COPPER AND ZINC MODIFIED CANADIAN NATURAL ZEOLITE AS A MEANS FOR REDUCING THE NUMBER OF E. COLI COLONY FORMING UNITS IN CONTAMINATED DRINKING WATER by Lon Kerr B. Sc., University of Northern British Columbia THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN NATURAL RESOURCES AND ENVIRONMENTAL STUDIES UNIVERSITY OF NORTHERN BRITISH COLUMBIA December 2022 © Lon Kerr 2022 Abstract Clean drinking water is essential for all life on Earth. Over 400, 000 people die annually from lack of clean drinking water, and the demand on water is predicted to increase in all sectors. The demand for safe drinking water presents an ongoing challenge for new water purification techniques. Since the 1950s, natural zeolite has been investigated as a means of water purification due to its stable, crystalline, porous structure and cation exchange capacities. Recent publications have indicated that natural zeolite modified with metal cations provides a stable treatment media for the elimination of E. coli bacteria in drinking water. In this series of analysis, Canadian natural zeolite from the Bromley River Valley, Kamloops BC, was modified with zinc and copper sulphates to create three novel water treatment options. Treatment of E. coli contaminated water was most effective with zinc modified zeolite, which also significantly lowered the pH in comparison to the copper and copper/zinc modified zeolite. Of the three zeolites, all released too much copper and zinc into solution that may be linked to the low cation exchange capacity (CEC) of the Canadian zeolite. E. coli colony forming units were reduced; however, they were not sufficient to meet drinking water standards. Future studies will focus on optimizing the ratio of modified zeolite needed to treat a given amount of pathogen in solution. ii Table of Contents Abstract ......................................................................................................................................................... ii Table of Contents ......................................................................................................................................... iii List of Figures ............................................................................................................................................... v List of Tables ................................................................................................................................................ v Glossary ....................................................................................................................................................... vi Acknowledgements ..................................................................................................................................... vii Chapter 1 - Introduction ....................................................................................................................... 1 1.1 Water ......................................................................................................................................... 1 1.2 Previous techniques for water purification ............................................................................... 2 1.3 Zeolites ...................................................................................................................................... 3 1.4 Mechanism of action on prokaryotes ........................................................................................ 6 1.5 Metal cations and consumption................................................................................................. 7 1.6 pH of Water............................................................................................................................. 10 1.7 Pathogen in water .................................................................................................................... 11 1.8 Summary ................................................................................................................................. 12 1.9 Research Question................................................................................................................... 13 1.10 Objectives ............................................................................................................................... 13 1.11 Hypothesis............................................................................................................................... 13 Chapter 2 - Materials and Methods .................................................................................................... 14 2.1 Collection of Zeolite Samples ................................................................................................. 14 2.2 Elemental Analysis ................................................................................................................. 14 2.3 Cation Exchange Capacity (CEC) ........................................................................................... 15 2.4 Modification of zeolite ............................................................................................................ 16 2.5 Ion Chromatography ............................................................................................................... 17 2.6 E. coli Acquisition and Storage ............................................................................................... 18 2.7 Minimum Inhibitory concentration (MIC): ............................................................................. 18 2.8 Timed desorption of cations .................................................................................................... 18 2.9 Colony counting ...................................................................................................................... 19 2.10 Cu and Zn Modified Canadian Zeolites on E. coli in Nutrient Broth ..................................... 19 2.11 Final pH of solution ................................................................................................................ 20 2.12 Data Analysis .......................................................................................................................... 20 Chapter 3 3.1 Results and Discussion.................................................................................................... 22 Leaching of Toxic Elements ................................................................................................... 22 3.1.1 Digestion .................................................................................................................................... 22 iii 3.1.2 Post Treatment Elemental Analysis. .......................................................................................... 23 3.1.3 Physical characterization............................................................................................................ 25 3.1.4 Cation Exchange Capacity (CEC) ....................................................................................... 26 3.2 Controlled Desorption of Treatment Ions ............................................................................... 27 3.2.1 Cation Exchange with Environments ......................................................................................... 27 3.2.2 Ion chromatography Sulphates ............................................................................................ 30 3.2.3 Final pH of Treated Solutions ............................................................................................. 31 3.3 Effects of Metal Modified Zeolite on E. coli ................................................................................ 32 3.3.1 Minimum Inhibitory Concentration (MIC) ................................................................................ 32 3.3.2 Comparing Canadian and Cuban Zeolites on E. coli .......................................................... 33 3.3.3 The Effect of Zn and Cu modified Zeolites on E. coli in Nutrient Broth ........................... 34 3.4 Avenues for Future Research or Improvement ............................................................................. 38 Chapter 4 - 4.0 Conclusion ................................................................................................................ 39 References ................................................................................................................................................... 40 iv List of Figures Figure 3-1: Scanning electron micrograph and XRF readings for the Canadian natural zeolite indicating clinoptilolite sample ..................................................................................................... 26 Figure 3-2:The mean and standard deviation for metal ion desorption of metal modified Cuban Zeolite (CubanZZ), zinc modified Canadian zeolite(CanZZ), natural Canadian zeolite (CanNZ), natural Cuban zeolite (Cuban NZ) and synthetic silver zeolite (n=10). ....................................... 29 Figure 3-3: The mean sulphate concentration( ppm)) for duplicate treatments in Tabor Lake water (n=10). ................................................................................................................................. 30 Figure 3-4: Mean and Standard deviation of final pH in nutrient broth after treatment with metal modified Canadian zeolite CanCu, CanCZ and CanZZ (n=16). ................................................................................................................. 31 Figure 3-5: The absorbance of 600nm wavelength light by growth media at 37 OC for increasing zinc sulphate concentration (n=8)...................................................................................................................................................... 