 3-2). The exposure chamber was









intended to simulate a home air duct, and was fabricated from a section of galvanized

steel ventilation duct of 0.6 by 0.15 meter in size. A constant speed fan provided air

circulation in the chamber, but as soon as ozone flow was started the normal airflow was

interrupted by temporarily redirecting the primary air-mover. The hinged opening of the

exposure chamber was securely sealed with an aluminum tape to prevent ozone leakage

to the ambient environment.

Ozone produced from the ozone generator (Figure 3-3) flowed through the

exposure chamber when required to carry out each experiment. Accidental back flow of

ozone in the airline during experiments was prevented by closing a damper in the airline

(Figure 3-4). Ozone was removed by purging air through the chamber, after which the

system was returned to the normal airflow operation. Ozone exhaust gas was removed

from the system by connecting the exhaust to a biological containment hood (Figure 3-5).

Copper tubings and fittings were used throughout the system because ozone has caustic

effects on rubber materials.

A data logger/process controller (CR-10) for this set up was connected to a laptop

computer equipped with PC208W software to carry out process control and datalogging

tasks. The programme written in PC208W-based software, depending upon preset

conditions of ozone treatment duration and frequency, was transferred to the data logger

(Appendix B). Once the connection between the two was established, the

datalogger/controller automatically controlled the complete process by continually

monitoring process conditions and transmitting control signals. Thus, the operation of the

method and apparatus was automatically controlled without requiring much attention.

Automation of the process had eliminated the need to be physically present to control the







ozone generator, fan, mechanical dampers and air switches. It also provided additional
flexibility by implementing a wider range of treatment duration times, which were not
available with used the time relays used earlier (Figure 3-6).
Wet towels or sponges were kept inside the chamber to maintain a humidity level
of 85-95 % which simulated actual air conditioning humidity environment and
prevented drying of inoculated samples during the experiment (Figure 3-7-A and Figure
3-7-B). Relative humidity was measured by using Dickson's temperature and humidity
handheld device during each experimental trial to confirm that sufficient humidity levels
were maintained in the exposure chamber.
% 1i I I I I


'l [

n I ( 1
,; ,


Figure 3-1. Lab scale experimental setup of ozone-treated air handling system.


~I












FLOW DIAGRAM


Figure 3-2. Process flow diagram of ozone-treated air handling system.


Figure 3-3. RGF-TURBOHYDROZONE ozone generators.


Figure 3-4. Mechanical damper in the inlet airline.


*aLET Ts


FLOWreiE























Figure 3-5. Biological containment hood.


r I


Figure 3-6. Process automation using CR 10 as a datalogger and process controller.














Figure 3-7. Sequence of steps taken to prepare humidity inside simulated air duct (A)
Wet sponges inside exposure chamber (B) Petri dishes in an exposure
chamber before starting experimental trial.









Preliminary Studies

Preliminary work for the purpose of method development had been divided into

three sections; preparation of inoculum, ozone generation and ozone measurement.

Preparation of Nutrient Media and Inoculum

Nutrient Media

A general-purpose tryptic soy agar was used to prepare one-tenth and full strength

nutrient media for the cultivation of microorganisms. One-tenth strength nutrient medium

was prepared by mixing 4 grams of tryptic soy broth (Bacto Tryptic Soy Broth without

Dextrose manufactured by DIFCO laboratories) with 15 grams of general agar (A-360

Purified Grade manufactured by Fisher Scientific) in one liter distilled water (Figure 3-8-

A) and full strength medium was prepared by mixing 40 grams broth in one liter distilled

water. The prepared medium was sterilized in an autoclave at 1210C for 15 minutes

(Figure 3-8-B) and was then allowed to cool down at room temperature before pouring

nutrient media into 100mm x 115 mm Petri dishes under a bacteria-free hood

environment (Figure 3-8-C) .Inverted plates were kept in plastic bags before storing them

in a refrigerator (Figure 3-8-D).

