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Reducing sediment and bacterial contamination in water using mucilage extracted from the Opuntia ficus-indica cactus

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Title:
Reducing sediment and bacterial contamination in water using mucilage extracted from the Opuntia ficus-indica cactus
Physical Description:
Book
Language:
English
Creator:
Buttice, Audrey Lynn
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Flocculant
Nopal
Prickly pear
Sustainability
Drinking water
Dissertations, Academic -- Chemical Engineering -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Throughout the past decade an increased amount of attention has been drawn to the water contamination problems that affect the world. As a result, a variety of purification methods targeted at communities in developing countries have surfaced and, although all have contributed to the effort of improving water quality, few have been accepted and sustained for long term usage. Case studies indicate that the most beneficial methods are those which use indigenous resources, as they are both abundant and readily accepted by the communities. In an attempt to make a contribution to the search for water purification methods that can serve in both developed and developing countries, two fractions of mucilage gum, a Gelling (GE) and a Non-Gelling (NE) Extract, were obtained from the Opuntia ficus-indica cactus and tested as a flocculating agent against sediment and bacteria suspended in surrogate ion-rich waters.Diatonic ions are known to influence both cell binding and mucilage properties, causing CaCl₂ to be tested as a flocculating agent alone and in conjunction with mucilage. Column tests were utilized to determine the settling rates of contaminant removal from the waters and the precipitated flocs were then evaluated. In columns employing Kaolin as a model for sediment removal, settling rates as high as 13.2 cm/min were observed using GE versus a control (suspensions with no treatment) settling at 0.5 cm/min. B. cereus tests displayed flocculation initiation up to 10 minutes faster than columns treated with calcium chloride (CaCl2) when using less than 10 ppm (GE) and 5 ppm (NE) of mucilage in addition to CaCl₂. B. cereus removal rates between 95 and 98% have been observed in high concentration tests (> 10⁸ cells/mL). Tests on E. coli flocculation differed slightly from those seen using B.cereus with control columns requiring 5 to 10 minutes longer to begin flocculation and mucilage treated columns displaying signs of flocculation much earlier. Mucilage is an ideal material for water purification and contaminant flocculation because it grows abundantly, is inexpensive and offers communities a sustainable technology.
Thesis:
Thesis (M.S.Ch.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Audrey Lynn Buttice.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 83 pages.

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University of South Florida Library
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University of South Florida
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Resource Identifier:
aleph - 002029602
oclc - 437000121
usfldc doi - E14-SFE0002944
usfldc handle - e14.2944
System ID:
SFS0027261:00001


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ABSTRACT: Throughout the past decade an increased amount of attention has been drawn to the water contamination problems that affect the world. As a result, a variety of purification methods targeted at communities in developing countries have surfaced and, although all have contributed to the effort of improving water quality, few have been accepted and sustained for long term usage. Case studies indicate that the most beneficial methods are those which use indigenous resources, as they are both abundant and readily accepted by the communities. In an attempt to make a contribution to the search for water purification methods that can serve in both developed and developing countries, two fractions of mucilage gum, a Gelling (GE) and a Non-Gelling (NE) Extract, were obtained from the Opuntia ficus-indica cactus and tested as a flocculating agent against sediment and bacteria suspended in surrogate ion-rich waters.Diatonic ions are known to influence both cell binding and mucilage properties, causing CaCl to be tested as a flocculating agent alone and in conjunction with mucilage. Column tests were utilized to determine the settling rates of contaminant removal from the waters and the precipitated flocs were then evaluated. In columns employing Kaolin as a model for sediment removal, settling rates as high as 13.2 cm/min were observed using GE versus a control (suspensions with no treatment) settling at 0.5 cm/min. B. cereus tests displayed flocculation initiation up to 10 minutes faster than columns treated with calcium chloride (CaCl2) when using less than 10 ppm (GE) and 5 ppm (NE) of mucilage in addition to CaCl. B. cereus removal rates between 95 and 98% have been observed in high concentration tests (> 10 cells/mL). Tests on E. coli flocculation differed slightly from those seen using B.cereus with control columns requiring 5 to 10 minutes longer to begin flocculation and mucilage treated columns displaying signs of flocculation much earlier. Mucilage is an ideal material for water purification and contaminant flocculation because it grows abundantly, is inexpensive and offers communities a sustainable technology.
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PAGE 1

Reducing Sediment and Bacterial Contamina tion in Water Using Mucilage Extracted from the Opuntia ficus-indica Cactus by Audrey Lynn Buttice A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical a nd Biomedical Engineering College of Engineering University of South Florida Major Professor: Norma Alcantar, Ph.D. Mark Jaroszeski, Ph.D. Joyce Stroot, Ph.D. Peter Stroot, Ph.D. Date of Approval: March 30, 2009 Keywords: flocculant, nopal, prickly pear sustainability, drinking water, kaolin, E. coli Bacillus cereus Copyright 2009, Audrey L. Buttice

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This work is dedicated to my mother Judy Smith, stepfather Mike Smith and brothers James and Jeffrey for their support, love and patience. W ithout a solid family structure to help promote and encourage my education I do not know where I would be or how I could ever have come this far. I would also like to dedicate this work to the people who are currently struggling with water contamination and to all of those who seek to help them.

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Acknowledgments I would like to start by thanking my majo r professor, Dr. Norma Alcantar for her support, faith and guidance over the last two years. For her time, input, for always understanding and for putting her students first, I will be forever thankful. I would like to thank Dr. Joyce Stroot fo r all of her guidance in the bacteria related work documented here. I have been privileged with an exquisite microbiology teacher. I extended my gratitude to Dr. Peter Stroot for his help and use of his facilities. Thank you to Dr. Mark Jaroszeski, and Dr Vinay Gupta for the assistance and usage of their laboratory equipment and Betty Loraamm for help with the TEM. Thank you to my fellow graduate students, and dear friends Eva Williams, Jeffy Jimenez, Cecil Coutinho, Bijith Mankidy and Samuel DuPont for all of their help and support. Thank you to my lab group, good lu ck all of your endeavors. The work documented in this thesis was funded by grant No. 0808002 from the National Science Foundation and Integrat ing Global Capabilities into STEM Education Critical Technologies, Strategi es for Meeting the UN’s Millennium Development Goals on Water and Sanitati on Grant Focus on Sustainable Healthy Communities: WATER from Graduate School USF and the State of Florida.

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i Table of Contents List of Tables iii List of Figures iv Abstract vi Chapter One: Introduction 1 1.1 Thesis Outline 1 1.2 Water Contamination and Regulation Policies 1 1.3 Current Removal Methods 6 1.3.1 Filtration Systems 7 1.3.2 Disinfectants 10 1.3.3 Coagulants/Flocculants 11 1.4 Project Objectives 13 1.4.1 Sediment Reduction from Ion-Rich Water Supplies 13 1.4.2 Bacteria Reduction from Ion-Ri ch Water Supplies 14 1.5 The Opuntia ficus-indica Cactus 14 1.5.1 Prevalence and Characterization 14 1.5.2 Past Studies of Contaminant Removal 18 1.6 Bacteria Studied 19 Chapter Two: Experimental Procedures 23

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ii 2.1 Mucilage Extraction and Characterization 23 2.2 Preparation of Synthetic Water and Calcium Chloride Solutions 26 2.3 Column Tests and Flocculation Evaluation 30 2.4 Bacteria Storage, Growth and Evaluation 32 2.5 Imaging Techniques 34 Chapter Three: Results and Discussion 37 3.1 Mucilage Extraction and Evaluation 37 3.2 Sediment Settling Tests 44 3.2.1 Kaolin Size Evaluation 44 3.2.2 Flocculation with Gelling Extract, Non-Gelling Extract and CaCl2 45 3.3 Bacteria Flocculation Tests 54 3.3.1 Bacillus cereus Flocculation and Evaluation 56 3.3.2 Escherichia coli Flocculation and Evaluation 67 Chapter Four: Conclusions and Future Work 73 4.1 Summary of Findings 73 4.2 Future Work Recommendations 75 4.2.1 Continued Bacteria Studies 75 4.2.2 Shelf-life Evaluation 76 4.2.3 Contaminant Combination Analysis 77 4.3 Final Remarks 77 References 78

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iii List of Tables Table 1. Characteristics of Bacillus cereus and Escherichia coli HB101 19 Table 2. Pad heating/liquidization methods and initial mass 25 Table 3. Synthetic water materials 27 Table 4. Concentrations of synt hetic water stock solutions 28 Table 5. Characteristics of stock solutions for mixing 5 L of soft water (SW) and hard water (HW) 30 Table 6. Materials used for column tests 31 Table 7. Materials used in bact eria growth and evaluation 33 Table 8. Materials and equipment used for imaging 35 Table 9. Summary of Gelling Extract (GE) and Non-Gelling Extract (NE) extraction 40 Table 10. Removal rates of B. cereus in soft water columns treated with GE, NE and 40 mM CaCl2 67

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iv List of Figures Figure 1. Schematic of a commonly used sand filter 7 Figure 2. Tunas and pads from the Opuntia ficus-indica cactus for sale at the Red Barn Flea Market in Bradenton, FL, USA 16 Figure 3. Outline proposed by F. Goycoolea and A. Crdenas for extracting NonGelling and Gelling Extract from the O. ficus-indica 17 Figure 4. Cell wall of gram-positive bacteria 21 Figure 5. Cell wall of gram-negative bacteria 22 Figure 6. Detailed outline of extr action method for mucilage evaluated 24 Figure 7. Images of extraction centrifugation and vacuum filtration 38 Figure 8. Images of extraction precipitation, drying and resulting mucilage 39 Figure 9. New mucilage tests on kao lin suspended in DI water (50g/L) 41 Figure 10. TEM images of the stock so lution of A) GE and B) NE 42 Figure 11. AFM scans of GE (A) and NE (B) stock solutions with imaged areas of 2 x 2 m x-y 43 Figure 12. Kaolin particle size evaluation using DLS and TEM 44 Figure 13. Full 60 minute plot of kaolin settling in DI water with GE 45 Figure 14. Truncated kaolin plot with linear curve fit slopes 46 Figure 15. Kaolin sedimentation measurements with NE, GE and Alum 48

PAGE 8

v Figure 16. Kaolin flocs seen in experimental columns 51 Figure 17. Microscope images of kaolin flocculation 52 Figure 18. TEM images of kaolin flocculation 53 Figure 19. Kaolin treated with CaCl2 in SW and HW 54 Figure 20. Image of Bacillus cereus settled flocs at the bottom of the test columns 56 Figure 21. B. cereus settling time versus CaCl2 concentration 57 Figure 22. B. cereus flocculation using Gelling Ex tract (GE) and Non-Gelling Extract (NE) concentration ranges in hard water (HW) with final CaCl2 concentrations of 20 mM 59 Figure 23. B. cereus settling times in SW with GE 62 Figure 24. Microscope images of B. cereus control columns 63 Figure 25. Microscope images of B. cereus columns treated with GE and NE in HW 64 Figure 26. Microscope images of B. cereus treated in SW columns 66 Figure 27. E. coli flocculation in HW at a ra nge of GE concentrations 68 Figure 28. Picture of flocs forming in treated E. coli columns under a UV light 70 Figure 29. Florescent images of the flocs formed in columns containing E. coli suspended in HW 72

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vi Reducing Sediment and Ba cterial Contamination in Water Using Mucilage Extracted from the Opuntia-ficus indica Cactus Audrey Lynn Buttice ABSTRACT Throughout the past decade an increased amount of attention has been drawn to the water contamination problems that affect the world. As a result, a variety of purification methods targeted at communities in developing countries have surfaced and, although all have contributed to the effort of improving wa ter quality, few have been accepted and sustained for long term usage. Case studies indicate that the most beneficial methods are those which use indigenous resour ces, as they are both abundant and readily accepted by the communities. In an attempt to ma ke a contribution to the search for water purification methods that ca n serve in both developed an d developing countries, two fractions of mucilage gum, a Gelling (G E) and a Non-Gelling (NE) Extract, were obtained from the Opuntia ficus-indica cactus and tested as a flocculating agent against sediment and bacteria suspended in surrogate ion-rich waters. Diatonic ions are known to influence both cell binding and mucilage properties, causing CaCl2 to be tested as a flocculating agent alone and in conjunction with mucilage. Column tests were utilized to determine the settling rates of contaminant removal from the waters and the precipitated

PAGE 10

vii flocs were then evaluated. In columns em ploying Kaolin as a model for sediment removal, settling rates as high as 13.2 cm/min were observed using GE versus a control (suspensions with no treatme nt) settling at 0.5 cm/min. B. cereus tests displayed flocculation initiation up to 10 minutes faster than columns treated with calcium chloride (CaCl2) when using less than 10 ppm (GE) and 5 ppm (NE) of mucilage in addition to CaCl2. B. cereus removal rates between 95 and 98% have been observed in high concentration tests (> 108 cells/mL). Tests on E. coli flocculation differed slightly from those seen using B. cereus with control columns requiring 5 to 10 minutes longer to begin flocculation and mucilage treated columns disp laying signs of flocculation much earlier. Mucilage is an ideal material for water pur ification and contaminant flocculation because it grows abundantly, is inexpensive and o ffers communities a sustainable technology.