32 Figure 3-6The relationship between the mass of metal modified Canadian zeolite treatment and the loss of colony forming units by E. coli after 24 hours of exposure..................................................................................................... 36 List of Tables Table 3-1:The difference between the mean of Canadian natural zeolite digested with HCl and NO 3 and EPA method 3050b( EPAFt 1996) to determine the difference in the results of zeolite that haven’t completely dissolved. ........................................................................................................................................................................ ……….23 Table 1-2 Analysis of modified zeolites and post bacterial trial zeolites for the elements listed column (mg element/kg of sample). …………...……………………………………………………………………………………………………………23 Table 1-3: Chemical composition of the clinoptilolite phase of Canadian natural zeolite tuff obtained by EDS analysis………………………………………………………………………………………………………………..25 Table 3-4 The mean cation exchange capacity (n=12) cation exchange capacity for natural and metal modified zeolites………………………………………………………………………………………………………………..26 Table 1-5 The effect of zeolite treatment on Tabor Lake water spiked with 2000 CFU E. coli after one hour and three hours of exposure. (n=10) ...……………………………………………………………………………………………………………...……….33 v Glossary CanNZ: Canadian Natural Zeolite from the Bromley River Valley CanZZ: Canadian zinc modified zeolite CanCZ: Canadian copper and zinc modified zeolite CanCU: Canadian copper modified zeolite CEC: Cation Exchange Capacity CFU: Colony Forming Units CubNZ: Cuban Natural Zeolite CubZZ: Cuban zinc modified zeolite ICP-OES: Inductively coupled plasma optical emission spectroscopy WHO: World Health Organization vi Acknowledgements I am so thankful for this opportunity and owe my success to my friends and family, Hossein for the opportunity to grow, my committee members, Dr. Li and Dr. Preston, for seeing me cross the finish line, the NALS team for their support efforts in the lab, the MATTERS team for deep friendships. MITACS for the funding and the invaluable training, UNBC for the space, funding, and great faculty who’ve been my mentors and are now friends. vii 1 Chapter 1 - Introduction 2 3 4 1.1 Water 5 Even in countries blessed with substantial water and land resources (e.g. Canada), water 6 is not always available when and where needed (Bereski et al. 2017). Sustainable development 7 requires protecting the resources for future generations, while meeting the needs of present 8 society. Taking into account climate change, social influences, and increasing demands on water 9 resources, the country is experiencing new challenges and competition for water. As demands on 10 water increase, effective management of water resources, including reuse of contaminated 11 surface and underground water streams, will be essential for a healthy environment. Thousands 12 of crises have dramatically affected water management and governance criteria. Waterborne 13 disease, such as Escherichia coli (E. coli) outbreaks in Walkerton, ON, Canada killed seven, and 14 induced illness in 2,300 people between 2000 and 2005 (Bradford et al. 2015). In this specific 15 case, studies showed that the source of the pollution was livestock manure that had been applied 16 on farmland as fertilizer. 17 Further, access to clean water within a country is not always consistent. In 2015, over 90 18 First Nations reserves had boil water advisories. Waterborne illness was 26 times higher on 19 reserves than the national average (Bradford et al. 2015) with 30% of reserve treatment facilities 20 actively posing a risk to community health (Bradford et al. 2015). Despite several million dollars 21 dedicated by the Government of Canada in 2020, there are still 58 communities without access to 22 reliable drinking water (Environment of Canada 2022). Some treatment options require constant 23 maintenance and chemical additives, such as chlorine, that have been shown to worsen common 24 skin conditions among reserve children (Warrick and Patrick 2019). Additionally, it seems that 1 25 the water in remote communities is often negatively impacted by local industry, such as mining 26 and manufacturing. In 2011, Robert J Patrick outlined that the issue with water treatment 27 extended beyond policy and funding to include a lack of appropriate water treatment technology 28 (Patrick 2011). To meet the water needs of small communities, a purification technology needs 29 to be affordable, require minimal maintenance and skill to operate, be reliable, and be able to 30 treat fecal matter pathogens and industrial by-products such as volatile organic compounds and 31 heavy metals (Warrick and Patrick 2019, Ravishnakar and Jamuna 2011, Bereskie et al. 2017, 32 Government of Canada 2017). 33 34 1.2 35 Previous techniques for water purification Reactive chemicals (e.g. chlorine) and/or ultraviolet (UV) radiation are usually used to 36 destroy water pathogens (KDF 2020). Several technical reports studied silver (Ag) and zinc (Zn) 37 oxide nanoparticles for treating microbiological contamination (Xiaolei et al. 2013, Aarestrup 38 and Hasman 2004, Ravishnakar and Jamuna 2011, Kallo 2001, Fuentes et al. 2014, Hrenovic et 39 al. 2012). Kinetic Degradation Fluxion (KDF) filters release small quantities of Cu and Zn (K. 40 Inc. 2020). Some articles have related the use of Ag/Zn zeolite for the same purpose (Orha et al. 41 2011, Aarestrep and Hasman 2004, Prabir et Wang 2019). The use of natural and modified 42 porous zeolitic minerals as multifunctional media is one of the efficient approaches for 43 decontamination of polluted water resources (Wang and Peng 2010). Zeolites are composed of a 44 durable alumino-silicate structure that forms a porous charged material. These unique chemical 45 and physical characteristics make zeolite appropriate for a multitude of environmental 46 applications where effective, low-cost materials are needed to bind, absorb, adsorb, fill, and filter 47 (Ravinshankar and Jamuna 2011, Boles et al. 1977, Orha et al. 2011, Fuentes et al. 2014). 48 Molecular sieve and ion exchange properties, as well as availability and relatively low cost, are 2 49 the major factors that make natural zeolites commercially attractive for environmental 50 remediation and industrial applications (Flanigen and Mumpton 1981). The discovery of zeolite 51 deposits with relatively high purity in Canada, United States, and other countries in the 1950s 52 marked the era of commerce for natural zeolite-based water filtration (Mumpton 1978). 53 54 55 56 1.3 Zeolites Natural zeolites are ubiquitous silicate formations found in cavities of basalt and trap 57 rock formations; however, commercially available zeolites are most often found in sedimentary 58 deposits (Hay and Sheppard 2001). Zeolites are crystalline hydrated alumino-silicates of alkaline 59 earth cations, capable of exchange with cations in solution (Mumpton 1978). Since the 1950s, 60 natural zeolites have been highly valued for their ability to remove toxic cations and rivals the 61 adsorption of synthetic sieves (Fuentes et al. 2007). Along with quartz and feldspar, zeolites are 62 tetra silicates that form a three-dimensional SiO4-4 tetrahedral, where all tetrahedral corner 63 oxygens are shared (Kitsopoulos 1999). The sharing of oxygen ions provides zeolites with 64 infinite (repeating units) three dimensional structures. The alumino-silica structures are 65 negatively charged and very strong in nature. This combination of properties allows cations to be 66 shared freely between the internal zeolite structure and its surrounding environment. For 67 example, two sodium ions with a positive charge each may be exchanged with a single calcium 68 ion with a two plus charge. It is apparent in zeolite studies that the ion affinity changes based on 69 the pore size and charge of the zeolite backbone (Boles et al. 1977, Ming and Dixon 1987). 70 71 Filtration and purification characteristics of natural zeolites can be applied to removing impurities existing in water in the form of insoluble, colloidal, and dissolved physical states, 3 72 which are of mineral, organic or biological origin (Kallo 2001). Zeolites were used to reduce the 73 levels of ammonium and other impurities in water treatment plants in Budapest (Hungary), 74 Colorado (USA), Tbilisi (Georgia) and Ukraine (Kallo 2001, Mumpton 1999). In both pilot- and 75 full-scale applications, the ion exchange and filtering properties of clinoptilolite-rich tuffs were 76 utilized with subsequent treatment systems. Practical applications of phillipsite-rich tuff from 77 Tenerife, Canary Islands, were also shown to favorably remove indicator bacteria and dissolved 78 organic matter from water in a packed percolator reactor (Harleman et al. 2009). A patented 79 micro-filtration system (i.e. Jossab Aqualite), functions by integrating clinoptilolite into an 80 appropriate technical set-up for purifying drinking water in emergencies where the requirement 81 of safe drinking water has been critical for public health (Hrleman et al. 2009). The technique 82 can filter out particles down to the size of 1–2 μm without any chemical additives. The 83 subsequent UV filter ensures complete removal of bacteria and parasites. For emergency 84 situations, the mobile units have a capacity ranging between 7-15 m3/h which corresponds to 85 fresh water for 5000–12000 persons per 8–10 hours of use or up to 20000–25000 persons for 20 86 hours of use, in compliance with the World Health Organization’s (WHO) standards 87 (Government of Canada 2017) (W.H.O. 2019). Mobile water purification units (Fig. 1) were 88 utilized between 1999- 2006 in Rosersberg, Sweden; Grozny, Chechneya; Belgrade, Kosovo for 89 rapid transportation to the point-of-need and immediate performance at the emergency site 90 (Figure1-1) (Harleman et al. 2009). This system is further proposed for the elimination of 91 radioactive fallout and for the removal of arsenic or geogenic pollutants from groundwater 92 (Harleman et al. 2009). 