Preparation of Inoculum

Petri plates containing nutrient media were inoculated with natural airborne

microorganisms to simulate actual air conditioning environment. Ambient building air of

old Rogers Hall was used to inoculate plates in previous work by Chynoweth. During this

work, the inoculation of Petri plates was first attempted using ambient air in Rogers Hall,

the new Agricultural and Biological Engineering Department building but with no

success. This building had been recently renovated with a new air handling system and

air ducts, which contained no appreciable air-borne microbial contamination. Thus, open









plates of both nutrient media strength were placed in a private home environment with an

older established air-handling system where natural micro flora could inoculate the Petri

dishes for 6, 8,12 hours with natural microorganisms found in an indoor setting (Figure 3-

9). Two replicates were carried out for each combination of nutrient strength and

inoculation duration. Plates were then closed and one replicate of each combination of

nutrient media strength and inoculation duration was kept for 72 hours of incubation

while another replicate was placed in an exposure chamber for ozone treatment. Plates

were treated with varying ozone concentrations and microbial growth on both nutrient

media strength plates was analyzed to decide inoculation duration and nutrient media

strength of plates to be used during experiments.













C



I B. ,."




Figure 3-8. Sequence of steps taken to prepare nutrient media (A) Nutrient media
prepared in a conical flask (B) Sterilization of nutrient media in a autoclave
(C) Solidification of nutrient media in Petri plate under bacteria free
environmental hood (D) Storage of Petri dishes in a refrigerator.


















Figure 3-9. Petri dishes inoculated from a natural indoor home flora.

Ozone Generation and Measurement

Ozone Generation

Ultravoilet (UV) radiation- based ozone generators were selected to produce on-

site ozone because they generally do not need air pretreatment and delivered

concentrations required to destroy airborne microbes for this research work. Two

different size large swimming pool RGF-TURBOHYDROZONE ozone generators were

used to attain varying levels of ozone concentration based on feed gas flow rate.

Specifications of generators are presented in Table 3-1.

Table 3-1. RGF-TURBOHYDROZONE ozone generator specifications.
MODEL 120 MODEL 60
20 scfm 1 cfm 5 cfm 20 scfm 1 cfm 5 cfm

mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm mg/hr ppm

1200 550 1700 383 3940 158 242 225 400 127 1500 63



Based on the ozone generator specifications, feed gas flow rate was varied from 1

cubic foot per minute to 5 cubic feet per minute in the generator. Flow meters connected

at the inlet and outlet of the ozone generator measured the flow rate of incoming feed gas

inside the generator and ozonated gas leaving the generator. Both compressed air and

pure oxygen were tested as a feed gas to the generator. Experiments were carried out to

measure the maximum and minimum levels of concentration that could be achieved by









using compressed air and pure oxygen with both ozone generators. A minimum possible

ozone concentration in the exposure chamber was obtained by diluting the ozonated air in

the chamber with a fixed flow rate of compressed air to the chamber. The purpose of

reaching lowest possible concentration in the chamber was to develop a microbial

inhibition design based on broader range of ozone concentration data.

Experiments were also conducted to measure the time required to stabilize ozone

concentration in the exposure chamber by collecting periodic samples of chamber

atmosphere every three minutes and measuring ozone concentration.

Ozone Measurement

Various methods were employed to measure ozone concentration in the exposure

chamber. Initial ozone concentration measurements were done with a UV photometric

ambient ozone analyzer (Model 49), but its narrow range of ozone concentration

measurement (Full scale range 0 to 1 ppm), insensitivity towards interference from other

gaseous constituents and high cost precluded its application for this research work. The

Standard lodometric method (Chemical Method) and Dragger pump method were used in

the study side by side to measure ozone concentration and later compared with one

another. Feed gas flow rates were varied in the range of 2.5 cfm to 5 cfm to meet

manufacturer's specifications and ozone concentration was measured on respective feed

gas flow rate using the Chemical and Dragger pump methods.

Procedure for Standard lodometric Method

Ozonated air from the exposure chamber was allowed to come in contact with 400

mL of 2% potassium iodide (KI) solution placed in two conical flasks (400 mL in each),

as shown in Figure 3-3. An open gas bubbling device (diffuser) was attached to the end

of tubing which brought the ozone in contact with potassium iodide solution for uniform









absorption of ozone in the KI solution. Both KI flasks were connected in series so that

unabsorbed ozone from the first flask could be transferred and absorbed in the second

flask. The sampling line was connected to the absorber and the gas containing ozone was

bubbled to the KI solution at a flow rate of 1 cubic foot per minute (cfm) for 3 minutes by

a vacuum pump. Unabsorbed gas was sucked from the vacuum pump and vented out to

the biological hood. The iodine formed in the solutions of KI, immediately after

acidification with 10 mL of IN sulphuric acid was titrated with a freshly standardized

0.2N sodium thiosulfate solution. This was followed by addition of 2 mL of the starch

indicator solution to the titrant just before the end point (recommended, but can be

optional, depending on skill and experience of the operators). The amount of sodium

thiosulfate needed to convert iodide ions of KI solution to iodine was measured, and the

necessary calculations were performed to determine ozone concentration.

