Investigation of Bioprocesses to Enhance Metal Extraction from Ores and Wastes in Mining Operations

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

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.

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.

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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

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

3.6 References ______62

Synthesis and Conclusions ______66

4.1 Overall Synthesis ______66

4.2 Circumneutral pH Bioleaching of Ni under Denitrifying Conditions ______66 4.4.1 Revival of Zhou’s Microcosms ______66 4.4.2 Total Bioleached Ni in the Microcosm Studies ______67 4.4.3 Mass Balance Study ______67

4.3 Cyanide Biodegradation in Gold Heap Leach Operations ______68 4.3.1 High-quality Environmental Sampling ______68 4.3.2 Evidence of Cyanide Biodegradation in Field and Chemical Parameters ______69 4.3.3 Identification of the Cyanide Degrading Microbe(s) ______69

Appendices ______70

Appendix A: Growth Medium Recipe ______70

Appendix B: pH and nitrate addition data from first and second generations after nitrate was added again in 2017 ______72

Appendix C: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from first and second generations after nitrate was added again in 2017 ______73

Appendix D: Sequencing data from first and second generations on day 181 after nitrate was added back to the bottles in 2017 ______74

Appendix E: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T10_LM and TSRA T11_LM ______75

Appendix F: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T12_LM and TSRA TN9_LM ______76

Appendix G: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation abiotic microcosm ______77

Appendix H: Total Ni leached as sum of Ni in solution and Ni re-dissolved in 0.25M H2SO4 __ 78

Appendix I: Bacterial cell count by qPCR ______79

Appendix J: Comparison between the microbial community in the TSRA bottles from day 220 in Zhou's work and day 181 after the inactive period ______80

Appendix K: 16S rRNA Sequencing results of the 3rd generation of microcosms. The colors indicate repeated microorganisms found in each bottle ______81

Appendix L: Field Data ______82

Appendix M: DNA extraction ______83

Appendix N: qPCR data ______84

Appendix O: Anions and nutrients ______85

Appendix P: Cyanide Species ______86

Appendix Q: Total dissolved metals ______87

Appendix R: Comparison between bacterial cell count, filtered volume and Hydrogenophaga relative abundance per sample ______90

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List of Tables

Table 1: Total Ni bioleached in first and second generations of microcosms determined by the sum of the Ni in solution and the re-dissolved Ni hydroxides in 0.25M H2SO4...... 25 Table 2: Material characterization by metal composition of the original and pre-washed UMFC materials ...... 26 Table 3: Theoretical molar ratios determined by each bioleaching scenario ...... 27 Table 4: Comparison between the theoretical molar ratios for each bioleaching scenario and the measured molar ratios in the first and second generation of microcosms...... 27 Table 5: Nickel recovery determined by dividing the total Ni leached by the expected Ni recovery from the nitrate consumption and sulfate production in the first and second generations of microcosms...... 28 Table 6: Ni recovery from total Ni available for leaching in comparison to the duration of the microcosm enrichment...... 29 Table 7: Total Ni bioleached in third generation of microcosms determined by the sum of the Ni in solution and the re-dissolved Ni hydroxides in 0.25M H2SO4...... 29 Table 8: Nickel recovery determined by dividing the total Ni leached by the expected Ni recovery from the nitrate consumption and sulfate production in the third generation of microcosms...... 30 Table 9: Comparison between the measured and theoretical molar ratios determined by the pre-washed UMFC bioleaching equation...... 31 Table 10: Theoretical molar ratios determined by each bioleaching scenario, including possible S0 oxidation. .... 31 Table 11: Parameters analyzed by the environmental laboratory (analysis description written by the laboratory)...... 47 Table 12: Illumina Data and NCBI information of the four clone representatives indicating how the organisms may grow...... 57

viii

List of Figures

Figure 1: Denitrifying microcosm study timeline from 2012 to 2019 showing four generations of microcosms indicated by different colors. Number of days indicates for how long the bottles were monitored...... 10 Figure 2: Microcosm design for new TSRA transfers showing all four generations of microcosms. Arrows indicate transfer direction of pelleted cells. Stars indicate bottles sacrificed for mass balance analysis...... 11 Figure 3: TSRA 10_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again...... 19 Figure 4: TSRA 11_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again...... 20 Figure 5: TSRA 12_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again...... 20 Figure 6: TSRA TN9_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again...... 5 Figure 7: Microscopy images of TSRA 10, 11, 12 and TN9 (100x magnification) indicating living cells...... 22 Figure 8: Evidence of bioleaching in third generation of microcosms...... 23 Figure 9: Plot of Thiobacillus relative abundance in the microbial community of each generation of microcosms. Green indicates the bottles originated from the first generation and blue shows transfers from the second generation...... 24 Figure 10: Yellow precipitate showing the elemental sulfur content in the original (A) and pre-washed (B) UMFC materials by carbon disulfide assay...... 32 Figure 11: Sampling locations at heap leach pad 7 (in bold): Pregnant solution from pad lateral lines (Sampler 3 and Sampler 45 to the left) and collecting pipes (Pad 7 North and Pad 7 South at the bottom)...... 45 Figure 12: Simplified process flow diagram indicating the sampling locations (bold)...... 45 Figure 13: Results for field parameters (measured directly in the field). Graph indicates the average from sample duplicates...... 51 Figure 14: Alkalinity, ammonia and anions concentrations. Graph indicates the average from sample duplicates...... 52 Figure 15: Concentrations of various cyanide species. Thiocyanate results were below the detection limit in all samples. Graph indicates the average from sample duplicates...... 52 Figure 16: Comparison of pH, gold and free cyanide concentrations and copies of 16S rRNA gene per mL across all samples. Error bars shows range of duplicate samples taken on consecutive days...... 53 Figure 17: Heatmap of the major Operational Taxonomic Units (OTUs) found in samples from the mine versus tree of sample clustering. This figure shows the remarkable dominance by the first OTU in the list, and the similarity between samples from similar locations...... 55

Figure 18: Nucleotide alignment of the 6 successful clones most similar to OTU#1 Hydrogenophaga-like 16S reference genes. Grey indicates identity at each nucleotide position; black lines are mismatched nucleotides in the sequences as compared to Clone5 and Clone7 ...... 56 Figure 19: Phylogenetic tree showing similarity of the Hydrogenophaga-like clones 5 and 2 and the best-hit organisms from NCBI BLAST, based on alignment of partial 16S rRNA gene containing 840 nucleotides...... 58 Figure 20: Plot of alkalinity, calcium concentration and pH as a function of Hydrogenophaga-like relative abundance in the samples analyzed...... 59

List of Appendices

Appendices ...... 70 Appendix A: Growth Medium Recipe ...... 70 Appendix B: pH and nitrate addition data from first and second generations after nitrate was added again in 2017 ...... 72 Appendix C: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from first and second generations after nitrate was added again in 2017 ...... 73 Appendix D: Sequencing data from first and second generations on day 181 after nitrate was added back to the bottles in 2017 ...... 74 Appendix E: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T10_LM and TSRA T11_LM ...... 75 Appendix F: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T12_LM and TSRA TN9_LM ...... 76 Appendix G: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation abiotic microcosm ...... 77

Appendix H: Total Ni leached as sum of Ni in solution and Ni re-dissolved in 0.25M H2SO4 ...... 50 Appendix I: Bacterial cell count by qPCR ...... 79 Appendix J: Comparison between the microbial community in the TSRA bottles from day 220 in Zhou's work and day 181 after the inactive period...... 80 Appendix K: 16S rRNA Sequencing results of the 3rd generation of microcosms. The colors indicate repeated microorganisms found in each bottle ...... 81 Appendix L: Field Data ...... 82 Appendix M: DNA extraction ...... 83 Appendix N: qPCR data ...... 84 Appendix O: Anions and nutrients ...... 85 Appendix P: Cyanide Species ...... 86 Appendix Q: Total dissolved metals ...... 87 Appendix R: Comparison between bacterial cell count, filtered volume and Hydrogenophaga relative abundance per sample ...... 90

List of Abbreviations

16S rRNA 16S ribosomal Ribonucleic Acid AMD Acid Mine Drainage BLAST Basic Local Alignment Search Tool CIC Carbon In Column DNA Deoxyribonucleic Acid DAPI Diamidino-2-phenylindol DO Dissolved Oxygen ICP-OES Inductively Coupled Plasma – Optical Emission Spectrometry IC Ion Chromatography LB Luri-Bertani NCBI National Center for Biotechnology Information OTU Operational Taxonomic Unit ORP Oxidation Reduction Potential PCR Polymerase Chain Reaction qPCR Quantitative Polymerase Chain Reaction TSRA Thompson Soil Recovery Area UMFC Ultramafic WAD Weak Acid Dissociable

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Introduction

1.1 General Introduction

Microbes have explored metals for billions of years, long before the Prehistoric man first began to use gold, silver, copper, tin, lead and iron, and historians named periods in human history after metals. In modern days, just like the first living microorganisms, man is intensively mining and processing a large amount of minerals to sustain a growing population. However, economic and high-graded ore deposits are becoming depleted, therefore low-grade and expensive-to-process ores became an alternative to meet the increasing global demand for metals. At the same time, mining companies are also facing environmental challenges with tailings stabilization, water supply and wastewater treatment.

Similar to humans in modern days, microbes also had to adapt to limited available resources to prosper in a rocky planet during the origin of life. The extreme metal-rich environment forced those creatures to develop mechanisms to obtain carbon and energy from peculiar sources, and thrive in a seemingly lifeless land. These microbial mechanisms of using metals have been studied to create biotechnologies for mineral processing and mining water and wastewater treatment and address geological and environmental issues in mining operations.

The present work describes remarkable interactions between minerals and bacteria found in iron sulfide tailings ponds at a nickel mine, and in cyanide solutions from a gold heap leaching mine. The goal of this research is to identify the microbial communities living in these extreme environments and understand how they thrive, to improve metal extraction from ores and wastes in both mines.

1 2

1.2 Thesis Outline and Objectives

This document is divided into four chapters. Chapter 1 describes the general introduction, the thesis outline and objectives. Chapter 2 begins with an overview of nickel extraction and mining operational concerns originated by the elevated serpentine content in low-graded ultramafic ores. A bioleaching alternative is suggested for nickel recovery under nitrate reducing conditions and circumneutral pH to reduce magnesium dissolution and increase nickel extraction. This project is a continuation of the Master thesis “Nitrate-Dependent, Neutral pH Bioleaching of Ni from an Ultramafic Concentrate” by Han Zhou from 2014, and aims to determine the total nickel bioleached recovery and mass balances in a long-term microcosm study.

Chapter 2 describes a cyanide biodegradation phenomenon occurring in a gold heap leaching process that prevents the cyanide solution from being recycled to the leach pads for further gold recovery. The methodologies for chemical and biological sampling and analyses are described. The results from biological analyses show that the microbial community at this mine site is peculiarly simple, mostly dominated by a Betaproteobacterium related to Hydrogenophaga. In addition, the chemical results suggest the ability of this bacterium to hydrolyze cyanide to products such as ammonium and formate.

Finally, Chapter 4 brings the overall synthesis and conclusions from Chapters 2 and 3.

Bioleaching of Nickel from Sulfidic Ores and Wastes under Denitrifying and Circumneutral pH Conditions: Long-term Microcosm Studies

Abstract

To investigate neutral bioleaching of nickel-rich ores and wastes as a sustainable leaching alternative for mining processes, a series of a microcosm studies were performed over the last six years. The experiments were carried out under both aerobic and denitrifying conditions and at pH close to 7. Indigenous environmental samples were obtained from natural waters within a mine site in Canada and used for enrichment. Dissolved nickel concentration, nitrate reduction, sulfide oxidation, and the microbial community were monitored during the experiments. The dissolved nickel concentration was found to be a fraction of the total nickel leached, since the nickel solubility is low at pH 6.5-7.5. Subsequent transfers were performed in order to better account for mass balances in the system, particularly in the anaerobic nitrate-amended enrichments, and the solid phase was also analyzed. The total nickel bioleached in this study confirmed a strong potential for microbial leaching under neutral pH.

3 4

2.1 Introduction and Specific Objectives

Acid mine drainage (AMD) is one of the largest environmental hazards generated by mining operations of sulfide ores. It is well known that natural oxidation of sulfide minerals and acidification are promoted by atmospheric oxygen and naturally occurring aerobic, acidophilic bacteria. In order to avoid this phenomenon, the mineral wastes produced are stored under water in tailing ponds, in an attempt to reduce contact with atmospheric oxygen, and therefore diminish acid production.

Very often, the materials disposed in tailings still have economic value, however are expensive to process through conventional technologies. In this case, an engineered accelerated AMD process known as bioleaching has the potential to provide a low cost and sustainable way to recover locked metal values from sulfidic tailings. Many bioleaching studies are conducted at low pH, however, it is important to investigate a neutral pH bioleaching potential, since this would reduce the costly neutralization requirements of the acid generation throughout the processes.

This research is a continuation of the Master thesis study “Nitrate-Dependent, Neutral pH Bioleaching of Ni from an Ultramafic Concentrate” by Han Zhou from 2014, when a series of microcosms were prepared under aerobic and anaerobic conditions utilizing mining water samples taken from tailings ponds and open pits, and ultramafic concentrate (UMFC) from the Thompson mine in Manitoba. The objective of the study was to investigate the nickel bioleaching potential under denitrifying conditions and neutral pH in which indigenous microbes were used as catalysts.

To better elucidate the bioleaching mechanism in this study, microbial consortia from selected first-generation cultures were used as inocula for a second enrichment, in which growth medium was used instead of mining waters. Additionally, a heated pre-treatment with sulfuric acid was performed on the UMFC donor in order to eliminate carbonates and magnesium oxides. All bottles were maintained at pH 6.5-7 to reproduce the native conditions of the indigenous microbes and reduce the dissolution of magnesium, while nickel was leached from the concentrate.

Zhou successfully demonstrated that nickel can be bioleached under denitrifying and circumneutral conditions by measuring the progress of bioleached nickel concentration in the aqueous solution, nitrate consumption and sulfate production. However, a critical difficulty in this research is that nickel precipitates at neutral pH (Warner et al., 1996), therefore it was not possible to determine the total bioleaching potential by only measuring nickel ions in solution. Consequently, the final leached solids needed to be analyzed by re-dissolving the precipitated nickel salts to account for all the nickel extracted from the concentrate, and lastly, to determine the mass balance in the system and recovery efficiencies.

This study comprised three objectives:

Objective 1: To revive the denitrifying indigenous cultures established by Han Zhou, after 4 years of inactivity by amending the bottles with nitrate as electron acceptor. This initial step of the study aims to evaluate if the cultures could be revived and also be successfully transferred to new bottles for further Ni bioleaching studies.

Objective 2: To examine the final ore material from the nitrate reducing microcosm experiments conducted by Han Zhou to try to account for the total bioleached Ni by re- dissolving any reprecipitated Ni salts in 0.25M H2SO4, and also determine the non-bioleached Ni in the remaining solids by Aqua Regia digestion.

Objective 3: To evaluate the mass balances in both the microcosms prepared by Zhou and in new transfers in order to compare the expected versus measured Ni recovery efficiencies based on cumulative nitrate consumed and sulfate produced, and leached Ni.

2.2 Literature Review

This section describes the nickel utilization and demand by society, the origins of nickel deposits and the conventional leaching process in hydrometallurgy. The operational issues resulting from the elevated serpentine content in ultramafic ores, and the bioleaching alternative for nickel recovery under nitrate reducing conditions and circumneutral pH, to reduce magnesium mobilization and increase nickel extraction will be discussed.

2.2.1 Nickel Sulfide Deposits

Nickel is an extremely versatile metal due to its resistance to corrosion, malleability and ductility. It is used for metal plating and the fabrication of coins and batteries, but most of the demand comes from stainless-steel manufacturing. The main nickel ore deposits are classified as sulfides and laterites. Lateritic deposits correspond to the world’s largest reserve of nickel and are formed from the deep weathering of exposed nickel containing rocks. Due to complex mineralogy, lateritic ores are usually uneconomic to mine, and consequently most of the nickel produced globally comes from sulfide ores in which the metal is mainly found in the pentlandite

(Ni,Fe)9S8 mineral form (Behera et al., 2015).