93 Due to the stability of zeolite and its cation exchange properties, researchers have been 94 attempting to produce zeolite-based water treatment apparatus (Mumpton 1999, Flanigan and 4 95 Mumpton 1977, Faghihian et al. 1999, Madji et al. 2015, Fuentes et al. 2014, Hrenovic et al. 96 2012). By incorporating zeolites into filtration units, it was possible to remove unwanted cations 97 and contaminants from solution. In other studies, the modification of zeolites with positively 98 charged molecules allowed for the delayed release of antimicrobial agents to treat 99 environmentally sourced water (Alsammarraie et al. 2018, Filali et al. 200, Fuentes et al. 2006, 100 Lalley et al. 2014). 101 102 103 104 (Figure 1-1) A mobile water purification unit based on natural zeolite, capacity: 4–7 m3/h, weight: 1800 kg. 105 Some natural zeolites are well known as physical adsorbents of pathogens, such as 106 Giardia, cryptosporidium, bacteria and their spores (Fuentes et al. 2014). Most of these 107 organisms and their spores are in the size range of 0.5-10 microns; therefore, the zeolite powder 108 can adsorb a high percentage of these microorganisms while the water passes through the zeolite 5 109 (Hughes 2003). In 1990, a team of researchers at the University of Havana developed a zinc- 110 modified zeolite that had microbicidal effects against bacteria, yeast, and protozoans (Fuentes 111 2014). According to their report, they purified natural zeolite and loaded Zn cations using zinc 112 sulphate. The product released zinc cations in a slow-release fashion, meeting the drinking water 113 standard of less than 5.0 mg/L recommended for levels of zinc (Government of Canada 2017). The applications for zeolite use in water treatment are abundant. The natural properties of 114 115 zeolite as cation exchangers and sieves are well documented for many zeolites in many 116 countries; however, the natural zeolites of the Bromley River Valley in Kamloops BC have never 117 been evaluated or tested in application for drinking water remediation (Kondo et al. 2019). By 118 establishing the natural properties of the zeolitic tuff and evaluating subsequent cation 119 modification, it may be possible to develop a system for the purification of contaminated water 120 (Djordie et al. 2011). 121 122 1.4 123 Mechanism of action on prokaryotes The cellular functions of bacterial pathogens are affected by at least three mechanisms 124 linked to metal modified zeolite. The first mechanism of action affects the optimum pH range for 125 bacterial reproduction (Aarestrup and Hasman 2004). By releasing metal ions into solution, the 126 resulting pH can impacted bacterial reproduction and prevented further propagation (Shameli et 127 al. 2011). A second mechanism (Sharma et al. 2009) indicated that free cations such as Ag and 128 Zn pass through the cell membrane and interact with the negatively charged DNA/RNA 129 molecules, preventing translation and transcriptional activities. The third potential mechanism 130 was damage done to the pathogen’s cell wall as it passed over or through the zeolite pore 131 (Hrenovic et al. 2012). 6 132 Transition metals in their ionic forms are known to bind with DNA, and zinc, 133 specifically, was utilized in zinc fingers as a regulatory element (Anupama et al 2014, Pierce 134 2020). In some studies, the binding for covalent ions, such as zinc, with DNA showed a strong 135 correlation and was likely interfering with the DNA transcription and regulation of the pathogens 136 being studied (Anupama et al. 2014, Khedr et al. 2011). 137 138 1.5 139 Metal cations and consumption Between 20 and 40mg of zinc is required daily for the activity of approximately 100 enzymes 140 in the human body and plays a role in the immune system, protein synthesis, wound healing, 141 DNA synthesis, and cell division (Anupama et al 2014, Plum et al. 2010). Zinc also supports 142 normal growth and development during pregnancy, childhood, and adolescence, and is required 143 for a proper sense of taste and smell (Plum et al. 2010). A daily intake of zinc is required to 144 maintain a steady state because the body has no specialized zinc storage system (Leda et al. 145 2019). Various studies have shown that zinc is effective against a wide variety of 146 microorganisms, especially those pathogens living in water that cause the gastrointestinal, 147 pulmonary, and skin infections that most commonly affect humans (Gomes et al. 2020). 148 According to the Guidelines for Canadian Drinking Water Quality, the removal of 149 microbiological contaminants, such as bacteria, protozoa and viruses is a high priority 150 (Government of Canada 2017). As a result of challenges with routine analysis of harmful 151 microorganisms that could be present in inadequately treated drinking water, the microbiological 152 guidelines focus on indicators (e.g. E. coli and total coliforms) and treatment goals. In addition to 153 microbiological guidelines, there are chemical and physical parameters for drinking water 154 (Government of Canada 2017, W.H.O. 2019): 155 1. health-based and listed as maximum acceptable concentrations (MAC); 7 156 2. based on aesthetic considerations and listed as aesthetic objectives (AO); or 157 3. established based on operational considerations and listed as operational guidance values 158 (OG). 159 Zinc has AO: ≤ 5.0 mg/L or 5 ppm (AO is based on taste), which is slightly higher than the 160 number from WHO guidelines (W.H.O. 2019). Water with zinc levels above the AO tends to be 161 opalescent and develops a greasy film when boiled. Therefore, it is important to control the 162 release of zinc from modified zeolite to water. Water with copper above AO will be blue in 163 appearance and will be explored as both an antimicrobial agent and indicator. 164 Copper is the third most abundant transition metal in the human body (Osredkar et al. 165 2011). Copper cations are involved in many biological processes including immune response, 166 nervous system maintenance (myelin sheaths) and defense against oxidative stress. Too much 167 copper in a diet is also linked to many diseases, including increased rates of diabetes, and 168 neurological impairments that vary depending on the age of the population (Leda et al. 2019). 169 Some sources recommend roughly 1mg/ day whereas others recommend avoiding copper 170 supplementations due to uncertain side effects of oversaturation. The B.C. safe water drinking 171 guidelines have a minimum acceptable concentration of less than 1 mg/L (1ppm) designated to 172 protect bottle feeding babies (Government of Canada 2017). Keeping the concentration below 2 173 ppm is required to avoid damage to kidneys (Government of Canada); however, other sources 174 report no side effects until water reaches a concentration of 6ppm Cu (Osredkar et al. 2011). 175 Some zeolite researchers have demonstrated that copper modified zeolite could reduce colony 176 forming units by almost 100% in drinking water and synthetic effluent but also indicate that 177 minimal amounts of copper were desorbed into solution (Hrenovic et al. 2012). 8 178 Copper ions have several proposed mechanisms for action against prokaryotic organisms. 179 One method involves the formation of reactive oxygen species (ROS) (Osredkar et al. 2011, 180 Angelova et al. 2011, Pavelkova et al. 2018) through the Haber–Weiss/Fenton reaction to form 181 OH- radicals (Pavelkova et al. 2018). 182 Fenton reaction Cu1+ + H2 O2 → Cu2+ + OH- + ·OH 183 Haber ─ Weiss Cu2+ + ·O2 → Cu+ + O2 184 These free radicals have been shown to cause damage by attaching and inhibiting DNA 185 transcription and regulation (Osredkar et al. 2011). In certain illnesses, the human host and the 186 bacterial infection will battle over the limited reserves of copper in the body, indicating that 187 copper is essential for a successful infection (Pavelokova et al. 2018). Evidently prokaryotes and 188 eukaryotes have developed systems for incorporating limited trace metal resources, and for 189 combating the risks associated with oversaturation (Leda et al 2019). 190 Due to the rarity of silver in natural environments, there are no guidelines addressing 191 silver in drinking standards; however, the BC guidelines for aquatic systems indicate that there 192 should be less than 2ug/L (2ppb) (Government of Canada 2017). Researchers have compared the 193 effect of silver, copper and zinc ions against E. coli and have found that silver is a substantially 194 superior antimicrobial (Prabir and Wang 2019, Janicijevic et al. 2020). Historically, silver has 195 been used for treating injuries to prevent infection (Rosabal et al. 2005, McEnvoy and Zhang 196 2014), and to treat contaminated water; however, in 2022, silver is 230 times the price of zinc, 197 and 75 times the price of copper, making silver-based treatment options extremely expensive. 198 Theoretically, the silver treated nanoparticles will outperform modified materials with copper 199 and zinc (Ravishankar and Jamuna 2011). It is still worth investigating copper and zinc, 9 200 however, as they are much more cost efficient. A tool or system designed with significantly 201 cheaper materials that can still eliminate the contaminants will be more accessible for smaller 202 communities (Warrick and Patrick 2019). 