The assembly for a Standard lodometric method for ozone product-gas

measurement is shown in Figure 3-10 below.

FROM OZONE OEfRWTC.H










Figure 3-10. Iodometric method test set-up for ozone concentration measurement.
Calculations


Concentration of ozone(C) in mg/L equals:

24 x volume of thiosulfate in L x Normality of thiosulfate/ V x 28.316
^SAA vltm METER


Figure 3-10. lodometric method test set-up for ozone concentration measurement.

Calculations

Concentration of ozone(C) in mg/L equals:

24 x volume of thiosulfate in L x Normality of thiosulfate/ V, x 28.316









where V,= corrected sample volume = V x (P- Pw)/ 760 x298/(273+T)

V = sample volume in cubic feet

P = Total Pressure (barometric pressure)

Pw= water vapor pressure corresponding to temperature T

T = Temperature

1 mg/L of ozone in air = 467 ppm ozone in air

The Dragger Pump consisted of a hand-held pump and a gas sample tube, which

received airflow from the exposure chamber and changed the color of indicator from

greenish-blue to yellow in the presence of ozone (Figure 3-11). The operating range of

the gas sample tube draggerr tube) was 20-300 ppm. As per manufacturer's

specifications, the ozone concentration measured by this method had a deviation of 10-15

%(+/-) in comparison to the Standard lodometric method.











Figure 3-11 .The Dragger pump.

Experimental Design

Each experimental trial consisted of exposing inoculated Petri dishes to a given

ozone concentration in the chamber for a specified period of time (duration) and at a

frequency of either 1, 2, or 3 times within a 24-hour period. Experiments were carried out

at 9 different ozone concentrations, and for time durations of either 15, 30, or 60 minutes

at each ozone concentration; as shown in Table 3-2.










Table 3-2. Experimental design matrix showing different combinations of treatment
duration, frequency and ozone concentration carried out in study.
Ozone 15 Minutes Treatment 30 Minutes Treatment 60 Minutes Treatment
Conc.
(ppm) Times per Day Times per Day Times per Day


1 2 4 1 2 4 1 2 4

2

3.5

5.7

9

11

17

30

35

45



At the completion of each experiment, the exposed Petri dishes were incubated to

promote outgrowth of surviving microorganisms for visual observation of colony forming

units and compared with untreated plates (Figure 3-12). Results were reported as either

growth (+) or no growth (-) for each treatment combination tested. Each experiment was

replicated a minimum of three times.






41

















Figure 3-12. Comparison of ozone treated plate (left) with untreated plate (right).














CHAPTER 4
RESULTS AND DISCUSSIONS

Findings from Preliminary Studies

Preliminary work for the purpose of method development had been divided into

three sections; ozone generation, ozone measurement, preparation of nutrient media and

inoculum.

Ozone Measurement and Generation

Ozone Generation

Selection of ozone generator and feed gas flow rate was based on the ozone

concentration levels needed to achieve microbial inhibition and cost of feed gas

respectively. Compressed air was selected as a feed gas for this study. Ozone

concentration data obtained with oxygen-fed generators were also collected to verify

the maximum concentration level that could be attained by both ultraviolet radiation

based ozone generators. Reducing air/oxygen flow rate through the ozone generators

gave higher ozone concentrations due to longer retention time of oxygen molecules in the

presence of ultraviolet light. Results ozone concentrations achieved under different flow

rate conditions are shown graphically in Figure 4-1.

Ozone Measurement

Ozone measurement was done by using the Standard lodometric method because

the percentage difference of ozone concentration measured by the Dragger method in

comparison to the Chemical method was much higher than the deviations mentioned by

the manufacturer. Also, the Chemical Method is a standard method and thus has greater









accuracy in comparison to other available methods. Although, ozone concentration data

were collected by using easy-to-use but less reliable Dragger method to compare ozone

concentrations obtained by two different measurement methods but only results obtained

by the Chemical method are shown. The results of ozone concentration levels are shown

in Table 4-1, 4-2, 4-3 and 4-4.