2.2.2 Hydrometallurgical Process of Low-graded Ultramafic Ores

Nickel sulfide ores are often associated with ultramafic and intrusive igneous rocks characterized by elevated iron and magnesium content. The magnesium content is often the major challenge when processing low-grade ores through conventional technologies. Since high-grade sulfide deposits are gradually becoming depleted, mining companies have been searching for alternatives to process low-graded ultramafic sulfide ores in order meet the increasing global demand for nickel (Cameron et al., 2009). In the conventional hydrometallurgical metal processing, rocks are crushed and ground to expose the nickel minerals. The material is then concentrated through flotation, where sulfide particles adhere to air bubbles and are separated from the gangue minerals. Although, hydrophobic magnesium particles tend to float with the sulfides, therefore reducing the separation efficiency. After the flotation step, the concentrate is leached to release the nickel ions or smelted to produce a nickel matte (Witney & Yan, 1996).

The elevated magnesium content in the concentrate increases the temperatures in the smelters where the nickel rich matte is roasted to be refined posteriorly. This process can be highly energy-intensive and thus raises operational costs. In comparison, recovering nickel from the magnesium-rich concentrate through leaching is also an arduous task, since magnesium oxides can be readily dissolved at low pH and consume the sulfuric acid required for nickel leaching. These difficulties are well known in the hydrometallurgical industry when exploring ultramafic

ore deposits through conventional mineral processing technologies, and extensive research has been done to lessen MgO handling expenses and increase nickel leaching yields.

2.2.3 The Leaching Mechanism for Metal Recovery

Leaching is a selective mechanism of metal transfer from a host mineral into an aqueous solution. Under ideal circumstances, undesirable components in the ore material will remain in the solid phase, whereas the metal of interest will be entirely dissolved into the liquid phase. The remaining solids are to be treated and disposed in tailings, and the metal-bearing solution will be further processed for metal removal.

The rate at which the metal is transferred from the mineral to the aqueous solution is the limiting factor in hydrometallurgy. In order to achieve an effective solid-liquid separation at minimum operational cost, the leaching solution should allow favorable chemistry that will maximize the targeted metal extraction while minimizing the dissolution of the gangue minerals. Therefore, in conventional hydrometallurgical processes, leaching is often performed under low pH conditions to provide satisfactory dissolution rates (Smit, 2001).

Nevertheless, processing low-graded ultramafic ores under acidic conditions decreases the selectivity of nickel from the unwanted magnesium species. Hence, an alternative approach is to perform leaching at a pH where nickel can be extracted, but magnesium dissolution is minimized. However, the degree of nickel transfer into the aqueous solution is reduced at elevated pH, thus the use of a catalytic agent, such as naturally growing microbes from mine sites, can increase the kinetics in the system.

2.2.4 The Bioleaching Alternative

This process of extracting metals from minerals utilizing microbes is called bioleaching. One example of this natural phenomenon is often seen in mine sites as Acid Mine Drainage (AMD) that occurs from the weathering of iron- and sulfur-rich rocks. Resident microbes enhance the

rate of these reactions and derive energy from the oxidation of sulfide to sulfate, solubilizing ferrous iron, which is then oxidized to ferric iron. The reduced iron and sulfide in the minerals are electron donors while oxygen is the electron acceptor in the metabolism of certain microbes. Microbially-accelerated mineral dissolution occurs either in a direct or indirect mechanism. In the direct mechanism, microbes directly utilize the sulfur or iron embedded in the mineral, while in the indirect mechanism, acid-dissolved and oxidized ferric iron initiates attach on the rock mineral and solubilizes sulfide and ferrous iron (Bosecker, 1997).

2.2.5 Nitrate Sources in Mining

In order to reduce the occurrence of AMD, mining waste rocks are disposed of in tailing ponds covered with water to minimize exposure to atmospheric oxygen. The pH is also adjusted to neutral conditions to stabilize the waste. These rocks and wastewaters often contain elevated nitrate levels due to the spillage or drainage of incompletely detonated, highly soluble ammonium-nitrate blasting agents used for underground mining, which can cause eutrophication in water bodies nearby (Pommen, 1983). Nitrates may also enter the tailings ponds from local agriculture.

A major effort in mine sites is to identify the potential sources of nitrate in blasting operations and diminish the nitrate dissolution into rocks and mine drainage waters, such as reducing explosive malfunctions, improving handling and usage practices, and the application of water- resistant emulsions. Some technologies for nitrate removal have also been investigated for mining wastewater treatment, including biological denitrification utilizing local microbial communities (Subedi, 2017 and Kiskira et al. 2017).

2.2.6 Nitrate-reducing Bioleaching of Ni at Circumneutral pH

Nitrate can also serve as an electron acceptor in microbial oxidation of iron or sulfur at neutral pH. This process could be of interest particularly for ultramafic ores for nickel bioleaching. At circumneutral pH, magnesium dissolution is reduced while nickel is unlocked from the mineral

host. The present study investigates the potential for biological leaching of nickel under denitrifying conditions at circumneutral pH, to improve the mining processing of a low-grade ultramafic ore deposit in Canada and to simultaneously treat possible nitrate contamination in the mine wastewaters.

2.3 Materials and Methods

2.3.1 Microcosm Revival from Zhou’s work

Master’s student Wendy Zhou initiated a microcosm study in 2012 to investigate neutral bioleaching of ultramafic ores (Zhou, 2014). In order to continue the work performed in the previous study, the denitrifying microcosm bottles containing ultramafic ore set-up in Zhou’s work were revived after a long period during which the bottles sat sealed and in the dark with no amendments of nitrate. (Figure 1). In 2017, a subset of microcosms were fed nitrate again and monitored for nitrate consumption, sulfate production and nickel dissolution which would indicate activity of the denitrifying microorganisms. Fluorescence microscopy using 4′,6- diamidino-2-phenylindol (DAPI) staining targeting DNA-rich regions was also performed to confirm presence of living cells and to measure cell abundance.

The bottles which showed activity as inferred from nitrate consumption, nickel solubilization and presented living cells stained by DAPI were selected for cell transfer to a third and fourth generations of microcosms. DNA was also extracted prior to these transfers to analyze how the microbial community evolved by the end of the 1st and 2nd generations.

*WZ *LM Figure 1: Denitrifying microcosm study timeline from 2012 to 2019 showing four generations of microcosms indicated by different colors. Number of days indicates for how long the bottles were monitored.

* Zhou’s microcosms (1st and 2nd generations in green and blue, respectively) are indicated as *WZ in the time axis, and the continuation of this study is indicated as *LM, followed by a 3rd and 4th generations of microcosms.

2.3.2 Microcosm Design for New Transfers

Four denitrifying active bottles from the first and second enrichments performed by Zhou (labelled TSRA: Thompson Soil Recovery Area) were selected for a third enrichment. A cell pellet from 10 mL of each culture was transferred to new microcosm bottles containing pre- washed UMFC as electron donor and growth medium (medium described in Section 2.3.4). Abiotic controls and duplicates of each bottle were also set-up. Subsequently, a fourth microcosm enrichment was set-up utilizing a cell pellet from 30 mL of TSRA TN9_LM as inoculum, and the original UMFC material as electron donor in growth medium (Figure 2). All cultures were maintained by KNO3 as electron acceptor.

Figure 2: Microcosm design for new TSRA transfers showing all four generations of microcosms. Arrows indicate transfer direction of pelleted cells. Stars indicate bottles sacrificed for mass balance analysis.

2.3.3 Ultramafic Material Preparation

The UMFC concentrate used in the previous study was kept sealed inside a Ziploc® bag at -20 °C to avoid oxidation. The material was thawed and submitted to a mild acid wash in 0.25M H2SO4 followed by Aqua Regia digestion to examine if the solids were still comparable to the original material used for the first microcosm enrichments, and to check if they were free of oxidized particles due to the minor possibility of contact with air throughout the years. Subsequently, an acid pre-wash treatment was performed. Wet ore (100g) was transferred to a 1L jacked reactor and washed with 800 mL of 1M sulfuric acid under 80°C and constant agitation at 250 RPM for 21h. The final material was cooled down, filtered and washed with 800 mL of 0.05 M sulfuric acid. In order to wash out the acid, the solids were re-pulped twice in a beaker with 1600 mL of MilliQ® water and then filtered. The final washed material was kept inside a Ziploc bag and frozen at -20°C to avoid oxidation, and a portion of it was dried to determine the moisture content and then digested in Aqua Regia for characterization.

2.3.4 Growth Medium and Stock Solutions Preparation

A defined mineral medium solution (1L) was prepared following a recipe adapted from Widdel and Bak (1992) as described in Appendix A. The phosphate buffer, redox indicator, and the

® NH4Cl and CaCl2.6H2O salts were added to MilliQ water and the solution was autoclaved for

30 min at 121°C and then purged for 1h with a N2/CO2 gas mix. The trace element and selenite- tungstate solutions were prepared separately and autoclaved and purged with N2 gas prior to their addition to the purged medium solution to avoid precipitation of salts. The saturated bicarbonate and vitamins stock solutions were also added after the medium was purged. The pH was measured and adjusted to 7 with HCl. Anaerobic sterile stock solutions of 1M KNO3, 1M NaOH and 1M HCl were also prepared for microcosm maintenance and pH adjustment.

2.3.5 Cell Transfer to New Microcosms

The 1st and 2nd microcosm enrichments were kept into a dark box inside an anaerobic glovebox

(Coy) with an atmosphere containing: 80%N2;10%H2;10%CO2. Prior to the transfer for the third set of transfers, the bottles were agitated then left to settle for 10 min. After the solids settled, 20 mL of the supernatant was transferred to an O-ring Nalgene® centrifuge tube using a sterile plastic pipette. The cap was sealed with anaerobic tape and the tube was brought out of the glovebox to be centrifuged. Two centrifugation steps were performed to guarantee that the cells would be pelleted down. The first centrifugation was performed using a swing bucket in the Avanti® J-E Centrifuge for 20 min at maximum speed (5300 RPM) and 22°C, under maximum acceleration. The second centrifugation was done using a fixed angle rotor for 20 min at 6000xG and 22°C under maximum acceleration.

Inside the glovebox, the supernatant was transferred to a 50 mL falcon tube to be analyzed by ICP and the cell pellets were resuspended in 20mL of anaerobic medium. UMFC pre-washed material (3 g dry wt.) was transferred to 160mL sterile microcosm bottles, followed by 90mL of medium, and the cells resuspended in 10mL of medium. Abiotic controls were also prepared and contained 3 g (dry wt.) of the pre-washed UMFC material and 100 mL of fresh medium.

Duplicates were made for both treatments. Sodium nitrate (0.2 mL of a 1 M stock) was added to all the bottles to a final concentration of 2 mM, which were sealed with a butyl rubber stopper and aluminum crimp.

The fourth enrichment duplicates were prepared following the same centrifugation steps described above using 60 mL of the TSRA TN9_LM resuspended in medium. The inoculum was transferred to a serum bottle containing 5g of the original UMFC and 70 mL of medium. Nitrate was added to the active and abiotic microcosms and the pH was adjusted to 6.5.

2.3.6 Microcosm and Transfer Culture Maintenance Aqueous samples from the microcosm supernatant were taken on a weekly basis to monitor the evolution of the denitrifying bioleaching mechanism, such as nitrate consumption and sulfate production by Ion Chromatography (IC), and Ni, S, Fe and Mg concentrations by Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES). To avoid nitrite accumulation in the system, 2mM of KNO3 was added each time that nitrate levels were low. The pH was maintained between 6.5 ± 0.2 by adding drops of HCl or NaOH and measuring the pH in each addition by an Oakton pH Spear Waterproof Pocket pH Testr®. The bacterial culture profile was also examined by DNA extractions for 16S rRNA sequencing and qPCR described in section 2.3.9.

2.3.7 Ultramafic Material Characterization by Aqua Regia Digestion

In order to determine the elemental composition of the original and pre-washed UMFC materials utilized for the microcosm setups, and of the final non-leached material by the end of the microcosm life, the solids were submitted to Aqua Regia digestion. Approximately 0.5 g of the material was dissolved in 15 mL of Aqua Regia solution, which is a combination of 70 wt.%

HNO3 and 35 wt.% HCl in a 1 to 3 ratio by volume.

The mixture was digested in a heated plate until a brownish liquid and grey solid particles (silica) were observed. The solution was then filtered with Q5 quantitative Fisherbrand® filter paper and

the solids were dried in a vacuum oven at 80 °C. The cooled filtrate was diluted with 5 wt.%

HNO3 in 100 mL volumetric flask and samples were taken for ICP analysis following the aqueous sample analytical procedures (section 2.3.8).

2.3.8 Aqueous Sample Analyses: Total Soluble Metals, Nitrate and Sulfate

Approximately once a week 1mL of aqueous samples were taken from the microcosms inside an anaerobic glovebox using a sterile syringe (Becton Dickenson & Co.®), and a sterile 22-gauge needle inserted through the bottle rubber stopper. The samples were brought out of the glovebox and immediately filtered using a PES 0.22 µm syringe filter (Pall Co. Acrodisc®) into an Eppendorf tube for metal and anion analyses.

® The aqueous samples were diluted in 5% HNO3 using an auto-diluter (Hamilton Microlab 500). Standards for Ni, S, Fe and Mg were prepared from stock solutions were similarly diluted to meet the ICP-OES, Agilent Technologies 700 calibration range. Prior to each analysis the equipment was stabilized by allowing the Argon plasma (1.2 kV) to run for approximately 20 min. The standards were run in every ICP analysis. Three to four wave lengths were chosen for each element and the calibration only passes if the measurement error for the element is below 10% and R2 is above 0.9999.

The same filtered samples utilized for ICP were also analyzed by Ion Chromatography for nitrate, nitrite, sulfate, phosphate and chloride. Each sample was diluted with MilliQ® water in a 1:50 ratio using pipettes. Standards were diluted to concentrations ranging from 0.005 to 1 mM and analyzed in each run for calibration. The analyses were performed by a Dionex Integrated Regenerative Ion Chromatography System 2100 set with an AS18 4×250mm anion column through where the samples were carried by a 23mM KOH eluent at 57mA and 30°C.

2.3.9 Biological Sample Analyses: DNA Extraction, qPCR and Amplicon Sequencing

Each DNA extraction was performed using the DNeasy® PowerSoil Kit by QIAGEN. Microcosm bottles were manually shaken to homogenize the slurry content and 1 mL of sample was extracted using a sterile syringe and transferred to an Eppendorf tube. The slurry was centrifuged for 15 min at 10,200 RPM, the supernatant was removed, and the pellet was placed into the PowerBead Tube provided in the kit by re-dissolving the pellet in the PowerBead Tube solution. The Quick-Start Protocol from the kit was followed to extract DNA and at the end of the procedure the pellet was eluted in 50 µL of Solution C6 and the DNA concentration was measured by NanoDrop® ND-1000 Spectrophotometer and Qubit® 3 Fluorometer. Subsequently aliquots of each DNA sample were used for 16S rRNA amplicon sequencing and quantification using quantitative Polymerase Chain Reaction (qPCR).

Quantitative Polymerase Chain Reaction (qPCR) was performed to quantify the abundance of total bacteria 16S rRNA gene sequences in each sample, and the universal bacterial primer set 1055f (5’-ATGGCTGTCGTCAGCT-3’) and 1392r (5’-ACGGGCGGTGTGTAC-3’) was used to target the 16S rRNA gene. The reaction mixture was made by adding 10μl of EvaGreen Mastermix, 0.5μl of each 10μM primer, and 7μl of ultrapure water. 2μl of each DNA sample was added to 18μl of the reaction mix to make the qPCR samples, which were run in triplicate and also included negative controls. A plasmid containing the almost complete 16S rRNA gene of Hydrogenophaga-like was prepared in the lab and used to make the standards. The reaction conditions were: 3 min at 95 °C followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s.

