Investigation of Bioprocesses to Enhance Metal Extraction from Ores and Wastes in Mining Operations By Laís Mazullo Mascarenhas Pereira A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Chemical Engineering and Applied Chemistry University of Toronto © Copyright by Laís Mazullo Mascarenhas Pereira 2019 Investigation of Bioprocesses to Enhance Metal Extraction from Ores and Wastes in Mining Operations Laís Mazullo Mascarenhas Pereira Master of Applied Science Chemical Engineering and Applied Chemistry University of Toronto 2019 Abstract Mine sites are a large source of genetic information that can be studied to explore ways of processing minerals using bacteria. This study investigates the bioleaching potential to extract nickel from ores and wastes at circumneutral pH and denitrifying conditions, and the cyanide biodegradation phenomenon at a gold heap leaching operation by examining naturally growing bacteria at two mine sites. Neutral pH bioleaching of nickel was achieved using nitrate as electron acceptor and oxidant. A mass balance revealed that about 20-30% of the original concentrate was leached over a period of more than 500 days. Faster rates could possibly be achieved by increasing biomass concentration. Thiobacillus was the dominant microbe identified in these microcosms. In gold heap leaching with cyanide, the microbial community was found to be almost entirely composed of a Hydrogenophaga with 16S sequence most similar to other alkaliphilic strains. This strain is presumably growing on the cyanide. ii Acknowledgments I would like to first express my deepest appreciation to my thesis supervisor Dr. Elizabeth Edwards for her continuous guidance of my Master research, patience, encouragement and immense knowledge. I cannot imagine having a better advisor for my Master study. Besides my supervisor, I would like to thank Dr. Vladimiros Papangelakis for his insightful comments and sharing expertise. I would also like to extend my profound gratitude to the Elements of Biomining Project (EBM) sponsors for providing funding and the environmental samples for this study. I cannot begin to express my thanks to Susie (Endang Susilawati), Line Lomheim and Georgiana Moldoveanu. Without their precious help and support in the lab this research would not have been possible. I would also like to thank Ivy (Minqing Yang) for her invaluable contribution with all the bioinformatics work in this study. I also wish to express my heartfelt thanks to Dr. Sávia Gavazza and Suzana Kraus for their valuable guidance during my academic trajectory, and sincerely thank Nadia Morson for being the second reader of this thesis and for her immeasurable support in these past two years. My sincere thanks also goes to my fellow lab mates and colleagues for the great discussions and all the fun we have had. Finally, I would like to express my most profound gratitude to my family for providing me with unceasing motivation and continuous support throughout my years of study. iii Table of Contents Acknowledgments __________________________________________________________________________________ iii Table of Contents___________________________________________________________________________________ iv List of Tables ______________________________________________________________________________________ viii List of Figures ______________________________________________________________________________________ ix List of Appendices __________________________________________________________________________________ xi List of Abbreviations ______________________________________________________________________________ xii Introduction ____________________________________________________________________________ 1 1.1 General Introduction ____________________________________________________________________________ 1 1.2 Thesis Outline and Objectives __________________________________________________________________ 2 Bioleaching of Nickel from Sulfidic Ores and Wastes under Denitrifying and Circumneutral pH Conditions: Long-term Microcosm Studies ____________________________________ 3 2.1 Introduction and Specific Objectives __________________________________________________________ 4 2.2 Literature Review _______________________________________________________________________________ 5 2.2.1 Nickel Sulfide Deposits _________________________________________________________________________________ 6 2.2.2 Hydrometallurgical Process of Low-graded Ultramafic Ores _______________________________________ 6 2.2.3 The Leaching Mechanism for Metal Recovery ________________________________________________________ 7 2.2.4 The Bioleaching Alternative ___________________________________________________________________________ 7 2.2.5 Nitrate Sources in Mining ______________________________________________________________________________ 8 2.2.6 Nitrate-reducing Bioleaching of Ni at Circumneutral pH ____________________________________________ 8 2.3 Materials and Methods __________________________________________________________________________ 9 2.3.1 Microcosm Revival from Zhou’s work ________________________________________________________________ 9 2.3.2 Microcosm Design for New Transfers _______________________________________________________________ 10 2.3.3 Ultramafic Material Preparation _____________________________________________________________________ 11 2.3.4 Growth Medium and Stock Solutions Preparation __________________________________________________ 12 2.3.5 Cell Transfer to New Microcosms ____________________________________________________________________ 12 2.3.6 Microcosm and Transfer Culture Maintenance _____________________________________________________ 13 2.3.7 Ultramafic Material Characterization by Aqua Regia Digestion ___________________________________ 13 2.3.8 Aqueous Sample Analyses: Total Soluble Metals, Nitrate and Sulfate _____________________________ 14 2.3.9 Biological Sample Analyses: DNA Extraction, qPCR and Amplicon Sequencing __________________ 15 iv 2.3.10 Final Solid Analyses ________________________________________________________________________________ 16 2.3.11 Elemental Sulfur Investigation ____________________________________________________________________ 17 2.4 Results and Discussion _________________________________________________________________________ 17 2.4.1 Revival of Zhou’s Microcosms ________________________________________________________________________ 18 2.4.2 Evidence of Living Cells _______________________________________________________________________________ 21 2.4.3 Dissolved Ni Evolution, Nitrate Consumption and Sulfate Production ____________________________ 22 2.4.4 Bacterial Cell Count and Microbial Community Profile _____________________________________________ 23 2.4.5 Mass Balance Study in Zhou’s Microcosm Bottles __________________________________________________ 25 2.4.6 Mass Balance Study in New Transfers _______________________________________________________________ 29 2.4.7 Elemental Sulfur Investigation _______________________________________________________________________ 31 2.5 Conclusions and Recommendations for Future Work _______________________________________ 32 2.6 References ______________________________________________________________________________________ 34 Identification of Cyanide Degrading Microorganisms in Heap Leach Operations for Gold Recovery _____________________________________________________________________________________ 36 3.1 Introduction and Specific Objectives _________________________________________________________ 37 3.2 Literature Review ______________________________________________________________________________ 38 3.2.1 The Chemistry of Gold Cyanidation __________________________________________________________________ 38 3.2.2 Cyanide Toxicity and Chemical Removal Technologies ____________________________________________ 40 3.2.3 Cyanide Biodegradation ______________________________________________________________________________ 41 3.2.4 Metabolic Pathways for Cyanide Biodegradation ___________________________________________________ 41 3.2.5 Identification of Cyanide-Degrading Microbes in Gold Heap Leach Operations __________________ 44 3.3 Materials and Methods _________________________________________________________________________ 44 3.3.1 Sample Collection and Field Analyses _______________________________________________________________ 44 3.3.2 Chemical Analyses: Anions, Nutrients, Cyanide Breakdown Products and Total Dissolved Metals ________________________________________________________________________________________________ 46 3.3.3 Biological Analyses: DNA Extraction, qPCR and Amplicon Sequencing ___________________________ 49 3.4 Results and Discussion _________________________________________________________________________ 50 3.4.1 Field and Lab Chemical Measurement Results ______________________________________________________ 50 3.4.2 Bacterial 16S rRNA Gene Copies in Relation to Free Cyanide Concentration and pH ____________ 53 3.4.3 Phylogenetic Analysis and Taxonomic Diversity ____________________________________________________ 53 3.4.4 Identification of the Cyanide-Degrading Microbes (s) ______________________________________________ 58 3.5 Conclusions and Recommendations for Future Work _______________________________________ 59 v 3.6 References ______________________________________________________________________________________ 62 Synthesis and Conclusions ____________________________________________________________