Ozone Concentration vs Feed Gas Flowrate


350

(- 300 -
0 2 -- Large Generator(Oxygen
0 250 feed gas)

S200 -- Small Generator(Oxygen
S150 -feed gas)

O 100 Large Generator(Air feed
Sgas)
o 50
N
0 0- Small Generator(Air feed
2.6 3.6 3.9 4.5 5.3 gas)
Feed Gas Flowrate(CFM)


Figure 4-1. Ozone concentration levels achieved under different feed gas flow rate
conditions.

Table 4-1. Ozone concentration vs. flow rate achieved by small RGF generator using
compressed air as feed gas.

Air Flow rate (cfm) Ozone Concentration
(ppm)

2.5 30

3.2 23

3.7 20

4.3 17









Table 4-2. Ozone concentration vs. flow rate achieved by small RGF generator using
pure oxygen as feed gas.

Oxygen Flow rate Ozone Concentration
(cfm) (ppm)


2.7 320

3.8 210

4.4 180

4.9 165


Table 4-3. Ozone concentration vs. flow rate achieved by large RGF generator using
compressed air as feed gas.

Oxygen Flow rate Ozone Concentration
(cfm) (ppm)


2.6 110

3.6 90

3.9 88

4.5 80

5.3 68

Table 4-4. Ozone concentration vs. flow rate achieved by large RGF generator using pure
oxygen as feed gas.

Air Flow rate Ozone Concentration
(cfm) (ppm)


2.6 50

3.7 45

4.2 40

4.5 35

4.9 32









The results of minimum possible ozone concentration level attained by diluting

exposure chamber with compressed air are shown in Table 4-5.

Table 4-5. Ozone concentration vs. flow rate achieved by small RGF generator using
compressed air as feed gas and diluting exposure chamber with compressed
air to achieve lower levels of ozone concentration for fixed air flow rate of 4
cfm to generator.

Air Pressure to Ozone
Chamber Concentration(ppm)
(psi)

3.5 11

4.5 09

6.5 5.7

7.5 3.5

9.0 2.0


The time required to stabilize ozone concentration in the exposure chamber was

determined by collecting periodic samples of chamber atmosphere and measuring ozone

concentration with the Standard lodometric method. A potassium iodide (KI)-ozone

sample was collected every three minutes (3-minutes interval) and subsequently titrated

with sodium thiosulfate solution to measure ozone concentration. These results show that

approximately 15 minutes were needed to stabilize ozone concentration in the chamber.

Based on these result, treatment durations of 15 minutes, 30 minutes and 60 minutes were

selected to prepare a treatment regime for the design of experiments. The results of ozone

concentration based on quantity of sodium thiosulfate consumed to titrate ozone-KI

solution are presented in Table 4-6.







46


Table 4-6. Ozone concentration over time in an exposure chamber.
Time based Sodium Thiosulfate Ozone Concentration
Samples consumed (mL) (ppm)
(minutes)
3 0.7 17


6 0.9 24


9 1.0 26.7


12 1.1 29.4


15 1.1 29.4


18 1.1 29.4


21 1.1 29.4


24 1.1 29.4


Ozone Concentration vs. Time


U -I-- I iI
0 5 10 15 20 25 30
Time (minutes)


Figure 4-2. Ozone concentration vs. time in an exposure chamber.


* *









Nutrient Media and Preparation of Inoculum

Visual observation of different nutrient media strength Petri dishes inoculated for 6,

8 and 12 hours when treated with ozone revealed that higher strengths of nutrient media

induced very fast population growth of microorganisms on plates that was unrealistic and

non representative of expected levels of contamination(Appendix C). Presence of such

high strength nutrients in an air handling unit via air flow under normal conditions was

also not possible. Thus, the appropriate nutrient media strength chosen to inoculate Petri

dishes with natural airborne microorganisms captured in an actual home setting was

found to be 10%.

Petri plates were inoculated for no longer than six hours because longer duration

caused undesirable rapid growth of microbes on plates. Overly frequent ozone treatments

(greater than 4 times per day) were also avoided because shut down of the air handling

unit too frequently would be inconvenient for any home or office setting. The results

from experiments on different nutrient media strength plates inoculated for 6, 8 and 12

hours are shown in Table 4-7.






