THE INFLUENCE OF O2 AVAIABILITY ON THE GROWTH OF FE(III) REDUCING BACTERIA IN COAL MINE-DERIVED ACID MINE DRAINAGE A Thesis Presented to The Graduate Faculty of the University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Zachary Santangelo August, 2019 THE INFLUENCE OF O2 AVAIABILITY ON THE GROWTH OF FE(III) REDUCING BACTERIA IN COAL MINE-DERIVED ACID MINE DRAINAGE Zachary Santangelo Thesis Approved: Accepted: __________________________ __________________________ Advisor Interim Dean of the College Dr. John Senko Dr. Linda M. Subich __________________________ __________________________ Faculty Reader Dean of the Graduate School Dr. Teresa Cutright Dr. Chand Midha __________________________ __________________________ Faculty Reader Date Dr. Linda R. Barrett __________________________ Department Chair Dr. David N. Steer II ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. John Senko, for pushing me and making me think about everything from every point of view. His research and ideas helped me think critically about research I never even considered. Dr. Senko constantly helped me become the best writer I could possibly be. I will always appreciate that in my work in the future. The members of my committee: Dr. Teresa Cutright and Dr. Linda Barrett, I offer my sincerest gratitude. I had a couple classes with Dr. Cutright, both of which I repeatedly thought back to when approaching my own research. She helped me not just think about problems, but truly understand what was happening. Dr. Barrett, I sincerely thank for her time in helping me move forward with my thesis, and making sure I knew what I was trying to explain properly. I must offer my thanks to Tom Quick. Without Tom, I surely would still be in the lab trying to get the dionex to work. Additionally, I would like to thank Elaine Butcher, who helped me with countless office questions as well as just enjoying conversation. Tom and Elaine deserve more thanks than I could possibly give them. Andrea Fodorne Mathe, from the Ohio State University, thank you for your help with the SEM. Setting up time and analyzing my samples with me not once but twice, Andrea was very helpful in helping me work with the machine that allowed me to retrieve images of the bacteria in my samples. To my fellow students, I thank you for all the time we spent working and laughing together. Bobby Miller and Shagun Sharma, thank you for your constant help and support III in the lab. With their help, I was able to learn how to use most equipment in the lab. With their support, I was able to confidently work on my research. Kayla Calapa, thank you for your help and answering my many questions. Nick Wander, R.J. McGinnis and Rebeccah DiPuccio, thank you all for the time you spent with me. These three had endless conversations with me that helped me keep my head on straight, and think my way out of any problem. Tim Schmucker, thanks for helping me back to reality time and again. I owe so much to every one of you. To Valerie Miller, thank you for your constant support. Valerie followed me to Akron without hesitation. Without her, none of this would have been possible. She knows how to get me fired up and take challenges head on, while also cooling my jets when I overreact. Our life continues to change and I never regret it. We continue moving forward, and while the future is uncertain, I remain excited to see it through with you. Finally, to my friends and family, I thank for their support. No matter where they were, I was constantly shown the love and support I needed to push through everything. Mom and Dad, you never doubted me, and continue to push me to do what I dream. I will continue to dream, thanks to them. Nick, Andrew, Luke, and Natalia, I wish I could have spent more time with all of you these past two years. So much has happened and you have all grown in ways I truly wish I could have been there for. My siblings have always been there for me, and I will always be there for them. Owen, Brendan, and Cece, thanks for your support and literally being here for me. I could not ask for better friends. Everyone has shown me nothing but love, support, and pride these past two years. It is thanks to all of you, I have been able to accomplish my goals. IV ABSTRACT Acid mine drainage (AMD) is an environmentally harmful outcome of coal mining. Mining exposes FeS2 to oxygen and results in low pH and iron oxidation. Fe(III) precipitates after Fe(II) is oxidized in AMD fluids that have become exposed to a combination of oxygen (O2) and water (H2O). Bacteria in AMD acquire energy through respiration. Aerobic respiration uses the most favorable terminal electron acceptor, oxygen. Once oxygen is depleted, the next thermodynamically favorable terminal electron acceptors can be used, including Fe(III) and sulfate. Fe(III) reducing bacteria are anaerobes, respiring in anoxic conditions. However, recent studies indicated that Fe(III) reduction occurred in oxic conditions. I hypothesized that Fe(III) reducing bacteria would not be inhibited by oxygen, under acidic conditions. Enrichment cultures in anoxic and oxic conditions with Fe(III)-containing media were tested for 159 days. Cultures were inoculated with material from an AMD contaminated site. Cultures were maintained through three transfers. After each transfer, Fe(III) reduction was observed by quantifying the accumulation of Fe(II). Fe(III) reduction occurred in both oxic and anoxic conditions. Culture samples were examined under a scanning electron microscope (SEM), where rod-shaped bacteria were observed in both anoxic and oxic cultures. 