PAGE 11

1 Chapter One: Introduction 1.1 Thesis Outline This thesis presents the flocculant cap abilities of a cactus common to many dry arid climates throughout the world. Two fractio ns of mucilage gum were extracted from the Opuntia ficus-indica (O. ficus-indica) cactus and tested for their ability to remove sediments and two types of bacteria from contaminated ion-rich water. Chapter One provides an introduction to th e current state of water contamination around the globe, discusses several relevant and currently used rem oval methods, introduces the O. ficusindica cactus and discusses the primary differences in the two types of bacteria that were studied during the course of this project. Chapter Two conveys details on the methodology and background used throughout the experimentation process. Chapter Three presents the results found using the techniques outlined in Chapter Two. Chapter Four summarizes the results and provides co nclusions and recommendations for future work on this project. 1.2 Water Contamination and Regulation Policies Over the past few decades an increasi ng amount of awareness has been drawn to the water contamination problems worldwide. Although water is a re newable resource, it is difficult to obtain for instance, in 2008 it wa s estimated that of the 70 percent of the

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2 Earth’s surface that is water, only one percent of is viable freshwater for drinking [1]. Water is vital to the health and life of ev ery known organism on the planet and with so little freshwater available for consumption, a significant amount of energy, time and money are spent maintaining, cleaning, and di stributing what little is available for consumption. In more developed countries t echnological advancements give way to new and more efficient, effective methods of cleaning water allowing large populations the health benefits of clean water access. Howe ver, less developed countries lack the money, technical equipment and education to build a nd sustain the same structures and continue to struggle with contaminated water supplies [2 ]. In these countries the quality of health is severely hindered by cont aminated wells, unforgiving st orage methods and a lack of proper sanitation [3]. The United Nations estimat ed that 1 billion people lacked access to potable water in 2006 and 2.6 billion people were neithe r educated regarding nor practiced safe sanitation t echniques [4]. With so many people living on the brink of illness and death a wealth of attention has been devoted to developing new and innovative methods of water purification that could mitigate the needs of people throughout the world [5]. Case studies have de monstrated that the most effective tools presented for sanitation have relied mainly on indigenous resources as they are available and accepted by the communities that use them [4]. Due to the common direct use of both ground water and runoff water, a lack of proper sanitation and poor water storage units a wide variety of contaminants have access to the community water supplies. Thes e contaminants include microorganisms, sediments, chemicals and heavy metals [6]. These materials are likely to be found in the

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3 same water supply demonstrating that the method of purification that is used needs to be capable of treating a combination of contaminants. Although all the contaminants that afflic t the world are danger ous and potentially life threatening, bacterial contamination is one pollutant that is commonly studied and assessed even in countries where water puri fication methods are of great resource and technology. The UN estimated in 2006 that an average of 1.8 million children die every year from diseases related to bacterial contamination which often cause severe diarrhea. Water is used in the household for activi ties ranging from bathi ng to growing crops, which give bacteria that have infiltrated the water supply direct access to the families that obtain water from nearby lakes, rivers or the community well [7]. By practicing inadequate sanitation, bacter ia is often brought into th e house and body through the water supply then systematically re-enters the gr oundwater where it will eventually reach the water supply again creating a cycle of bacteria l contamination [8-11]. As a result of the connection between sanitation and bacteria contaminated water supplies, health organizations have gradually expanded water purification attempts to include education regarding the importance of pr acticing proper sanita tion. Due to this cycle many sources responsible for safe water efforts, such as the World Health Orga nization (WHO), discuss the required need of community support and ed ucation leading to a variety of regulation and outreach programs [4, 12, 13]. For the past half century, the United States’ water sources, treatment facilities, and distribution systems have been highly regulated by the Environmental Protection Agency (EPA), founded in 1970, under a series of policies and statues. For instance, the

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4 Water Pollution Control Act was developed by the EPA in 1948 and re gulates the quality of various bodies of water [14]. This act ma rked the beginning of a multitude of water quality control acts, including the Safe Dri nking Water Act (SDWA), instituted in 1974, and the Clean Water Act (CWA), developed in 1977 [3]. To prepare the standards for microbiological contamination limits, the EP A did a series of case studies comparing several contaminants found in water supplies to local health, and a statistical analysis of the association between contaminant and disease rate resulted in the set limits. The CWA regulates the quality of surface water that is used for recreational activities. Escherichia coli ( E. coli) and Enterococci both found in the large intestine of mammals, were introduced as indicator organisms to devel op standards and indicate whether or not the water had been polluted with fecal contamin ants [7]. These standards were set at 126 cells/100 mL for E. coli and 33 cells/100 mL for Enterococci The regulations outlined by the SDWA are naturally more stringent th an those provided by the CWA because the regulated water sources will be consumed by the urban and rural communities. For the most part, the information provided by the SD WA is not exact limits to which regulations are upheld, but rather are methods and tech niques required to reduce the amount of biological contaminant as much as possible ai ming for zero viable cells [2, 3, 7]. One of the very few standards that are listed is for fecal coliforms, which restricts the number of positive tests to five percent when more than forty samples are taken in a month [15]. Both of these Acts also regulate the frequency that the water in question is to be tested and offers many suggestions to both clean and test the water samples. From the time of

PAGE 15

5 their enactment, these guidelines have evolve d with additional laws to better fit the improved technology and microbiological asse ssments of the current time [14]. Although not as strict as these U.S. laws organizations such as the United Nations (UN) have also developed se veral programs designed to help developing countries gain access to clean water. The UN was developed in 1945 with the goal of spreading peace and helping the world gain basic human right s. A branch of the UN called the World Health Organization (WHO) was formed in 1948 and focuses on the health and quality of life of people throughout the world. A logical part of th e WHO’s contribution to the developing world is focused on water and sanitation improvements. Documents supported by the WHO report to tal coliform standards in a very similar manner to the EPA. For example, up to 95 percent of untr eated water must test negative for these coliforms to meet the criteria for safe consumption [3]. In treated water, any positive detection is cause for reevaluation because additional contaminating sources may be present or the purification system bei ng used is not performing optimally. The Millennium Declaration was deve loped by the UN in 2000 which spawned the Millennium Development Goals (MDG). These goals outline eight human rights, including environmental sustainability and clean water access for developing countries. The goal devoted to sustainability and wate r aims to cut the population living without clean water and effective sanita tion in half by the year 2015. Progress reports released in 2007 and 2008, composed by the UN, outline th e progress accomplished in reaching the goals at the halfway point. It was discusse d in the reports that for the goal of basic sanitation and clean water resources to be met, the success rate needs to increase

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6 dramatically. Several areas s howed signs of improvement while others were actually considered to have worsened [12, 13]. In addition to the WHO and MDG, the UN also began to organize Human Development Reports in 1990 [ 16]. Each yearly report is published with information relative to the most pressing issues of th e year. The 2003 report wa s dedicated to the MDG and discussed in great detail the goals, the difficulties that would be faced, how they could possibly be achieved and the aspect s of life that would be affected. The report also discusses the importance of individual country ownership of the goals, which has shown to play an extremely vital role in the use and maintenance of the purification systems installed [16]. The 2006 report focused on the quest to bring water to countries that struggle with contamination. Topics included the relationshi p between economics and water availability, progress witnessed in the MDG, strategies for significant impacts, water providers, and the im portance of practicing good sani tation techniques [4]. 1.3 Current Removal Methods In the US, centralized water treatment systems are in place and utilized in water delivery to households and consist of fairly intricate technologica l purification methods. Developing countries do not ha ve access to a centralized wa ter system and are limited by a number of factors including low energy so urces, unavailability of chemicals and equipment, and many have not been educated regarding water purif ication and sanitation. In response to these limitations a variet y of purification methods have been developed and tested for use in developing c ountries. Many of these systems fit in to one

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7 of three different categories, including filtration, disinfection and coagulation/flocculation or are composed of a combination of these three methodologies [17]. 1.3.1 Filtration Systems Many filters designed for use in developing countries consist of raw materials that are naturally found in the targeted area. Thes e materials include sand, rice hull, and coal. In their simplest form these filters are fair ly simple, inexpensive, and easy to build and maintain, and have been found to be an eff ective tool for removi ng bacteria and other microorganisms from contaminated water. To extend the filters lifetime, it is recommended that the user allows the water to sit for up to four days prior to filtration, allowing turbidity in the water to settle natu rally and microbiological content to die [17]. Figure 1 shows a schematic of the commonly used slow sand filter. Schmutzdecke Sand Gravel Water Inlet Water Outlet Figure 1. Schematic of a commonly used sand filter Water enters in the top of the unit and is cleaned mostly by the schmutzdecke layer before passing through the sand and gravel then exiting from the bottom of the unit.

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8 Almost any sand filter operates by introduci ng water into the filtration unit at the top of the column. A layer of bacteria, algae and various other living contaminants called the schmutzdecke gradually builds above the sand portion of the filter, which is added mainly for support. Case studies indicate that the schmutzdecke layer requires approximately three weeks to build to a sufficient level [18]. This layer is considered to remove a fairly large amount of living contaminants before the water even reaches the sand portion of the fi lter. Tests with Escherichia coli (E. coli) have shown that a significant amount of bacteria removal occurs during the m ovement of the water through the schmutzdecke layer, but the amount that is removed is greatly im pacted by the design, and operational state of the unit [17-19]. Ot her work has shown a 39 percent increase of coliphage, viruses that infect bacteria, removal in sand filtration columns to 99 percent after the schmutzdecke has had time to build itself on the top of the packed sand. A limiting factor of slow sand filter use is that as the layer builds, it begins to inhibit the flow of water and has to be removed appr oximately every three months causing the performance of the filter to drop periodically [20]. In addition to slow sand filtration, rapid sand filtration systems have also been te sted, but have proven to be more difficult to set up and function at a less efficient rate compared to slow sand filtration units [17]. Often the addition of coagulants is necessa ry to obtain removal rates equal to those observed with the slow sand filtration me thodology, causing these systems to be less effective in developing communities [19]. Rice hull, which is another commonly used material for filter beds, has been known to contain approximately 90 percent si lica which provides the ability to purify

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9 contaminated water and reduce turbidity. These filters also demonstrate slightly higher removal rates of E. coli (90 to 99 percent removal) when compared to slow sand filters (60 to 96 percent removal) [17]. Coal and activated carbon, which has been treated with high temperatures and a lack of oxygen, are both materials that are also studied for their abilities to remove contaminants from drinking water [5]. In add ition to being tested alone for removal, these materials are also often impregnated with other materials that are known to reduce contaminants, for instance aluminum sulfate (also known as Alum) or lime. Studies of coal impregnated with Alum the removal rate s of viruses, rotaviruses and polioviruses were between 95 and 99 percen t in certain pH ranges [17]. Activated carbon is also commonly used in water treatm ent due to its high porosity and high reactivity, which aids it in targeting water suspended organic substa nces and improve taste, odor, and turbidity [21]. A practical up-flow filtration system was designed by the United Nations Children’s Fund (UNICEF) in 1987, which comb ines carbon and sand filtration into one unit that is easy to operate, maintain and produces a signif icant amount of clean drinking water. These filters, however, need to be cleaned regularly and often require the water to be free of harmful pathogens prior to filtration [17]. While the filter systems discussed so far are mainly composed of raw materials found in the community, other methods have also been developed using materials that the communities produce and use regularly. One such filter is used in Bangladeshi villages and utilizes a sari cloth. Studi es showed that when folded between four a nd eight times,