Aliquots of the extracted DNA were sent to Genome Quebec Canada where Illumina MiSeq technology was applied to generate amplicon reads with primers 926f (AAACTYAAAKGAATWGRCGG) and 1392r (ACGGGCGGTGWGTRC) to target the variable regions V6-V8 of the 16S rRNA genes. Raw sequences were then processed with the online pipeline MetaAmp (http://ebg.ucalgary.ca/metaamp/) to obtain the microbial composition of each sample in terms of operational taxonomic unit (OTU) present in each community.

Blue-fluorescent DAPI (4′,6-diamidino-2-phenylindole) nucleic acid stain was used for microscopy imaging of the bacterial cells in the microcosms. The bottles were shaken, and 1 mL

sample was taken with a syringe. One drop (approximetly10 µL) of the sample was loaded into the 4 mm wells of the sterile microscopy slides. The samples were fixed to the slide by slightly heating it over a flame. The samples were then gently washed with MilliQ® water, and 10 µL of a 1µg/mL stock solution of DAPI was added to each sample well.

The slides were covered in a dark container to avoid DAPI degradation by light while drying for 10 min prior to a last gentle wash with MilliQ® water to remove the stain. A droplet of immersion oil was placed on each well and the slides were observed using an epifluorescent microscope (BX 51, Olympus) with a 100 x UPlan Apochromat objective lens, 150 W xenon lamp (Opti Quip), a blue excitation filter cube (excitation band pass 372 nm; emission barrier filter 456 nm) and 10x focusing eyepiece to check for uniformity of cell morphology (1,000-fold magnification). Pictures were taken using the HAMAMATSU camera ORCA-Flash4.0 LT PLUS using the CellSens software.

2.3.10 Final Solid Analyses

After an aliquot of cells from 1st and 2nd generation microcosms was used to create transfer cultures, the remaining solids from these microcosms were filtered and weighed, and the material was submitted to a mild acid wash with 50 mL of 0.25M H2SO4 at pH 3 to re-dissolve any precipitated nickel salts. Two acid washes were performed to guarantee complete dissolution. Subsequently, the solids were washed three times with MilliQ® water to eliminate the sulfuric acid and account for any remaining re-dissolved metals, and a portion of this material was dried in a vacuum chamber (-750mm Hg) with desiccant for 1 day.

Finally, 0.5 g of the dried material was digested by Aqua Regia, and both the nickel-rich acidic solutions and the Aqua Regia samples were analyzed by ICP. The total nickel was determined as the sum of the nickel mass from each acid and water washes (i.e. bioleached nickel), and the non-bioleached nickel remaining in the concentrate. The nickel recovered dissolved in the 0.25M

H2SO4 solution and in the remaining concentrate were compared to the total amount of nickel originally added to the bottles.

Two active bottles and one abiotic control from the third generation were also selected for a

0.25M H2SO4 wash after a period of enrichment when sulfate concentration achieved similar levels as the second generation and nitrate was being actively consumed. The same procedure as the 0.25M H2SO4 wash for the first and second generations was followed.

2.3.11 Elemental Sulfur Investigation

The acid pre-wash of the UMFC material was performed to eliminate gangue materials and make the sulfide minerals easily available for microbial leach. However, under the pH and temperature conditions where this pre-wash was performed, sulfur could be unlocked from pyrrhotite dissolution and precipitated in the elemental form instead of volatilizing as H2S (Dyson and Scott, 1976). The products of the acid wash would then be pyrite, pentlandite and elemental sulfur (instead of pyrrhotite) and this would affect the bioleaching ratios since the microbes could use nitrate to oxidize this elemental sulfur to sulfate and nickel would not be leached.

In order to determine if elemental sulfur was formed during the acid wash, 4 g of both original and pre-washed UMFC materials were added to pre-weighed beakers covered with a watch glass and dissolved in 40 mL of carbon disulfide solution in a stirring plate for 1h. The solution was filtered and left in a fume hood until complete volatilization of the carbon disulfide, leaving behind a yellow solid product corresponding to elemental sulfur. The beakers containing elemental sulfur were weighed and the measured mass was compared to the available sulfur in the initial material. The results were used to determine elemental sulfur to nitrate ratios for mass balance calculations.

2.4 Results and Discussion

The following sections describe the microcosm revival from Zhou’s work after a period of 4 years of no nitrate addition, and how the microbial community changed during this time. The total Ni leached in the first and second generations are determined from a series of mild acid washes with 0.25M H2SO4 to re-dissolve the precipitated Ni hydroxides. The total Ni recovery

was determined by comparing the total Ni leached with how much Ni was estimated to be extracted from the cumulative nitrate consumption and sulfate production in the bottles.

The successful cell transfer from Zhou’s bottles to a third generation of microcosms is shown in section 2.4.3. The bacterial cell count and the microbial community are discussed in section 2.4.4. The solids from the third generation were also submitted to a mild acid wash with 0.25M

H2SO4, and the total Ni recovery was determined. An elemental sulfur assay was performed to explain why almost no Ni was recovered in the third generation and the low Ni recovery in the second generation.

2.4.1 Revival of Zhou’s Microcosms

The subsequent graphs (Figure 3, Figure 4, Figure 5 and Figure 6) show the data from Zhou’s TSRA first and second microcosm experiments from 2014 (indicated as *WZ) followed by 4 years of no nitrate addition. During these four years, anaerobic conditions were maintained, and since there was plenty of sulfate from the prior oxidation of sulfide, sulfate-reduction occurred. During this phase, the pH was not controlled, and the microcosms reached basic conditions at pH 8.

When nitrate was added again to the bottles (indicated as *LM, raw data in Appendix B and Appendix C), sulfur re-oxidation to sulfate was observed, which plateaued below the maximum levels from 2014, and indicated that no more nickel could be unlocked from the concentrate under those conditions. As shown, nickel precipitated with sulfide ions and as nickel hydroxides during the reducing inactive phase, and some sulfur was solubilized as S2- and then re-oxidized to sulfate after nitrate was added.

The microbial community analysis of TSRA 11_WZ (Figure 4) indicated that the microbes were growing under methanogenic conditions during the inactive period after the all the sulfate had reduced. Methanogenic microorganisms, such as Methanosarcina and Methanospirillum, were consuming dead biomass as electron donor and producing methane (Boone and Mah, 1986 and Ferry & Boone, 1974). The elevated abundance of Anaerolineaceae also indicated methanogenic

conditions, since growth is enhanced in the presence of methanogens in this family (Yamada et al. 2006). Whereas Anaerolineaceae was found in low abundance in TSRA TN9_WZ, because the organic matter was eliminated from the UMFC after the pre-wash treatment.

A comparison between the microbial community in day 220 from the sequencing data in Zhou’s work (Zhou, 2014) and day 181 after the inactive period is shown in Appendix J. The dominance of Thiobacillus in the community increased with time, indicating that Thiobacillus is the most important microbe in the bioleaching mechanism. The complete analysis of this data will be subject of future work.

Figure 3: TSRA 10_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again.

Figure 4: TSRA 11_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again.

Figure 5: TSRA 12_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again.

Figure 6: TSRA TN9_WZ Cumulative nitrate consumed and sulfate produced, pH and dissolved Ni before and after inactive period. Microbial community at day 180 after nitrate added again.

2.4.2 Evidence of Living Cells

The microscopy images in Figure 7 indicated that living cells remained after the period of inactivity. The long filamentous microorganisms are believed to be methanogens and Anaerolineaceae, as compared to the Chloroflexi genus imaged by Yamada et al. (2006) using DAPI staining. The absence of this microorganism in the microscopy images of TSRA TN9_WZ confirmed the sequencing results (Appendix D). The small and rod-shaped bacteria are likely to be Thiobacillus, as described by Kelly and Wood (2000).

Figure 7: Microscopy images of TSRA 10, 11, 12 and TN9 (100x magnification) indicating living cells.

2.4.3 Dissolved Ni Evolution, Nitrate Consumption and Sulfate Production

Figure 8 indicates the nickel bioleaching mechanism in the third generation of microcosms (raw data in Appendix E and Appendix F). The abiotic control did not present significant sulfate production and demonstrated slight nitrate consumption (Appendix G). The increase in sulfate levels and nitrate consumption in the active bottles indicated successful cell transfer from previous microcosms and effective bioleaching. Nickel salts re-dissolution began to be observed when the pH was adjusted below 6.5 after a period of microbial adaptation to circumneutral conditions.

Figure 8: Evidence of bioleaching in third generation of microcosms.

2.4.4 Bacterial Cell Count and Microbial Community Profile

The bacterial cell count for these microcosm studies are presented in Appendix I. Bacteria can be found in the supernatant and also attached to the solids, so it is challenging to account for the total bacteria in the microcosms. Another challenge is that metals can interact with the solutions from the DNA extraction kit. A test was performed extracting DNA from the supernatant after shaking bottles and letting the solids settle, and from the slurry right after the bottles were fully mixed. The results indicate that the metals in the slurry inhibited the DNA extraction. Therefore, the qPCR results only indicate the minimum copies of cells per mL of culture, not the total bacterial count.

The 16S rRNA sequencing results of the DNA samples extracted from the 3rd generation of microcosms are presented in Appendix K. The selectivity for Thiobacillus in each transfer is evident in Figure 9. The relative abundance of Thiobacillus increased from maximum of 26% in TSRA 10_WZ from the first generation to 85% in TSRA T10_LM from the third generation. These results confirm that Thiobacillus is the major player in the bioleaching mechanism. The complete analysis of this data will be subject of future work.

100%

80%

60%

40% Relative Abundance (%) Relative

20% Thiobacillus 0% TSRA 10_LM TSRA 11_LM TSRA 12_LM TSRA TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TSRA N9_LM TSRA TSRA T10_LM TSRA T11_LM TSRA T12_LM TSRA T10_LM TSRA T11_LM TSRA T12_LM TSRA TSRA TN9_LM TSRA TN9_LM TSRA 1st Generation at Day 1st and 2nd Generations at 3rd Generation at Day 189 3rd Generation at Day 280 220 (Zhou, 2014) Day 181 After Inactive Period

Figure 9: Plot of Thiobacillus relative abundance in the microbial community of each generation of microcosms. Green indicates the bottles originated from the first generation and blue shows transfers from the second generation.

2.4.5 Mass Balance Study in Zhou’s Microcosm Bottles

After the successful cell transfer to a third generation of microcosms, the final solids from the first and second enrichments maintained by Zhou were analyzed by a series of mild acid washes with 0.25M H2SO4 to re-dissolve the precipitated nickel salts and account for the total bioleached nickel. Mass balances were determined by comparing the theoretical molar ratios in the original

25 and pre-washed UMFC materials with the measured cumulative nitrate consumed and sulfate produced, as well as with the total Ni leached.

The total nickel bioleached was determined by the sum of the dissolved ions in the supernatant and the re-dissolved nickel salts in the acid washes (Appendix H). The remaining nickel in the concentrate material is the difference between the initial nickel available in the microcosms and the total nickel bioleached. The results show that only a small fraction of the total nickel bioleached was being measured by nickel ions in the aqueous phase. Most nickel (90-99%) had indeed precipitated and the bioleaching recovery was greater than expected from the supernatant measurements (Table 1).

Table 1: Total Ni bioleached in first and second generations of microcosms determined by the sum of the Ni in solution and the re-dissolved Ni hydroxides in 0.25M H2SO4.

Ni in solution Ni in 0.25M H2SO4 Total Ni bioleached Remaining Ni in the Microcosm Treatment (mmol/bottle) washes (mmol/bottle) (mmol/bottle) solids (mmol/bottle) TSRA 10_WZ 1st Active 0.00 1.47 1.47 4.0 TSRA 11_WZ 1st Active 0.01 1.24 1.25 4.3 TSRA 12_WZ 1st Active 0.02 0.87 0.89 4.6 TSRA TN9_WZ 2nd Active 0.03 0.43 0.46 3.9 TSRA ABIO TN3_WZ 2nd Abio 0.00 0.03 0.01 4.4

Denitrifying bioleaching equations were determined by the pentlandite theoretical formula in Equation 1, and defining molecular formulas for the original and pre-washed UMFC concentrates (Equations 1 and 2) based on the nickel, iron and sulfur composition in the material characterization and their respective molar masses (Table 2). The stoichiometry was determined on the basis of 1 mol of sulfur, and the theoretical molar ratios are shown at Table 3.

Table 2: Material characterization by metal composition of the original and pre-washed UMFC materials.

Metal Original UMFC (wt%) Pre-washed UMFC (wt%) Ni 7.9% 11.2% Mg 8.3% 0.4% Fe 23.7% 23.3% S 18.2% 27.1% Soluble Si 0.1% 0.2% Ca 0.5% 0.1% Mn 0.1% 0.0% Co 0.3% 0.3% Cu 0.4% 0.4% Zn 0.1% 0.0% Cr 0.0% 0.0% Oxygen, carbonates 40.4% 37.1% and silica

Pentlandite theoretical:

0 2 &0 &2 !"#.%&'()#.'* + ,. -'!./ + #. &'1&. + 1 → #. '()(#1)/ + 7.8 + #. 99!&(:) + #. %&'!" Equation 1

Original UMFC:

0 2 &0 !"#.8/(),./* + &. ,'!./ + ,. 881&. + ,. #,1 → ,. /()(#1)/ + 7.8 + ,. #9!&(:) + #. 8/!"&2 Equation 2

Pre-washed UMFC:

0 2 &0 !"#.8,()#.9%* + ,. 99!./ + #. ;81&. + #. -1 → #. 9%()(#1)/ + 7.8 + #. ;8!&(:) + #. 8,!"&2 Equation 3

Table 3: Theoretical molar ratios determined by each bioleaching scenario.

*Theoretical ratios determined by X, Y and Z from the bioleaching equations on the basis of 1 mol of S.

** The colors indicate the bioleaching scenario for each microcosm generation.

Theoretical molar ratios were determined according to each bioleaching scenario and compared to the measured Ni:NO3, Ni:SO4 and NO3:SO4 ratios in Table 4. The cumulative nitrate consumed and sulfate produced by the end of each microcosm study, as well as the total nickel leached determined by the hydroxides dissolution in 0.25M H2SO4 are presented in Table 5. The percentage of nickel recovery was calculated by dividing the total Ni leached by the Ni expected from the nitrate consumed and sulfate produced.

Table 4: Comparison between the theoretical molar ratios for each bioleaching scenario and the measured molar ratios in the first and second generation of microcosms.

*Theoretical molar ratios determined from the theoretical equations in Table 3

** Measured molar ratios determined by the total Ni leached, cumulative nitrate consumed and cumulative sulfate produced

Table 5: Nickel recovery determined by dividing the total Ni leached by the expected Ni recovery from the nitrate consumption and sulfate production in the first and second generations of microcosms.

The Ni:NO3 results from the first generation closely match the theoretical molar ratio of the original UMFC, indicating that most Ni was recovered from the nitrate added. The Ni:SO4 is similar to the pentlandite theoretical molar ratio, which shows that most sulfate produced originated from the bioleaching of pentlandite. However, the NO3:SO4 levels were higher than predicted values. This indicated that nitrate could have been used as an electron acceptor by other electron donors in the concentrate. Since these bottles were set-up with unwashed concentrate and mining waters, it is possible that they contained some pre-oxidized Ni, which is shown in the recoveries over 100%.

The NO3:SO4 mass balance in the second generation closely matches the theoretical ratio since other electron donors were eliminated during the pre-wash treatment, which means that nitrate was the sole electron acceptor for sulfur oxidation. Although, less nickel was leached when compared to the first enrichment, and the Ni:NO3 and Ni:SO4 ratios were lower than the expected for the pre-washed UMFC. This showed that some sulfur was released from the UMFC material during the pre-wash treatment and was available for oxidation by nitrate during bioleaching. The abiotic control showed minor nickel recovery, and low nitrate and sulfate concentrations, demonstrating that the microbes substantially enhanced nickel leaching under denitrifying conditions.

Table 6 shows the Ni recovery from the total Ni available in the bottles. The total recovery was greater than when only the Ni in the supernatant was being considered. However, the duration of

the experiments indicated that bioleaching occurred at a slow rate. Faster bioleaching rates could possibly be achieved by increasing biomass concentration.