16S rRNA abundance of sequences indicates that Bacilli were the majority of the Fe(III) reducing bacteria in oxic cultures and Clostridia were the majority of the Fe(III) reducing bacteria in anoxic cultures. In acidic conditions, Bacilli reduced Fe(III) without being inhibited by oxygen. V TABLE OF CONTENTS List of Figures..................................................................................................................vii List of Tables………………………………………………………………….………..viii Chapter I. Introduction……………………………………………………………………………..1 1.1 Acid Mine Drainage…………………………………………………………..1 1.2 Oxidation-Reduction Reactions and Terminal Electron Acceptors…………..2 1.3 Iron Mounds…………………………………………………………………...5 1.4 Previous Studies……………………………………………………………….7 1.5 Hypothesis……………………………………………………………………..8 II. Materials and Methods...……………………………………………………………...10 III. Results..……………………………………………………………………………....16 3.1 Approach……………………………………………………………………..16 3.2 First Round…………………………………………………………………..19 3.3 Second Round………………………………………………………………..21 3.4 Third Round………………………………………………………………….22 3.5 Scanning Electron Microscopy………………………………………………26 3.6 Taxonomic Composition of Cultures………………………………………...34 IV. Discussion..…………………………………………………………………………..37 4.1 Fe(III) Reduction in AMD…………………………………………………...37 4.2 AMD Sulfate Reduction……………………………………………………..41 4.3 Change in pH………………………………………………………………...42 4.4 Bacteria Abundances………………………………………………………...44 4.5 Iron Mound Applications…………………………………………………….45 V. Conclusion...………………………………………………………………………….47 References….…………………………………………………………………………50 VI List of Figures 1 Photograph of AMD flowing over the iron mound at the Mushroom Farm..…6 2 Culture transfer process……………………………………………………...12 3 Fe(II) concentrations, sulfate concentrations, glucose concentrations, and pH values of the anoxic and oxic cultures over 159 day period. ………………..17 4 SEM of the uninoculated medium that contains Fe(III) hydroxide particles...27 5 SEM of an oxic culture……………………………………………………...28 6 SEM of an oxic culture………………………………………………………29 7 SEM of an anoxic culture……………………………………………………30 8 SEM of a culture in a medium consisting of glucose, but no Fe(III)………...31 9 16S rRNA gene abundance of sequences at the class-level………………….36 VII List of Tables 1 Table of Redox couples……………………………………………….…………..3 VIII CHAPTER I INTRODUCTION 1.1 Acid Mine Drainage Acid mine drainage (AMD) is the product of sulfide-bearing material that is exposed to a combination of oxygen (O2) and water (Akcil and Koldas, 2004). Oxygen oxidizes sulfide minerals (Eq. (1)). 2- 2+ + FeS2 + 3.5 O2 + H2O → 2 SO4 + Fe + H (1) Pyrite is a mineral commonly associated with coal seams (Akcil and Koldas, 2004). This process can occur naturally, but mining enhances the sulfide minerals exposure to oxygen (Akcil and Koldas, 2004). The most common metal sulfide exposed in mining is pyrite 2+ 2- + (FeS2) (Baker and Banfield, 2003). Sulfur oxidation (Eq. (1)) yields Fe , SO4 , and H , contributing to the amount of total dissolved solids already within natural waters (Akcil and Koldas, 2004). AMD has high concentrations of sulfate and iron from oxidation of sulfide-containing minerals of mines (Kusel et al., 1999). The characteristics of AMD include low pH, high conductivity, and high metal concentrations (Eby, 2004). 2+ + 3+ 14 Fe + 3.5 O2 + 14 H → 14 Fe + 7 H2O (2) Fe(II) oxidation by oxygen (Eq. (2)) reacts at a relatively slow rate at low pH (Baker and Banfield, 2003). Bacteria can accelerate Fe(II) oxidation (Baker and Banfield, 2003). Fe(II) oxidation will increase the concentration of Fe(III) produced (Eq. (2)) (Akcil and Koldas, 2004). Fe(III) may become reduced, to oxidize additional pyrite-S (Eq. (3)) (Akcil and Koldas, 2004). 3+ 2+ 2- + FeS2 + 14Fe + 8H2O → 15Fe + 2SO4 + 16H (3) 1 Sulfide and Fe(II) will serve as electron donors when used for respiration by microorganisms (Sawyer et al., 2003). Fe(II) oxidizing bacteria use oxygen as a terminal electron acceptor (Eq. (2)) in order to create the energy needed for cell synthesis and maintenance (Sawyer et al., 2003). However, AMD that has high pH (5.5-8.0) will be unsuited for bacteria that prefer acidic conditions. AMD enters circumneutral streams and high pH enhances the rate of abiotic Fe(II) oxidation and subsequent hydrolysis of Fe(III) and precipitation of Fe(III) hydroxides (Eq. (4)), hydroxides that are known as “yellowboy” (Akcil and Koldas, 2004; Senko et al., 2008). 3+ + Fe + 3H2O → Fe(OH)3 + 3H (4) The precipitation of Fe(III) hydroxides and production of hydrogen ions (Eq.