48


Table 4-7. Results of experimental trials on Petri dishes containing different
combinations of nutrient media strength and inoculation duration.
Ozone Concentration
(ppm) Inoculation Treatment Treatment Results for media strength
Dragger Chemical Duration Duration Frequency
Method Method (hrs) (min.) (per day)
10% 100%
240 210 12 1 30


* All data points are from a minimum of 3 replicates.
'+' Represents Growth and '-'Represents No Growth.









Exposure Treatments

Preliminary experimental data obtained prior to this study demonstrated that partial

inhibition of Serratia and Aspergillus inoculum was achieved using lower levels of ozone

concentration in a simulated air conditioning environment. However, higher ozone

concentration levels were not tested and no fixed treatment duration time and frequency

were maintained to prepare a database upon which a defined treatment regime could be

specified. This study involved a design of experiments with several trials covering a wide

range of combinations of ozone concentration, predetermined treatment duration and

frequency. Results of this work are summarized in Table 4-8, which is divided into two

parts namely treatment zone of effective treatment and treatment zone of ineffective

treatment. The effective treatment zone shows combinations of ozone concentration,

treatment duration and frequency required to achieve complete microbial inactivation

while only partial microbial inhibition was achieved in treatment zone of ineffective

conditions.

These results reveal that treatment duration during ozone exposure is more

important than frequency in affecting the rate of microbial inactivation. The results reveal

that at 9 ppm and lower levels of ozone concentration frequent treatments are effective to

inactivate microbial growth but as ozone concentration levels are increased from 1 Ippm

and higher treatment duration play a major role to inhibit microbial growth.

Combinations of 30 minutes or longer treatment duration with concentration levels as low

as 12 ppm achieve similar inhibition results. The results also show that one 15-minute

treatment per day of exposure for ozone at a concentration of 45 ppm is effective to

achieve microbial inactivation.









Table 4-8. Inhibition design matrix based on different combinations of treatment
duration, frequency and ozone concentration.
Ozone 15 Minutes Treatment 30 Minutes Treatment 60 Minutes Treatment
Concentration Times Per Day Times Per Day Times Per Day
(ppm) 1 2 4 1 2 4 1 2 4

02 + + + + + + + + +

3.5 + + + + + + + + +

5.7 + + + + + + + + +

09

11

17

20

23

30

35

45


Treatment Zone of Treatment Zone of
Ineffective Conditions Effective Conditions

All data points are from a minimum of 3 replicates.
Pictures in Appendix A
'+' represents Growth
'-'represents No Growth

An intermittent ozone treatment regime limited biofilm regrowth potential because

it provided only partial residual protection against recontamination. Under suitable

environmental conditions after ozone treatment, regrowth of bacteria and fungi on the

Petri dishes took place but at a much slower rate. Petri plate treated with 45ppm

concentration for 15 minutes at a treatment frequency of one time per day did not show










any growth but when the same plate was kept in the indoor environment for the next 48

hours initiation of microbial growth was quite visible. Therefore, regular ozone treatment

of air-handling systems at least once per day is necessary to achieve complete microbial

inhibition.

The experimental results of intermittent application of ozone have indicated the

importance of adopting an effective ozone dose that is capable of inhibiting biofilm

formation in an air handling system. Figure 4-3, 4-4 and 4-5 show that effective microbial

inactivation of airstreams with airborne microbial contamination can be achieved with

ozone treatment. The results presented here suggest that an ozonation system capable of

disinfecting condensation coil surfaces and duct pans of an air conditioning system can

be developed based upon the treatment conditions given in the effective region of Table

4-8.


15 Minutes Treatment Duration
50
45
40
35
30
S25
20
15
10
S5

1 2 4
Treatment Fre que ncy(pe r day)
Growth E No Growth

Figure 4-3. Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 15 minutes of treatment
duration.











30 Minutes Treatment Duration
35

30 -

25 -

20

15

S10 -

5
0

1 2 4
Treatment Frequency(per day)

E Growth E No Growth

Figure 4-4. Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 30 minutes of treatment
duration.



60 Minutes Treatment Duration


30
--

& 25

.2 20

S15

10

5
0
0 -


Treatment Frequency (per day)

Growth U No Growth


Figure 4-5. Microbial inactivation in a simulated air conditioning duct based on varying
ozone concentration, treatment frequency and 60 minutes of treatment
duration.