Table 6: Ni recovery from total Ni available for leaching in comparison to the duration of the microcosm enrichment.

2.4.6 Mass Balance Study in New Transfers

After 245 days of enrichment, the third generation of microcosms was sacrificed and the solids were washed with 0.25M H2SO4 following the same methodology performed for the first and second generations to account for the total nickel leached in the biotic and abiotic bottles.

The final nickel bioleached determined by the mild acid washes with 0.25M H2SO4 was low and highly similar to the abiotic control output, as shown in Table 7.

Table 7: Total Ni bioleached in third generation of microcosms determined by the sum of the Ni in solution and the re-dissolved Ni hydroxides in 0.25M H2SO4.

Ni in solution Ni in 0.25M H2SO4 Total Ni bioleached Remaining Ni in the Microcosm Treatment (mmol/bottle) washes (mmol/bottle) (mmol/bottle) solids (mmol/bottle) TSRA TN9_LM 3rd Active 0.01 0.02 0.03 4.4 TSRA T12_LM 3rd Active 0.04 0.02 0.05 4.4 TSRA ABIO_LM 3rd Abio 0.00 0.03 0.03 4.4

However, the elevated sulfate production and considerable nitrate intake in the active bottles from the third generation of microcosms suggested high nickel bioleaching (Table 8 and Table 9). These results indicate that nitrate was used for sulfate production from pre-oxidized sulfur particles in the pre-washed UMFC material, instead of oxidizing sulfide from nickel-bearing minerals (Table 10).

Additional sulfur could have been liberated through pyrrhotite dissolution during the concentrate pre-wash under high temperature and acidic conditions and created a coating barrier on the nickel sulfide minerals. The pre-oxidized sulfur, in the elemental form, was then easily available for the denitrifying microorganisms as electron donor (Equation 4), while the nickel-rich mineral was buried underneath the sulfur layer. Direct bioleaching was inhibited by the lack of accessibility of bacteria with the surface of sulfide minerals and nickel leaching yield was drastically reduced.

S0 oxidation:

# 0 &0 2 7 + ,. &!./ + #. 81&. → 7.8 + #. %!&(:) + #. 91

Equation 4

Table 8: Nickel recovery determined by dividing the total Ni leached by the expected Ni recovery from the nitrate consumption and sulfate production in the third generation of microcosms.

Table 9: Comparison between the measured and theoretical molar ratios determined by the pre-washed UMFC bioleaching equation.

Theoretical molar ratios Measured molar ratios Microcosm Donor Treatment Ni:NO3 Ni:SO4 NO3:SO4 Ni:NO3 Ni:SO4 NO3:SO4 TSRA TN9_LM Pre-washed UMFC 3rd Active 0.01 0.01 0.9 TSRA T12_LM Pre-washed UMFC 3rd Active 0.2 0.4 1.9 0.02 0.02 0.9 TSRA ABIO_LM Pre-washed UMFC 3rd Abio - - -

*Theoretical molar ratio determined from Equation 3, however sulfate is being produced by S0 oxidation following Equation 4.

** Measured molar ratios determined by the total Ni leached, cumulative nitrate consumed and cumulative sulfate produced

Table 10: Theoretical molar ratios determined by each bioleaching scenario, including possible S0 oxidation.

*Theoretical ratios determined by X, Y and Z from the bioleaching equations on the basis of 1 mol of S.

** The colors indicate the bioleaching scenario for each microcosm generation.

2.4.7 Elemental Sulfur Investigation

A carbon disulfide assay was performed in the original and pre-washed UMFC to identify the presence of elemental sulfur (Figure 10). The original material contained 6 mg of S0 in 4 g of sample, whereas the pre-washed UMFC contained 134 mg. These results correspond to 0.2 mmol S0/bottle in the initial original ore mass added to the microcosm, and 3.1 mmol S0/bottle in the pre- washed UMFC bottles. This finding revealed that elemental sulfur was formed during the UMFC acid wash pre-treatment, and showed that the sulfate generated in the third generation of microcosms had not surpassed the S0 available for leaching.

These results also explain why the nickel recovery was lower than expected from the nitrate and sulfate levels in the second generation. Similar pyrrhotite dissolution might have occurred during the UMFC pre-treatment used in the second generation, but to a lesser extent.

Figure 10: Yellow precipitate showing the elemental sulfur content in the original (A) and pre-washed (B) UMFC materials by carbon disulfide assay.

2.5 Conclusions and Recommendations for Future Work

This long-term microcosm study proved that Ni can be bioleached from ultramafic ores and wastes under denitrifying conditions at circumneutral pH. The mass balances in these studies were closed after accounting for the Ni precipitated as hydroxides.

The UMFC acid pre-wash, in the second and third generations, was an attempt to minimize basic conditions introduced by the prominent magnesium content, and therefore lessen pH adjustments throughout the experiments. It also enabled better investigation of NO3:SO4 ratios since only the nitrate would be used to oxidize sulfur. This proved that the excess nitrate consumed in the first

generation of microcosms was due to organics and other electron donors utilizing nitrate as electron acceptor.

The elemental sulfur analysis also showed that excess of sulfate produced in the second and third generations of microcosms came from pre-oxidized sulfur originated by the pyrrhotite dissolution in the acid pre-wash treatment performed in the UMFC. For this reason, a fourth generation of microcosms was set-up using the original UMFC to maintain the active culture and eventually be sacrificed to determine its mass balance and compare the results with the first generation.

Despite the advantage of reducing magnesium mobilization at circumneutral pH bioleaching, the non-dissolution of magnesium oxides can slow down the bioleaching progression since the sulfides are surrounded by minerals from the serpentine group and cannot be reached by the microbes. The circumneutral pH also introduces a passivation difficulty since leached metals precipitate on top of the concentrate, which reduces mass transfer and diffusion. pH control is key to guarantee ideal solubility conditions, and a slightly agitated system could allow the dispersion of particles and enhance mass transfer, at the same time that the microbes can access the minerals for direct bioleaching to occur.

2.6 References

Cameron, R. A., Lastra, R., Mortazavi, S., Gould, W. D., Thibault, Y., Bedard, P. L., … Kennedy, K. J. (2009). Elevated-pH bioleaching of a low-grade ultramafic nickel sulphide ore in stirred-tank reactors at 5 to 45 °C. Hydrometallurgy, 99(1–2), 77–83. https://doi.org/10.1016/j.hydromet.2009.07.001

Bak F., & Widdel F. (1992). Gram-negative mesophilic sulfate-reducing bacteria. The Prokaryotes, 4(2), 3352-3378.

Behera, S. K., & Mulaba-Bafubiandi, A. F. (2015). Advances in microbial leaching processes for nickel extraction from lateritic minerals - A review. Korean Journal of Chemical Engineering, 32(8), 1447–1454. https://doi.org/10.1007/s11814-015-0085-z

Boone D.R., & Mah R.A. (1986). Methanosarcina. Bergey’s manual of systematic bacteriology. Springer 2001, 269-276.

Bosecker, K. (1997). Bioleaching: Metal solubilization by microorganisms. FEMS Microbiology Reviews, 20(3–4), 591–604. https://doi.org/10.1016/S0168-6445(97)00036-3

Dyson, N.F., & Scott, T.R. (1976). Acid leaching of Nickel sulphide concentrates. Hydrometallurgy, 1, 361-372

Ferry J.G., & Boone D.R. (1974). Methanospirillum. Bergey’s manual of systematic bacteriology. Springer 2001, 264-268.

Kelly, D. P., & Wood, A. P. (2000). Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. International Journal of Systematic and Evolutionary Microbiology, 50(2), 511–516. https://doi.org/10.1099/00207713-50-2-511

Kiskira, K., Papirio, S., van Hullebusch, E. D., & Esposito, G. (2017). Fe(II)-mediated autotrophic denitrification: A new bioprocess for iron bioprecipitation/biorecovery and

34 35

simultaneous treatment of nitrate-containing wastewaters. International Biodeterioration and Biodegradation, 119, 631–648. https://doi.org/10.1016/j.ibiod.2016.09.020

Pommen, L.W., The effect on water quality of explosive use in surface mining, Vol. 1, B.C. Ministry of Environment Technical report No. 4; May 1983.

Smit, D. S. (2001). The Leaching Behavior Of A Ni-Cu-Co Sulphide Ore In An Oxidative Pressure - Acid Medium, 4–32. Retrieved from http://dspace.nwu.ac.za/bitstream/handle/10394/9626/Smit_DS_TOC.pdf?sequence=1

Subedi, G., Taylor, J., Hatam, I., & Baldwin, S. A. (2017). Simultaneous selenate reduction and denitrification by a consortium of enriched mine site bacteria. Chemosphere, 183, 536–545. https://doi.org/10.1016/j.chemosphere.2017.05.144

Warner, T. E., Rice, N. M., & Taylor, N. (1996). Thermodynamic stability of pentlandite and violarite and new Eh-pH diagrams fro the iron-nickel-sulphur aqueous system. Hydrometallurgy, 41, 107–118. https://doi.org/10.1016/0304-386X(95)00081-Q

Witney, J.Y. & Yan D.S. (1996). Reduction of magnesia in nickel concentrates by modification of the froth zone in column flotation. Minerals Engineering, 10 (2), 139-154

Yamada, T., Sekiguchi, Y., Hanada, S., Imachi, H., Ohashi, A., Harada, H., & Kamagata, Y. (2006). Anaerolinea thermolimosa sp. nov., Levilinea saccharolytica gen. nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov., novel filamentous anaerobes, and description of the new classes Anaerolineae classis nov. and Caldilineae classis nov. in the bacterial phylum Chloroflexi. International Journal of Systematic and Evolutionary Microbiology, 56(6), 1331–1340. https://doi.org/10.1099/ijs.0.64169-0

Zhou, H., (2014). Nitrate-Dependent, Neutral pH Bioleaching of Ni from an Ultramafic Concentrate (Master’s thesis). University of Toronto, Toronto, Canada.

Identification of Cyanide Degrading Microorganisms in Heap Leach Operations for Gold Recovery

Abstract

A gold mine extracts gold through conventional cyanide heap leaching. After gold is removed from the pregnant solution, the barren cyanide solution is recycled to the ore pad. It has been observed that cyanide degrades during this process. Cyanide solution is applied at a concentration of approximately 100 mg/L and is degraded to approximately 10 mg/L. As a result, this process cannot rely on a steady recycle of cyanide solution for further gold recovery. An unpublished study determined that microbes were responsible for this degradation. The present investigation aimed to identify possible cyanide degrading microorganisms. Biological and chemical samples were obtained from the pregnant and barren solutions from the site. The microbial community at this mining site is remarkably simple, dominated largely by a single

Betaproteobacterium related to Hydrogenophaga. A few closely related microbes are known to have the ability to hydrolyze cyanide to products such as ammonium and formate.

36 37

3.1 Introduction and Specific Objectives

This work investigates the cyanide biodegradation phenomenon occurring at a gold heap leaching process from a mine located in southern California, U.S. Sodium cyanide solution is applied in heap pads via dripping lines placed on top of ore piles. The barren solution percolates through the pad, where cyanide complexes with gold and the pregnant solution is then processed through carbon columns in a carbon-in-column approach (CIC) to adsorb gold, and the barren solution is recycled to the heap pads for further gold recovery.

This mine is experiencing unexpected increase in cyanide consumption at its heap leaching operation, and consequently, the process cannot depend exclusively on continuous recycle of the barren cyanide solution and continual addition of NaCN is required. As a result, the company has been facing an annual increase in the sodium cyanide reagent costs to maintain gold production.

Previous studies conducted on-site concluded that cyanide consumption was enhanced by microbial activity in the heaps and/or process water, however the microbes responsible for this activity were not identified.

For Objective 1, field parameters, chemical and biological samples were obtained from the pregnant and barren solutions at the mine site in study. An environmental laboratory performed chemical analyses for anions, nutrients, total dissolved metals and cyanide species. Environmental DNA samples were extracted from filtered cyanide solution at the site. This initial phase in the project is to provide high-quality environmental samples for the investigation of cyanide biodegradation.

Objective 2 is to inspect any evidence in the chemical and field parameters that can indicate means at which cyanide biodegradation might be occurring, such as cyanide transformation products that can suggest specific biodegradation pathways. These findings can elucidate strategies for the inhibition of microbial activity of cyanide degradation at the mine site.

Lastly, Objective 3 of this study is to identify the microbes involved in this process. The microbial community identified by DNA sequencing can reveal microorganisms that can adapt to highly basic environments, elevated concentration of toxic compounds, and thrive utilizing

38 unusual sources of energy. The enzymatic mechanism and biochemistry of cyanide degradation will be investigated.

3.2 Literature Review

This section provides an overview of the chemistry of gold cyanidation in heap leaching operations, cyanide toxicity and removal technologies in mining operations, including degradation via microbial activity.

3.2.1 The Chemistry of Gold Cyanidation

Cyanide is a chemical compound formed by a triple bond between a carbon and a nitrogen atom (C≡N) known as cyano group. It can be found in the environment in various forms: as inorganic salts, such as sodium or potassium cyanide, as metal complexes, in the organic form, or in the gaseous form as hydrogen cyanide, which is a highly volatile product of the acidification of the cyanide salts (Luque-Almagro, 2005).

The solubility of gold in cyanide solutions was acknowledged by Scheele in 1783 and the role of oxygen in this reaction was investigated by Elsner in 1846. In 1888, MacArthur and the Forrest brothers patented the cyanidation process for gold extraction from ores, and it was quickly commercialized (Bautista, 1974). New practices of gold extraction for industrial applications started to be applied during the 1970s, particularly heap leaching, which was greatly benefited from the concurrent technology of gold recovery from solution through carbon-in-columns (CIC’s) followed by electrowinning (Marsden & House, 2006).

Heap leaching is a metal recovery technology in which a leach solution percolates through a bed of ore material and releases the target metal from the solid particles into the liquid (Petersen, 2016). The pregnant solution is subsequently carried out from the bed and the metals are recovered through conventional hydrometallurgical techniques. The barren solution is then transported back to the leach bed and the cycle is repeated. 39

Sodium cyanide (NaCN) solution is the main reagent utilized for gold extraction in heap leaching. This solution is prepared by dissolving sodium cyanide salt in water forming Na+ and CN- ions as shown in the equation below.

NaCN ↔ Na+ + CN- Equation 5

When cyanide ions are hydrolyzed, hydrogen cyanide (HCN) is generated and hydroxyl (OH-) ions are formed. Hydrogen cyanide is a weak acid, and total cyanide dissociates in water as half hydrogen cyanide and half free cyanide ions at pKa 9.2 (Beck, 1987).

CN (aq)̄ + H2O(l) ↔ HCN(aq) + OH (aq̄ ) Equation 6

+ – HCN(aq) + H2O(l) ↔ H (aq) + CN (aq) Equation 7

Gold dissolution in cyanide solution only happens in the presence of oxygen, as shown by Elsner (Bautista, 1974) and the reaction occurs in two oxidation steps. First, gold complexes with cyanide when the oxygen is reduced forming hydrogen peroxide, which then becomes the oxidizing agent in the second complexation step.

̄ 2Au + 4CN ̄ + O2 + 2H2O → 2Au(CN)2 ̄ + H2O2 + 2OH Equation 8

2Au + 4CN ̄ + H2O2 → 2Au(CN)2 ̄ + 2OH ̄ Equation 9

The Elsner’s equation summarizes the equations above:

4Au + 8CN ̄ + O2 + 2H2O → 4Au(CN)2 ̄ + 4OH ̄ Equation 10

The leach reactions occur by the diffusion of the cyanide ions and the dissolved oxygen through the solution layer on the surface of solid particles. Therefore, the cyanide leaching process is affected by several factors including particle size, cyanide concentration, oxygen concentration, solution temperature and pH, which need to be closely studied.

Cyanate (CNO-) can also be formed when hydrogen cyanide and free cyanide react with oxygen. Cyanate formation is undesirable in leaching operations since it does not bind to gold and decreases the free cyanide availability in the solution.