203 204 1.6 205 pH of Water The pH of water plays a significant role in its properties. Commercially sold water ranges 206 from pH 6 - pH 10 (Khan and Chohan 2010. For drinking water purposes, some researchers 207 propose that drinking more basic water will prevent certain illnesses such as cancer (Renal et al. 208 2011) while lower pH beverages might be the result of flavour additives such as lemons and 209 limes. In terms of water quality, pH lower than 6.5 and above 8 will taste different and may be 210 unappealing. Few studies exist; however, sheep have been observed preferring tap water over 211 neutralized AMD indicating a taste difference (Horvath 1985). 212 In natural water tables, the pH greatly affects which organisms thrive and are present in 213 the ecosystem. For example, a shift towards a higher pH will result in more cyanobacteria, which 214 create bad odors, tastes and can be toxic to humans and animals (Government of Canada 2017). 215 Low pH bodies of water are a risk to wildlife, as many species of plants and animals are not able 216 to survive sudden or prolonged exposure to highly acidic water. Microorganisms are also 217 challenged by changes in pH. Extremely basic and acidic solutions are beneficial as 218 antimicrobials (Lee et al. 1997). Cleaners such as vinegar, with a pH of 2-3, and bleach, with a 219 pH of 11-13, are common household cleaners valued for their ability to kill germs and eliminate 220 bad odors. 221 A final consideration is that the free cations and anions in solution will affect the final pH 222 (Zhang et al. 2018). Acidic solutions are known to increase the solubility of transition metals 223 ((Król at al. 2020). There seems to be no significant indication that pH influences the speed of 10 224 dissociation; however, the total amount of cations desorbed will change (Zhang et al. 2018). As 225 the zeolite and the cation treatment dissociate, the competition between H+ and the dissolved 226 metals for ligands (e.g., OH−,CO32-, SO42-, Cl-, S2-, and phosphates) becomes more and more 227 significant (Lee et al. 1997, Zhang et al. 2019). This system may not be comparable as the zeolite 228 framework will not completely dissolve in solution. The ideal pH range for the resulting water 229 will be between 6 and 8 (Government of Canada 2017) and will be monitored by pH probe. 230 231 1.7 232 Pathogen in water There are many water borne pathogens that exist across the world (Fuentes et al. 2014) 233 Among the most common is E. coli, a member of the Enterobacteriaceae family. E. coli is a 234 gram-negative bacterium that is typically found in the guts of mammals and wastewater effluent. 235 As an environmental pathogen, the concentration of E. coli is notably less in lakes and rivers 236 than in ponds, swamps, and municipal wastewater (Hrenovic et al. 2012). The dangers associated 237 with E. coli are specifically present in bodies of water contaminated with fecal material. Rain 238 and snow melt may drive fecal matter into water streams and wells. Natural disasters, such as 239 hurricanes and flooding, can compromise entire wastewater treatment facilities, creating 240 potential for potation to spread within cities and water treatment systems. Most water treatment 241 facilities are effective at removing E. coli from drinking water; however, it is during times of 242 natural disaster or system overload when the risks to human health are the highest. 243 This research elected to use a less infectious strain of E. coli then is typically found in 244 contaminated water. The use of a less infectious strain comes at the risk that this organism might 245 not be a good representative organism for other coliforms, or pathogenic micro-organisms. A 11 246 benefit to using this organism means that the lab equipment and procedures needed for handling 247 the BLS1 organism translate into cost savings for the project and safety for the community. 248 249 1.8 Summary Water resources continue to be fundamental to human existence, and water security is 250 251 important for healthy communities. Despite a multitude of techniques for water treatment being 252 developed to purify contaminated drinking water, there is no singular solution for all situations. 253 This research will test the feasibility of a low cost, easily operated filtration system using a metal 254 modified Canadian zeolite. Zinc and copper modifications are a logical option to pursue, as they 255 have demonstrated some anti-microbial properties and are significantly cheaper than silver. 256 Therefore, silver nano-particles will only be used in certain comparison studies. E. coli is not the 257 only water borne pathogen of concern; however, due to its simplicity to cultivate and the 258 existence of a BSL1 strain, it is a good organism to start with. E. coli is also a ubiquitous 259 organism; therefore, it will provide the largest scope of the possible infectious organisms to 260 target. 261 A group of Cuban researchers have patented a water purification system using zinc 262 modified zeolite (Fuentes et al. 2014). This research team from the Zeolites Engineering 263 Laboratory of Havana have shared some of their zeolite sample, and modified zeolite for 264 comparison during this study. The Cuban natural zeolite (CubanNZ) and Cuban zinc modified 265 zeolite (CubanZZ) will be used as benchmarks for comparisons to the Canadian natural zeolite 266 (CanNZ) and Canadian zinc-modified zeolite (CanZZ). In addition to zinc modification CanNZ 267 will be modified into a copper (CanCu), and zinc/copper hybrid form (CanCZ). 268 12 269 270 271 272 273 274 275 276 277 278 279 280 281 1.9 Research Question 1) Do Canadian natural zeolites from the Bromley River Valley leach unwanted heavy metal contaminants (arsenic, cadmium, lead, mercury, etc.) into water? 2) Will Canadian natural zeolite modified with copper and zinc be able to maintain a steady concentration of treatment ions in solution? 3) Will the metal modified Canadian zeolites be able to eliminate the growth of E. coli over a short period of time (1hour-24hours)? 1.10 Objectives 1) Evaluate the Canadian natural zeolite for undesirable impurities via acid digestion and elemental analysis by ICP-OES 2) Modify the Canadian natural zeolite using pre-established methodologies for metal 282 loading and determine their effects on the elemental composition of the metal (zinc and 283 copper) modified Canadian zeolite. 284 285 286 287 3) Determine if the metal modified Canadian zeolites have the capacity to reduce the colony forming units of E. coli. 1.11 Hypothesis 288 Water treated with Canadian natural zeolites that have been modified with copper and zinc 289 will be able to effect the E. coli colony forming units in solutions over a 24-hour period, while 290 maintaining a concentration of metal ions that meets drinking water safety guidelines . 13 Chapter 2 - Materials and Methods 291 292 293 294 2.1 Collection of Zeolite Samples 295 Zeolite samples were collected from the Bromley River deposit in Kamloops, BC, and analysed 296 in the Northern Analytical Lab (NALS) at the University of Northern British Columbia. Bromley River 297 zeolite deposits are open pit; therefore, representative samples were collected using surface sampling and 298 hand-dug excavation methods. Prior to modification, particles were between powdered and 4mm (-10+18 299 mesh). Samples were sieved into 0.5-1mm, 1-3mm and 3-5mm diameters. At UNBC, characterization 300 tests were performed using X-ray diffraction (XRD), scanning electron microscopy with electron 301 dispersive spectroscopy (SEM EDS) and inductively coupled plasma optical emission spectroscopy (ICP- 302 OES). 303 X-ray fluorescence (XRF) was used to determine the classification of zeolite (Alver et Sakizci 304 2010). X-ray diffraction (XRD) was performed using a Rigaku Miniflex 300 to identify the crystalline 305 structures of the zeolite sample. The surface morphology of the Canadian zeolite was determined using 306 scanning electron microscopy (SEM) operating at 10 keV of acceleration voltage and coupled with energy 307 dispersive X-ray analysis (EDAX). 308 309 2.2 Elemental Analysis 310 Zeolites are characteristically durable and, as such, require strong acids to fully dissolve the 311 structure (Peru and Collins 1993). The use of hydrofluoric acid (HF) is required to destroy a zeolite; 312 however, HF requires special considerations for laboratory safety. Hydrochloric acid and nitric acid are 313 used to digest most soil samples and is well established in EPA Method 3050 (EPA 1996). Powdered 314 Bromley sample was digested along with coarse Bromley sample (1mm-3mm) and a commercial 315 comparator, ZeoDigest. Zeolite samples were digested in solutions of 4NO3:1HCl, 1NO3:4HCl, 316 2.5NO3:2.5HCL and 3NO3:1.5HCl. The tubes were then topped to 15 mL using deionized water (DI). The 14 317 resulting solutions were prepared for analysis using ICP-OES to compare elemental values. The 3.5 NO3: 318 1.5HCl mixture was selected for the subsequent digestions. Ground zeolite samples were dried at 100 oC 319 for 24 hours, digested using the modified EPA method 3050b (EPA 1996) with 2.5ml hydrochloric acid 320 and 2.5mL nitric acid (based on results of the above digestion investigation) and analyzed for elemental 321 composition using ICP-OES (ECS 4010 CHNS-O Analyzer, Costech Analytical Technologies Inc.) with 322 a modified EPA method 200.2 (EPA 1994). 