CHAPTER 5
CONCLUSIONS AND SUGGESTIONS

Conclusions

The results presented in this research demonstrate that airborne ozone is an

effective disinfectant to achieve microbial inhibition with promising applications to treat

air handling systems. This research reconfirmed the results obtained by previous work of

Chynoweth that an ozone concentration of at least 9 ppm for duration of 30 minutes with

two times treatment frequency per day can result in a total inhibition of natural airborne

microorganisms. The results reveal that one 15-minute treatment per day of exposure for

ozone at a concentration of 45 ppm is effective to achieve total microbial inactivation.

The results also show that at 11 ppm and higher ozone concentration treatment duration is

more important than frequency in affecting the rate of microbial inactivation while at

lower levels of ozone treatment frequency has more impact on microbial inhibition.

Definitive inhibition design is developed to optimize conditions for applying gas-

phase ozone to inhibit microbial contamination, to obtain maximum decontamination by

applying a correlation between ozone concentration, exposure time and treatment

frequency and to suggest practical treatment conditions to be applied to disinfect air

conditioning systems.

The treatment regime suggested in this research to disinfect air handling systems is

not a replacement to the general cleaning of the system. A clean and air leak proof system

are prerequisite to obtain best disinfection results and to avoid any ozone leakage in the

indoor air environment respectively. Ozone treatment in night time or early morning may









be effective to achieve microbial inhibition and convenient to the building residents as

their routine work will not be affected by the shutdown of air handling system.

Recommendations

Based on the results of testing different combinations of ozone concentration,

treatment frequency and treatment duration, the most practical conditions suggested by

inhibition design matrix to achieve total microbial inhibition in an air conditioning

systems treatment are

* One 15-minute treatment per day of ozone exposure at a concentration of 45 ppm,

* Alternatively, combination of 30-minutes treatment with concentration levels as
low as 25 ppm one time per day.


Suggestions for Future Work

The studies here open some areas for expansion of this research. Further research

on studying the effect of ozone on specific microorganisms like Bacillus cn/il ,t i, and

plotting death curves to determine the exact number of log cycle reduction of microbes

achieved by airborne ozone in the air conditioning systems can be done.

Further modification in the process can be done to treat exhaust ozone gas from air

conditioning system by reducing the ozone concentration to an acceptable maximum

level/ or decomposing ozone into environment friendly oxygen for discharge to the local

environment. This can be accomplished thermally and/or by catalytic means, or by ozone

passage through wet granular activated carbon beds.

Cost economics study can be further done to check the commercial feasibility of

this research project. The cost of ozone disinfection system will primarily be dependent

on the area of the air conditioning system and duct work to be disinfected.















APPENDIX A
INHIBITION DESIGN EXPERIMENT PICTURES












15 Minutes Ozone Treatment Duration


1 Time / Day 2 Times / Day 4 Times / Day

















1 T;i / TDo,


mI ay /CL


t4 limes / uay


- -. ,. m~ I ~


2 Times / I)av


10 nnm











1 Time / Day 2 Times / Day 4 Times / Day














30 Minutes Ozone Treatment Duration


1 Time / Day 2 Times / Day 4 Times / Day
? nnm


; 7 nnm














1 Tii / TDxr


- -. ,. ~-v -- I ~


2 Times / I)av


4 limes / Day


I11 ppm

11ppm


G oz











1 Time / Day 2 Times / Day 4 Times / Day


35 DDm












60 Minutes Ozone Treatment Duration


1 Time / Day 2 Times / Day 4 Times / Day











1 Time / Day 2 Times / Day 4 Times / Day
9 DDm


No Gnro hll













1 Timp / Dav


2 Times / Day


4 Times / Day


17 Dom


I













1 Time / Day 2 Times / Day 4 Times / Day

35 ppm
No Growth No Growth No Growth













APPENDIX B
PROCESS CONTROL PROGRAMS

15 Minutes Ozone Treatment
One Time Treatment per Day
*Table 1 Program

01: 60 Execution Interval (seconds)

1: If time is (P92)

1: 900 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

2: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2:0110 C4..C1 =low/high/high/low

3: End (P95)

4: Volt (Diff) (P2)

1: 1 Reps

2: 3 25 mV Slow Range

3: 2 DIFF Channel

4: 1 Loc [ ]

5: 1.0 Mult

6: 0.0 Offset

5: If time is (P92)

1:915 Minutes (Seconds --) into a









2: 1440 Interval (same units as above)

3:30 Then Do

6: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

7: End (P95)

8: Do (P86)

1:10 Set Output Flag High

9: Real Time (P77)