- 4HCN + 3O2 ↔ 4CNO + 2H2O Equation 11

- - - 3CN +2O2 +H2O↔3CNO +2OH Equation 12

Cyanide can also bind to transition metals and form stable cyano-metal complexes, which reduces the efficiency of gold heap leaching applications.

3.2.2 Cyanide Toxicity and Chemical Removal Technologies

Free cyanides (HCN and CN-) are extremely toxic, and these compounds are usually known for their poisoning effect. Due to its high affinity to metals, cyanide can bind to iron in the cytochrome c oxidase enzyme and inhibit the electron transport chain within the cell in aerobic organisms. Anaerobic microorganisms are also susceptible to cyanide intoxication caused by the inhibition of metalloproteins (Luque-Almagro, 2005).

Due to the hazards related with cyanide, effluents containing this compound must be treated before discharging into water bodies. Cyanide-contaminated waters are most often treated by chemical oxidation or alkaline chlorination processes. Chemical oxidation can be an expensive treatment option since it requires large amounts of chlorine to fully transform cyanide into nitrogen gas and carbon dioxide (Dash et.al, 2009).

This process can only be applied to free cyanide and has a great disadvantage of increasing the total solids content of the residual water, which also becomes high in chlorine, which is toxic for aquatic life, and can react with organic substances and form carcinogenic chlorinated compounds. Therefore, this water cannot be recycled and requires further treatment prior to discharge.

Another vastly used cyanide treatment technology is oxidation by sulfur dioxide. The advantage of this technique is that SO2 is an inexpensive reagent. However, sulfates are generated in this process and need to be removed before the water is release to the environment. Other alternatives, such as oxidation by hydrogen peroxide, reverse osmosis, ozonation, etc., require expensive reagents or equipment that may not be financially feasible.

3.2.3 Cyanide Biodegradation

Despite the toxicity of cyanide for many microorganisms, some have adapted to cyanide-rich environments and can utilize this compound as a nitrogen and/or carbon as source for growth, while degrading or transforming the cyano group. These microorganisms are often found in cyanide-containing wastewaters from mining, metal-processing and jewelry industries.

Biological degradation is a relatively inexpensive and environmentally-safe approach that is becoming more extensively used. Therefore, studying and understanding the metabolic pathways for cyanide biodegradation is of great importance. To be able to identify reaction products that can indicate the activity of key cyanide-degrading enzymes would also be beneficial. Then, once the metabolic reactions are identified, techniques can be applied to stimulate microbial degradation in cyanide-rich wastewaters, or inhibit enzymatic activities when undesirable cyanide biodegradation occurs in leaching operations.

3.2.4 Metabolic Pathways for Cyanide Biodegradation

There are four general pathways for the biodegradation of cyanide: hydrolytic, oxidative, reductive, and substitution/transfer (Dash et.al, 2009; Ebbs, 2004).

Cyanide degradation by hydrolytic reactions is catalyzed by the enzymes cyanide hydratase and cyanidase acting on HCN, or nitrile hydratase and nitrilase using nitriles following the reactions below, where R either represents an aliphatic or aromatic group.

Hydrolytic:

Cyanide hydratase

HCN + H2O → HCONH2 Equation 13

Cyanidase

HCN + 2H2O → HCOOH + NH3 Equation 14

Nitrile hydratase

R-CN + H2O → R-CONH2 Equation 15

Nitrilase

R-CN + 2H2O → R-COOH + NH3 Equation 16

The reductive reaction follows two steps that results in methane and ammonia production, and the enzymes that catalyze this pathway are rare.

Reductive:

Nitrogenase

+ − HCN + 2H + 2e → CH2=NH + H2O → CH2=O Equation 17

+ − + − CH2=NH + 2H + 2e → CH3=NH + 2H + 2e → CH4 + NH3 Equation 18

Cyanide monoxygenase converts cyanide to cyanate, which is then catalyzed by cyanase to

- - + ammonia and carbon dioxide in the bicarbonate-dependent reaction: CNO + HCO3 + 2H →

NH3 + 2CO2. Cyanide dioxygenase directly transforms cyanide into ammonia and carbon dioxide.

Oxidative:

Cyanide monooxygenase

+ + HCN + O2 + H + NAD(P)H →HOCN + NAD(P) + H2O Equation 19

Cyanide dioxygenase

+ + HCN + O2 + 2H + NAD(P)H →CO2 + NH3 + NAD(P) Equation 20

Cyanide can also be converted into β -cyanoalanine by cyanoalanine synthase using cysteine or O-acetylserine (OAS) as substrate. Cyanide can also react with thiosulfate by cyanide sulfurtransferase to form thiocyanate and sulfite. The thiocyanate produced by sulfurtransferase enzymes can then be biodegraded in two different pathways involving either carbonyl sulfide (COS) or cyanate as intermediates as shown below.

Substitution/transfer:

Cyanoalanine synthase

- Cysteine + CN → β-cyanoalanine + H2S Equation 21

- - OAS + CN → β-cyanoalanine + CH3COO Equation 22

Cyanide sulfurtransferase (thiosulfate)

- 2- - 2- CN + S2O3 → SCN + SO3 Equation 23

Thiocyanate biodegradation:

Carbonyl pathway (thiocyanate hydrolase)

- - SCN + 2H2O → COS + NH3 + OH Equation 24

Cyanate pathway (cyanase)

- - - - 2- + SCN + 3H2O + 2O2 → CNO + HS → HS + 2O2 → SO4 + H Equation 25

- + - + CNO + 3H + HCO → NH4 + 2CO2 Equation 26 44

3.2.5 Identification of Cyanide-Degrading Microbes in Gold Heap Leach Operations

The highly alkaline and calcium carbonate-rich environment in gold heap leaching operations and the elevated concentration of cyanide compounds can set challenging conditions for life. Although, unique microorganisms capable of using non-evident electron acceptors and carbon sources can thrive in those environments. This research aims to identify microbes that can use cyanide as a resource for life and understand how this compound is being degraded at a gold mine site.

3.3 Materials and Methods

3.3.1 Sample Collection and Field Analyses

In order to investigate the cyanide degradation occurring in the gold mine operation, field sampling was conducted to collect liquid and solid samples from the active leach pad (leach pad 7) and process area.

The following samples were collected: fresh water used to make the sodium cyanide solution, the barren cyanide solution before being added to the pad, the pregnant solution coming out of the pad lateral lines, including the collecting pipes north and south. The pregnant solution before entering the carbon columns was also sampled, as well as the barren solution after gold was adsorbed in the new and old carbon columns (see Figure 11 and Figure 12). Replicate samples were taken after a day to verify if the parameters changed with time and temperature.

Figure 11: Sampling locations at heap leach pad 7 (in bold): Pregnant solution from pad lateral lines (Sampler 3 and Sampler 45 to the left) and collecting pipes (Pad 7 North and Pad 7 South at the bottom).

Figure 12: Simplified process flow diagram indicating the sampling locations (bold).

Dissolved oxygen (DO), pH, conductivity, oxidation reduction potential (ORP), and temperature were measured in-situ by the handheld multiprobe YSI 556 MPS. The multiprobe was introduced into the water or, when this was not possible, a sample was taken using a clean beaker and the measurement was performed immediately.

Approximately 4L of liquid sample was taken from each location, stored in 1L sterile plastic bottles and kept inside a cooler with ice. The samples were transported to the laboratory facility at the site and filtered through a 0.22 µm sterile EMD Millipore Sterivex® filter to collect the cells for later DNA extraction. A filtration apparatus was built utilizing a vacuum pump connected through plastic tubes to two Sterivex® filters which were attached to two sterile 60 mL syringe barrels. Three liters of each liquid sample was progressively poured into the syringe barrels and, once the full 3L filtration was complete, the Sterivex® filters were labeled and each placed inside a sterile 50 mL falcon tube and stored on ice.

All samples were kept on ice before being shipped to the environmental laboratory and the University of Toronto on the same day the samples were taken. The material was shipped inside a cooler with ice packs and kept refrigerated throughout the approximate 24h journey. The filters were kept inside a -80°C freezer upon arrival at the University of Toronto until processed, whereas the liquid samples were analyzed by the environmental laboratory according to the respective analysis holding time.

3.3.2 Chemical Analyses: Anions, Nutrients, Cyanide Breakdown Products and Total Dissolved Metals

The sampling bottles for the chemical analyses were provided by the environmental laboratory that also conducted the analyses. Liquid samples (approximately 1L) were collected in plastic, glass or amber bottles and preserved with acid or base according to the type of analysis and holding time as determined by the laboratory.

The parameters and analyses are described in Table 11 below: 47

Table 11: Parameters analyzed by the environmental laboratory (analysis description written by the laboratory). Parameter Analysis Description

Alkalinity This analysis is carried out using procedures adapted from APHA Method 2320 "Alkalinity". Total alkalinity is determined by potentiometric titration to a pH 4.5 endpoint. Bicarbonate, carbonate and hydroxide alkalinity are calculated from phenolphthalein alkalinity and total alkalinity values. Ammonia This analysis is carried out, on sulfuric acid preserved samples, using procedures modified from J. Environ. Monit., 2005, 7, 37 - 42, The Royal Society of Chemistry, "Flow-injection analysis with fluorescence detection for the determination of trace levels of ammonium in seawater", Roslyn J. Waston et al. Nitrite Inorganic anions are analyzed by Ion Chromatography with conductivity and/or UV detection. Nitrate Inorganic anions are analyzed by Ion Chromatography with conductivity and/or UV detection. Sulfate Inorganic anions are analyzed by Ion Chromatography with conductivity and/or UV detection. Cyanate This analysis is carried out using procedures adapted from APHA method 4500-CN "Cyanide". Cyanate is determined by the Cyanate hydrolysis method using an ammonia selective electrode Free Cyanide This analysis is carried out using procedures adapted from ASTM Method 7237 "Free Cyanide with Flow Injection Analysis (FIA) Utilizing Gas Diffusion Separation and Amperometric Detection". Free cyanide is determined by in-line gas diffusion at pH 6 with final determination by colourimetric analysis. Thiocyanate This analysis is carried out using procedures adapted from APHA Method 4500-CN- M "Thiocyanate" Thiocyanate is determined by the ferric nitrate colourimetric method. 48

Water samples containing high levels of hexavalent chromium, cyanide (together with sulfide), reducing agents, or hydrocarbons may cause negative or positive interferences with this method.

Total Cyanide This analysis is carried out using procedures adapted from ISO Method 14403:2002 "Determination of Total Cyanide using Flow Analysis (FIA and CFA)". Total or strong acid dissociable (SAD) cyanide is determined by in- line UV digestion along with sample distillation and final determination by colourimetric analysis. Method Limitation: This method is susceptible to interference from thiocyanate (SCN). If SCN is present in the sample, there could be a positive interference with this method, but it would be less than 1% and could be as low as zero. Weak Acid This analysis is carried out using procedures adapted from APHA Method Dissociable 4500-CN I. "Weak Acid Dissociable Cyanide". Weak Acid Dissociable Cyanide (WAD) cyanide is determined by in-line sample distillation with final determination by colourimetric analysis. Total Au This analysis is carried out using procedures adapted from "Standard Methods for the Examination of Water and Wastewater" published by the American Public Health Association, and with procedures adapted from "Test Methods for Evaluating Solid Waste" SW-846 published by the United States Environmental Protection Agency (EPA). The procedures may involve preliminary sample treatment by acid digestion, using either hotblock or microwave oven (EPA Method 3005A). Instrumental analysis is by inductively coupled plasma - mass spectrometry (EPA Method 6020A). Total Metals Water samples are digested with nitric and hydrochloric acids, and analyzed by CRC ICPMS.

Method Limitation (re: Sulfur): Sulfide and volatile sulfur species may not be recovered by this method.

3.3.3 Biological Analyses: DNA Extraction, qPCR and Amplicon Sequencing

DNA was extracted from the filters using the DNeasy® PowerSoil Kit by QIAGEN at the University of Toronto. Both ends of the Sterivex® filter cartridge were cut off with a sterilized tubing cutter, and the filter was placed on a sterile Petri-dish and cut into small strips with a razor blade. Then the strips were placed into the PowerBead Tube provided in the kit and the Quick- Start Protocol was followed to extract DNA. DNA was eluted in either 50 or 100µL of Solution

C6 and the DNA concentration was measured by NanoDrop® ND-1000 Spectrophotometer and Qubit® 3 Fluorometer. Subsequently aliquots of each DNA sample were used for 16S rRNA amplicon sequencing and quantification with quantitative Polymerase Chain Reaction (qPCR).

Because the community was found to be of low diversity, the DNA sample collected from Pad 7 was used to obtain the 16S rRNA gene sequence of dominant taxa using primers 8f (AGAGTTTGATCMTGGCTCAG) and 1392r (ACGGGCGGTGTGTAC). The PCR product was cleaned up using the Sigma GenElute® PCR Clean-Up Kit, before ligating amplified DNA into the Invitrogen’s PCR2.1 vector and transforming into “One-Shot TOP10 Chemically Competent” E. coli cells. Clones were selectively grown overnight on agar plates containing Luri-Bertani (LB) broth and Kanamycin in an incubator at 37℃. Ten positive colonies were randomly selected and transferred to a LB broth containing Kanamycin, and grown overnight in a shaker at 200rpm and 37℃. The plasmid DNA in the grown E. coli was extracted using the GenElute® Plasmid Miniprep Kit, and subsequently sent to The Centre of Applied Genomics (TCAG) for Sanger sequencing. The sequencing results were processed and trimmed in the 50 program Geneious, and the final sequences were then compared with the known 16S rRNA gene sequences in the database of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence alignment and the construction of the phylogenetic tree were also performed in Geneious.

For qPCR, to quantify the abundance of 16S rRNA gene sequences corresponding to all bacteria in each sample, the following universal bacterial primer set was used to target the 16S rRNA gene: 1055f (5’-ATGGCTGTCGTCAGCT-3’) and 1392r (5’-ACGGGCGGTGTGTAC-3’). The reaction mixture contained 10μl of EvaGreen Mastermix, 0.5μl of each 10μM primer, and 7μl of ultrapure water, and each sample contained 18μl of the reaction mix and 2μl of DNA sample. The samples were run in triplicate and negative controls were included in the plate. The reaction conditions were as follows: 3 min at 95 °C followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s. Plasmid DNA from Clone #7 containing the 16S rRNA gene obtained from the cloning experiments described in the previous paragraph was used as the qPCR standard.

3.4 Results and Discussion

Data obtained include measurements of physical and chemical properties of each sample, as well as total bacterial abundance (from qPCR) and community composition (from amplicon sequencing). Full length 16S rRNA genes were obtained for 10 representative clones for more detailed phylogenetic analysis. Raw data is provided in the Appendix L to Appendix R. The bioinformatics for the phylogenetic analysis and taxonomic diversity were performed by Yang (Ivy) Minqing.

3.4.1 Field and Lab Chemical Measurement Results

The field parameters, anions, nutrients and cyanide species results are shown below (Figure 13, Figure 14 and Figure 15). The data is presented in the process flow direction: from where the barren solution is applied to the heap pad to where the pregnant solution is processed for gold extraction. The field parameters indicate a slight pH decrease in the pregnant solution. Nitrate

levels are elevated in all the samples, and alkalinity is high in the Samplers due to calcium carbonate scaling formation in the drip lines. Figure 15 shows the significant drop in the cyanide levels in the barren and pregnant solutions, and cyanate is formed. The highly alkaline environment suggests the presence of microbes that thrive using calcium carbonate as carbon source, and the cyanate formation can indicate the metabolic pathway at which these microbes degrade cyanide.

25 pH DO (mg/L) ORP/10 T (C) Conductivity (mS/cm^2)

20 ) 2

Conductivity (mS/cm 5 pH, DO pH, (mg/L), DO ORP/10, T (C) and

0 Raw Water Barren Sampler 3 Sampler 45 Pad 7 Pad 7 Before After New After Old Solution North South CIC's CIC's CIC's

Process flow direction

Figure 13: Results for field parameters (measured directly in the field). Graph indicates the average from sample duplicates.