323 324 2.3 325 Cation Exchange Capacity (CEC) Cation exchange capacity (CEC) was determined using established ammonium acetate procedures 326 (Kitsopoulos 1999). Powdered zeolite was dried in a 100OC oven for 24 hours. Then, 10 mL of 1 molar, 327 pH 7 solution of ammonium acetate was added to 1 g of each zeolite sample. The ammonium acetate 328 solution was changed once every 24 hours for three days. At each exchange, the supernatant was 329 discarded. After 72 hours, 10 mL isopropanol was used to wash the samples to remove excess ammonium 330 acetate. The zeolite samples were then dried at room temperature for 24 hours. Ammonium ions were 331 replaced within the zeolite structure by washing six times with 10 mL of 10% NaCl. The wash was saved 332 for ICP- OES analysis of sodium ion concentration. 333 Zeolites possess a natural positive charge due to the structure of the aluminum, oxygen and silica 334 tetrahedron structure. This charge allows the material to readily exchange ions with the surrounding 335 medium. Analyzing the zeolite’s ability to exchange ions was part of the initial characterization and 336 allows for an estimation of potential loading and desorbing. The cation exchange method was based off 337 previous work from Greek zeolites (Kitsopoulos 1999) and employed a variation of the Berthelot method 338 adapted for the AA3 auto analyzer (Garcia and Baez 2012) (Kanda 1995) (Ming and Dixon 1987) 339 (Hendershot and Laland 1993). 340 Zeolite samples were ground to a powder less than 125 um in diameter. Between 100 and 150mg 341 of dried sample was transferred to 10mL of 2M NH4OAc pH7. The zeolite was placed on a shaker and the 15 342 solution was changed every 24 hours, three times, to ensure each ion exchange site was filled with 343 ammonium. The NH4OAc solutions were saved for later analysis by ICP-MS for cations Na, K, Ca and 344 Mg. The resulting rinse solutions were diluted in HCl to match the matrix solution for the ICP and 345 compared for the cations mentioned above. Each rinse solution was centrifuged at 10, 000 rpm for 5 346 minutes to separate the supernatant and zeolite components. After the ammonium loading, the zeolites 347 were rinsed five times with warm DI- and five times with isopropanol. Each wash was shaken by hand 348 and centrifuged at 5000 rpm for 10 minutes before decanting. The final samples were left to dry in the 349 oven at 30OC for 24 hours before continuing. 350 The dried zeolite samples were weighed and transferred into new 15 mL falcon tubes. The zeolite 351 samples were exposed to six rinse solutions of 10 mL 10% NaCl acidified to 0.005M HCl. The rinse 352 solutions were saved in 100mL volumetric flasks. The final CEC analysis was performed using ICP-OES 353 following EPA method 9081 (EPA 1986). Commercially available natural zeolite products from United 354 States (e.g. KMI from Nevada) and from Cuba were used as benchmark samples to be compared with the 355 Canadian natural zeolite and modified zeolites (Table 3-2). 356 357 2.4 358 Modification of zeolite The selected sample was purified using a sodium chloride rinse (0.5M NaCl) in order to remove 359 water-soluble impurities. The zeolites were washed in a 1:10 mass to mass (m/m) ratio for 24 hours at 360 room temperature. The resulting zeolites were rinsed with 10 mL of DI on repeat until the solution was 361 clear of sodium (roughly 30-50 mL) (Yeasmin, et al. 2016). Sodium was detected through the use of 362 silver nitrate drops, which formed white precipitate in solution with sodium cations. 363 Canadian natural zeolites, converted into sodium form following the above method, were used to 364 create zinc-modified Canadian natural zeolites following the methods outlined by the Cuban ZZ project 365 (Fuentes et al. 2014). The reaction was allowed to take place at 100oC for 24 hours. The resulting 16 366 supernatant was decanted and the modified zeolites were washed several times with DI until the rinse was 367 clear. The newly formed zeolites were then transferred to a metal tray and dried at 100oC for 24 hours. A 0.5M ZnSO4•7H2O solution was prepared for zeolite modification. The mass-to-mass ratio of 368 369 solution and sodium form zeolite was 10:1. Six Erlenmeyer flasks were used to soak 10 g of zeolite in 370 100 mL of the zinc sulfate solution. The flasks were then placed in a temperature-controlled shaker for 24 371 hours at 30oC. Three different zinc sulfate solutions were created in 250 mL volumetric flasks. 50 mL of 372 solution was saved from each flask for elemental analysis to determine the efficiency of the loading. 373 After the zinc sulfate and zeolite exposure had occurred, the Erlenmeyer flasks were decanted 374 into falcon tubes for further elemental analysis. The zeolite was then rinsed three times with 50 mL of DI 375 water and dried at 100oC. 1.00 g of sample was saved from each reactor flask for future analysis, and the 376 other zeolites were mixed into one homogeneous sample. A 0.5M CuSO4•5HO solution was prepared 377 and used in the same manner as the 0.5 M zinc solution for modification. A mixture of 0.5M CuSO and 378 0.5M ZnSO was prepared following the same procedure as above. 379 Sample Calculations (Skoog 2014): 380 (287.56g/mol ZnSO*7HO) x (0.5mol/1000mL)x (250mL/1 solution)=35.95gZnSO*7HO 381 (249.68g/mol CuSO*5HO) x (0.5mol/1000mL)x (250mL/1 solution)=31.21g CuSO*5H0 382 383 384 2.5 Ion Chromatography Ion chromatography was utilized to determine the amount of sulphate present in solution after 385 modified zeolite had been combined with E. coli treated water. Nitrates, carbonates and nitrites were also 386 investigated using ion chromatography. CanNZ, CanZZ, CubanNZ, CubanZZ and SSZ were place in a 387 1:100 m/m ratio with water collected from Tabor Lake, Prince George, BC. Samples were placed on a 388 shaker for 24 hours, then filtered for particulate matter and transferred into ion chromatography tubes. 17 389 The analysis was performed with a pump speed of 1.5 mL/min running 23mmol KOH eluent with 390 suppression ASRS of 4 millimeters at 86 milliamps. 391 392 2.6 E. coli Acquisition and Storage 393 E. coli is often used as an indicator organism for safe water drinking (Hrenovic et al. 2012) 394 (Fuentes et al. 2014). E. coli strain ATCC8793 was ordered from The American Type Culture Collection 395 (ATCC) in an 8-mini pack (Product number ATCC-8739-mini-PACK). The bacteria were stored as per 396 supplier instructions at -20oC. One of the eight packs was propagated into nutrient broth and incubated for 397 18 hours at 37oC. Glycerol was added to the nutrient broth (Difco BD 234000) to form a 10% volume to 398 volume (v/v) mixture. 200 uL of E. coli were transferred to sterile Eppendorf tubes and re-frozen at -20C 399 for use in the anti-bacterial trials. 400 401 2.7 Minimum Inhibitory concentration (MIC): 402 The effect of zinc on the growth rate of E. coli, had to be verified. A small test was performed 403 using an Optical Emission Spectrometer (OES) and E. coli grown in increasing concentrations of zinc 404 solution. A sample of frozen zeolite was thawed and transferred into 10 mL of sterile nutrient broth. The 405 nutrient broth was then inoculated with E. coli and spiked with zinc sulphate. The range of zinc was 0, 406 0.2, 0.5, 1, 2, 3, 4 and 5ppm in each test tube. The test tubes were incubated at 37oC for 24 hours, then 407 analyzed for optical density at 600nm wavelength. 408 409 2.8 410 Timed desorption of cations The rate and profiles of the zeolite desorption had to be established prior to application on the 411 target organism. 1 g of CanNZ, CanZZ, Cuban ZZ and SS zeolites were placed in 100 mL of sterile Tabor 412 Lake water. The flasks were shaken for 24 hours and sampled at 10, 30, 60 180 300 720 and 1440 413 minutes. Triplicates were performed for mean and standard deviation.Antimicrobial assay in Tabor Lake 414 water with metal modified zeolites 18 415 416 To compare the effectiveness of modified zeolites on E. coli growth, 0.1 g of CanZZ, CanNZ, 417 synthetic silver zeolite or CubanZZ were added to 100 mL of autoclaved Tabor lake water. The lake water 418 was then spiked with approximately 2000 cfu/mL of E. coli. The resulting solution was incubated at 37oC. 419 One set of samples was analysed after one hour of exposure and another set of samples was analysed at 420 three hours. The procedure was performed in triplicate to compare mean and standard deviation (n=10). 421 Samples were analysed for E. coli and coliforms with the IDexx quant-tray 2000 technique (Rice, et al. 422 2012). 423 424 2.9 425 Colony counting Bactericidal effects of the developed media on micro-organisms present in drinking water will be 426 studied using reference strain ATCC 8793 for E. coli. The contact time was varied (30min, 1 hour, 2 427 hours), and the amount of zeolite in solution was held constant (1g/100mL). The most efficient media was 428 then used to test actual water samples. The Colilert and membrane filtering techniques were used for 429 bacteriological studies (Rice et al. 2012). The Colilert method simultaneously detects and quantifies both 430 total coliforms and E. coli 24 hours after sampling. The known amount of zeolite was in contact with un- 431 modified and modified zeolite at a given temperature and pH. At a given time, the zeolites were removed 432 from the treated water. The post copper, zinc and copper/zinc modified Canadian zeolites were dried at 433 100oC for 24 hours, and digested using the modified EPA method 3050b for elemental analysis (EPA 434 1996). 435 436 2.10 Cu and Zn Modified Canadian Zeolites on E. coli in Nutrient Broth 437 The effects of CanCU, CanZZ, and CanCZ, on E. coli in nutrient broth was analysed using the 438 IDEXX Quanti-Tray 20, 000 (Rice et al. 2012). A sample of E. coli was inoculated into 100 mL of 439 nutrient broth for 18 hours then 1 mL of grown culture was transferred into sterile 100 mL portions of 440 nutrient broth. Four replicates for 0.5 g, 1.0 g, 2.0 g, and 2.5 g of CanCU, CanZZ, and CanCZ were mixed 19 441 into individual flasks of inoculated broth and incubated at 37OC for 24 hours. All cultures of E. coli were 442 analysed using the IDEXX Quanti-Tray 20, 000 method and compared using mean, standard deviation 443 and data analysis described below. 