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10 the cloth resulted in a filter with a pore size of approximately 20 m. The case study targeted cholera infection and with the use of the folded sari as a filtration mechanism infections decreased by 38 percent. It was also observed that 90 percent of the villagers used the sari with little opposition from the users [22]. 1.3.2 Disinfectants Disinfection is a water treatment that is commonly used in both developed and developing countries and works by chemically inactivating microorganisms [7]. Chlorine is the most commonly used chemical and is used in developing countries at concentrations of 0.2 to 0.5 mg/L of treated wa ter. Clay pots availabl e in the communities are filled with sand and the ap propriate amount of chlorine a nd are then submerged in the water that is to be treated for at least one week [17]. Case studies in Uzbekistan have shown that in homes where chlorine was us ed as a disinfectant for drinking water, diarrhea cases decreased 67 percent among ch ildren compared to those without the disinfectant [23]. When using disinfectant chemicals alone, bacteria can attach to the surface of the pipes or containers in which the water is transferred or stored leading them to gain resistance to the treatm ent. By attaching to these surfaces, or to other particles in the water, the bacteria can pot entially be shielded from the disinfectant allowing it to survive and grow. In studies with Klebsiella pneumonia surface attachment was among one of several factors, incl uding age of the biofilm, encapsu lation and growth conditions, that inhibited the inactivation with chlorine by 2 and 10 times the rate of unattached cells [24]. Similar results are s hown for the chlorination of Bacillus anthracis and Bacillus

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11 thuringiensis spores [25]. Many times chlorine is ut ilized in conjuncti on with a filtration system to assist with turbidity reduction an d the removal of materi al that is not living however, most natural materials will de grade with chlorine contact [17]. One concern associated with disinfectants is the possibility of disease from residual chemicals. In the past, chlorine has been monitored for its potential threat as a carcinogenic and possible effects on the reproduc tive tract [7]. Disinfection also does not remove the particles that are in the water. In developing countries sola r radiation is also commonly used to disinfect bacteria living in feces and water. Clear plastic bottles are filled with drin king water and then put in the sun between six hours and two days, depending on the climate at the given time. This can raise the temperature of the water to 55 C reducing the amount of active organisms [7]. 1.3.3 Coagulants/Flocculants The final major form of water decontam ination is coagulation and filtration. Coagulation involves an additive designed to change the chemi cal charge of the particles contaminating the water. By doing this the pa rticles can be floccula ted, creating a large volume of connected particles that will then be settled to the botto m of the column under the influence of gravity [26]. Although some particles settle due to gravitational forces alone, the establishment of aggregated par ticles can increase the sedimentation rate drastically. There are four di fferent types of sedimenta tion currently being studied including discrete particle settling, floccu lant settling, hindered settling and compression

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12 settling. Discrete particle settling occurs wh en the suspended partic les settle naturally without the addition of a coagul ant or flocculant. When par ticles begin settling naturally but flocculate as they descend through the wate r, flocculant settling is considered to be taking place. Hindered settling occurs when th e settling of the particles influences the settling of other particles in the solution a nd compression is considered to take place when there are a large number of particles pr esent in the solution and settling is hindered. It is also common for more th an one of these settling types to occur in a given system [26, 27]. Aluminum Sulfate (often called Alum) is commonly used as a coagulant in many developed countries as a method of water pur ification; however, it is not as easily accessed in developing communities. With many case studies indicating the bene fits of using indigenous materials for flocculation, attention has been drawn to the long time use of plant materials and clays as coagulating agents. The seeds fr om several plants, including the Moringa oleifera Moringa stenopetala and nirmali have traditionally been used by some communities for several hundred years and are beginning to be tested in laboratory settings for their abilities to remove contaminants [17, 28]. Removal with these organic materials have shown 90 percent reductions of tu rbidity in waters with suspended kaolin clay and 40 to 50 percent reduction of bacteria using concen trations as low as 2 ppm. Generally, the inner surface of the containers that hold contam inated water are coated with the material and a reduction of suspended particles can be observed overnight [17, 29].

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13 Current studies of bacteria adhesion using ions, such as calcium (Ca2+), have suggested that by introducing m onovalent and divalent ions to solutions containing cells that a increase in binding was obs erved. In studies on the adhesion of Lactobacillus (L.) reuteri DSM 12246, L. plantarum Q47, L. rhamnosus GG, and L. johnsonii NCC 533 to epithelial cells from mammals, significant increases in binding were observed with the addition of calcium ions [30]. In addition, the importance of Ca2+ on the stability of activated sludge has also been studied. When Ca2+ is removed from the sludge, flocculation decreases as does the filterabilit y, while turbidity increases, indicating that the Ca2+ plays a fairly considerable role in the binding of the sludge contents [31]. 1.4 Project Objectives The main objective of the project documented in this thesis is to test the use of mucilage gum, extracted from the Opuntia ficus-indica (O. ficus-indica) as a flocculating agent for developing countries. Because th e cactus is commonly found throughout the world, it offers the potential of serving peopl e in many countries without the risk of community opposition. Treatment on two types of contaminants were tested separately and evaluated in surrogate hard and soft wa ters that mimic natural water sources. 1.4.1 Sediment Reduction from Ion-Rich Water Supplies The first goal of this project was to evalua te the effects that ion-rich water has on the mucilage and its ability to reduce turbidity suspended solids, in water. Kaolin clay was used as a representation of sediments and was suspended in deionized (DI), hard

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14 (HW) and soft (SW) waters for treatment with two fractions of mucilage gum. The resulting flocculati on was evaluated. 1.4.2 Bacteria Reduction from Ion-Rich Water Supplies The second goal of the project was to study, and compare, the ability of mucilage to remove gram-positive and gram-negative bacteria from ion rich water. The ionconcentration, mucilage type and flocculati on was studied and compared briefly to the removal ability witnessed w ith sediments. Gram-positive Bacillus cereus (B. cereus) and gram-negative Escherichia coli (E. coli) HB101 that has been transformed to contain a plasmid with a gene encoding the green flor escence protein (GFP), were used to study removal and possibly also serve as surrogate s for similar types and sizes of bacteria. 1.5 The Opuntia ficus-indica Cactus 1.5.1 Prevalence and Characterization The O. ficus-indica also known as the N opal or Prickly Pear, is a cactus that is found in most areas of the globe that offer dry arid climates. A lthough native to Mexico, the O. ficus-indica has spread throughout the world a nd can currently be found growing in many regions including Sout h America, North America, In dia, Africa and many of the countries surrounding the Mediterranean Sea [ 32]. Not only can the cactus be found all over the world, but it also grow s at an extremely fast rate A case study on a Nopal farm just outside of Mexico City repo rted that the fruit from the ca ctus could be harvested in as little as two to three months after the cactus is planted. In addition, this study also has

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15 reported vegetation production (dry weight) from the plant to be as much as 20,00050,000 kg/ha/yr (1ha = 1 hectare = 10,000m2) and fruit production of 8,00012,000kg/ha/yr [32]. Scientists predict that the pads (nopalitos) and fruit (tunas) were consumed as a food source by the community dating as fa r back as 9,000 – 12,000 years ago [33]. In addition to use as a food source, the cactus has also served many other uses in the community and has gained attention from the scientific world. The fruit from the cactus is often used to create dyes and indigenous knowledge indicates that the pads have been used as a water purification method. Past rese arch and knowledge has also suggested that the pads could potentially serve a pu rpose in the medical field [33, 34]. Since the cactus grows abundantly in many ar eas and is currently used as a food source, the pads and fruit can be found at many local markets and is generally inexpensive. Figure 2 shows the tunas and pads from the O. ficus-indica for sale at the Red Barn Flea Market in Bradenton, Florida. The fruits are sold here three for a dollar and the pads, which contain mucilage are sold five for a dollar.

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16 Figure 2. Tunas and pads from the Opuntia ficus-indica cactus for sale at the Red Barn Flea Market in Bradenton, FL, USA. The Opuntia genus in the cacti family has been known for its large production of mucilage, a complex used by the cactus to stor e water. Mucilage serves many purposes in the food industry. It has been used as an addi tion to house paint and is the product of the cactus that is used by some communities as a water purific ation method [34]. As shown in Figure 3, a method has been developed to extract two different fractions of mucilage gum, a Non-Gelling (NE) and Gelling (GE) Extract, from the O. ficus-indica [34].

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17 Figure 3. Outline proposed by F. Goycoolea and A. Crdenas for extracting Non-Gelling and Gelling Extract from the O. ficus-indica [34] The chemical contents of the mucilage gum from the O. ficus-indica has been studied in the past and, although there have been some discrepancies with the reported contents, several main components have been identified. This mucilage is thought to

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18 consist of approximately 55 sugars, mainly arabinose, galactose, rhamnose, xylose and uronic acids, the percentage of which varies with mucilage type [34-36]. Studies with the addition of divalent cations, such as Mg2+ and Ca2+, to mucilage have resulted in property changes, such as increases in viscosity [ 34, 36]. While NE is reported to display higher viscosities than GE without a ny additions, GE was shown to ha ve a higher percentage of uronic acids, which is thought to provide the extract with st ronger gelling properties that are witnessed in the presence of mon ovalent and divalent cations [34]. 1.5.2 Past Studies of Contaminant Removal Young et al. demonstrated the use of NE and GE mucilage fractions for removal of sediment in DI water and concluded that bot h mucilage fractions act faster in sediment removal than the controls containing no floccu lating agent, and solutions treated with the commonly used Alum [37-39]. Thes e tests also concluded that in order to obtain the same settling rate as the GE extrac t, 300 times as much Alum would need to be used. Residual turbidity was also evaluated in these tests and appeared to rise with increasing mucilage concentration. At very low concentrations of mucilage treatment, however, the residual turbidity was relatively low. In the same study arsenic removal with GE was evaluated and suggested that the arsenic is some how transported to the top of the column by the mucilage resulting in a 33 – 45% removal rate [37-39]. Further ev aluation regarding effectiveness of the mucilage to remove heavy metals is currently unde r investigation.

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19 In this thesis the work done by Young et al. was expanded upon by the testing of the removal of the sediment kaolin from DI a nd ion-rich water. In addition, the spectrum of contaminants that are being studie d was broadened to include bacteria. 1.6 Bacteria Studied For this project, two bacteria types were evaluated for flocculation when treated with mucilage. Two non-pathogenic bacteria, Bacillus cereus (B. cereus) a gram-positive bacterium, and Escherichia coli (E. coli) HB101 a gram-negative bacterium were utilized. Table 1 lists some char acteristics of these bacteria. Table 1. Characteristics of Bacillus cereus and Escherichia coli HB101. Bacillus cereus Escherichia coli Type gram-positive gram-negative Size, Shape 1 x 3 m, Rod 1 x 2 m, Rod Location Soil Mammal Feces Spore-forming YES NO Optimal Growth Conditions Temperature: 35-37 C, Stirring: 200 rpm Temperature:35 C, Stirring: 200 rpm Pathogenic NO NO These bacteria were chosen for testing becau se of their availab ility, ease of use in the laboratory setting and becau se of their locations. Both of these bacteria could be a

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20 point of concern in dri nking water contamination. B. cereus commonly found in soil, could potentially be washed into the a quifers that supply wa ter with rain, and E. coli can easily enter water supplies if safe sanitation is not practic ed [40, 41]. As previously discussed, drinking water is monitored for E. coli as part of the safe drinking water regulations, and is used as an indicator or ganism to detect fecal contamination. These bacteria could possibly also act as surrogates for other bacteria cont aminants of similar size and characteristics if the flocculation aff ects of the mucilage is a result of surface interactions. Since this is commonly the case with flocculants, the surface characteristics of gram-positive and gram-negative bacteria was studied and compared. Figure 4 shows a schematic of the cell wa ll of gram-positive bacteria. The outer layer of the cell wall consists of peptidog lycan, a combination of polysaccharides and amino acids, and contains teichoic acids, a nother kind of polysaccharide that links to lipids and maintain the attachment with the cell membrane [42].