700 Alkalinity Ammonia Nitrate Nitrite Sulfate

600

500

400

300 Sulfate (mg/L) 200

100 Alkalinity, Ammonia, Nitrate, Nitrite and 0 Raw Water Barren Sampler 3 Sampler 45 Pad 7 Pad 7 Before After New After Old Solution North South CIC's CIC's CIC's

Process flow direction

Figure 14: Alkalinity, ammonia and anions concentrations. Graph indicates the average from sample duplicates.

120 Cyanide (WAD) Total Cyanide Cyanate Free Cyanide

100

20 and Free Cyanide (mg/L)

Cyanide (WAD), Total Cyanide, Cyanate Raw Water Barren Sampler 3 Sampler 45 Pad 7 Pad 7 Before After New After Old Solution North South CIC's CIC's CIC's

Process flow direction

Figure 15: Concentrations of various cyanide species. Thiocyanate results were below the detection limit in all samples. Graph indicates the average from sample duplicates.

3.4.2 Bacterial 16S rRNA Gene Copies in Relation to Free Cyanide Concentration and pH

Figure 16 provides the 16S rRNA gene copies per mL of sample for each of the samples analyzed. This number is a proxy for cell count. The data are plotted along with cyanide concentration and pH showing that the major cyanide loss occurs in the pad, where a significant pH drop, from 10-11 to 8-9, is also observed.

Studies have shown that cyanide complexes are more resistant to biodegradation than free cyanide (Aronstein, et al., 1994). This could suggest that free cyanide is being degraded as opposed to complexed cyanide, which means that the degradation is more likely to be occurring before cyanide complexes with metals in the pad.

Figure 16: Comparison of pH, gold and free cyanide concentrations and copies of 16S rRNA gene per mL across all samples. Error bars shows range of duplicate samples taken on consecutive days.

3.4.3 Phylogenetic Analysis and Taxonomic Diversity

The amplicon sequencing data revealed that the samples did not present a vastly diverse community due to the highly alkaline environment that creates extreme selective conditions for microbe survival. An OTU related to Hydrogenophaga was the most abundant microbe present in all cyanide containing samples (Figure 17). Some microbes of this type have been shown to utilize hydrogen as energy source for growing autotrophically (fixing CO2) and potentially can use cyanide as a source of energy and nitrogen (Suzuki et al., 2017, Belykh et al., 2016). The next most abundant microbes (<~5%) at the site also belong to Betaproteobacteria, within the Rhodocyclaceae, including the genus Azoarcus. These organisms tend to be facultative anaerobes, able to use nitrate or nitrite as electron acceptors when oxygen is limiting. Often, cyanide-degrading microorganisms coexist with nitrifying microbes, which can utilize the ammonia produced in cyanide oxidation as electron donor and oxygen as electron acceptor. Some nitrifying microbes were identified in the samples (e.g. Nitrosomonas OTU#9). These microorganisms are chemoautotrophs and utilize inorganic carbon as carbon source and therefore have a slow growth yield, which can also be inhibited by the toxicity of cyanide to their respiratory chain (Kapoor, et al., 2016). This may explain their low abundance in the samples shown in Figure 17, especially in the barren solution where the cyanide level is the highest.

Figure 17: Heatmap of the major Operational Taxonomic Units (OTUs) found in samples from the mine versus tree of sample clustering. This figure shows the remarkable dominance by the first OTU in the list, and the similarity between samples from similar locations.

Because of the dominance by one OTU (OTU#1, related to Hydrogenophaga), the full length of the 16S rRNA gene sequence was retrieved. Primers that target the full sequence length were used for amplification, and resulting amplicons were cloned into E. coli in order to amplify and sequence each of them. Ten clones were sequenced by the Sanger method to get long reads. The

56 sequences from these clones were aligned with the shorter OTU sequences obtained from Illumina data, and six of them matched with OTU#1 (Figure 18).

Basic Local Alignment Search Tool (BLAST) was used to compare two representative clone sequences from the Sanger sequencing information (i.e., Clone 2 and Clone 5) to the NCBI database. The best matches including both isolates and uncultured bacterial clones in the database were identified and were aligned with Clone 2 and Clone 5 to calculate the similarity, which can be visualized in the phylogenetic tree shown in Figure 19. Based on the alignment results, the 16S rRNA sequence of Clone 5 is identical to that of the uncultured bacterium (AM777979.1) collected from a high pH groundwater associated with serpentinization (Tiago et al, 2013), and Comamonadaceae bacterium B1 (AP014569) isolated from a highly alkaliphilic serpentinizing site (Suzuki et al., 2014). The sequences of Clone 2 and Clone 5 were also highly similar to an uncultured Hydrogenophaga (LK392396) found in the process water from ore heap leaching process with high concentrations of cyanide and thiocyanate (Belykh et al., 2016). Table 12 presents the Illumina data and NCBI information obtained for each clone and indicates how the most closely related organisms may grow. 57

Table 12: Illumina Data and NCBI information of the four clone representatives indicating how the organisms may grow.

Figure 19: Phylogenetic tree showing similarity of the Hydrogenophaga-like clones 5 and 2 and the best-hit organisms from NCBI BLAST, based on alignment of partial 16S rRNA gene containing 840 nucleotides.

*The accession numbers and the sources of the reference organisms are also included in the tree tip labels.

**The horizontal scale bar represents the number of differences between sequences (e.g., 0.1 means 10 % differences between two sequences).

3.4.4 Identification of the Cyanide-Degrading Microbes (s)

Suzuki et al. (2014) described three unique highly alkaliphilic strains capable of autotrophic growth with hydrogen as electron donor, oxygen as electron acceptor and calcium carbonate as source of carbon, and proposed to place their isolated strains in a new genus called “Serpentinomonas”. Figure 20 plots pH, alkalinity and calcium concentration as a function of the relative abundance of the Hydrogenophaga-like OTU#1. There does appear to be a slight relationship that is consistent to what Suzuki et al. (2014) reported, and may provide some ideas for suppressing the growth of this microorganism by managing these parameters. The authors have also completed the full genome sequence of these “Serpentinomonas” strains (including strain B1 that is most similar to the Hydrogenophaga-like OTU#1) and concluded that they can

use nitrate in addition to oxygen as electron acceptors, and also benefit from other organic carbon sources, such as addition of acetate.

Further studies need to be performed under controlled conditions in the laboratory to confirm the function of the Hydrogenophaga-like OTU#1. Lab experiments combined with what is known from the literature provide good avenues to test to control its growth based on these chemical parameters and the investigation of its whole genome sequence.

Figure 20: Plot of alkalinity, calcium concentration and pH as a function of Hydrogenophaga-like relative abundance in the samples analyzed.

3.5 Conclusions and Recommendations for Future Work

The microbial community at this mining site is remarkably simple, dominated largely by a single Betaproteobacterium in the Comamonadaceae, related to Hydrogenophaga. A few closely related microbes are known to have the ability to hydrolyze cyanide to products such as ammonium and 60 formate. The products of this hydrolysis can be used as energy, carbon and nitrogen sources by a variety of bacteria. The ammonium is readily oxidized to nitrite and nitrate when oxygen is present. Nitrite and nitrate are electron acceptors when oxygen is limiting and other electron donors are available.

There is evidence for all of these processes based on the microbial community analysis. However, what is most striking is the extreme dominance by the single Hydrogenophaga-like microbial phylotype. This type of organism has been found to dominate in highly alkaline environments (optimum pH ~11), and to use oxygen or nitrate as electron acceptors (Suzuki et al. 2014). The best information is that these organisms use H2 from serpentinization of ultramafic rock and that the organisms are adapted to high pH and calcium carbonate from which they derive carbon. In the case of this study, it could be that the electron donor is some form of cyanide or other organic compounds in the recirculated water.

While Hydrogenophaga have been found in mine leachate (Belykh et al., 2016), their role in the cyanide degradation in this study was not established. The likely metabolism is oxidation or hydrolysis of cyanide to formate and ammonium, presumably carried out by the Hydrogenophaga-like organisms.

Given the simplicity of the microbial community, the recommendations moving forward are: 1) To carry out some bottle tests (Microcosms) using the cyanide solution and ore samples coming from this mine site to establish the products and stoichiometry of cyanide degradation, monitor the microbial community evolution over time, and to evaluate conditions (pH, organics) that could reduce the rate of CN degradation in the leach beds. This would establish that the Hydrogenophaga-like organism is indeed the cyanide degrader, grow the culture to sufficient volume to help to identify which conditions will lead to higher or lower growth rates. Assays for “cyanidase” activity could be conducted on crude extracts to quantify activity and evaluate possible enzyme inhibitors.

2) Sequence the genome of the dominant Hydrogenophaga-like organism to establish which cyanide degrading genes it contains (nitrilases, hydratases, or oxygenases) to better devise a means to inhibit the activity. Initial ideas to try to limit activity would include

pH adjustment or removal of oxygen or other organics to limit microbial growth, or to identify a specific inhibitor of the cyanide-degrading enzymes identified either through genome sequencing and biochemical assays.

3.6 References

Aronstein, B. N., Maka, A., & Srivastava, V. J. (1994). Chemical and biological removal of cyanides from aqueous and soil-containing systems. Applied Microbiology and Biotechnology, 41(6), 700–707. http://doi.org/10.1007/BF00167288

Beck, M. T. (1987) Critical survey of stability constants of cyano complexes. Pure and Applied Chemistry 59(12):1703–1720.

Belykh, M. P., Petrov, S. V., Chikin, A. J., & Belkova, N. L. (2016). Genetic diversity of bacteria adapted to cyanide-bearing compounds in the technogenic ecosystems as detected by 16S rDNA sequences. Contemporary Problems of Ecology, 9(5), 563–573. http://doi.org/10.1134/S1995425516050012

Bautista, R. G. (1974). Hydrometallurgy. Advances in Chemical Engineering, 9(1), 18-20. New York, NY: Academic Press, Inc.

Crespo-Medina, M., Twing, K. I., Kubo, M. D. Y., Hoehler, T. M., Cardace, D., McCollom, T., & Schrenk, M. O. (2014). Insights into environmental controls on microbial communities in a continental serpentinite aquifer using a microcosm-based approach. Frontiers in Microbiology, 5(NOV). http://doi.org/10.3389/fmicb.2014.00604

Dash, R. R., Gaur, A., & Balomajumder, C. (2009). Cyanide in industrial wastewaters and its removal: A review on biotreatment. Journal of Hazardous Materials, 163(1), 1–11. https://doi.org/10.1016/j.jhazmat.2008.06.051

Ebbs, S. (2004). Biological degradation of cyanide compounds. Current Opinion in Biotechnology, 15(3), 231–236. https://doi.org/10.1016/j.copbio.2004.03.006

Fukuda,A., Hagiwara,H., Ishimura,T., Kouduka,M., Ioka,S., Amano,Y., Tsunogai,U., Suzuki,Y. and Mizuno,T. (2010). Geomicrobiological properties of ultra-deep granitic groundwater from the Mizunami Underground Research Laboratory (MIU), central Japan. Microbial Ecology. 60 (1), 214-225 http://doi.org/10.1007/s00248-010-9683-9

62 63

Gihring,T., Moser,D.P. and Onstott,T.C. The distribution of microbial taxa in the subsurface water of the Kalahari Shield, South Africa (2006). Geomicrobiology Journal, volume 23, issue 6, 415-430. http://doi.org/10.1080/01490450600875696

Guzmana, L., Segarrac, M., Chimenosc, J. M., Cabotb, P. L., & Espiell, F. (1999). Electrochemistry of conventional gold cyanidation, 44, 2625–2632.

Hurek, T., & Reinhold-Hurek, B. (2003). Azoarcus sp. strain BH72 as a model for nitrogen- fixing grass endophytes. Journal of Biotechnology, 106(2–3), 169–178. http://doi.org/10.1016/j.jbiotec.2003.07.010

Kanjanasuntree, R., Kim, J.-H., Yoon, J.-H., Sukhoom, A., Kantachote, D., & Kim, W. (2018). Arenimonas halophila sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology, 68(7), 2188–2193. http://doi.org/10.1099/ijsem.0.002801

Kapoor, V., Elk, M., Li, X., & Santo Domingo, J. W. (2016). Inhibitory effect of cyanide on wastewater nitrification determined using SOUR and RNA-based gene-specific assays. Letters in Applied Microbiology, 63(2), 155–161. http://doi.org/10.1111/lam.12603

Kim, M.S., Roh,S.W., Nam,Y.D., Yoon,J.H. and Bae,J.W. (2009). Alishewanella jeotgali sp. Nov., isolated from traditional fermented food, and emended description of the genus Alishewanella. International Journal of Systematic and Evolutionary Microbiology, 59, 2313–2316.

Luque-Almagro, V. M., M.-J. Huertas, M. Martínez-Luque, C. Moreno-Vivián, M. D. Roldán, L. J. García-Gil, F. Castillo and R. Blasco (2005). Bacterial degradation of cyanide and its metal complexes under alkaline conditions. Applied and Environmental Microbiology, 71: 940-947.

Luque-Almagro, V. M., P. Cabello, L. P. Sáez, A. Olaya-Abril, C. Moreno-Vivián and M. D. Roldán (2018). "Exploring anaerobic environments for cyanide and cyano-derivatives microbial degradation." Applied Microbiology and Biotechnology, 1067–1074. 64

Marsden, P.J. and House, L. (2006). The Chemistry of Gold Extraction, Littleton, Society of Mining, Metallurgy and Exploration Inc., p. 19.

Petersen, J. (2016). Heap leaching as a key technology for recovery of values from low-grade ores – A brief overview. Hydrometallurgy, 165, 206–212. https://doi.org/10.1016/j.hydromet.2015.09.001

Reinhold_Hurek, B., Hurek, T., Gillis, M., Hoste, B., Vancanneyt, M., Kersters, K., De Ley, J. (1993). Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth), and description of two species, Azoarcus indigens sp. nov. and Azoarcus communis sp. nov. International Journal of Systematic and Evolutionary Microbiology, 43: 574-584

Springer, N., Ludwig, W., Philipp, B., Schink, B. (1998) Azoarcus anaerobius sp. nov., a resorcinol-degrading, strictly anaerobic, denitrifying bacterium. International Journal of Systematic Bacteriology, 48(3): 953-6.

Suzuki, S., Kuenen, J. G., Schipper, K., Van Der Velde, S., Ishii, S., Wu, A., … Nealson, K. H. (2014). Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site. Nature Communications, 5(May). https://www.nature.com/articles/ncomms4900

Tiago, I., & Veríssimo, A. (2013). Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization. Environmental Microbiology, 15(6), 1687– 1706. http://doi.org/10.1111/1462-2920.12034

Watson, R., Butler, E.C. V., Clementson, L., Berry, K. M. (2005). Flow-injection analysis with fluorescence detection for the determination of trace levels of ammonium in seawater. Journal of Environmental Monitoring, 7(1): 37-42.

Xing, W., Li, J., Li, D., Hu, J., Deng, S., Cui, Y., & Yao, H. (2018). Stable-isotope probing reveals the activity and function of autotrophic and heterotrophic denitrifiers in nitrate removal from organic-limited wastewater. Environmental Science and Technology, 52(14), 7867–7875. research-article. http://doi.org/10.1021/acs.est.8b01993

Zhou, J., Fries, M., Chee-Sanford, J.C., Tiedje, J.M. (1995). Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth on toluene and description of Azoarcus tolulyticus sp. nov. International Journal of Systematic Bacteriology. 45: 500-506.

Synthesis and Conclusions

4.1 Overall Synthesis

This work provides good understanding of fundamental knowledge of how microbes interact with minerals in mining processes. Both projects studied autotrophs that use minerals as a solid surface to grow on, and were identified using low-cost and high-throughput DNA sequencing techniques. Understanding how these microbes thrive in mining environments is crucial to develop bioprocesses for mining operations as an alternative to succeed in limited geological, economical, and environmental circumstances. The following sections describe the main findings and future work of both projects presented in this thesis.