1: 221 Day,Hour/Minute, Seconds (midnight = 2400)

10: Sample (P70)

1: 1 Reps

2: 1 Loc [ ]

*Table 2 Program

02: 0.0000 Execution Interval (seconds)

*Table 3 Subroutines

End Program

Two Times Treatment per Day
Table 1 Program

01: 60 Execution Interval (seconds)

1: If time is (P92)

1: 625 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do









2: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2:0110 C4..C1= low/high/high/low

3: End (P95)

4: If time is (P92)

1: 640 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

5: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

6: End (P95)

7: If time is (P92)

1:1345 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

8: Set Port(s) (P20)

1: 0000 C8..C5 = low/low/low/low

2:0110 C4..C1 =low/high/high/low

9: End (P95)

10: If time is (P92)

1:1360 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)









3:30 Then Do

11: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

12: End (P95)

13: Do (P86)

1:10 Set Output Flag High

14: Real Time (P77)

1: 221 Day,Hour/Minute, Seconds (midnight = 2400)

15: Sample (P70)

1: 1 Reps

2: 1 Loc [ ]

*Table 2 Program

02: 0.0000 Execution Interval (seconds)

*Table 3 Subroutines

End Program

Four Times Treatment per Day
*Table 1 Program

01: 60 Execution Interval (seconds)

1: If time is (P92)

1: 1015 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

2: Set Port(s) (P20)









1: 0000 C8,C7,C6,C5 Options

2:0110 C4..C1= low/high/high/low

3: End (P95)

4: If time is (P92)

1:1030 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

5: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

6: End (P95)

7: If time is (P92)

1: 1375 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

8: Set Port(s) (P20)

1: 0000 C8..C5 = low/low/low/low

2:0110 C4..C1 =low/high/high/low

9: End (P95)

10: If time is (P92)

1:1390 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do









11: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

12: End (P95)

13: Iftime is (P92)

1: 295 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

14: Set Port(s) (P20)

1: 0000 C8, C7, C6, C5 Options

2:0110 C4..C1= low/high/high/low

15: End (P95)

16: If time is (P92)

1: 310 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

17: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

18: End (P95)

19: If time is (P92)

1: 655 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)









3:30 Then Do

20: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2:0110 C4..C1= low/high/high/low

21: End (P95)

22: If time is (P92)

1: 660 Minutes (Seconds --) into a

2: 1440 Interval (same units as above)

3:30 Then Do

23: Set Port(s) (P20)

1: 0000 C8,C7,C6,C5 Options

2: 0001 C4..C1 = low/low/low/high

24: End (P95)

25: Do (P86)

1:10 Set Output Flag High

26: Real Time (P77)

1: 221 Day,Hour/Minute, Seconds (midnight = 2400)

27: Sample (P70)

1: 1 Reps

2: 1 Loc [ ]

*Table 2 Program

02: 0.0000 Execution Interval (seconds)






73


*Table 3 Subroutines

End Program














APPENDIX C
PETRI PLATE PICTURES TO SELECT NUTRIENT MEDIA AND INOCULATION
DURATION

Comparison of 10% (right) with 100 %( left) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 4
hours and incubated for 2 days.


* Comparison of 10% (left) with 100 %( right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 6
hours and incubated for 2 days.









* Comparison of 10% (left) with 100% (right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 8
hours and incubated for 2 days.


* Comparison of 10% (left) with 100% (right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 12
hours and incubated for 2 days.









* Comparison of 10% (left) with 100% (right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 4
hours and incubated for 3 days.


* Comparison of 10% (right) with 100% (left) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 6
hours and incubated for 3 days.









* Comparison of 10% (left) with 100% (right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 8
hours and incubated for 3 days.


* Comparison of 10% (left) with 100% (right) nutrient media strength plates
when both the plates were inoculated in natural home micro flora for 12
hours and incubated for 3 days.















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BIOGRAPHICAL SKETCH

Aakash Khurana was born in Sitapur city of Uttar Pradesh state in India. He was

awarded a Bachelor of Science degree in chemical engineering by Dr. Babasaheb

Ambedkar Marathwada University, India, in 1998. He worked as a Chemical Process

Engineer in Vam Organic Chemical Limited, India for two and a half years before

coming to United States for higher studies. He expects to be awarded a Master of

Engineering degree in agricultural and biological engineering from the University of

Florida in August 2003 and plans to pursue a career in the food or pharmaceutical

industry.