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21 Teichoic acid lipoteichoic acid Integral protein Cell Wall (Peptidoglycan layer) Cell/Plasma Membrane Gram-Positive Bacteria Periplasmic Space Figure 4. Cell wall of gram-positive bacteria. The outer layer consists of a thick peptidoglycan layer which is exposed to the external environment. In Figure 5, components of the gram-nega tive bacteria that are different than those found in gram-positive bacteria are shown in red while those that are relatively the same are shown in black. Gram-negative walls also have a layer of peptidoglycan, however, it is much thinner than those found in gram-positive walls and it is not directly exposed to the environment outside of the cell. Gram-negative bacteria have an additional bilayer that consists of phospholipids, channel proteins an d an outer layer that consists of lipids attached to sugars, called lipopolysacharides or LPS.

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22 LPS Layer Cell Wall (Peptidoglycan layer) Cell/Plasma Membrane Outer Membrane Gram-Negative Bacteria Polysaccharide Lipid A Figure 5. Cell wall of gram-negative bacteria The outer cell wall consists of a lipopolysacharides layer, wh ich is exposed to the ex ternal environment. Lipid A, which is attached to the polysac charide, is the cause of illness when gram-negative bacteria are killed while insi de of the body. This ex tra layer in the cell wall also makes gram-negative bacteria more difficult to kill with antibiotic treatment, as it could potentially immobilize the moveme nt of drug into the cell [43-45]. The differences shown in the outer por tion of the bacteria cell walls could potentially be responsible for differences in the ability of mucilage to remove the bacteria.

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23 Chapter Two: Experimental Procedures 2.1 Mucilage Extraction and Characterization Mucilage was extracted for use in this pr oject using a method very similar to that outlined by F. Goycoolea and A. Crdenas disc ussed in section 1.5.1 of this thesis [34]. O. ficus-indica pads were originally purchased from Living St ones Nursery in Tucson, Arizona then replanted and grown in Tampa, Fl orida. Figure 6 shows a detailed outline of the method used for this purific ation and the alterations made to the previously discussed methodology.

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24 Figure 6. Detailed outline of extraction method fo r mucilage evaluated. Main differences from the protocol described by Goycoolea et al. include heating method, liquidization method, filter size, and precipitation and washing chemicals. 14(1:1 vol ratio)

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25 A total of four pads and four different heating/liquidi zation methods were used to determine the method that would produce the hi ghest yield percent. Table 2 outlines the pads by number, the methodology used, and th eir mass both before and after cleaning. Table 2. Pad heating/liquidization methods and initial mass. PAD Heating and Maceration Method Initial Mass (g) Mass after Cleaning (g) 1 Boiled with Salt and Blended 346.2 335.9 2&3 Steamed and Blended with Water 543.5 534.4 4 -1 Boiled and Macerated 439.5 438.5 4 2 Boiled and Blended Total 1329.2 1308.8 Aside from the heating and liquidizat ion method, all pads follow the outline shown in Figure 6 to provide GE and NE mucilage fractions. The final step of the extraction involves grinding the dried muc ilage with a mortar and pestle to provide a fine powder for its use in mucilage experiments. Prior to experimentation this powder wa s added to DI water, with a final concentration of 500 ppm, and mixed with a tissue grinder to produce an even suspension. This solution was then diluted accordingly for experimentation a nd was stored in the refrigerator sealed with wax film until it was used.

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26 Once the extraction was complete, the mucilage was tested with kaolin and compared to a control without mucilage, in or der to indicate whether or not the mucilage could produce increased settling as observed in the work done by Young et al. [37-39]. 2.2 Preparation of Synthetic Water and Calcium Chloride Solutions In an attempt to closely evaluate how mucilage would react when used as a purification method in the ionrich environment that is commonly found in real bodies of water, surrogate ion-rich waters were prep ared for kaolin and bacteria suspensions. Smith, Davison and Hamilton-Taylor offer reci pes for three major freshwater surrogates including hard, soft and acidic water [46]. For the purposes of this work, hard water (HW) and soft water (SW) were prepared and utilized in comparison with deionized water (DI). The water preparations presented by these authors are based off of samples taken from Esthwaite Water Lake (SW mode l) and Rostherne Me re Lake(HW model) both found in England. The preparation of these waters include the mixing of several solutions of salts dissolved in DI water, resulting in high ion-concentration water (HW) and low ionconcentration water (SW). The materials used during the preparatio n of the surrogate waters can be found listed in Table 3.

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27 Table 3. Synthetic water materials. Name Manufacturer Catalog # Lot # Description Calcium Nitrate Tetrahydrate (Ca(NO3)24H2O MP Biomedicals 193800 6343E 100g, Ultra Pure Potassium Phosphate Acros 205920025 A02409 51 2.5kg, 99+%, Ultra Pure Sodium Sulfate Anhydrous Sodium Sulfate (Na2SO4) Acros 354250010 B01234 72 1kg, Granular Potassium Bicarbonate MP Biomedicals 152557 5477H 1000g, Reagent Grade Calcium Chloride Hexahydrate (CaCl26H2O) Acros 389250010 A02315 11 1kg, Extra Pure Sodium Bicarbonate (NaHCO3) Fisher Scientific S233-500 073814 500g Magnesium Chloride Hexahydrate (Cl2Mg6H2O) Acros 197530010 A02452 68 1kg, 99% Magnesium Sulfate Heptahydrate Acros 423905000 A02370 24 500g, 98+% Calcium Chloride (CaCl2) MP Biomedicals 153502 9645E 500g 500 mL Filter Fisher Scientific S66128 638611 0.20 m Cellulose Nitrate membrane, sterile 50 mL Filter Corning 430320 302085 05 0.22 m Cellulose Acetate membrane, Sterile Compressed Nitrogen Airgas NI HP300 Minimum Purity 99.995%, gas cylinder Compressed Air Airgas AI UZ300CT Ultra High Purity, Certified Compressed Carbon Dioxide Airgas CD R300 Research Grade Deionized Water (DI) Millipore System A10 F4BN7 4788 DI Feed Water, Ion-Exchange, Activated Carbon

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28 The stock solutions prepared are outlined in Table 4 along with the amount of chemical added, the final concentration a nd the final ion-concentrations. The mixing concentrations and final pH is shown in Table 5. Table 4. Concentrations of synthe tic water stock solutions. Chemical Amount (g) Conc (M) SS Ion Conc (g/L) Cz+ AzSoft Water (SW) Stock Solution 1: 1 L prepar ed, 1/1000 dilution factor Magnesium Chloride Hexahydrate 12.17 0.060 1.455 4.244 Calcium Chloride Hexahydrate 17.49 0.080 3.200 5.661 Clacium Nitrate Tetrahydrate 3.541 0.015 0.601 1.859 Stock Solution 2: 5 L prepared, 1/1.1 dilution factor Calcium Oxide 0.094 0.0003 0.014 0.001 Stock Solution 3: 1 L prepared, 1/1000 dilution factor Sodium Sulfate 16.34 0.115 5.290 11.05 Potassium Bicarbonate 2.508 0.025 0.999 1.528 Sodium Bicarbonate 1.681 0.020 0.460 1.220 Hard Water (HW) Stock Solution 1: 1 L prepared, 1/100 dilution factor Calcium Chloride Hexahydrate 7.497 0.034 1.372 2.426 Calcium Nitrate Tetrahydrate 1.189 0.005 0.202 0.624 Stock Solution 2: 5 L prepared, 1/1.1 dilution factor Calcium Oxide 0.458 0.002 0.066 0.005

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29 Table 4. (Continued). Stock Solution 3: 1 L prepared, 1/100 dilution factor Sodium Sulfate 2.820 0.020 0.913 1.907 Potassium Bicarbonate 0.763 0.008 0.304 0.465 Sodium Bicarbonate 2.265 0.027 0.620 1.645 Potassium Phosphate 0.47 0.003 0.108 0.262 Stock Solution 4: 1 L prepared, 1/100 dilution factor Magnesium Sulfate 10.05 0.041 0.991 3.915 Stock solutions 1, 3, and 4 (for HW only) re quire the salts to be added to DI water using a stir bar. Smith et al. advises that, if kept in a cool shaded place, these solutions will not expire and can be used for future water preparations. Because Calcium Oxide (CaO) is harder to dissolve th an the other salts a nd requires the water to be stripped of carbon dioxide (CO2), the water was bubbled with compressed nitrogen (N2) gas for one hour using a nitrogen blanket created with a grocery bag. The bubbling was achieved using standard tubing with holes punctured ap proximately six inches up from the bottom using a screw. This tube was submerged in the water while it was stirred with a stir bar allowing the bubbles to fill the container. The appropriate amount of CaO was then added to the water and was then bubbled with N2 for another hour until the CaO was completely dissolved. The solution was then bubbled for 10 minutes, decreasing the pH and preventing the formation of any unwanted precipitates as outlined by Smith et al. These stock solutions were then mixed according to Table 5, which also indicates the final pH of the solution after bubb ling with compressed air.

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30 Table 5. Characteristics of stock solutions for mixing 5 L of soft water (SW) and hard water (HW). SS 1 (mL) SS 2 (mL) SS 3 (mL) SS 4 (mL) DI Water (mL) Total Vol (mL) Final pH Soft Water 5 4545 5 -445 5000 7.43 Hard Water 50 4545 50 50 305 5000 8.34 Upon completion, the water was stored on th e bench top at room temperature and, prior to use, 500 mL of the waters were filtered using a vacuum pump and bottle top filters with 0.20 m membranes, sterilizing the water for use. The SW and HW were also used to produce calcium chloride (CaCl2) solutions that were tested with kaolin and used in bacteria tests. Stock solutions of CaCl2 were prepared in both HW and SW and were then filtered using a vacuum pump and bottle top filters with 0.22 m membranes, sterilizing the solutio n for later use. The solution was then diluted accordingly for each experiment. 2.3 Column Tests and Flocculation Evaluation Column tests were used to evaluate the flocculation and removal of sediment and bacteria suspended in water. The column array was set up using 10 mL Fisherbrand pipettes which were broken between the -1 and -2 mL markers. The bottom of the pipettes were closed using parafilm and th e column was taped together and hung in front of a dark sheet of paper so that the flo cculation could be easily seen. Because of the flocculating abilities of the diatonic ion Ca2+, and its affects on the mucilage, CaCl2

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31 solutions of various conc entrations were prepared and test ed with kaolin and bacteria. For both kaolin and bacteria test s the column contents were added together in 10 mL centrifuge tubes, then vortexed before being poured into the column array. Table 6 lists the materials utilized during column test experiments. Table 6 Materials used for column tests. Name ManufacturerCatalog # Lot # Description Tissue Grinder Fisher Scientific 08-414-10B Medium grind, 7 mL Kaolin Fisher Scientific S71954 200009608 500g Aluminum Sulfate (Al2(SO4)3)18H2O Fisher Scientific S70495 200305504 500g Phosphate Buffer Saline (PBS) Sigma P-3813 047K8207 0.01 M, pH 7.4 Kaolin particle size was unknown from the manufacturer and was determined using Dynamic Light Scattering (DLS) a nd Transition Electron Microscopy (TEM). Kaolin suspensions with final concentrations of 50 g/L were used in the column tests evaluated in this thesis and mimic the possible mud-like conditions in water storage units. At this concentration, the kaolin solutions formed a clear interface while settling, allowing its height to be read every minut e for sixty minutes. The settling rates were determined and plots were generated for co mparison between different waters, mucilage type and concentrations. Tests were run with varying mucilage concentration in SW, HW and DI waters. In addition, the use of the common flocculant Alum was also evaluated for comparison.