444 445 2.11 Final pH of solution 446 The initial and final pH of solutions treated with metal modified Canadian natural zeolite was 447 assessed using an Orion pH probe. The pH probe was calibrated three times with pH 3, pH7 and pH 10 448 solution. A solution of DI was used to rinse the probe between samples (Orion 2011). 449 450 2.12 Data Analysis 451 The final pH of solution was analyzed for normality using a Shapiro-Wilk. Due to non-normal data, a 452 Kendall- Theil regression was applied. Following, a Levene’s F-test a Krustal Wallis was applied to 453 determine the effects of final pH on E. coli cfu. 454 The effects of metal modified Canadian zeolite were analysed for normality using a Shapiro- 455 Wilk. Following the normal data analysis, a linear regression (R2) was determined along with a Cook’s 456 distance less than 1. A Levene’s F test was then applied, followed by a 1-way ANOVA. 457 The effect of the metal modified zeolites on the colony forming units of E. coli in inoculated growth 458 media was analysed statistically using SPSS software. The entire dataset for post treatment E. coli colony 459 forming units was normalized (log base 10). The data was then analyzed for normality using both a 460 Shapiro-Wilk test (a=0.05). Once the data was determined to be normal, a regression analysis was 461 performed with an investigation for collinearity between the mass of zeolite and the type of modification 462 used. Probability plots were formed as well to investigate the distribution of values along with the 463 Standard Residual. Cook’s Distance was investigated with a limit of 1.0. This data set (n=48) was then 464 analyzed using a Pearson-Correlation and Linear Regression. To determine if there were differences 465 between the effects of the metal modified Canadian zeolites, a Levene’s F Test was applied to determine 20 466 homogeneity of variance. This data set did not posses equal variances; therefore, a Krustal-Wallis analysis 467 was applied. 468 21 469 Chapter 3 - Results and Discussion 470 471 472 3.1 Leaching of Toxic Elements 473 474 3.1.1 Digestion 475 Zeolites are characteristically stable structures and therefore typical digestion methods use 476 hydrofluoric acid (HF) (Boles et al 1977). Barring access to HF, several combinations of nitric acid and 477 hydrochloric acid were attempted (Table 3-1). The implication of incomplete digestion is that small 478 amounts of cations locked within the silicate structure of the zeolite might not be represented during 479 analysis. These protected ions would impair the accuracy of the elemental analysis. EPA method 200.2 is 480 typically used to digest soils and sludge, while only employing (NO3) and (HCl)(EPA 1994). Two 481 replicates were digested per mixture of acid (n=8). The mean was calculated, and all variations were 482 compared to the EPA method 200.2(EPA 1994). Visually, there were still intact masses of zeolite in the 483 base of the acid bath; however, the elemental analysis in (Table 3-1) allows for some comparison of the 484 digestion methods. Table 3-1 indicates that toxic elements; Pb, As, Cr and Ti are recovered in comparable 485 quantities despite the mixture of HCl and NO3. Given the consistent measurements (Table 3-1) the 486 applied digestion method of 1.5HCl:3.5HNO3 was acceptable (Figure 3-1). 487 488 489 490 491 492 22 493 494 Table 3-1:The difference between the mean of Canadian natural zeolite digested with HCl and NO3 and EPA method 3050b( EPA 1996. COLUMN1 As B Ba Be Ca Cd Ce Co Cr Cu Fe K Mg Mn Na Ni Pb Ti Zn 4HNO3 :1HCL 2mm 0.00 0.97 86.59 0.32 1480.45 0.14 10.14 0.54 0.86 1.67 859.95 4486.47 320.16 20.00 1501.56 0.96 2.69 15.97 15.69 1HNO3 :4HCL 2.5HNO3 :2.5HCL 2mm -1.14 -0.54 26.05 0.19 348.93 0.05 9.80 0.56 0.09 0.56 -659.43 2925.22 35.11 10.98 1024.78 0.24 2.92 -66.36 2.50 2mm -0.65 0.71 62.38 0.28 1098.04 0.07 6.32 0.36 -0.30 1.51 45.93 3176.02 90.22 12.35 1199.66 0.45 5.16 -13.85 7.61 4HNO3 :1HCL 1HNO3 :4HCL 2.5HNO3 :2.5HCL Ground -0.71 0.80 76.38 0.17 1190.59 0.11 -20.92 0.14 2.36 0.67 998.29 2109.09 328.04 9.21 530.57 0.12 1.79 39.47 12.00 Ground 0.37 -1.91 -28.44 -0.02 -371.5 -0.15 46.34 -0.58 -49.33 -3.20 -1703.80 378.30 -173.24 -5.56 -2.80 -1.55 -1.13 -105.26 -14.31 Ground 0.71 -0.80 -76.38 -0.17 -1190.59 -0.11 20.92 -0.14 -2.36 -0.67 -998.29 -2109.09 -328.04 -9.21 -530.57 -0.12 -1.79 -39.47 -12.00 495 496 497 3.1.2 Post Treatment Elemental Analysis . 498 The Canadian metal modified zeolites were analysed pre- and post-bacterial treatment for 499 comparison to the natural zeolite. Table 3-2 shows the copper and zinc modification greatly increased the 500 amount of copper and zinc from the natural Canadian zeolite, and that only a small portion of the 501 additional cations are used in this treatment. 502 503 504 23 505 506 Table 3-2 Analysis of modified zeolites and post bacterial trial zeolites for the elements listed column (mg element/kg of sample). Metals (mg/kg) Zn Zeolite As B Ba Ca Cr Cu Fe Hg K Mg Mn Na Ni P Pb S Zn <9.00 2.00 373.53 3342.00 0.97 8.00 1410.00 <3.75 13728.00 1058.00 8.00 8167.00 1.00 47.50 10.40 111.00 11526.00 Zn Cu Zeolite Cu Zeolite Zeolite Bio Bio <9.00 <9.00 15.00 3.00 2.00 2 331.23 417.08 378.67 3308.00 2998.00 2844.00 1.24 1.43 1.29 11.00 11945.00 6088.00 1539.00 1476.00 1739.00 <3.75 <3.75 <3.75 18666.00 14234.00 22601.00 939.00 1060.00 984.00 7.00 7.00 9.00 6321.00 7571.00 7162.00 1.00 1.00 2.00 268.50 272.60 <14.00 10.10 25.80 29.60 89.00 577.00 231.00 10103.00 55.00 49.00 Cu/Zn Zeolite <9.00 2.00 331.89 2963.00 1.23 7195.00 1884.00 <3.75 12665.00 903.00 11.00 7054.00 2.00 <14.00 23.10 2001.00 7083.00 Cu/Zn Zeolite Bio 17.00 2.00 411.07 3355 0.98 2942.00 1658 <3.75 20011.00 1105.00 5.00 5929.00 2.00 260.30 24.00 98.00 4203.00 507 508 Comparing initial readings with the post modified zeolite and the post application zeolite (Table 509 3-1 with Table 3-2) demontrated, toxic ions did not leach into solution. The elements As, Cd, Cr and Ti 510 were extracted during the conversion of Canadian natural zeolite into metal modified Canadian zeolite 511 (CanZZ, CanCu and CanCZ). The element Pb, however, did not reduce in quantity from initial analysis 512 (Table 3-1). Instead, the concentration of Pb in solution increased when treated with metal modified 513 Canadian zeolite (Table 3-5). This suggests that the CanNZ has a high affinity for Pb, and that initial 514 digestion may not have revealed the full extent of toxic cations present within the zeolite structure. Table 515 3-1 showed that there is a low amount of lead available in the zeolite structure; however, Table 3-2 516 showed that metal modified zeolite possessed more of this toxic ion (2-5ppm vs 10-25ppm). It was clear 517 from this comparison, that the digestion method used to analyze the natural zeolite did not fully represent 24 518 the composition of the material. The increase in total cations indicated that HF digestion methods were 519 more appropriate for zeolite analysis (EPA 1994). Additionally, it was important to recognize the 520 competition between cations within the zeolite structure (Orha et al. 2011) because the copper and zinc 521 cations might have out competed the lead cations for binding sites. The increase in lead detection was 522 likely due to the zeolite’s preference for copper and zinc cations over lead cations. This preference 523 allowed a more favourable set of conditions for lead cations to release into the acid bath (Orha et al. 524 2011). Consequently, the digestion of silicate-based material using hydrochloric and nitric acid was 525 insufficient (EPA 1994). 526 One limitation of this thesis is the lack of replicates and statistical application on early 527 experiments. More replicates would have provided a stronger empirical data set from which to draw 528 conclusions. Including a known silicate-based standard in digestion would have informed the completion 529 of a digestion method, which would address the issues encountered using hydrochloric and nitric acid. 530 The use of a known standard would have allowed for some form of correction value to be generated in the 531 case of discrepancy (Ming and Dixon 1987). Further replication would improve the empirical nature of 532 the digestions by bringing the mean calculations closer to the true value of the zeolite material, and could 533 be applied in predictive models. 534 535 3.1.3 Physical characterization 536 The undigested material was analyzed using SEM-EDS and XRF (Figure 3-1,Table 3-3). 537 Chemical composition of the clinoptilolite phase of Canadian natural zeolite tuff was obtained by EDS 538 analysis. The aluminum silica ratio of 5.8 was determined by SEM-EDS (Table 3-3) and supported the 539 identification of Bromley River Valley zeolite as clinoptilolite (Boles, et al. 1977). Additionally, the XRF 540 (Figure 3-1) matched database comparisons (Alver and Sakizci 2010). The energy dispersive X-ray 541 spectroscopy (EDS) of the Canadian natural zeolite sample provided the chemical composition of the 542 Canadian natural zeolite. In comparison with known literature, this sample matched the chemical 25 543 composition of clinoptilolite, shown in Table 3-3 (Boles et al. 1977, Hrenovic et al. 2012). The scanning 544 electron micrograph, when applied to the Canadian natural zeolite, revealed its crystalline structure and 545 the X-ray fluorescence read out matched the layout of clinoptilolite, as shown in Figure 3-1 (Alver and 546 Sakizci 2010). 