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© 2021 Kayla Calapa All Rights Reserved © 2021 KAYLA CALAPA ALL RIGHTS RESERVED HYDROLOGIC ALTERATION AND ENHANCED MICROBIAL REDUCTIVE DISSOLUTION OF FE(III) (HYDR)OXIDES UNDER FLOW CONDITIONS IN FE(III)-RICH ROCKS: CONTRIBUTION TO CAVE-FORMING PROCESSES A Thesis Presented to The Graduate Faculty of the University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Kayla Calapa May, 2021 HYDROLOGIC ALTERATION AND ENHANCED MICROBIAL REDUCTIVE DISSOLUTION OF FE(III) (HYDR)OXIDES UNDER FLOW CONDITIONS IN FE(III)-RICH ROCKS: CONTRIBUTION TO CAVE-FORMING PROCESSES Kayla Calapa Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Dean of the College Dr. Hazel Barton Dr. Joe Urgo _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. John Senko Dr. Marnie Saunders _______________________________ _______________________________ Committee Member Date Dr. Ira Sasowsky _______________________________ Department Chair Dr. Stephen Weeks ii ABSTRACT Our previous work demonstrated that microbial Fe(III)-reduction contributes to void formation within Fe(III)-rich rocks, such as banded iron formation (BIF), iron ore and canga (a surficial duricrust), based on field observations and static batch cultures. Microbiological Fe(III) reduction is often limited when biogenic Fe(II) passivates further Fe(III) reduction, although subsurface groundwater flow and the export of biogenic Fe(II) could alleviate this passivation process. Given that static batch cultures are unlikely to reflect the dynamics of groundwater flow conditions that are in situ, we carried out comparative batch and column experiments to extend our understanding of the mass transport of iron and other solutes under flow conditions, and its effect on community structure dynamics and Fe(III)-reduction. We developed a synthetic media based on the porewater chemistry of caves that form in the Fe(III)-rich rocks (synthetic porewater; SPW). This SPW was amended with 5.0 mM lactate as a carbon source. Under anaerobic conditions, microbial Fe(III) reduction was enhanced in flow-through column incubations, compared to static batch incubations. During incubation, the microbial community profile in both batch culture and columns shifted from a Proteobacterial dominance to the Firmicutes; however, there was a shift from dominance by members of the Clostridiaceae, Peptococcaceae and Veillonellaceae, the latter of which has not previously been associated with Fe(III)-reduction activities. The bacterial Fe(III) reduction altered the advective properties of canga-packed columns and enhanced permeability. Our data demonstrate that iii removing inhibitory Fe(II) via mimicking hydrologic flow of groundwater increases reduction rates and overall Fe-oxide dissolution, while suggesting that reductive weathering of Fe(III)-rich rocks such as canga, BIF, and iron ores may be more substantial than previously understood. iv ACKNOWLEDGEMENTS I am extremely appreciative to all that were involved in helping to shape my graduate school experience, for the research opportunities I’ve been given the past few years, and to my family and friends that have been a major support system for me throughout it all. I would first like to thank my advisor, Dr. Hazel Barton, and committee members, Dr. John Senko and Dr. Ira Sasowsky, for bringing me into the Iron Caves Research Group, and allowing me to work in their labs and work on such a cool project. I’d also like to thank the National Science Foundation, Dr. Augusto Auler and his group, as well as Anglo-American mining company for making our research and sample sites possible and accessible. Thank you to the members of the Barton and Senko labs that have been apart of my time at the University of Akron. A special thanks to my anaerobic microbiology sensei, Ceth Parker. Ceth helped to shape a solid foundation for me in the anaerobic realm of microbiology, and he is both a great scientist and friend. I’d also like to thank Olivia Hershey, who helped to foster my initial research skills, and she is one of the reasons I sought out an advanced degree and continued research in our lab. Thank you to Melissa Mulford who completed the batch experiments in this study, and to my former undergraduate student, Tyler Rieman, for assisting with sample analysis. This was made possible because of your efforts and contributions. Thank you to the many collaborators and individuals helping our research group. Thanks to Drs. Ken Kemner and Max Boyanov for letting our research group shoot X-ray beams at our samples. Not many people get the opportunity to use a synchrotron, and for v that, I am forever grateful. Thank you to Steve Gerbetz over in the mechanical engineering department, who helped me custom cut the column rack, so I didn’t have to struggle with endless amounts of utility clamps and ring stands in that anaerobic glove bag. You’re a life saver. Dr. Chris Ziegler in the chemistry department here at the university allowed me to take an advanced chemistry course as a microbiologist, which was an opportunity both tough and rewarding. He made chemistry fun, funny, and understandable (which isn’t an easy thing to do when discussing things like molecular orbital diagrams). Thank you for peaking my interest in the analytical chemistry world – it’s a fun place to be. And last, but most definitely not least, thank you to Mr. Tom Quick. You are a gem in the geology department at the University of Akron, even post retirement. Thanks for being kind, always willing to lend a hand, and having patience for the million questions I always have. It’s been a long road getting here, but thank you to everyone who’s played a part in my journey thus far. vi TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ...............................................................................................................x CHAPTER I. INTRODUCTION ...........................................................................................................1 II. MATERIALS AND METHODS ...................................................................................4 Sample collection and preparation for batch incubations and column experiments. ....4 Batch Incubations ..........................................................................................................5 Column assembly and operation ....................................................................................6 Sample processing and analytical techniques ................................................................7 Microbial community analysis ......................................................................................8 III. RESULTS AND DISCUSSION .................................................................................10 Fe(III) reduction in static incubations .........................................................................10 Fe(III) reduction in column incubations. .....................................................................13 Microbial communities in column incubations. ..........................................................16 Microbially induced hydrologic alterations of canga columns ...................................17 Biogeochemical implications ......................................................................................18 REFERENCES .................................................................................................................26 APPENDICES .................................................................................................................. ix APPENDIX A. pH OF CAVE SAMPLES .................................................................34 APPENDIX B. WATER CHEMISTRY OF CAVE SAMPLES................................35 vii APPENDIX C. ANION CONTENT OF WEEKLY EFFLUENT SAMPLES ..........36 APPENDIX D. X-RAY DIFFRACTION ANALYSIS OF PH 4.75 COLUMNS .....37 APPENDIX E. X-RAY DIFFRACTION ANALYSIS OF PH 6.8 COLUMNS .......38 viii LIST OF FIGURES Figure Page 1: Batch cultures of Fe(III) reduction in SPW canga. ................................................21 2: Illumina sequencing results of phylum-level community diversity in batch and column cultures ......................................................................................................22 3: Illumina sequencing results of genus-level community diversity within the Firmicutes from the batch and column cultures ....................................................23 4: Fe(III) reduction and changes in sulfate and pH in the column experiments ........24 5: Bromide breakthrough curves of sub muros-inoculated and uninoculated columns after 63 d of operation ............................................................................................25 ix LIST OF TABLES Table Page 1: Post mortem analysis of column contents. .............................................................20 x CHAPTER I INTRODUCTION The Southern Espinhaço Mountain Range (SE) of southeastern Brazil contains commercially important, high-grade iron ore hosted by the Serra da Serpentina Group; a stratigraphic unit which includes the iron-rich Serra do Sapo Formation (Auler et al. 2019). These sedimentary units were formed by the precipitation of Fe(III) and Si phases from solution during the Proterozoic Eon (Rosière et al., 2019; Silveira Braga et al., 2021; Weber et al., 2006). Iron ores can include magnetite
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