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32 Bacteria tests were evaluated with high bacteria con centrations of 108 cells/mL, in order to make the effect of mucilage addition easier to see. Unlike kaolin columns, bacteria columns do not form a clear interface, but rather small flocs, which can be seen forming and falling in the otherwise turbid water. The time from which these flocs began to form to the time that they completed th eir descent was recorded and compared for various treatments conditions. All results shown are the average and stan dard deviation of at least three settling tests and all statistics were calculated using Origin 8. 2.4 Bacteria Storage, Growth and Evaluation B. cereus and E. coli were grown and stored on gla ss beads with a mixture of LB media (including ampicillin and arabinose concentrations of 5 mg/mL and 100 g/mL for E. coli ) and glycerol at -80 C. When ready for use, cultures were started from these beads on LB media agar plates. The plates were grown for 12 hours at 35 C and were then stored in the refrigerator for future use. One colony was then selected from the plate using a sterile loop and immerged in a 5 mL LB media tube that was incubated at 37 C shaking at 200 rpm for at l east 9 hours. After 9 hours a 75 mL LB broth culture was inoculated from the 5 mL culture using a 1:1000 dilution. This culture was incubated over night at 35 C shaking at 200 rpm and removed for use approximately 15 hours later at the same optical density reading for every experiment providing a stock solution concentration of 109 cells/mL. The bacteria were th en washed once in PBS using a centrifuge running at 4,000 rpm for 5 min and a mini vortexer. Once washed, the final

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33 stock solution cell count was determined usi ng a direct counting chamber from Nexcelom Bioscience. Table 7 lists the materials that were used during ba cteria growth and evaluation. Table 7. Materials used in bacteria growth and evaluation. Name Manufacturer Catalog # Bacillus cereus Frozen stock already in lab ATCC 10876 Escherichia coli pGLO Bacterial Transformation Kit BioRad 1660003EDU ATCC 33694 Yeast Extract Fisher Scientific BP1422500 076094 500g Tryptone Acros 611845000 B0124145 500g Sodium Chloride (NaCl) Acros 424295000 B0113819 99+% Agar MP Biomedicals 100262 8388F USP Grade, 80-100 mesh Ampicillin Sodium Salt (C16H18N3O4SNa) MP Biomedicals 194526 R21558 Crystalline, Cell Culture Reagent L-(+)-Arabinose (C5HO6) MP Biomedicals 100706 8590J Crystalline, Purity: >98% Petri-dishes Acros 0875798 100x15mm Orbital Incubator Shaker Amerex Gyromax 727 20-420 rpm Ambient Temp +580 C Cellometer Cell Counting Chamber Nexcelom Bioscience CP2-002 Plastic Disposable Counting Grid

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34 In bacteria tests where removal percen tage was evaluated, plate counts were performed according to standard microbiol ogy procedures [43-45]. One mL of solution was taken from the top of the column and dilu tions of this sample were plated and the colonies counted 24 hours later. This count was subtracted from the initial count and divided by the initial count to provide the percentage of the bacteria that were removed from the solution. The E. coli HB101 used in this stud y was transformed into E. coli GFP using a pGLO transformation kit purchas ed from Biorad. By heat sh ocking the bacteria with the pGLO plasmid present, the PGLO en ters the cell wall resulting in E. coli cells that exhibit green fluorescence under UV light. In order to grow the transfor med cells, the sugar arabinose and antibiotic ampicillin is added to the LB media or agar that is used. With final concentrations of 5 mg/mL arabinose th e bacteria continued to produce the pGLO when multiplying and with the addition of 100 m/mL of ampicillin, b acteria that did not contain the pGLO plasmid were eradicat ed from the media by the antibiotic. 2.5 Imaging Techniques A combination of microscopy tec hniques, including Transition Electron Microscopy (TEM), Atomic Force Micros copy (AFM) and Optical Microscopy (OM), were utilized in the evaluation of the mucilage and floc formation of the particles evaluated. The equipment and materials used in sample preparation are listed in Table 8.

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35 Table 8. Materials and equipment used for imaging. Name Manufacturer Catalog # Lot # Description Optical Microscope Leitz Diaplan Wetzlar 10, 40 and 100 objectives Camera for Microscope Diagnostic Instruments Inc. Model 15.2 64 Mp Shifting Pixel Serial # 241632 Microscope Imaging Software Diagnostic Instruments Inc. SPOT version 4.5 Microslides Corning 2948-75 x 25 18907003 Single frosted, precleaned 75x75mm Formvar/Carbon 150 Mesh Copper Grids Electron Microscopy Sciences FCF150Cu-50 Formvar coated grid, 150 mesh Transition Electron Microscope (TEM) FEI Company Morgagni 268 D Resolution: 35280000x High Voltage Range 40 to 100 kV Mica Axim Mica ASTM D351-97 Scratch Free, V1 quality Aluminum Cantilevers Budget SensorsTAP300AlAl reflex coating, 30 nm thick, Resonance Frequency-300 kHz, Force Constant – 40N/m Atomic Force Microscope (AFM) PSIA XE-100 Research-Grade AFM; Max scan range The topography of the mucilage stock solu tion (500 ppm) was evaluated using the AFM. Scans of the mucilage were obtaine d using aluminum TAP300Al cantilevers in tapping mode on a fresh cleaved mica surface.

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36 Internal mucilage structure and sample s from taken from the top of kaolin columns were evaluated using a TEM. A 20 L sample of the solution was deposited on a copper grid and left to adsorb for five mi nutes. The remaining liquid was then removed and the grid left to dry for appr oximately 1 hour prior to imaging. Kaolin was removed from the column at the interface between the settled flocs and the water using a glass pipette and bulb and imaged on the optical microscope. Flocs were extracted from the bottom of the columns containing bacteria, using a small valve, and were also imaged by optical microscopy. For both kaolin and bacteria approximately 7 L of sample was deposited on a glass micr oscope slide and covered with a glass coverslip for imaging. E. coli images were taken on the microscope using a GFP tube filter to produce florescent images.

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37 Chapter Three: Results and Discussion 3.1 Mucilage Extraction and Evaluation As discussed in section 2.2, four pads were processed providing a Gelling (GE) and Non-Gelling (NE) Extract for testing. Fi gures 7 and 8 exhibit images taken during the extraction process. The lett ers located in the upper left hand corners of the pictures correspond with the steps described on th e flow chart provided in Figure 6. Image A corresponds with step five and s hows the precipitant that was used for the GE, left, and the supernatant which will produce the NE, right (step 5 in Figure 6). Image B demonstrates the filters, made by cu tting circular shapes out of fabric, used during the precipitate filtration. Image C show s the precipitate before, during and after filtration and image D shows the supernatant as it is emerging from the filter funnel (steps 11 and 14 in Figure 6).

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38 A B D C Figure 7. Images of extraction centrifugation a nd vacuum filtration. Image A shows the precipitate (produces GE) a nd supernatant (produces NE) obtained from centrifugation step of extraction method. B demonstrates the fabric filters that were cut for use in filtering the precipitate as shown in imag e C. Image D shows the supernatant as it emerges from the filter funnel. Figure 8 shows images of th e latter part of the extr action method where acetone and ethanol (volume ratio 1:1) were added to both mucilage fractions and left to precipitate (step 15 in Figure 6). Images A a nd B show the mucilage being drawn out of the solution as the water is evaporated. The mucilage was left to sit in this solution for two days before it was removed and wash ed with isopropanol. Image C shows the mucilage spread out on Petri dishes while being dried. The image on the bottom right (D)

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39 shows the final powder state of the mucilage once it has been dried and ground with a mortar and pestle (step 17 in Figure 6). All steps shown were carried out in a fume hood. A B C D Figure 8. Images of extraction precipitation, dr ying and resulting mucilage. Image A and B shows the mucilage being drawn out of so lution as the water is evaporated using ethanol and acetone. Image C shows the washed mucilage spread on Petri dishes to dry and D shows the final product of the extraction. Table 9 presents the results from this extraction including the mass of both GE and NE extracted as well as the percent yiel d, which represents the mass of the dried mucilage over the initial pad mass.

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40 Table 9. Summary of Gelling Extract (GE) a nd Non-Gelling Extract (NE) extraction. PAD NE Extracted (g) % Yield GE Extracted (g) % Yield 1 1.3835 0.40 N/A N/A 2&3 2.7488 0.51 0.801 0.15 4-1 3.1976 1.46 N/A N/A 4-2 2.2384 1.02 N/A N/A Total 9.5683 0.73 0.801 0.0612 In some instances, the GE amount obtaine d was small and during the evaporation step of the extraction procedure it was unint entionally dried completely and was therefore discarded as shown by N/A in the Table 9. Although the mass of extracte d mucilage appears to be relatively low, the amount of mucilage that was obtained has the potential of treating a large amount of water. The percent yield presented is also low which is mostly because the pad is composed of a large percentage of water and other materi al that is removed during the extraction process. As seen later in this documen t, the amount of mucilage preferred for contaminant removal is approximately 2 ppm (2 mg/L), which indicates that an extracted weight of 1 g is capable of cleaning up to 500 L of water. Figure 9 shows the removal rates of kaolin (50g/L) when treated with the mucilage obtained from the extraction with a final concentration of 2 ppm.

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41 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Mucilage Type from ExtractionSettling Rate (cm/min) Mucilage Type from Extraction Settling Rate (cm/min) Figure 9. New mucilage tests on kaolin suspended in DI water (50g/L). Settling rates suggest that all mucilage induces settling faster than no treatment. From Figure 9 it is seen that all of the mucilage obtained from the extraction induced higher settling rates in kaolin than the untreated control. The differences in settling rates could potentia lly be contributed to th e purity of the extraction. For comparison purposes, it was desire d to test removal with GE and NE mucilage fractions obtained from the same pad and extraction me thod. Therefore the GE

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42 and NE obtained from Pads 2&3, the only met hod that resulted in both GE and NE, was used for evaluation in all experiments. In an attempt to better unde rstand the different properties observed between the GE and NE, images were obtained of the st ock solution using both the TEM and AFM. Figure 10 shows the images taken with the TEM of the GE (A) and NE (B) with a magnification of 28,000x. The image of the GE di splays an orderly ch ain-like structure with almost the same angle of orientation. C onversely, NE images show a denser net-like structure with cell sizes of approximately 200 nm. 500 nm A 500 nm B Figure 10. TEM images of the stoc k solution of A) GE and B) NE. GE displays an orderly structure with nearly the same a ngle of orientation while NE shows a much denser net-like structure. Samples extracte d from 500 ppm stock solutions of mucilages and deposited on copper grids. The solution was a llowed to sit for five minutes before the remaining water was removed and the grid left to dry for approximately one hour prior to imaging.

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43 Figure 11 displays AFM scans of both GE (A ) and NE (B) with imaged areas of 2 x 2-m x-y and 0.5 x 0.5 m x-y. The AFM pict ures shown here are consistent with the internal differences shown in the images obtained from the TEM. Maximum heights of 2.01 nm (GE scans) and 1.42 nm (NE scans) were recorded by the AFM. GE NE GE NE Figure 11. AFM scans of GE (A) and NE (B) stock solutions with imaged areas of 2 x 2m x-y. Zoom-in images provide a 0.5 x 0.5 m x-y scan that is not necessarily from the indicated area of the larger scan.

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44 The structural differences observed here could potentially be the cause of behavioral differences that were seen in the remaining results f ound in this section. 3.2 Sediment Settling Tests 3.2.1 Kaolin Size Evaluation Figure 12 shows the DLS output with a part icle diameter determined to be 518 30 nm. The intensity is determined to be 100 percent which indicates that there is a relatively small, if any, distribution of si zes in the kaolin suspension. The TEM image located in the upper right hand corner confirms this size. Diameter (nm) = 518 30 % intensity = 100 Figure 12. Kaolin particle size evaluation us ing DLS and TEM. Particle size was determined to be approximately 51830 nm.