547 Table 3-3: Chemical composition of the clinoptilolite phase of Canadian natural zeolite tuff obtained by EDS analysis. SiO2 TiO2 Al2O3 Fe2O3 MnO MgO CaO K2O Na2O P2O5 BaO SrO L.O.I. Total(%) 70.18 0.15 12.09 1.40 0.00 0.56 1.70 3.90 0.86 0.01 0.05 0.02 8.83 99.75 548 549 550 Figure 3-1: Scanning electron micrograph and XRF readings for the Canadian natural zeolite indicating clinoptilolite sample 551 552 3.1.4 Cation Exchange Capacity (CEC) 553 The cation exchange capacity of the Canadian natural and Canadian modified zeolites was low in 554 comparison with the comparator zeolites (Table 3-4). The zeo-digest and the KMI zeolites possessed a 555 CEC more than twice as high as the Canadian zeolites. The Cuban zeolites measured at a closer CEC to 556 the Canadian materials; however, the CubanNZ was still 1.5x that of the CanNZ. The difference in CEC 557 may have contributed to the differences in desorption profiles. The Cuban zeolite displayed a slow release 558 of zinc cations (Figure 3-3). The Canadian zinc modified zeolite released more zinc rapidly, despite being 559 modified using the same material and methodology. 26 560 Ion preference may have also played a significant role in the desorption of treatment ions. Table 561 3-4 indicated that the CanZZ adsorbed more of the Mg in solution than the CubanZZ. If the Canadian 562 zeolite significantly prefers the ion mixture of Tabor Lake water, then it will readily exchange more 563 cations than the CubanZZ. Further investigation into the Canadian zeolite’s ion preferences could lead to 564 discovering preferred environmental conditions that facilitate a lower total zinc desorption and a slower 565 desorption profile (Kistsopoulos 1999). 566 The Cation Exchange Capacity (CEC) of Canadian zeolite and its zinc modified forms were 567 compared with (a) a zeolite food additive, ZeoDigest, (b) the Cuban natural zeolite, (c) Cuban zinc 568 modified zeolite and (d) KMI Nevada deposit. The CEC values obtained for the comparators were much 569 higher than the CEC values for the Canadian zeolites. The Canadian Natural Zeolite had a lower CEC 570 value than any of the comparators. Finer granules of zeolite also registed a smaller CEC value (Table 3-4) 571 Table 3-4 The mean cation exchange capacity (n=12) cation exchange capacity for natural and metal modified zeolites. Sample Name Zeodigest meq/100g 293.25 CubanNZ CubanZZ 148.66 113.58 KMI CanNZ powder CanNZ 2mm 220.44 94.47 103.57 572 573 574 3.2 Controlled Desorption of Treatment Ions 575 576 3.2.1 Cation Exchange with Environments 577 The elemental desorption for CanNZ, CanZZ, CubanZZ and Synthetic Silver zeolites were 578 compared over a 24-hour period (Figure 3-2). Figure 3.2 displays the natural Cuban and Canadian zeolites 579 in comparison with their zinc modified formats against the background elemental composition of Tabor 580 Lake and a synthetic silver zeolite. There was no detectible silver analyzed in any other sample than the 581 ones containing the synthetic silver zeolite, used as a baseline comparator. 582 The Synthetic Silver zeolite performed as expected by eliminating all E. coli CFU in solution 583 (Table 3-5). There was no Ag in solution unless SS zeolite was present, and quickly after contact, the 27 584 concentration of Ag increased rapidly (Figure 3-2). As the zeolite performed ion exchange with the 585 solution, the Ag concentration plateaued due to complete desorption or a limit on exchanged had been 586 reached (Alsammarraie et al. 2018) (Ravishankar and Jamuna 2011) (Orha et al. 2011). In Figure 3-3, the 587 concentration of Ag plateaued as the concentration of magnesium approached 0ppm, indicating that the 588 synthetic silver zeolite is reached an ion exchange limit with the Tabor Lake water which prevented 589 further Ag cations from being released. It is possible that there was more silver that could be desorbed but 590 didn’t have a counter ion for exchange (Orha et al. 2011) (Filali et al. 2000). 591 Magnesium was one of the best exchange ions for clinoptilolites, as demonstrated in Figure 3-2. 592 The silver zeolite had the most exchange occurring with the magnesium, followed by the CanNZ. The 593 zinc modified zeolites demonstrated the least amount of exchange. Sodium was another readily available 594 cation for exchange and appeared to increase in solution for every zeolite except for the CanZZ, which 595 indicated a release of sodium by the zeolitic material. 596 The CubanZZ released zinc into solution; however, the CubanZZ maintained a lower 597 concentration of zinc in solution over time. The Cuban ZZ maintained roughly a 5ppm concentration of 598 zinc which is consistent with their documentation (Fuentes et al. 2014). This concentration is acceptable 599 for drinking water standards (The Government of Canada 2017). The release of zinc into solution by the 600 CanZZ was three times greater than standards and continued to increase over time (Figure 3-2). The 601 minimum zinc concentration was above the drinking water standard and possibly indicates insufficient 602 washing during the modification process (Figure 3-2). The CubanZZ also did not consume all of the 603 available cations for exchange such as Mg. This zeolite seemed to maintain a 5ppm equilibrium with 604 solution that is desirable for drinking and long-term storage. 28 Concentration of Ag (ppm) 120 100 80 60 40 20 0 0 10 20 Concentration of Mg (ppm) B A 140 4 3.5 3 2.5 2 1.5 1 0.5 0 CanNZ SilverZeo CubanZZ CanZZ 0 Exposure Time (Hours) D 30 20 15 10 5 0 0 5 10 15 Time of Exposure (Hours) 20 Concentration of Zn (ppm) Concentration of Na (ppm) C 25 607 608 609 20 Exposure time (Hours) 605 606 10 25 20 15 10 5 0 0 10 20 Time of exposure (Hours) Figure 3-2:The mean and standard deviation for metal ion desorption of metal modified Cuban Zeolite (CubanZZ), zinc modified Canadian zeolite(CanZZ), natural Canadian zeolite (CanNZ), natural Cuban zeolite (Cuban NZ) and synthetic silver zeolite (n=10). 610 The Cuban zinc modified and natural zeolite released a large amount of sodium into solution 611 (Figure 3-2). The expectation was that there would be limited sodium desorption from the zinc modified 612 zeolite; however, this sodium may have been left over from the modification process. In this 613 concentration of 20-25ppm, sodium is closer to oceanic salt water than desired drinking water conditions. 614 20ppm NaCl is above the tolerance of many prokaryotic species including E. coli. It is possible that the 615 CubanZZ includes NaCl in its mechanism of action as a disruption to cell membranes by lysis (Pierce 616 2020). 617 The CanZZ had a very sudden release of zinc into solution, and it provided more zinc as time 618 passed; however, it did not plateau as rapidly and may have taken longer than 24 hours to reach 619 equilibrium with solution. The CanZZ also did not fully exchange with Mg in solution, indicating that 620 there were still viable cations for exchange. With this information, it would not be considered safe to 29 621 drink water treated with the CanZZ in a volume ratio of 1g per 100mL, instead, it would be appropriate to 622 perform further tests with a larger volume of water to see if the CanZZ could maintain a concentration of 623 Zn in solution that meets drinking water guidelines (The Government of Canada 2017). 624 625 3.2.2 Ion chromatography Sulphates 626 Ion chromatography was utilized to determine the amount of sulphate present in solution after 627 modified zeolite had been combined with E. coli treated water. Figure 3-3 displays the change in sulphate 628 concentration in Tabor Lake water after the addition of natural and metal modified zeolite. The Cuban 629 zeolites leeched sulphate in solution up to 15ppm for the natural zeolite, and over 25ppm for the Cuban 630 zinc modified zeolite. There was no change in sulphate quantities detected in assays with synthetic silver 631 zeolite, CanNZ or CanZZ. Nitrates, carbonates, and nitrites were also investigated using ion 632 chromatography; however, no changes were observed. Sulphate (ppm) 30 25 20 15 10 5 0 CanNZ 633 634 CanZZ CubanNZ Zeolite CubanZZ SilverZeolite Figure 3-3: The mean sulphate concentration( ppm)) for duplicate treatments in Tabor Lake water (n=10). 635 Sulphate concentration of solutions can have an effect on the microbial community in solution 636 (Aarestrup and Hasman 2004). The modification for the treated zeolites was performed using zinc and 637 copper sulphates and, therefore, it was pertinent to determine if the Canadian modified zeolites were 638 releasing sulphate into solution. Figure 3-3 shows that the CanNZ, CanZZ and SS zeolites released trace 639 amounts of sulphate into solution. In contrast, the CubanNZ and the CubanZZ released more sulphate into 640 solution. 30 641 The Canadian and Cuban zeolites were clinoptilolites and were modified with zinc following very 642 similar reaction conditions (Fuentes et al. 2014). Sulphates attached to CubanZZ and not the CanZZ 643 indicated potential disparities in the washing and loading procedures performed by the two different 644 research groups. Further collaboration between the two research groups may identify the origin of the 645 sulphates in the CubanZZ, or a method for maintaining the sulphate within the CanZZ for use as an anti- 646 microbial. 647 648 3.2.3 Final pH of Treated Solutions 649 The final pH of nutrient broth was analysed for normality using the Shapiro-Wilk and for equal 650 variances using Levene’s F-test. The data collected from the final pH was neither normal (Shapiro-Wilk 651 p=0.022,431 >2,431 <1 <1 >2,431 >2,431 3 hour exposure Mean E.coli Mean coliform cfu/100mL cfu/100mL <1 <1 >2,431 >2,431 <1 <1 488 461 <1 <1 <1 <1 >2,431 >2,431 >2,431 >2,431 709 710 In contrast, the CanZZ was more successful at killing the E. coli within the first hour of contact. 