4.2 Circumneutral pH Bioleaching of Ni under Denitrifying Conditions

This study provided good knowledge of bioleaching of nickel from sulfidic ores and wastes under nitrate reducing conditions and circumneutral pH. The ultramafic nature of the material in this work creates processing challenges due to the elevated magnesium content that requires excessive acid addition in the leaching stage to unlock the Ni sulfides. The acid requirement and the waste generation are reduced when operating at circumneutral pH. Another advantage of this process is that nitrate-rich waters from blasting operations can also be treated while Ni is leached.

4.4.1 Revival of Zhou’s Microcosms

Objective 1 of this study was to revive the denitrifying cultures started by Han Zhou, after 4 years of inactivity, by amending the bottles with nitrate. The microscopy and sequencing results indicated that the main microorganisms found by Zhou were still active in the bottles and

66 67

Thiobacillus was the major player in the bioleaching mechanism. The cells from Zhou’s microcosms were successfully transferred to new bottles for further Ni bioleaching studies, and an increase in the selectivity for Thiobacillus was observed in each transfer. The relative abundance of Thiobacillus increased from maximum of 26% in TSRA 10_WZ from the first generation to 85% in TSRA T10_LM from the third generation. These results confirm that Thiobacillus is the major player in the bioleaching mechanism. The sequencing data from all microcosms will be the subject of future work.

4.4.2 Total Bioleached Ni in the Microcosm Studies

Objective 2 was to inspect the final ore material from the nitrate reducing microcosm tests performed by Han Zhou and from the new transfers. The solubility of Ni is limited at circumneutral pH and the leached Ni was likely precipitating in the bottles. The goal was to account for the total bioleached Ni by re-dissolving any reprecipitated Ni salts in 0.25M H2SO4 solution, and also determine the non-bioleached Ni in the remaining solids by Aqua Regia digestion. The results show that only a small fraction of the total nickel bioleached was being considered by only measuring the nickel ions in the aqueous phase. Most nickel (90-99%) had in fact precipitated and the bioleaching recovery was greater than expected from the supernatant measurements in Zhou’s work. However, the duration of the experiments shows that the leaching occurred at a slow rate. An increase in the biomass concentration can possibly enhance bioleaching rates.

4.4.3 Mass Balance Study

Objective 3 was to evaluate the mass balances in the microcosms prepared by Zhou and in the new transfers, and compare the measured Ni with the recovery expected from the cumulative nitrate consumption and sulfate production. Once the precipitated Ni was taken into account, the mass balance was closed in Zhou’s microcosms. The excess nitrate consumed in the first generation was due to the presence of some organics and other electron donors in the mining waters used as inocula. And the lower Ni recovery efficiencies in the second and third 68 generations were due to the production of elemental sulfur in the heated acid wash pre-treatment of the UMFC material. A fourth generation of microcosms using the original UMFC is currently being maintained for future mass balance calculations.

4.3 Cyanide Biodegradation in Gold Heap Leach Operations

This project investigated the cyanide biodegradation phenomenon occurring at the heap leach operation of a gold mine site. The large losses in cyanide prevents the effective reuse of the leach solution for further gold recovery, as a result, additional cyanide solution is required to maintain the production, which increases the costs of gold extraction.

At the same time, strict discharging limits for cyanide species to tailings ponds and receiving waters, and the increasing requirement for higher cyanide levels to process complex gold ore bodies due to geological limitations, has aroused the interest in cyanide removal technologies. Biological treatments are often more cost-effective than chemical and physical alternatives, and safe for the environment.

This study aimed to identify the cyanide-degrading microbe(s) living at this mining site to inhibit their activity in the processing waters, and also provide an alternative to eliminate cyanide in the tailings ponds via biological degradation.

4.3.1 High-quality Environmental Sampling

Objective 1 of this work was to provide high-quality environmental samples for the examination of cyanide biodegradation. Field parameters, chemical and biological samples were collected from the pregnant and barren solutions at the gold mine site. The chemical analyses for anions, nutrients, total dissolved metals and cyanide species were performed by an environmental laboratory. DNA samples were also extracted from filtered cyanide solution at the site.

4.3.2 Evidence of Cyanide Biodegradation in Field and Chemical Parameters

Objective 2 was to examine the chemical and field parameters data for any evidence of cyanide biodegradation. The highly alkaline environment suggests that microbes can use calcium carbonate as carbon source, and the increase in cyanate while free cyanide is degraded can indicate the metabolic pathway at which this phenomenon is happening.

4.3.3 Identification of the Cyanide Degrading Microbe(s)

Lastly, the Objective 3 of this study was to identify the microbes involved in the cyanide biodegradation phenomenon. The DNA sequencing data revealed a simple microbial community, dominated largely by a Hydrogenophaga-like microorganisms in the Comamonadaceae family. This type of microorganism has been found to be adapted to high pH and calcium carbonate from which they derive carbon. It is possible that the electron donor in the case of this study is a cyanide specie or other organic compounds in the leaching solution. Bottle tests will be performed to confirm the role of this microbe in the cyanide degradation at the mine site, and the genome of the dominant Hydrogenophaga-like will be sequenced.

Appendices

Appendix A: Growth Medium Recipe

1. Trace element mixture (Adapted from Widdel & Bak, 1992): 1mL

• Distilled Water: 987 mL • HCl (25% = 7.7 M): 12.5 mL

• FeSO4.7H2O: 2102.90 mg

• H3BO3: 30.47 mg

• MnCl2.4H2O: 100.61 mg

• CoCl2.6H2O: 189.69 mg

• NiCl2.6H2O: 23.80 mg

• CuCl2.2H2O: 2.29 mg

• ZnSO4.7H2O: 144.46 mg

• Na2MoO4: 30.44 mg

2. Selenite-tungstate solution (Adapted from Widdel & Bak, 1992): 1mL

• Distilled water: 1L • NaOH: 408.94 mg

• NaSeO3: 3.89 mg

• Na2WO4.2H2O: 7.81 mg

3. Phosphate buffer – MM1 EdLab Mineral Medium: 10mL

• Final concentration (2mM) • KH2PO4: 20.96 g or K2HPO4: 42.85 g in 1L of distilled water • Adjust pH to 7.0

4. Redox indicator – MM5 EdLab Mineral Medium: 1mL

• Resazurin 1 g/L

5. Saturated Bicarbonate – MM6 EdLab Mineral Medium: 10mL

• Add 20 g of NaHCO3 into 100 mL of distilled water • Pour slurry into 160-ml serum bottle, cover it with foil and autoclave • After autoclaving, purge the bottle with O2-free N2 for a least 15 minutes while cooling • Seal the bottle with sterile black butyl rubber stopper and crimp it • The solution will have undissolved NaHCO3 in the bottom

6. Vitamins – MM7 EdLab Mineral Medium: 10mL

• Biotin: 0.02 g • Folic acid: 0.02 g • Pyridoxine HCl: 0.1 g • Riboflavin: 0.05 g • Thiamine: 0.05 g • Nicotinic acid: 0.05 g • Pantothenic acid: 0.05 g • PABA: 0.05 g • Cyanocobalamin (vitamin B12): 0.05 g • Thioctic (lipoic) acid: 0.05 g • Coenzyme M: 1.0 g

Make up to 1 L and adjust pH to 7. Dilute the stock 1/100 to get a 100x stock to be used. Filter- sterilize the 100x stock into sterile 160-ml serum bottle and purge the solution with sterile O2-free N2 for 15 minutes. Seal with the bottle with sterile black butyl rubber stopper and crimp it.

7. Salts

• NH4Cl: 535 mg

• CaCl2.6H2O: 700mg

72 Appendix B: pH and nitrate addition data from first and second generations after nitrate was added again in 2017

pH Nitrate Additions Date Day TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TN9_WZ TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TN9_WZ 2017-09-21 0 2017-10-05 14 2017-10-13 22 7.07 6.98 7.07 7.46 2017-10-20 29 + + + 2017-10-26 35 2017-11-01 41 2017-11-17 57 + + + 2018-01-16 117 7.51 7.70 7.21 2018-01-22 123 2018-01-24 125 + + + + 2018-02-01 133 7.69 2018-02-09 141 2018-02-13 145 7.91 2018-02-20 152 2018-02-28 160 7.98 7.92 7.66 2018-03-16 176 2018-03-21 181 (DNA) 2018-05-16 237 + + + 2018-06-01 253

73 Appendix C: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from first and second generations after nitrate was added again in 2017

Cumulative Nitrate Consumed (mmol/bottle) Cumulative Sulfate Produced (mmol/bottle) Dissolved Ni (umol/bottle) Date Day TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TN9_WZ TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TN9_WZ TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ TSRA TN9_WZ 2017-09-21 0 0.65 0.68 0.60 0.57 6.36 29.23 115.08 1.25 2017-10-05 14 1.11 0.77 0.63 7.08 14.07 80.77 2017-10-13 22 2017-10-20 29 8.2 5.2 4.2 5.13 24.70 57.96 2017-10-26 35 9.53 32.56 88.83 2017-11-01 41 2017-11-17 57 8.4 5.4 4.4 0.77 0.56 0.50 13.74 39.97 89.19 2018-01-16 117 2018-01-22 123 2018-01-24 125 8.6 5.6 4.6 3.4 0.79 0.65 0.51 1.14 9.40 46.35 91.34 1.73 2018-02-01 133 0.79 0.59 0.46 1.07 9.76 47.73 90.01 6.81 2018-02-09 141 0.81 0.61 0.47 1.14 11.79 51.37 91.10 9.24 2018-02-13 145 2018-02-20 152 0.76 0.58 0.44 1.03 11.58 49.46 80.16 12.4 2018-02-28 160 0.74 0.55 0.43 0.98 13.42 56.35 86.37 14.16 2018-03-16 176 0.77 0.54 0.44 1.00 21.32 2018-03-21 181 (DNA) 2018-05-16 237 8.8 5.8 4.8 0.69 0.49 0.38 0.91 10.89 50.19 71.66 2018-06-01 253 0.66 0.46 0.35 0.82 12.25 38.98 68.34 29.54

74 Appendix D: Sequencing data from first and second generations on day 181 after nitrate was added back to the bottles in 2017

Microcosm Phylum Class Order Family Genus Microorganism OTU Relative Abundance (%) Proteobacteria(100) Betaproteobacteria(100) Hydrogenophilales(100) Hydrogenophilaceae(100) Thiobacillus Thiobacillus OTU_2 56.42 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_8 13.23 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_4 8.98 Proteobacteria(100) Gammaproteobacteria(100) Chromatiales(100) Ectothiorhodospiraceae(100) Acidiferrobacter Acidiferrobacter OTU_26 4.61 TSRA 10_WZ Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Azospira Azospira OTU_11 3.57 Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Sulfuritalea Sulfuritalea OTU_21 2.42 Microgenomates Microgenomates OTU_41 2.35 OTU<2% 8.42 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_4 15.96 Euryarchaeota(100) Methanomicrobia(100) Methanomicrobiales(100) Methanospirillaceae(100) Methanospirillum Methanospirillum OTU_13 10.42 Proteobacteria(100) Betaproteobacteria(100) Hydrogenophilales(100) Hydrogenophilaceae(100) Thiobacillus Thiobacillus OTU_2 10.04 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_10 6.72 Proteobacteria(100) Betaproteobacteria Betaproteobacteria OTU_17 5.95 Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Sulfuritalea Sulfuritalea OTU_21 5.35 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_36 5.02 Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Azospira Azospira OTU_11 4.13 TSRA 11_WZ Proteobacteria(100) Gammaproteobacteria(100) Chromatiales(100) Ectothiorhodospiraceae(100) Acidiferrobacter Acidiferrobacter OTU_24 2.96 Bacteria OTU_30 2.77 Chloroflexi(96) Anaerolineae(84) Anaerolineales(84) Anaerolineaceae Anaerolineaceae OTU_38 2.70 Bacteria OTU_35 2.54 Microgenomates Microgenomates OTU_40 2.51 Euryarchaeota(100) Methanomicrobia(100) Methanosarcinales(100) Methanosarcinaceae(100) Methanosarcina Methanosarcina OTU_51 2.43 Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Burkholderiaceae(100) Limnobacter Limnobacter OTU_113 2.22 OTU<2% 18.27 Proteobacteria(100) Betaproteobacteria(100) Hydrogenophilales(100) Hydrogenophilaceae(100) Thiobacillus Thiobacillus OTU_2 36.17 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_4 23.59 Chloroflexi(100) Anaerolineae(100) Anaerolineales(100) Anaerolineaceae Anaerolineaceae OTU_10 8.05 Proteobacteria(100) Gammaproteobacteria(100) Chromatiales(100) Ectothiorhodospiraceae(100) Acidiferrobacter Acidiferrobacter OTU_24 5.63 TSRA 12_WZ Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Azospira Azospira OTU_11 4.77 Proteobacteria(100) Betaproteobacteria Betaproteobacteria OTU_17 4.14 Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Burkholderiaceae(100) Limnobacter Limnobacter OTU_113 2.25 Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Sulfuritalea Sulfuritalea OTU_21 2.08 OTU<2% 13.31 Proteobacteria(100) Betaproteobacteria(100) Hydrogenophilales(100) Hydrogenophilaceae(100) Thiobacillus Thiobacillus OTU_2 45.06 Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Comamonadaceae(100) Comamonas Comamonas OTU_9 13.98 Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Burkholderiaceae(100) Limnobacter Limnobacter OTU_113 11.69 TSRA TN9_WZ Actinobacteria(100) Actinobacteria(100) Micrococcales(100) Intrasporangiaceae(100) Intrasporangium Intrasporangium OTU_12 8.56 Proteobacteria(100) Betaproteobacteria(100) Rhodocyclales(100) Rhodocyclaceae(100) Azospira Azospira OTU_11 5.23 OTU <2% 15.46

75 Appendix E: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T10_LM and TSRA T11_LM

TSRA T10_LM TSRA T11_LM Day pH NO3 (mmol/bottle) SO4 (mmol/bottle) Dissolved Ni (umol/bottle) Day pH NO3 (mmol/bottle) SO4 (mmol/bottle) Dissolved Ni (umol/bottle) 0 0.00 0.00 0.00 0 7.23 0.00 0.04 0.00 6 7.08 0.00 0.12 0.96 6 0.23 0.14 0.00 14 0.29 0.31 0.17 14 0.26 0.92 19 7.25 0.32 0.03 19 0.32 27 0.47 0.23 0.06 27 7.13 0.45 0.06 34 6.86 0.48 0.05 34 0.60 0.55 0.11 41 6.45 0.64 0.05 41 6.72 0.37 0.05 54 6.51 0.64 0.03 54 0.78 0.75 0.23 62 6.71 0.80 0.87 0.04 62 6.72 0.83 0.09 69 6.44 0.84 0.05 69 6.35 0.96 0.73 1.76 76 6.05 0.93 0.91 3.40 76 6.48 1.01 1.76 88 0.96 5.72 88 1.09 1.15 3.79 104 1.03 1.07 8.28 104 6.45 1.18 4.69 136 6.65 1.02 12.95 136 6.25 1.31 1.21 9.84 158 6.51 1.15 1.38 15.16 158 1.25 12.05

Appendix F: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation bottles TSRA T12_LM and TSRA TN9_LM

TSRA T12_LM TSRA TN9_LM Day pH NO3 (mmol/bottle) SO4 (mmol/bottle) Dissolved Ni (umol/bottle) Day pH NO3 (mmol/bottle) SO4 (mmol/bottle) Dissolved Ni (umol/bottle) 0 7.03 0.00 0.06 0.00 0 7.03 0.00 0.07 0.00 9 0.18 0.22 0.00 9 0.14 0.25 0.00 15 0.27 1.73 15 0.26 0.12 23 23 29 6.97 0.46 0.06 29 7.03 0.44 0.02 37 0.57 0.66 0.15 37 0.51 0.64 0.12 42 6.98 0.73 0.03 42 7.08 0.67 0.04 50 0.78 0.91 0.34 50 0.68 0.83 0.16 57 6.69 0.92 0.13 57 6.77 0.83 0.32 64 6.28 1.00 2.86 64 6.52 0.85 0.11 77 6.42 1.13 0.14 77 6.57 0.98 0.06 85 1.15 1.34 1.63 85 1.01 1.23 0.11 92 6.51 1.29 0.86 92 6.55 1.20 0.08 99 5.98 1.33 1.40 5.17 99 6.07 1.15 1.27 1.52 111 5.99 1.29 7.03 111 6.18 1.28 2.58 127 5.88 1.52 1.68 12.52 127 6.27 1.28 1.46 4.55 159 6.28 1.62 15.86 159 6.17 1.35 7.32 181 6.54 1.66 1.90 16.97 181 6.41 1.55 1.79 10.44 190 2.37 190 1.98 233 2.01 2.64 233 2.04 2.40 245 36.39 245 6.53 11.85