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45 3.2.2 Flocculation with Gelling Extr act, Non-Gelling Extract and CaCl2 Experiments of settling using kaolin result in plots similar to the plot shown in Figure 13. The kaolin height is plotted as a function of time for several different mucilage concentrations. This plot was generated using the data collected from an experiment of kaolin suspended in DI water (50 g/L) that was treated with GE and is used here a representation of only one run of data a nd is shown primarily as an example. 010203040506 0 0 2 4 6 8 10 12 14 16 18 20 22 0ppm 5ppm 10ppm 15ppm 25ppm 50ppmKaolin Height (cm)Time (min) Figure 13. Full 60 minute plot of kaolin se ttling in DI water with GE. Due to the dynamics of column tests, the kaolin reaches a point in the column where it slows its settling and begins to co mpress into the bottom of the column. From the plot shown in Figure 13, it is suggested that the mucilage columns settled faster as concentration increased. Figure 14 shows the same results as the above plot but here the

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46 plots were truncated where the compression bega n leaving only a straight line. The dotted lines represent the data, while the red lines display the linear curve fit as calculated using Origin. The slopes of the lines represent the se ttling rate of the kaolin in cm/min, and are provided here for each c oncentration of GE. 024681012 0 2 4 6 8 10 12 14 16 18 20 0ppm 5ppm 10ppm 15ppm 25ppm 50ppmKaolin Height (cm)Time (min) 0.43 1.04 1.23 1.99 2.95 5.5 min cm Figure 14. Truncated kaolin plot with linear curv e fit slopes. Resulting slope represents the settling rate of the kaolin in cm/min. All kaolin data was processed in this manner and the corresponding settling rates were plotted together in orde r to evaluate the consequence of different concentrations, water type and flocculants as seen in Figur e 15. All standard deviations calculated are

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47 comparisons of the settling rates determined according to the modeling described in Figure 14. The NE and GE plots show the kaolin se ttling rates as a function of mucilage concentration ranges from 0 to 100 ppm in DI, SW and HW. The bottom graph shows the removal rate of kaolin suspended in HW when treated with the commercially used flocculant Alum. Alum was tested with kaolin suspended in HW (50g/L) because this is the water type that exhibited the best results with GE and NE. Three characteristics of the mucilage induced settling can be determined from the first two plots. First of all, regardless of the water type, the kaolin settles at the same rate of approximately 0.5 cm/min without the addition of mucilage. This is the first point on the plots, and indicates that any further differences in the settling are a result purely of the mucilage interaction with the kaolin clay and ions in the water.

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48 0 2 4 6 8 10 12 14 Concentration (ppm)NE020406080100 0 2 4 6 8 10 12 14 GEKaolin Settling Rate (cm/min)0100200300400500 0 2 4 Al2(SO4)3 -HW-SW-UP Figure 15. Kaolin sedimentation measurements with NE, GE and Alum. Top plot: Kaolin suspensions in hard water (HW), soft water (SW) and Deionized water (DI) treated with NE at a range of concentrations (50 g/L). Mi ddle plot: Kaolin suspensions in hard HW, SW and DI water treated with GE at a ra nge of concentrations. Bottom Plot: Kaolin suspension in HW treated w ith aluminum sulfate Alum. DI

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49 Figure 15 shows the effect of ion-concentr ation in the water with NE, which is observed to significantly influence the kaol in settling rate. Columns containing HW exhibit settling rates higher than the SW, whic h in turn settled faster than the kaolin suspended in DI water. In columns treated w ith GE (Figure 15 middle) the same effect is observed concerning th e ion-concentration of the wate r, however, here it is not as significant as in NE treated columns. The settling rate of the kaolin is a f unction of the mucilage concentration, both NE and GE, and increases with concentration regardless of the water type. Initially the relationship between concentra tion and settling rate appears to be directly proportional, but as the concentration of mucilage increases, the settling rate eventually reaches a point where it does not react as dramatically to increases in mucila ge concentration. Finally, the NE has a more significant e ffect in removing the sediment kaolin from contaminated water than GE. In HW columns treated with 100 ppm NE, an average settling rate of 13.2 cm/min was achieved, wh ile columns under the same conditions but treated with GE only reached an averag e settling rate of 11.0 cm/min. Columns containing SW and DI water display similar differences. The variation in settling capabilities between the mucilage types observed here could possibly be attributed to the way that the different mucilage structures discussed in section 3.1 interact with both the kao lin and the ions in the water. Figure 15 (bottom plot) also shows the settli ng rate of kaolin columns treated with Al2(SO3)4. Kaolin suspended in HW was used to generate these results, as HW yielded the best results in columns treated with mucilage. Experimental results with Al2(SO3)4

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50 concentrations ranging from 0-500 ppm show litt le to no increase in settling rate when compared to the control, indicating that in the conditions provided mucilage is a more efficient and effective flocculating agent for sediment contaminated waters. In columns treated with higher mucilage concentrations (approximately 15 – 100 ppm) textural changes of the kaolin were observed from the flocculation effects. The increased settling rates discu ssed above are a result of this flocculation. Fi gure 16 shows a photograph of this consistency difference. Column 1 is a control containing no mucilage, while columns 2, 3 and 4 are columns containing GE at concentrations of 15, 25 and 50 ppm. All columns shown are of kaol in suspensions in DI water, however, the same consistency changes were observed in columns of SW, HW and columns treated with NE.

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51 1 2 3 4 Figure 16. Kaolin flocs as seen in experimental columns. Column 1 presents a control column with kaolin suspended in DI water wi th no treatment. Column s 2, 3 and 4 are test columns containing kaolin suspended in DI wa ter when treated with Gelling Extract (GE) concentrations of 15, 25 and 50 ppm. Flocculatio n can be observed in the columns treated with GE. In an attempt to visualize the floccu lation effects of the mucilage, TEM and optical microscopy images were prepared. Figure 17 illustrates microscope images of kaolin in HW both alone and treated with 50 ppm NE. Image A is an image of a sample taken from the liquid kaolin interface at th e bottom of the control column. This column was not treated with any flocculating agent and the kaolin particles are seen to be freely floating in the solution. Image B shows the same liquid kaolin interface taken from a column that was treated with a final NE concentration of 50 ppm Here, the flocs of kaolin can be seen confirming what wa s originally seen as flocculation.

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52 20 m 20 m A B Figure 17. Microscope images of kaolin flocculation. Samples taken from columns containing kaolin suspended in hard water (H W) untreated (A) and with the addition of 50 ppm NE (B). This confirms the textural differences observed in the columns during experimentation. Figure 18 confirms the flocculation observe d in the columns and with the optical microscope and shows TEM imaging of the flocs formed during settling. The TEM images shown in Figure 18 were generated using samples taken from the top of the settled columns containing kaolin suspended in DI water both untreated (A) and with the addition of 50 ppm GE (B). Due to the di fference in extraction points, the size and amount of the flocs and kaolin illustrated he re are slightly differe nt from those above. The image in Figure 18 (A) shows the sample taken from the top of the control column that was not treated with any flocculanting agent. All columns imaged of the control

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53 exhibited similar images of lone kaolin particles. This indicates that there is no flocculation of kaolin, and the few particles that are left in the solu tion are freely floating. 1000 nm 1000 nm A B Figure 18. TEM images of kaolin flocculation. Samples taken from the top of columns containing kaolin suspended in DI water both untreated (A) and w ith the addition of 50 ppm GE (B). The control column exhibits no flocculation, while aggregation is observed in the mucilage treated column The image on the right shows a sample fr om the top of a kaolin column treated with 50 ppm GE. Although this image was genera ted from a column th at was treated with GE in place of NE and DI wate r in place of ion-rich, the same flocculation is observed. In order to determine whethe r or not the diatonic ion Ca++ has an effect on kaolin binding as it has been projected to have on bacteria, CaCl2 solutions were prepared and added to columns containing kaolin suspended in SW and HW with final concentrations of 0-50 mM. Figure 19 shows the results from these tests prepared using the same method

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54 as the previously plotted kaolin results. Alth ough at some concentrat ions the settling rate appears to have increased slightly compared to the control, these increases are not significant when compared to the increases observed with the addition of mucilage. 01020304050 0 2 4 HW0 2 4 SWCaCl 2 Concentration ( mM ) Kaolin Settling Rate (cm/min) Figure 19. Kaolin treated with CaCl2 in SW and HW. Results demonstrate the use of calcium chloride on the settling rate of kaolin suspended in soft water (SW) and hard water (HW). The addition of CaCl2 is not seen to play a significant role on the settling rate when compared to the untreated column 3.3 Bacteria Flocculation Tests In tests evaluating the removal of bacter ia suspended in synthetic waters, it was observed that the mucilage alone did not display any signi ficant settling. CaCl2 did induce flocculation when tested alone and al so when used in combination with both

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55 fractions of mucilage. Because of this, al l tests discussed here are treated with a combination of CaCl2 and mucilage, and compared to a control column treated with only CaCl2 and no additional flocculants. Unlike the kaolin settling discussed in section 3.3, th e bacteria flocculation studied does not form a clear interface that can be recorded every minute. Instead the treated bacteria form small white flocs in th e otherwise turbid water, which then fall to the bottom of the column as they are forme d. Figure 20 shows the flocs at the bottom of an experimental column that contains B. cereus in HW that has been treated with CaCl2 and GE. Due to the difference, the mucilage evaluation discussed in this section was slightly different. Here, box plots are used to show the beginning (bottom of the box), duration (the space in between), and the completion (top of the box) of the floc formation and decent in the column. Dotted lines are used to represent the beginning (bottom dotted line) and the end (top dotted line) of the cont rol column that contains no mucilage and only a specified amount of CaCl2.

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56 Figure 20. Image of Bacillus cereus settled flocs at the bottom of the test columns. From left to right, the columns contain the following treatments for B. cereus in hard water (HW). 1: no treatment, 2-7 all contain CaCl2 at a concentrati on of 20 mM, and 3-7 contain the following concentrations of a dded Gelling Extract (GE): 3: 25 ppm, 4: 50 ppm, 5: 2 ppm, 6: 3 ppm and 7:4 ppm. 3.3.1 Bacillus cereus Flocculation and Evaluation There are many factors that could potentially affect the removal rate of B. cereus from contaminated water such as CaCl2 concentration, mucilage concentration and the ion-content of the water. Fi gure 21 provides a plot of B. cereus settling with CaCl2 concentrations from 10-35 mM and these same concentrations with the addition of NE. From this plot it is observed that as CaCl2 concentrations increase, so does the settling rate of the B. cereus Results from columns treated with mucilage in addition to CaCl2, exhibit flocs beginning and completing mo re quickly than columns containing only CaCl2. This indicates that although the CaCl2 causes flocculation, when combined with mucilage the speed of the reaction increases because of the GE or NE addition. All 1 2 3 4 5 6 7

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57 mucilage treated columns shown contained a final NE concentration of 2 ppm, which indicates that only a small amount of mucilage is needed to increase the flocculation time by up to 10 minutes. 1015203035 0 5 10 15 20 25 30 35 40 45 50 CaCl2Concentration ( mM ) Time (minutes) Figure 21. B. cereus settling time versus CaCl2 concentration Results represent the time at which flocs formed in the columns (the bottom of the box) and the time that they finished their decent to the bottom of th e column (top of the box). The dotted lines represent the control begin and end time. Columns contained 108 cells/mL suspended in hard water (HW) treated with a range of CaCl2 concentrations both alone and with the addition of 2 ppm Non-Gelling Extract (NE)

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58 Figure 21 represents the flocculation of B. cereus as a function of the CaCl2 concentration while the mucilage content is held constant. In order to isolate the effects of the mucilage on the flocculation, CaCl2 concentration was held constant over a ra nge of mucilage con centrations. Figure 22 provides a plot of the settling of B. cereus for GE and NE concentration ranges of 0-50 ppm in HW.