711 This might be explained by the different release profiles of the zeolites (Figure 3-3). The CanZZ had a 712 much more immediate release of zinc into solution and maintained at a higher concentration than the 713 CubanZZ (Figure 3-3). 714 715 3.3.3 The Effect of Zn and Cu modified Zeolites on E. coli in Nutrient Broth 716 The effect of metal modified zeolite on the mean Log10CFU of E.coli, was strong, as 717 demonstrated by the R2 values in (Table 3-6). Based on the Krustal-Wallis analysis there was no 718 significant difference between the effects of metal modified zeolite on the target organism. Despite the 719 strong R2 values, the E. coli numbers never reached values acceptable for human consumption 720 (Government of Canada 2017). In addition to the concerns with E. coli cells, the treated water did not 721 maintain a safe drinking standard for either zinc or copper. The copper concentration reached over ten 722 times the safe drinking standards, and the zinc concentration was more than double. The CanZZ zeolite 723 displayed the highest R2 value; however, the CanCZ treatment desorbed double the amount of zinc. If 724 zinc were the main active ingredient in E. coli reduction, then the CanCZ should have performed much 725 better than the CanZZ treatment. Additionally, some researchers have published on the success of copper 726 modified filtration units using substantially lower copper concentrations (Ayben and Ulku 2004). 34 727 E. coli typically thrives in the bodies of mammals and enters the environment through biological 728 secretion, where it is found in high concentrations (Hunter 2003). Testing metal modified zeolite on the 729 elimination of E. coli required an environment that was more favorable than the autoclaved Tabor Lake 730 water. Copper and zinc modified zeolites were tested on E. coli in nutrient broth and strong antimicrobial 731 effects were observed (Figure 5-6); however, the metal modified zeolite desorbed more zinc into solution 732 than is acceptable by drinking water guidelines (Government of Canada 2017). In all three trials with 733 metal modified Canadian zeolite, the concentrations of copper and zinc were far more elevated than is 734 acceptable by drinking water standards. Based on the regression analysis performed, the CanCu zeolite 735 had the strongest effect on the E. coli (Table 3.5 R2=0.771>0.495>0.446); however, the Kruskal-Wallis 736 analysis revealed that there was no significant difference between groups (p=0.054>a=0.05). 737 35 738 739 A Zinc concentration (ppm) Log10 mean of n=4, CFU/100mL E.coli 12.00 10.00 8.00 6.00 4.00 2.00 R2=0.49 0.00 0 1 20 18 16 14 12 10 8 6 4 2 0 2 0.5 Mass of CanZZ (g) R2=0.77 160 140 120 100 80 60 40 20 0 0.5 1 2 1 2 2.5 Mass of CanCU (g) C Log10 mean of n=4, CFU/100mL E.coli Concentration in solution (ppm) Mass of CanCU (g) 9 8 7 6 5 4 3 2 1 0 742 743 744 2.5 Mass of CanZZ(g) Cu concentration (ppm) 10 9 8 7 6 5 4 3 2 1 0 0 741 2 B Log10 mean of n=4, CFU/100mL E.coli 740 1 2 R =0.45 0 1 Mass of CanCZ (g) 2 120 100 Copper Zinc 80 60 40 20 0 0.5 1 2 2.5 CanCZ (g) Figure 3-6The relationship between the mass of metal modified Canadian zeolite treatment and the loss of colony forming units by E. coli after 24 hours of exposure. 745 36 746 The success of the CanZZ on solutions containing 2000cfu of E. coli in Tabor Lake water, as 747 shown in Table 3-3, justified testing solutions more favourable to E. coli. The line graph in Figure 3-6 748 displays the mean and standard deviations (n=16) E. coli cfu after 24 hours of exposure with metal 749 modified Canadian zeolite. The associated bar graphs demonstrate the mean and standard deviations 750 (n=16) of the active cations in solution after 24-hours of treatment. The copper and zinc concentrations 751 achieved in solution were far above the safe drinking standards and the E. coli was not reduced to zero 752 colony forming units in any of the trials (Breskie et al. 2017, W. H. O. 2019, Environment Canada 2022). 753 The zinc leached in nutrient broth (Figure 3-6) was comparable with the quantity of zinc leached 754 into Tabor Lake water by CanZZ (Figure 3-2). After 24 hours, 1 gram of CanZZ released 12ppm zinc 755 into nutrient broth (Figure 3-6) while 1 gram of CanZZ released 17ppm zinc in Tabor Lake water. This 756 variation may be due to a difference in counter ions for which zinc could exchange or it may be due to 757 natural variation within the zeolite sample (Orha et al. 2011, Kallo 2001, Filali et al. 2000). The E. coli 758 bacteria was spiked at a higher concentration than in Tabor Lake trials (108 to 1010 cfu) which may have 759 played a factor in the survival rate. A comparable paper testing zinc modified Croatian zeolite (Hrenovic 760 et al. 2012) commenced their trials with 106-108 cfu E. coli. They reported that their zeolites were able to 761 eliminate up to 100% of the E. coli over the period of 24 hours. They also indicate that minimal changes 762 in pH were observed; however, they do not report their final cation concentrations in solution (Hrenovic 763 et al. 2012). 764 The antibacterial activity recorded during the Tabor Lake and MIC experiments follows 765 expectations outlined by previous researchers (Hrenovic et al. 2012, Fuentes et al. 2014). Table 3-5 shows 766 that the metal modified, and silver synthetic zeolites can reduce the quantity of E. coli to zero in a very 767 short time; whereas, the Canadian natural zeolite has next to no effect. 768 769 37 770 771 772 3.4 Avenues for Future Research or Improvement Smaller inoculations of E. coli are recommended for analysis in future studies. Large quantities of 773 organism required extended periods for growth in stable conditions and lead to large differences in 774 organism counts. During the metal modified Canadian zeolite trials on E. coli, it was challenging to 775 maintain a consistent amount of starting organism. The CanCu, CanZZ and CanCZ zeolites were tested 776 against different starting concentrations of E. coli, which might have had an impact on the effectiveness 777 of three treatments. Additionally, working with elevated colony counts required multiple dilutions, 778 inoculations and related transfers between glassware and media, which may have compounded errors, 779 expressing themselves in large standard deviations such as those in Figure 5-5. The volume of solution 780 per sample was also challenging. Large volumes of nutrient broth required long periods of autoclaving 781 and substantial volume to develop a sample size from which to draw statistical significance. 782 Colony counting techniques are widely used and considered a good technique for quantifying the 783 number of coliforms in environmental solutions, however; the process required substantial large frames 784 for analysis to ensure that the counts would fall within detection (Burlage et al. 1998). The method 785 applied during the MIC determination addressed concerns of timing and volume. The spectrophotometer 786 detected the density of cells and can be related to CFU/mL and OD600nm, prepared in advance. The costs 787 were low, requiring only disposable or washable cuvettes. Using smaller solution sizes of 10mL in test 788 tubes, instead of 100mL in Erlenmeyer flasks, would allow the experimenter to perform ten times the 789 analyses for a similar amount of broth prepared. 790 In a colony counting design, timing is important as the organisms will continue to grow or be 791 eliminated as time progresses. If a sample falls outside of the range covered by the plates, then it is 792 challenging to redo a sample. However, if a tube of broth were too thick with target organism, a dilution 793 could be performed immediately and analysed immediately after. The rapidity of this correction would 794 protect the researcher from errors due to growth and time discrepancies. Relating the OD600nm to a 38 795 CFU/mL would only require one round of plating to create a relationship curve between cells and 796 OD600nm in a certain solution. It is recommended that future studies apply the use of a 797 spectrophotometry either in conjunction with colony counting, or in lieu of, to simplify the research. 798 799 Chapter 4 - Conclusions 800 Canadian Natural zeolite could play a role in water filtration as it does not leach any toxic metal 801 or sulphates into solution in water treatment studies. The treated zeolite revealed more lead during the 802 digestion procedure than the natural zeolite, indicating that digestion methods with hydrochloric acid and 803 nitric acid were insufficient at complete digestion. The low CEC suggested that the zeolite may not have 804 been effective at removing environmental contaminants, but further investigation will be needed for 805 certainty. Areas of future inquiry include cation preference and determining an optimal ratio of zeolite 806 used per volume of solution treated. The metal modified Canadian zeolite from Bromley deposit 807 displayed a bactericidal effect on E. coli in both lake water and in nutrient broth; however, the amount of 808 metal cations desorbed into solution was too high for safe consumption. The pH of solution may have 809 played a significant role along with the metal cations in solution; however, the pH data set and the cation 810 treatment data set did not share enough qualities for simple comparison. Analysis of comparator zeolites 811 revealed that international zeolite samples released large volumes of sulphate and sodium into solution, 812 bringing into question the main mechanism of action. 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