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Removal of Soluble Selenium in the Presence of Nitrate from Coal Mining-Influenced Water by Frank Nkansah - Boadu BSc., Kwame Nkrumah University of Science and Technology, 2003 MASc., The University of British Columbia, 2013 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2019 © Frank Nkansah - Boadu, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Removal of Soluble Selenium in the Presence of Nitrate from Coal Mining-Influenced Water submitted by Frank Nkansah-Boadu in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemical and Biological Engineering Examining Committee: Susan Baldwin, Chemical and Biological Engineering Supervisor Vikramaditya Yadav, Chemical and Biological Engineering Supervisory Committee Member Troy Vassos, Adjunct Professor, Civil Engineering Supervisory Committee Member Anthony Lau, Chemical and Biological Engineering University Examiner Scott Dunbar, Mining Engineering University Examiner ii Abstract Biological treatment to remove dissolved selenium from mining-influenced water (MIW) is inhibited by co-contaminants, especially nitrate. It was hypothesized that selenium reducing microorganisms can be obtained from native mine bacteria at sites affected by MIW due to the selection pressure from elevated selenium concentrations at those sites. Enrichment of these microorganisms and testing of their capacity to remove dissolved selenium from actual coal MIW was the objective of this dissertation. Fifteen sediments were collected from eleven different vegetated or non-vegetated seepage collection ponds and one non-impacted natural wetland. Nine sediments achieved greater than 90% dissolved selenium removal within 72 hours when inoculated into selenate-reducing bacteria growth medium. To find microorganisms capable of removing dissolved selenium in the presence of nitrate, six of the sediments were inoculated into two different types of growth media; one with selenate as the sole electron acceptor and the other with both nitrate and selenate as electron acceptors. Both media were otherwise identical and contained lactate as the electron donor. Decrease in dissolved selenium concentration was observed in all enrichments, but the effect of nitrate on the rate and extent of removal was variable. Nitrate inhibited dissolved selenium removal rates in four of the enrichments. However, in one instance, microorganisms enriched from a natural vegetated marsh receiving coal MIW (Goddard Marsh) were not inhibited by nitrate and the dissolved selenium removal rates were similar in both media. In another instance, the presence of nitrate enhanced dissolved selenium removal by enrichments from a pond receiving coal mine waste seepage (Lagoon A). When enrichments from Lagoon A and Goddard Marsh, respectively, were tested for dissolved selenium removal from actual coal MIW, the former achieved greater (40%) than the latter (10%). Through 16S and whole genome sequencing studies, species capable of removing selenate in the enrichments were classified as Bacteroides, Serratia, Clostridium and Methanosarcina. However, most of these species did not survive in the MIW. The dominant species in the MIW were classified as Sulfurospirillium, Veillonella, Pseudomonas and Bacteroides, which were shown to be capable of reducing selenium, based on putative metagenome assembled genomes (MAGs) obtained for these species. iii Lay Summary Some mining-influenced water (MIW) contains elevated concentrations of dissolved selenium that could be harmful to downstream ecosystems. Biological treatment to remove dissolved selenium through reduction to insoluble forms is the current preferred method. Its effectiveness is challenged by the presence of competing constituents, especially nitrate, which is a common co-contaminant in MIW because of explosives used in mining. In this dissertation, consortia of bacteria were enriched from different sites impacted by coal mine waste seepage. These enriched bacteria were tested for their capacity to remove dissolved selenium from actual MIW. The study found that, even though bacteria with the capability to remove dissolved selenium survived in the MIW, the extent of dissolved selenium removal was lower in the actual MIW than in the enrichment media. Possibly, other constituents of the MIW negatively affected the selenium reducing bacteria or essential nutrients for these bacteria were missing from the MIW. iv Preface This research project would not have been completed without the invaluable support from my research advisors. The supervisory team greatly assisted in the overall completeness of the research scope, identification of the broad research objectives, examination and interpretation of the data and revisions of the chapters. The Chapters 2 - 6 have been revised into three manuscripts for publication. The manuscripts prepared for publication have been strengthened by contribution from the supervisory team, Dr. Susan Baldwin, Dr. Troy David Vassos and Dr. Vikramadiyta Yadav. I critically reviewed relevant literature, designed and performed the experiments, conducted laboratory analyses, analyzed the data, organized and presented the results and prepared draft manuscripts. Jon Taylor performed the sequencing of DNA from the early experiments. David Gurr, Ido Hatam and Susan Baldwin performed the bioinformatic analyses of the 16S and whole genome sequencing data. v Table of Contents Abstract……………………………………………………………………………………... iii Lay Summary ………………………………………………………………………………..iv Preface ………………………………………………………………………………………...v Table Contents ………………………………………………………………………………vi List of Tables ………………………………………………………………………………...xi List of Figures ………………………………………………………………………………xiv List of Abbreviations ……………………………………………………………………...xvii Acknowledgements …………………………………………………………………………xx Dedication. …………………………………………………………………………………xxii Chapter 1 : Introduction ..................................................................................... 1 1.1 Background .................................................................................................. 1 1.2 Research Goal .............................................................................................. 5 1.2.1 Research Questions .....................................................................................................5 1.3 Structure of the Dissertation ......................................................................... 6 1.4 Literature Review ......................................................................................... 7 1.4.1 Selenium Chemistry ....................................................................................................7 1.4.2 Biogeochemical Selenium Cycle .................................................................................9 1.4.3 Selenium Oxyanion Reduction Pathways by Bacteria ................................................ 14 1.4.4 The Effect of Nitrate on Dissimilatory Selenate Reduction ....................................... 15 1.4.5. Effect of Salinity on Nitrate Reduction and Selenate Reduction ................................ 18 1.4.6 Microbial Species Capable of Selenium Oxyanion Reduction and the Enzymes involved ............................................................................................................................. 20 1.4.7 Specific Dissimilatory Selenate Reductase Enzyme ................................................... 24 vi 1.4.8: Denitrification Pathway ............................................................................................ 27 1.4.9 Dissimilatory Sulfate Reduction Pathway .................................................................. 29 1.4.10 Bioreactors used for Removal of Dissolved Selenium .............................................. 31 1.4.11 Challenges with using Bioreactors for Removal of Dissolved Selenium from MIW . 35 1.4.12 Sources of Inocula used to Achieve Dissolved Selenium Removal in Bioreactors .... 37 1.4.13 Microbial Diversity in Selenate Reduction Bioreactors ............................................ 39 Chapter 2 : Assessment of Dissolved Selenium Removal Potential of Mine Site Sediment Bacteria ...............................................................................................41 2.1 Synopsis ......................................................................................................41 2.2 Materials and Methods ................................................................................43 2.2.1 Sites, Sampling and Chemical Characteristics ............................................................ 43 2.2.2 Growth Medium and Culturing .................................................................................. 46 2.3 Results ........................................................................................................48 2.3.1 Sample Location Characteristics ................................................................................ 48 2.3.2 Batch Studies of Dissolved Selenium Removal ......................................................... 52 2.3.3 Correlation of Dissolved Selenium Removal with Chemical Characteristics .............. 53 2.4 Discussion ...................................................................................................55