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59 0.50.7511.52345102550 0 5 10 15 20 25 60 65 NEMucilage Concentration (ppm) ( ) 0 5 10 15 20 25 50 55 60 GE 0 5 0 5 10 15 20 25 50 55 Time (min) Figure 22. B. cereus flocculation using Gelling Extract (GE) and Non-Gelling Extract (NE) concentration ranges in ha rd water (HW) with final CaCl2 concentrations of 20 mM. Experimental columns treated with GE exhibit increased flocculation time with concentrations ranging from 0.5 to 5 ppm. In columns treated with NE flocculation times occurring faster than the control were observed in columns treated with concentrations between 0.5 and 4 ppm. The higher mucilage concentrations (25 and 50 GE and 10, 25 and 50 NE) did not show signs of flocculati on during the time frame of the experiment.

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60 From the plots in Figure 22, it is obser ved that there is a concentration of mucilage, both GE and NE, where the floccula nt is no longer more efficient or effective as the control. In columns treated with GE, the mucilage caused flocculation at a faster rate than the control until a concentration of 10 ppm was reached. At this concentration, the column treated with mucilage begins to form flocs earlier than the control, treated with only CaCl2, but then the floc formation and se ttling continues after the control is complete and is still not finished after an hour. At GE concentrations of 25 and 50 ppm, flocculation had not even begun in the tim e scope of the experiment. This critical concentration of mucilage could be caused by a number of things in cluding over activity of the mucilage when introduced to the column. The second plot provided shows the settling time of columns treated with NE under the same water and CaCl2 conditions. These plots display similar concentration results as those seen in the GE treated columns, however, the mucilage stops settling more rapidly than the control at 5 ppm. At concentrations ranging from 0.5 to 4 ppm, th e mucilage treated columns begin and end faster than the control but at concentrations of 5 ppm, the treated column takes more time to completely settle than the control, although it s till begins faster a nd ends in the scope of the experiment. Then, at 10, 25 and 50 ppm, the mucilage fails to draw a reaction from the B. cereus in the time frame of the experiment. This effect is noticed to begin at much lower concentrations in columns treated with NE rather than GE. This is potentially due to the structural differences that were discu ssed in section 3.1 and could be caused by the size difference in the mucilage structure. A dditionally, the lack of flocculation observed in high concentrations test could be caused by the mucilage interaction with itself at the

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61 higher concentration. This plot also outlines the low concentr ations of mucilage that are required, and actually preferred, for bacteria settling. At only 0.5 mg/L, effective and rapid flocculation is observed gr anting the mucilage extracted from one pad the ability to possibly treat a large amount of water. It was observed that the SW requi red a higher concentration of CaCl2 in order to provide similar results to those observed in HW at 20 mM, which is most likely caused by the ion difference in the waters. The HW contains a higher level of Ca2+ ions after it is prepared than the SW, leading it to need a smaller concentration of CaCl2 during experimentation. In SW column s containing only 20 mM CaCl2 and treated with mucilage ranges from 2 to 25 ppm, flocculation o ccurred slowly if at all. This observation implies that in order for the GE to be as eff ective as possible, additional ions may need to be added to the water with the mucilage. B ecause of this difference, HW columns were treated with 20 mM CaCl2 while SW columns contained a final concentration of 50 mM. Figure 23 provides a plot of settling ti me versus GE concentration in SW.

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62 0.50.7511.52345102550 0 5 10 15 20 25 30 35 GEMucila g e Concentration ( pp m ) Time (minutes) Figure 23. B. cereus settling times in SW with GE. So ft water columns treated with GE display similar results to hard water columns treated under the same conditions. Here, in the presence of 50 mM CaCl2, GE concentrations of 0.5 to 10 ppm exhibit flocculation more rapidly then the c ontrol containing only CaCl2. Concentrations of 25 and 50 display no flocculation in the time frame of the experiment. This plot shows similar results to the pl ot shown in Figure 22 of the flocculation with GE. The main differences observed are th at the column contai ning 10 ppm complete settling faster than the control, and it is not until concentrations of 25 and 50 are used that the mucilage fails to work as well as the control. This is most likely caused by the higher CaCl2 concentrations used in tests with SW.

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63 In regards to better understanding the di fferences observed in the settling times provided in Figures 21, 22 and 23, microscope images were ta ken of the control columns and the columns treated with mucilage. Fi gure 24 shows microsc ope images of two control columns. The first image (A) shows cells from a column that contained untreated B. cereus in HW and the second image (B) is from the same suspension as above, but treated with 20 mM CaCl2. 20 m 20 m Control 1: No Mucilage, No CaCl2,Control 2: HW, No Mucilage, CaCl2, : 20 mM A B Figure 24. Microscope images of B. cereus control columns. Final cell concentration of 108 cells/mL. Image A taken of a sample obtai ned from an untreated column while the image B is cells from a column treated with CaCl2. In untreated columns bacteria are dispersed and freely floating. W ith the addition of 20 mM CaCl2 small flocs are observed.

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64 Figure 24A shows that in the absence of CaCl2 and mucilage, there is no flocculation and the bacteria are still freel y floating in solution. Figure 24B shows that with the addition of CaCl2 at a final concentration of 20 mM small flocs are formed, causing the settling that was discussed ear lier. Figure 25 presents images of the B. cereus under the same conditions as the control column s, but solutions treated with GE (A) and NE (B) with final concentrations of 2 ppm. 20 m 20 m GE: 2 ppm, CaCl2, : 20 mM NE: 2 ppm, CaCl2, : 20 mM A B Figure 25. Microscope images of B. cereus columns treated with GE and NE in HW. The images here show the flocculation e ffects that the mucilage has on the B. cereus Both images are from columns containing 20 mM of CaCl2 and 2 ppm of GE (A) and NE (B). The flocs observed here are much larger th an those seen in columns with only CaCl2 treatment.

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65 The column containing only CaCl2 showed some flocculation, the effect was not as large or tightly packed as those observed in columns treated with GE and NE. Similar flocculation was observed in columns cont aining SW and can be seen in Figure 26. Image A shows the flocculation caused by the CaCl2 at a concentration of 50 mM. The flocculation phenomena observed here is si milar to that shown above in the columns containing only 20 mM CaCl2, indicating that at a concentr ation more than double larger and more stable flocs are not formed. The images below show the flocs formed in columns treated with GE (B) a nd NE (C) with final concentra tions of 2 ppm, and are very similar to those seen in Figure 25. This indicates that the flocs formed are generally much larger and more stable in both HW and SW than their CaCl2 counterparts.

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66 Control 2: SW, No Mucilage CaCl2,: 50 mM 20 m GE: 2 ppm, CaCl2,: 50 mM 20 m NE: 2 ppm, CaCl2,: 50 mM 20 m A B C Figure 26. Microscope images of B. cereus treated in SW columns. Image A shows cells extracted from a column tr eated with 50 mM of CaCl2 and no mucilage. Here, as in previous images, flocs are present, but they ar e small and do not appear to be very stable. GE (B) and NE (C) treated bacteria with a final concentration of 2 ppm show the flocs that are larger and more defined than those observed in CaCl2 control columns. Table 10 provides removal rates of B. cereus suspended at high concentrations (108 cells/mL) in SW with final CaCl2 concentrations of 40 mM. GE and NE treatment concentrations evaluated include 0, 2, 3, 4 and 5 ppm. All removal rates are in the 9599% range including those of th e column containing only CaCl2.

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67 Table 10. Removal rates of B. cereus in soft water columns treated with GE, NE and 40 mM CaCl2. Mucilage Type Mucilage Concentration 0 ppm 2 ppm 3 pp m 4 ppm 5 ppm GE 98.21 % 98.50 % 98.06 % 98.59 % 95.69 % NE 98.21 % 98.11 % 98.07 % 98.59 % 98.51 % These results show that, although th e addition of mucilage increases the flocculation reaction that takes place between the B. cereus and the CaCl2, the resulting removal rate does not differ significantly. Alth ough these removal rate s appear high, the water treated in these experime nts is not yet fit to consume. Due to the high initial cell count concentrations, removal rates of 99% s till results in a significant number of viable cells in the solution. 3.3.2 Escherichia coli Flocculation and Evaluation In the testing of mucilage for E. coli removal, HW, CaCl2 concentrations of 20 mM, and GE were used due to their capabilities observed in B. cereus tests. Figure 27 provides a plot of flocculation time as a func tion of GE concentration in columns of HW with 20 mM CaCl2.

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68 0.50.7511.52345102550 0 5 10 15 20 25 50 60 Time (min)Mucila g e Concentration ( ppm ) GE 2 Figure 27. E. coli flocculation in HW at a range of GE concentrations. In columns containing 108 cells/mL, E. coli flocculation with the additi on of mucilage was observed to be more efficient with the addition of 0.5 to 10 ppm GE. The control required twice as much time after column inoculation to show si gns of settling when compared to the same test with B. cereus Figure 27 shows that for final GE con centrations of 0.5 to 10 ppm, the columns treated with mucilage begin and end much faster than th e control. From this plot differences between B. cereus and E. coli can be observed. In columns of B. cereus in HW the control column flocculation was obser ved to begin in 7.5 minutes. Conversely, in

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69 columns containing E. coli flocculation does not begin in the control column until 14 minutes after column inoculation. B. cereus columns were also seen to complete settling 24 minutes into the experiment while E. coli columns took slightly longer. Both bacteria exhibit differences to th e kaolin studies performed with the same mucilage. In kaolin columns the higher mucila ge concentrations wo rked better and an optimal concentration as reached where sett ling no longer increase d. In columns of bacteria treated with mucilage the opposite e ffect is observed. The lower concentrations work better and at higher concentrations, no r eaction is seen in the columns. This is potentially due to a number of things in cluding the size and surface characteristic differences between the contaminant types. In kaolin suspensions, the ion concentration was observed to affect the settling rate; however, the mucilage did not rely on the presence of ions for the flocculation to o ccur with kaolin as it does with bacteria. Figure 28 shows a picture of the floc s forming in columns that contain E. coli suspended in HW and treated with 20 mM CaCl2 and GE concentrations of 0 ppm (1), 10 ppm (2), 25 ppm (3), 50 ppm (4), and 2 ppm (5) under UV light.

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70 1 2 3 4 5 Figure 28. Picture of flocs forming in treated E. coli columns under a UV light. All columns contain E. coli suspended in hard water a nd treated with 20 mM CaCl2. GE mucilage addition as follows: (1) 0 ppm, (2) 10 ppm, (3) 25 ppm, (4) 50 ppm, (5) 2 ppm. Columns 2 and 5 display signs of flocculation. Since the E. coli were transformed to contain a pG LO plasmid, the cells fluoresce under UV light making the flocs formed easier to see. In Figure 28, columns 2 and 5 have begun to form flocs and the water around th e flocs appears relatively clear when compared to columns 1, 3 and 4 that have not yet began to flocculate. In addition to using the E. coli GFP’s florescent qualities help to assess the removal from the water as the flocs are formed, but they also allow the flocs to be viewed under the microscope using a florescent filt er. Figure 29 shows florescent microscope images E. coli suspended in HW and treated with no flocculating agent (A), with only 20

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71 mM CaCl2 (B), and with 20 mM CaCl2 and 2 ppm GE (C). In Image A, of a column that was not treated, single E. coli bacteria can be seen floati ng freely in the solution. When treated with 20 mM CaCl2 (B) flocculation can be observed, however, the flocs are not very large and do not contain a lot of bacteria. In the final image (C), from a sample treated with 2 ppm GE in the presence of 20 mM CaCl2, a large cluster of bacteria are present, and covers the entire image. Here, th e size and high bacteria content of the flocs formed using mucilage can be observed. The flocculation observed in bacteria columns is similar to that seen in suspensions of kaolin treated with mucilage. The gathering of particles observed in both cases causes the density of the contaminating ma terial to change, as it is becoming larger, and gravity induced settling occurs.

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72 10 m 10 m Control 1: No Mucilage, No CaCl2Control 2: No Mucilage, CaCl2: 20 mM Control 2: GE: 2 ppm, CaCl2: 20 mMA BC10 m Figure 29. Florescent images of the flocs formed in columns containing E. coli suspended in HW. Not treated (A), treated with 20 mM CaCl2 (B) and with the addition of 2 ppm GE (C). By comparing the images the differences in the size of the flocs formed can be observed.

