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University of Cincinnati UNIVERSITY OF CINCINNATI Date: 12-Nov-2009 I, Colin P White , hereby submit this original work as part of the requirements for the degree of: Master of Science in Biological Sciences It is entitled: Molecular Microbial Ecology and Operational Evaluation of a Full-scale and Pilot-scale Biologically Active Filter for Drinking Water Treatment Student Signature: Colin P White This work and its defense approved by: Committee Chair: Ronald Debry, PhD Ronald Debry, PhD 6/21/2010 273 Molecular Microbial Ecology and Operational Evaluation of a Full- scale and Pilot-scale Biologically Active Rapid Sand Filter for Drinking Water Treatment A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science In the Department of Biological Sciences of the McMicken College of Arts and Sciences By Colin Patrick White B.S. Biological Sciences University of Cincinnati April 2007 Committee Chair: Ronald W. DeBry, Ph.D. Abstract Nitrification in drinking water distribution systems is a problem prevalent throughout the world, and it has become more pertinent since chloramination has become a popular disinfectant technique. Because nitrification requires ammonia, removing ammonia in source waters prior to treatment would benefit both the utility and consumers. Biologically active filtration is a well known technology in Europe but its reliability, and thus implementation, is questioned in the United States. In this study, natural microbial flora from a full-scale treatment plant in Greene County, Ohio was used to seed two pilot scale rapid sand filters. These filters were evaluated for their ability to oxidize ammonia-nitrogen. Molecular techniques, including 16S ribosomal RNA and amoA gene sequencing and denaturing gradient gel electrophoresis (DGGE) analysis, were used to phylogenetically identify and fingerprint the isolates. In addition to investigating nitrification, microbial arsenic oxidation was also investigated in pilot-scale filters. Chemical analysis and microbial ecology is compared and discussed in terms of operational changes and water chemistry. 2 Dedication To my parents Acknowledgements First, I would like to thank my advisory committee in the Department of Biological Sciences: Drs. Ronald DeBry and Jodi Shann, for their support, guidance, and patience over the years. Next, I would like to acknowledge the Environmental Protection Agency, Office of Research and Development for which funding for this research via the UC/EPA Traineeship was awarded. Special thanks goes to Dr. Darren Lytle, who arranged for this opportunity and was an invaluable resource as well as a great mentor. Under his expert tutelage I gained an appreciation for collaborative and applied research. Without his mentorship and constant encouragement I would never be where I am today. Credit is also due to members of the US EPA Treatment Technology Evaluation Branch, especially Daniel Williams and Christy Muhlen, for their endless help and guidance both in the lab and in life. In addition, I would like to thank my laboratory mates, Amy Reed, Andrea Galloway, Alissa O’Donnell, Jermaine Conover, Jennifer Liggett, and David Hauber for their help and friendship. 3 TABLE OF CONTENTS List of Tables Table 3.2.1. Sample/Monitoring Strategy…………………………….………….34 Table 3.2.2. Sampling/Analytical Procedures…………………………..………..35 Table 3.5.1. Primers and References to Reaction Conditions……………………39 Table 3.5.2. PCR Reaction Setup………………………………………………..40 Table A1. QA Objectives for Method Detection Limit, Precision, Accuracy, and Completeness…………………………………………………...108 Table A2. Summary of QC Checks…………………………………………….110 List of Figures Figure 1. Nitrogen Fertilizer Loading in the United States……………………….15 Figure 2. USGS National Water Quality Assessment of Filtered Ammonia in Source Water………………………………………………………..16 1. Introduction 1.1 Background…………………………………………………………………….6 1.2 Objectives……………………………………………………………………...9 1.3 Approach……………………………………………………………………...9 1.4 OvervieW of Thesis………………………………………………………..…10 2. Literature Review 2.1 Overview of Ammonia Behavior in Water…………………………………...11 4 2.1.1 Ammonia Chemistry……………………………………………….11 2.1.2 Ammonia Distribution……………………………………………..13 2.2 Ammonia Health Effects……………………………………………………..16 2.2.1 Carcinogenic Effects and Risks…………………………………….16 2.2.2 Non-Carcinogenic Effects and Risks……………………………….17 2.2.2.1 Acute Exposure………………………………………….17 2.2.2.2 Chronic Exposure………………………………………..17 2.2.3 Beneficial Effects…………………………………………………..18 2.3 Treatment Technology Options……………………………………………..18 2.3.1 Overview…………………………………………………………..18 2.3.2 Breakpoint Processes………………………………………………18 2.3.3 Biological Processes………………………………………………..19 2.3.4 Ion Exchange Processes…………………………………………….21 2.3.5 Membrane Processes……………………………………………….22 2.3.6 Aeration Processes…………………………………………………23 2.4 Microbial Processes in Ammonia Cycling, Oxidation, and Reduction…….