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Microbial Arsenic Transformation in Near Subsurface Environments Robert James Reiter

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Microbial Arsenic Transformation in Near Subsurface Environments Robert James Reiter Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 2015 Microbial Arsenic Transformation in Near Subsurface Environments Robert James Reiter Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Reiter, R. (2015). Microbial Arsenic Transformation in Near Subsurface Environments (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1094 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. MICROBIAL ARSENIC TRANSFORMATION IN NEAR SUBSURFACE ENVIRONMENTS A Thesis Submitted to the Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctorate of Philosophy By Robert Reiter December 2015 Copyright by Robert Reiter 2015 MICROBIAL ARSENIC TRANSFORMATION IN NEAR SUBSURFACE ENVIRONMENTS By Robert Reiter Approved November 6, 2015 ___________________________ ________________________ Dr. John F. Stolz Dr. Peter Castric Professor of Biology Professor of Biology (Committee Chair) (Committee Member) ___________________________ ________________________ Dr. Nancy Trun Dr. Partha Basu Associate Professor of Biology Professor of Chemistry (Committee Member) (Committee Member) ___________________________ Dr. Philip Reeder Dean and Professor Bayer School of Natural and Environmental Sciences iii ABSTRACT MICROBIAL ARSENIC TRANSFORMATION IN NEAR SUBSURFACE ENVIRONMENTS By Robert Reiter December 2015 Dissertation supervised by Dr. John F. Stolz. In the first part of this study, environmental bacterial arsenic transformation was investigated in anthropogenically arsenic contaminated subsurface sediments from the former Vineland Chemical Company in Vineland, NJ. Subsurface sediments from the vadose (371 mg/kg arsenic) and aquifer (81 mg/kg arsenic) zones and an off-site surface sediment (0.7 mg/kg arsenic; control) were used as inocula for enrichment cultures with selective media to assess for microbial arsenic metabolic activity. Arsenite (As(III)), monomethyl arsenic acid (MMA) and dimethyl arsenic acid (DMA) were provided as electron donors, while arsenate (As(V)) was provided as an electron acceptor. An anion exchange chromatography method with conductivity and UV/Vis detection was developed and utilized to investigate arsenic transformations under each selective condition. iv While demethylation and arsenite oxidation were not observed, there was strong As(V) reduction to As(III) activity in the enrichments amended with lactate and As(V). Polymerase chain reaction (PCR) was used to amplify arrA and arsC genes, followed by cloning and sequencing. Both vadose and aquifer enrichments gave positive results for arrA (respiratory arsenate reductase) and arsC (arsenic resistance), while only arsC amplified from the off-site. Both 16S rRNA genes and the ribosomal intergenic spacer (RIS) genes were used to assess community diversity. The results indicate a diverse community with As(V) respiring bacteria. In the second part of this study, the utility of ion chromatography (IC) in speciating inorganic (As(V), As(III)) and organic (MMA, DMA) was assessed. The As(V) and roxarsone respiring Alkaliphilus oremlandii OhILAs was cultured and the spent medium analyzed. Under the conditions used, OhILAs failed to produce any As(III) and As(V) when grown on roxarsone and lactate; however, As(V) to As(III) reduction was observed when grown in the presence of lactate and As(V). In the third part of this study, microcosms were used to assess bacterial arsenic transformation in cores from a coal ash impoundment. A medium was formulated based on the water chemistry of the system. As(V) respiration was detected at all four depths tested (0-2m; 2-4m, 4-6m, and 8-10m), with the surface sediments showing the greatest rates. v DEDICATION I would like to dedicate this work to my beloved and late father, Robert Reiter. vi ACKNOWLEDGEMENTS I would like to thank Dr. Stolz and the entire Stolz lab for allowing me to experience years of lab research in such a fascinating field of study and great working environment. More specifically, I would like to thank Rishu Dheer, Lucas Eastham, Jennifer Rutter, Samir Joshi and Oliver Dugas for their assistance in multiple facets of my thesis research. I would like to thank my committee members, Dr. Basu, Dr. Castric and Dr. Trun for all the support, helpful advice and guidance they provided. I would also like to thank Dr. Chuck Welsh for being such a supportive mentor and friend during my time working under him as a lab instructor. Additionally, I would like to thank Dr. Kyle Bibby from the University of Pittsburgh for allowing me to perform high-throughput 16S rDNA sequencing in his laboratory. Lastly, thank you to the Duquesne University Department of Biological Sciences for funding my education, teaching and research, as well as all of the faculty and staff within the department that have provided me with teaching, support and guidance. vii TABLE OF CONTENTS Chapter I. Arsenic Background...................................................................................... 1 A. Chemistry of Arsenic.................................................................................................. 1 B. History of Arsenic ...................................................................................................... 2 C. Arsenic in the Environment ....................................................................................... 7 D. Arsenic Toxicity ....................................................................................................... 10 E. Arsenic metabolism ................................................................................................. 17 i. Methylation............................................................................................................. 17 ii. Arsenate reduction ................................................................................................. 22 a. Arsenic resistant microbes ................................................................................. 22 b. Dissimilatory arsenate reducing prokaryotes ..................................................... 23 iii. Arsenite oxidation ................................................................................................ 24 iv. Methylarsenic demethylation ............................................................................... 26 v. Bacterial arsenic mobilization ............................................................................... 27 Chapter II. Development of Thermo Scientific/Dionex ICS-1100 anion exchange chromatography method ................................................................................................ 33 A. Abstract and Hypotheses .......................................................................................... 33 B. Background ............................................................................................................... 34 C. Materials and Methods ............................................................................................. 34 i. Dionex ICS-1000 system features .......................................................................... 34 ii. Chromeleon LE 7.0 software ................................................................................. 35 a. Chromeleon Console .......................................................................................... 35 b. Calibrating standards ......................................................................................... 38 c. Data Processing .................................................................................................. 39 iii. Standard preparation ............................................................................................ 39 a. Vineland enrichments......................................................................................... 41 b. Coal combustion byproduct disposal site microcosms ...................................... 41 c. Alkaliphilus oremlandii OhILAs cultures .......................................................... 42 D. Results ...................................................................................................................... 42 i. System optimization ............................................................................................... 42 viii ii. Standard Preparation ............................................................................................. 53 G. Discussion ................................................................................................................ 53 Chapter III. Vineland Chemical Company enrichment culture analyses ................. 58 A. Abstract and Hypotheses .......................................................................................... 58 B. Background ............................................................................................................... 60 C. Methods and Materials ............................................................................................ 61 i. Sediment Excavation .............................................................................................. 61 ii. Media preparation .................................................................................................
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