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73 Chapter Four: Conclusions and Future Work 4.1 Summary of Findings The work presented in this thesis demonstrates that both the Gelling (GE) and Non-Gelling (NE) mucilage extracts obtained from the O. ficus-indica cactus are capable of removing sediment and bact eria. Different fractions of mucilage are more efficient with different contamination species, which is possibly due to the structural differences between the two mucilage compounds. Structura lly, the GE exhibits a fiber like structure that is orderly and directional while th e NE has a denser net like structure. Kaolin has been observed to form a very cl ear interface as it se ttles that could be read every minute and plotted to observe th e settling nature of the column. In water prepared at high ion-concentrations, referred to as HW, settling rates of kaolin increased with increasing concentrations of both NE a nd GE at a faster rate than SW, surrogate water with lower ion-concentration, which in tu rn settled faster than kaolin suspended in DI water. The concentration of both NE and GE gradually reach a point where the settling rate begins to level off and the change in rate versus conc entration is no longer significant. In columns containing NE averag e settling rates of 13.5 cm/min were reached and in columns treated with GE average rates of 11.2 cm/min were observed. Columns containing Al2(SO4)3 at high concentrations exhibited little increase in settling rate above the control settling of 0.5 cm/min.

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74 Images of samples taken from column s of high GE and NE concentrations showed a clear aggregation where samples fr om the control columns showed particles that were free floating and se parated. The flocculation phenomena is the cause of the increased settling rates with the addition of mucilage. Gram-positive B. cereus demonstrated flocculation similar to kaolin when exposed to GE and NE coupled with CaCl2. Bacteria flocculation, unlike kaolin was not observed to form a clean interface, rather the flocculation beginning and end time were recorded and evaluated compared to a control. Here, the mucilage wa s more effective at lower concentrations, which is opposite to wh at was observed in kao lin columns. For NE, concentrations of 0.5 to 4 ppm produced flocs that both developed and settled faster than the control column that contained only CaCl2. At concentrations of 5 ppm the flocs took a greater amount of time to settle then those formed in the control column. At concentrations of 10, 25 and 50 ppm, signs of fl occulation did not even appear in the time scope of the experiment. Columns treate d with GE worked at slightly higher concentrations than the columns treated with NE and were not slower than the control until concentrations of 10 ppm. Concentrations of 25 and 50 ppm showed no signs of removal. In addition, the flocculation ti me frame was shown to decrease as CaCl2 concentration increased over 10-35 mM, as we ll as columns ran with the addition of 2 ppm NE to these CaCl2 concentrations. Images of B. cereus untreated showed that the bacteria were still freely floating in solution and that no aggregation had occurre d. In columns containing HW with 20 mM CaCl2 and SW with 50 mM CaCl2, a small amount of flocculation was observed, but it

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75 appeared to be small and loosely stru ctured. In images of HW (20 mM CaCl2) and SW (50 mM CaCl2) and the addition of 2 ppm NE or GE, flocs were observed to be both larger and seemingly more stable due to the tightness of the bacteria packing. From these treated columns removal rates of 95-99 percent were observed. Settling results for gram-negative E. coli proved to be similar to those for B. cereus using HW, CaCl2 concentrations of 20 mM and GE concentrations ranging from 0.5-10 ppm. Control columns were observed to require twice as long to begin settling after inoculation and mucilage columns did not appear to require more time. When observed under UV light, the water appeared to clear significantly while the flocs were being formed and in microscope images usi ng florescence, large flocs were observed in columns treated with 2 ppm GE and 20 mM CaCl2 when compared to those treated with only 20 mM CaCl2 and a column that was untreated. 4.2 Future Work Recommendations 4.2.1 Continued Bacteria Studies All work provided here focused on the use of a Gelling and Non-Gelling Extract from the O. ficus-indica Although previous work with the third fraction of mucilage, Combined Extract (CE), suggest s that its use will result in a removal somewhere between the two fractions studied in this work, it would be valuable to test these contaminants using this mucilage fraction as well. The testing of this extract could also potentially illustrate how the mucilage will work when it is used in developing countries as this is what would be most easily obtained from the pads.

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76 In this work, only suspensions of B. cereus were evaluated fo r bacteria removal rates. Removal rates of E. coli also should be tested and both should be evaluated at low initial cell concentrat ions. In environmental bodies of water, concentrations of 105 cells/mL are more likely to occur and need to be evaluated to determine the effectiveness of mucilage at these low concentrations. The concern of whether or not E. coli and B. cereus could feed on the sugars found in the mucilage will also need to be addressed. 4.2.2 Shelf-life Evaluation The results provided in this thesis demons trate the small amount of mucilage that is required for contaminant removal from su rrogate waters. Because of the relationship between the amounts of mucilage used to th e amount obtained from a single pad through the extraction process, it would be valuable to consider the shelf-life of the mucilage product. This would determine whether or not the fast settling rates observed above would change over time, and if so to what de gree. The GE and NE used to generate the results described by Young et al. were extracted in 2004 and still have the ability to remove sediment suspensions from water, alth ough it may not be as effective as it was at the time of its extraction. In order to eval uate and account for the possible differences in structure that occurs over ti me, prepared mucilage should be tested at intervals to determine whether or not efficiency is lost This evaluation could also be done with equipment designed to evaluate the contents of the mucilage, as it structural differences could be measured there as well.

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77 4.2.3 Contaminant Combination Analysis This work studied two contaminants sepa rately and provides data that suggests mucilage is a useful flocculating agent for both. However, because it is highly unlikely that a single contaminant will occur in a given body of water, it would be interesting and provide a better understanding of how the mucilage would suffice in real world situations if the mucilage was tested on a column that contains a combination of two or more contaminants. 4.3 Final Remarks From this work it can be concluded that mucilage has the potential of removing some of the most common contaminants from ion-rich water supplies. The amount of mucilage that is required to cause significant flocculation is very lo w and a single cactus pad offers the ability to clean a large amount of water. Not only does the cactus provide a green technology for use in water purification, but it also avoids controversy that is often observed with current methods including community opposition, energy requirements, and inconsistent results. Due to its common use and abundant growth, the O. ficus-indica cactus could offer an inexpensive, easy to use and extremely valuable flocculant to countries that struggle w ith water contamination.

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78 References 1. Thornton, J., Water Loss Control Manual McGraw-Hill Professional: 2002. 2. Bartram, J.; Howard, G., Drinking-wate r Standards for the Developing World. In The Handbook of Water and Wa stewater Microbiology Mara, D.; Horan, N., Eds. Academic Press: San Diego, CA, 2003. 3. Gleeson, C.; Gray, N., The Coliform Index and Waterborne Disease: Problems of Microbial Drinking Water Assessment E & FN Spon: Boundary Row, London, 1997. 4. UN Human Development Report 2006; Beyond Scarcity: Power, poverty and the global water crisis ; EPA Office of Water: New York City, 2006. 5. Geldreich, E. E.; Reasoner, D. J., Home Treatment Devices and Water Quality. In Drinking Water Microbiology McFeters, G. A., Ed. Springer-Verlag New York, Inc.: New York City, 1990 pp 147-167. 6. EPA Bacterial Water Quality Standards for Recreational Waters ; Office of Water: Washington DC, 2003. 7. Bitton, G., Wastewater Microbiology 3 ed.; John Wiley & Sons, Inc: Hoboken, NJ, 2005.

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79 8. Jensen, P. K.; Ensink, J. H. J.; Jayasi nghe, G.; Hoek, W. v. d.; Cairncross, S.; Dalsgaard, A., Domestic Transmission routes of pathogens: The problem of in-house contamination of drinking water duri ng storage in developing countries. Tropical Medicine and International Health 2002, 7 (7), 604-609. 9. Mintz, E.; Bartram, J.; Lochery, P.; We gelin, M., Not Just a Drop in the Bucket: Expanding Access to Point-of-Use Water Treatment Systems. American Journal of Public Health 2001, 19 (10), 1565-1570. 10. Quick, R. E.; Venczel, L. V.; Mintz, E. D.; Soleto, L.; Aparicio, J.; Grionaz, M.; Hutwagner, L.; Greene, K.; Bopp, C.; Maloney, K.; Chavez, D.; Sobsey, M.; Tauxe, R. V., Diarrhoea prevention in Bolivia through point-of-use water treatment and safe storage: a promising new strategy. Epidemiol. Infect 1999, 122 83-90. 11. Curtis, V.; Cairncross, S.; Yonli, R., Review: Domestic hygiene and diarrhoea pinpointing the problem. Tropical Medicine and International Health 2000, 5 (1), 22-32. 12. UN The Millennium Development Goals Report ; United Nations: New York City, 2008. 13. UN The Millennium Development Goals Report ; United Nations: New York City, 2007. 14. Miller, E. W.; Miller, R. M., Water Quality and Availability ABC-CLIO, Inc: Santa Barbara, CA, 1992. 15. EPA National Primary Drinking Water Standards ; EPA Office of Water: 2003.

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81 23. Semenza, J. C.; Roberts, L.; Hender son, A.; Bogan, J.; Rubin, C. H., Water Distribution System and Dia rrheal Disease Transmission: A Case Study in Uzbekistan. American Journal of Tropic al Medicine and Hygiene 1998, 56 (6), 941-946. 24. LeChevallier, M. W.; Cawthon, C. D.; Lee, R. G., Factors Promoting Survival of Bacteria in Chlorinated Water Supplies. Applied Environmental Microbiology 1988, 54 (3), 649-654. 25. Morrow, J. B.; Almedia, J. L.; Fitzgera ld, L. A.; Cole, K. D., Association and Decontamination of Bacillus Spores in a Simulated Drinking Water System. Water Research 2008, 42 (20), 5011-5021. 26. Parsons, S. A.; Jefferson, B., Introduction to Potable Wa ter Treatment Processes Blackwell Publishing Ltd: Oxford, UK, 2006. 27. Montgomery, J. M., Water Treatment Principles and Design John Wiley & Sons: New York City, 1985. 28. Ndabigengesere, A.; Narasiah, K. S., Quality of Water Treated by Coagulation using Moringa Oleifera Seeds. Water Research 1997, 32 (3), 781-791. 29. Babu, R.; Chaudhuri, M., Home Water Treatment by Direct Filtration with Natural Coagulant. Indian Journal of Water and Health 2005, 3 (1), 27-30. 30. Larsen, N.; Nissen, P.; Willats, W. G. T ., The effect of Calcium Ions on Adhesion and Competitive Exclusion of Lactobacillus ssp. and E. coli 0138. International Journal of Food Microbiology 2007, 114 (1), 113 119.

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83 39. Young, K. A.; Anzalone, A.; Pichler, T.; Picquart, M.; Alcantar N. A., Using the Mexican Cactus as a New Environmentally Benign Material for the Removal of Contaminants in Drinking Water. 2005, 2005 (93), 965-966. 40. Geldreich, E. E., Microbiological Qualit y of Source Waters for Water Supply. In Drinking Water Microbiology McFeters, G. A., Ed. Springer-Verlag New York, Inc.: New York City, 1990; pp 3-31. 41. Priest, F. G., Isolation and Identificat ion of Aerobic Endospore-Forming Bacteria. In Bacillus Harwood, C. R., Ed. Plenum Pre ss: New York City, 1989; pp 27-56. 42. Archibald, A. R., The Bacillus Cell Envelope. In Bacillus Harwood, C. R., Ed. Plenum Press, New York: New York City, 1989. 43. Bauman, R. W., Microbiology Pearson Education, In c.: San Francisco, 2004. 44. Ingraham, J. L.; Ingraham, C. A., Indroduction to Microbiology 2 ed.; Brooks/Cole: Pacific Grove, CA, 2000. 45. Lim, D., Microbiology 2 ed.; WCB/McGraw-Hill: 1998. 46. Smith, E. J.; Davison, W.; Hamilton-Tayl or, J., Methods for Preparing Synthetic Freshwaters. Water Research 2002, 36 (5), 1286-1296.