…24 2.4.1 Microbial Oxidation………………………………………………..24 2.4.2 Microbial Reduction………………………………………………..26 3. Materials and Methods 3.1 Pilot Plant Design and Operation…………………………………………….28 3.2 Water Chemistry……………………………………………………………..32 3.3 Microbial Culturing…………………………………………………………..35 5 3.4 Isolation of Nucleic Acids……………………………………………………37 3.5 amoA and 16S Gene Library Construction and Phylogenetic Analysis…......38 4. Results 4.1 Manuscript I…………………………………………………………..……..41 4.2 Manuscript II…………………………………………………………..…….64 5. Discussion…………………………………………………………………….…...….93 6. Future Work…………………………………………………………………...…..…94 7. References……………………………………………..………………………….….95 Appendix………………………………………………………………………………....99 6 1. INTRODUCTION 1.1. BACKGROUND With the exception of mixing ammonia-cleaning agents with bleach, ammonia is + rarely associated with human health problems. Ammonia in its ionized form NH4 is called ammonium and is not toxic to aquatic life whereas in its un-ionized form NH3, it is extremely toxic to aquatic life. As the primary ionization state of ammonia is as ammonium, its presence in pretreated water and finished drinking water is not regarded as a contaminant by the United States Environmental Protection Agency (US EPA). Though the compound proper of ammonia does not call for alarm, it is in the chemistry of ammonia where problems arise in drinking water treatment. Ammonia has the ability to provide organisms with a great source of energy as it is oxidizable by a group of prokaryotes residing within the !-Proteobacteria and Chrenarcheota. It is the oxidized forms of ammonia that threaten human health. In pre-treated drinking water these forms of oxidized ammonia, nitrite and nitrate, have a maximum contaminate limit (MCL) set by the US EPA at 1 mg.L and 10 mg/L, respectively. When ingested, nitrate is reduced to nitrite and binds oxyhemoglobin converting it to methylhemoglobin. Methylhemoglobin is not capable of carrying oxygen and can be lethal. Blue baby syndrome is a common concern in areas with high nitrite/nitrate levels in drinking water. In addition to direct impact on human health, ammonia has indirect impact as well. As ammonia is primarily a groundwater contaminant, its presence is usually accompanied by iron and arsenic from natural mineral deposits in the aquifer. The physicochemical removal of arsenic can be coupled concurrently to the iron removal 7 process, a common and cost effective method for iron removal. The primary oxidization state of arsenic in ground water requires it to be oxidized to sorb to oxidized iron particles for subsequent removal by filtration. The presence of ammonia in source waters can interfere with this process by chemically consuming the added oxidant required to oxidize the arsenic, potentially exposing consumers to arsenic levels above the US EPA MCL of 10 ug/L. The consumption of chlorine increases the chlorine demand of the system leading to a decrease in the free chlorine residual in the distribution system. The lower disinfectant residual may allow biological growth in the distribution system and lead to nitrification events and disease outbreaks. It is therefore beneficial to remove ammonia in source waters prior to treatment. Technologies that exist for ammonia removal include ion exchange, air stripping, membrane processes, biological processes, and breakpoint chlorination. These technologies all have strengths and weaknesses, but of particular interest are biological processes as their potential benefits outweigh the weaknesses compared to other technologies. Biological processes have been used in Europe and wastewater industry for years, but have only recently been considered in the United States. The slow acceptance in the U.S. is primarily due to negative perceptions related to microorganisms as well as a poor understanding of the complex microbial community and its role related to filter reliability. Herein, I attempt to elucidate the microbial community of a full-scale biologically active filter, demonstrate its effectiveness at oxidizing ammonia, as well as test its reliability and ease of implementation at the pilot-scale. 8 1.2. OBJECTIVES As ammonia in source waters leads to several operational and distribution system issues in the drinking water industry, it is my hypothesis that a rapid sand filter can become biologically active. After an initial seeding event a period of time will be required to establish a population of microorganisms capable of complete nitrification. These organisms responsible for nitrification will not be negatively affected by changes in operational parameters,
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