77 Appendix G: Cumulative nitrate consumed, cumulative sulfate produced and dissolved Ni data from third generation abiotic microcosm

TSRA ABIO_LM Day pH NO3 (mmol/bottle) SO4 (mmol/bottle) Dissolved Ni (umol/bottle) 9 7.02 0.00 0.06 0.00 15 0.01 0.08 0.00 23 0.06 0.15 29 37 7.18 0.06 0.09 42 0.19 0.06 0.09 50 7.19 0.06 0.01 57 0.19 0.06 0.08 64 6.94 0.06 0.12 77 6.91 0.35 0.10 85 6.88 0.02 0.02 92 0.34 0.10 0.10 99 7.48 0.05 0.04 111 7.03 0.34 0.04 0.04 127 6.80 0.03 0.42 159 6.84 0.34 0.03 0.12

78 Appendix H: Total Ni leached as sum of Ni in solution and Ni re-dissolved in 0.25M H2SO4

Microcosm Donor Treatment Ni in SN (mg) Ni in AW1 (mg) Ni in AW2 (mg) Ni in WW1 (mg) Ni in WW2 (mg) Ni in WW3 (mg) Total Ni leached (mg) TSRA 10_WZ Original UMFC 1st Active 0.19 75.15 9.56 1.17 0.11 0.06 86.25 TSRA 11_WZ Original UMFC 1st Active 0.62 64.32 7.47 0.79 0.08 0.03 73.31 TSRA 12_WZ Original UMFC 1st Active 1.09 48.65 1.87 0.63 0.05 0.02 52.31 TSRA TN9_WZ Pre-washed UMFC 2nd Active 1.81 23.84 0.99 0.47 0.03 0.03 27.17 TSRA TN3 ABIO_WZ Pre-washed UMFC 2nd Abio 0.01 0.86 0.30 0.24 0.17 1.58 TSRA TN9_LM Pre-washed UMFC 3rd Abio 0.67 0.41 0.26 0.21 0.10 1.64 TSRA T12_LM Pre-washed UMFC 3rd Active 2.06 0.36 0.24 0.13 0.18 2.97 TSRA ABIO_LM Pre-washed UMFC 3rd Active 0.02 0.87 0.52 0.43 1.84 * SN: supernatant, AW: Acid wash, WW: water wash

79 Appendix I: Bacterial cell count by qPCR

Sample DNA concentration Date Generation Day Sample SQ Mean (copies/mL of culture) Supernatant TSRA TN9_WZ 6.28E+04 6.28E+06 Supernatant TSRA 10_WZ 1.45E+04 1.45E+06 181 days Supernatant TSRA 11_WZ 5.61E+03 5.61E+05 1st and 2nd after Supernatant TSRA 12_WZ 6.03E+03 6.03E+05 21-Mar-18 generations inactive Slurry TSRA TN9_WZ 1.97E+03 1.97E+05 period Slurry TSRA 10_WZ 2.64E+01 2.64E+03 Slurry TSRA 11_WZ 4.48E+00 4.48E+02 Slurry TSRA 12_WZ 2.69E+00 2.69E+02 Supernatant TSRA TN9_LM_1 7.78E+06 7.78E+08 Supernatant TSRA T10_LM_1 1.57E+06 1.57E+08 04-Dec-18 3rd 188 Supernatant TSRA T11_LM_1 1.61E+06 1.61E+08 Supernatant TSRA T12_LM_1 2.21E+06 2.21E+08 Supernatant ABIO_LM_1 2.38E+02 2.38E+04 Supernatant TSRA TN9_LM_2 1.78E+06 1.78E+08 Supernatant TSRA T10_LM_1 4.58E+06 4.58E+08 06-Mar-19 3rd 280 Supernatant TSRA T10_LM_2 4.08E+06 4.08E+08 Supernatant TSRA T11_LM_1 6.35E+05 6.35E+07 Supernatant TSRA T12_LM_2 4.88E+06 4.88E+08

80 Appendix J: Comparison between the microbial community in the TSRA bottles from day 220 in Zhou's work and day 181 after the inactive period

Pyrotag: Wendy (without Chimera Check). Day 220 from Zhou's work. Max OTU > 1% TSRA 10_WZ TSRA 11_WZ TSRA 12_WZ Old OTU ID in Wendy's Pyrotag Betaproteobacteria_GU208255 32.29% 9.84% 4.85% PyroOTU7494, 4380,1393,2504 Sulfuritalea_FM207947 10.73% 28.21% 20.15% PyroOTU408 Thiobacillus_FM212993 26.40% 19.13% 2.12% PyroOTU3487 Gammaproteobacteria_AY955089 2.64% 0.18% 20.05% PyroOTU5381 Gallionellaceae_HE587245 0.13% 5.70% 10.90% PyroOTU7062 Bacteroidetes_GQ347194 0.15% 0.02% 9.98% PyroOTU600 Sideroxydans_GQ261317 0.18% 1.60% 9.44% PyroOTU343, 183 Rhodocyclaceae_AB795487 0.11% 4.99% 0.45% PyroOTU2838 Betaproteobacteriales_NEWOTU4035 0.03% 2.90% 0.01% PyroOTU4701 Anaerolineaceae_NewOTU2414 2.66% 0.50% 2.46% PyroOTU3690 Cystobacteraceae_AF414588 1.93% 1.59% 0.57% PyroOTU7853 Betaproteobacteria_JN038589 1.63% 0.00% 0.00% PyroOTU7588 Betaproteobacteria_NewOTU1056 0.07% 1.62% 0.00% PyroOTU1811 Betaproteobacteria_GU127809 0.30% 1.39% 1.09% PyroOTU5214 Cryomorphaceae_NewOTU2107 0.04% 0.06% 1.00% PyroOTU1742

Illumina: Lais by MetaAmp. Day 181 after inactive period. Max OTU > 1% TSRA 10_LM TSRA 11_LM TSRA 12_LM TSRA N9_LM Old OTUID in Metaamp Thiobacillus_FM213008 56.53% 10.08% 36.36% 45.08% IllOTU_2 Anaerolineaceae_GQ203640 8.99% 16.02% 23.71% 0.00% IllOTU_4 Anaerolineaceae_NewOTU2954 13.26% 1.71% 0.27% 0.00% IllOTU_8 Methanospirillum_AB447872/JN397878 0.05% 10.46% 0.10% 0.00% IllOTU_13 Anaerolineaceae_NewOTU2414 0.40% 6.75% 8.09% 0.23% IllOTU_10 Intrasporangium_D85486 0.00% 0.00% 0.03% 8.57% IllOTU_12 Betaproteobacteria_GU127809 0.54% 5.97% 4.16% 0.09% IllOTU_17 Comamonas_NewOTU732 0.00% 0.00% 0.00% 13.99% IllOTU_9 Betaproteobacteria_AY945879 0.13% 2.23% 2.27% 11.70% IllOTU_113 Gammaproteobacteria_AY955089 0.33% 2.97% 5.66% 0.02% IllOTU_24 Sulfuritalea_FQ658953 2.42% 5.37% 2.09% 0.16% IllOTU_21 Anaerolineaceae_JX221849 0.01% 5.04% 0.10% 0.00% IllOTU_36 Azospira_FN794258 3.58% 4.15% 4.79% 5.23% IllOTU_11 Gammaproteobacteria_JN039017 4.62% 0.01% 0.02% 0.00% IllOTU_26 Simplicispira_HQ178724/GQ389171 0.11% 0.33% 0.34% 7.11% IllOTU_231 Bacteria_KC540954 0.20% 2.78% 1.25% 0.00% IllOTU_30 Anaerolineae SJA-15__NewOTU3114 0.00% 2.71% 0.00% 0.00% IllOTU_38 Bacteria_JX505360 1.06% 2.55% 0.00% 0.00% IllOTU_35 Bacteria__NewOTU2505 0.00% 2.52% 0.00% 0.00% IllOTU_40 Methanosarcina_EU420708 0.00% 2.44% 0.00% 0.00% IllOTU_51 Candidatus Roizmanbacteria RIFOXYD1__NewOTU267 2.35% 1.26% 0.00% 0.00% IllOTU_41 Methanoregula_AB329661 0.00% 1.63% 0.00% 0.00% IllOTU_59 Gallionellaceae_GQ388795 0.05% 0.78% 1.59% 0.02% IllOTU_77 Bradyrhizobium_FQ659783 0.09% 0.41% 1.16% 0.21% IllOTU_67 Ignavibacteria OPB56_AY218625 0.00% 1.13% 0.00% 0.00% IllOTU_68

*Raw sequence reads can be found in supplementary file: Raw-DNA-Sequence_1st-and-2nd-generations_Illumina-Pyrotag-Comparison_Chapter2.xlsx

81 Appendix K: 16S rRNA Sequencing results of the 3rd generation of microcosms. The colors indicate repeated microorganisms found in each bottle

Day 189 (05-Dec-18) Day 280 (06-Mar-19) Sample Microorganism OTU>1% Sample Microorganism OTU>1% Thiobacillus 68.84% Thiobacillus 84.34% Simplicispira 10.12% Simplicispira 8.92% TSRA T10_LM_1 Simplicispira 7.65% Simplicispira 1.38% Azospira 4.14% Leifsonia 1.37% TSRA T10_LM_1 Intrasporangium 1.10% Thiobacillus 74.97% Leifsonia 1.92% Simplicispira 14.41% Nocardioides 1.31% TSRA T10_LM_2 Simplicispira 3.80% uncultured bacterium 1.09% Azospira 1.50% Thiobacillus 74.04% Leifsonia 1.86% Simplicispira 1.99% Thiobacillus 84.91% Thiobacillus 1.90% Simplicispira 1.10% TSRA T11_LM_1 Azospira 6.20% Azospira 2.10% Intrasporangium 3.52% TSRA T11_LM_1 Intrasporangium 1.69% Burkholderiaceae 5.85% Burkholderiaceae 4.29% Leifsonia 1.88% Leifsonia 1.51% Thiobacillus 50.33% Limnobacter 1.34% Simplicispira 42.41% Thiobacillus 68.31% TSRA T12_LM_1 Simplicispira 1.36% Simplicispira 19.74% Intrasporangium 2.14% TSRA T12_LM_1 Simplicispira 1.17% Thiobacillus 50.43% Azospira 1.38% Simplicispira 17.56% Intrasporangium 2.98% TSRA TN9_LM_1 Thiobacillus 25.41% Thiobacillus 68.84% Azospira 1.93% Simplicispira 10.12% Leptolinea 1.76% Simplicispira 7.65% Azospira 4.14% TSRA TN9_LM_2 Intrasporangium 1.10% Leifsonia 1.92% Nocardioides 1.31% uncultured bacterium 1.09%

*Raw sequence reads can be found in supplementary file: Raw-DNA-Sequence_3rd-generation_TSRA_Chapter2.xlsx

82 Appendix L: Field Data

Sampling DO Conductivity Sample Size Sample Location Time pH ORP T(C) Date (mg/L) (mS/cm^2) (L)

Raw Water Downstream Process 19-Apr-18 1:05PM 7.04 5.91 157 25 1.81 3.00 Rep. 1 Barren Solution 8" Pipe Along West Slope of PAD7 18-Apr-18 2:40PM 10.16 3.08 119 27 7.80 2.96 Rep. 2 Barren Solution 8" Pipe Along West Slope of PAD7 19-Apr-18 7:30AM 10.29 4.15 97 21 7.76 2.96 Rep. 1 Sampler 3 West Slope of PAD7 18-Apr-18 10:50AM 11.94 5.93 46.5 31 9.77 3.06 Rep. 2 Sampler 3 West Slope of PAD7 19-Apr-18 7:00AM 11.98 8.51 110 12 9.54 2.98 Rep.1 Sampler 45 West Slope of PAD7 18-Apr-18 10:41AM 11.61 7.28 86.3 23 8.00 3.06 Rep. 2 Sampler 45 West Slope of PAD7 19-Apr-18 6:45AM 11.61 7.68 86.7 15 7.64 3.02 Rep. 1 Pad 7 North Sampler Box, South of PAD7 18-Apr-18 9:49AM 9.00 7.84 183 24 3.96 3.35 Rep. 2 Pad 7 North Sampler Box, South of PAD7 19-Apr-18 6:30AM 9.08 5.87 193 21 7.59 3.01 Rep. 1 Pad 7 South Sampler Box, South of PAD7 18-Apr-18 9:45AM 8.60 6.16 203 23 7.46 3.00 Rep. 2 Pad 7 South Sampler Box, South of PAD7 19-Apr-18 6:20AM 8.54 5.94 209 21 7.52 3.02 Rep. 1 Before CIC's Downstream Process 18-Apr-18 3:00PM 9.25 4.16 181 24 7.44 3.12 Rep. 2 Before CIC's Downstream Process 19-Apr-18 12:30PM 9.33 4.45 188 23 7.46 3.02 Rep. 1 After New CIC's Downstream Process 18-Apr-18 3:15PM 9.24 4.21 147 25 7.39 2.98 Rep. 2 After New CIC's Downstream Process 19-Apr-18 12:45PM 9.32 3.93 125 23 7.47 3.10 Rep.2 After Old CIC's Downstream Process 19-Apr-18 12:35PM 9.30 4.41 141 23 7.49 3.09

83 Appendix M: DNA extraction

DNA conc. Extraction Volume Elution volume Total DNA Sample from Qubit Date Filtered (L) (uL) (ng/L) (ng/uL)

Raw Water 09-Jul-18 1.50 50 3.32 111 Rep. 1 Barren Solution 30-Jul-18 1.48 50 74.60 2520 Rep. 2 Barren Solution 09-Jul-18 1.48 50 44.00 1486 Rep. 1 Sampler 3 30-Jul-18 1.53 50 0.70 23 Rep. 2 Sampler 3 30-Jul-18 1.49 50 4.36 146 Rep.1 Sampler 45 30-Jul-18 1.53 50 0.33 11 Rep. 2 Sampler 45 09-Jul-18 1.51 50 0.30 10 Rep. 1 Pad 7 North 30-Jul-18 1.68 50 72.60 2167 Rep. 2 Pad 7 North 09-Jul-18 1.51 50 20.60 684 Rep. 1 Pad 7 South 30-Jul-18 1.50 50 72.60 2420 Rep. 2 Pad 7 South 09-Jul-18 1.51 50 29.60 980 Rep. 1 Before CIC's 30-Jul-18 1.56 50 30.40 974 Rep. 2 Before CIC's 09-Jul-18 1.51 50 17.30 573 Rep. 1 After New CIC's 30-Jul-18 1.49 50 56.20 1886 Rep. 2 After New CIC's 09-Jul-18 1.55 50 22.80 735 Rep.2 After Old CIC's 09-Jul-18 1.55 50 38.20 1236

84 Appendix N: qPCR data

Absolute Volume Bacterial copies Elution volume Sample Analysis date Dilution Factor Concentration Filtered (mL) (copies/uL) (uL) (Copies/mL)

Raw Water 16-Aug-18 1500 1.14E+06 50 1 3.80E+04 Rep. 1 Barren Solution 16-Aug-18 1480 1.27E+07 50 1 4.29E+05 Rep. 2 Barren Solution 16-Aug-18 1480 1.14E+07 50 1 3.86E+05 Rep. 1 Sampler 3 16-Aug-18 1530 8.45E+04 50 1 2.76E+03 Rep. 2 Sampler 3 16-Aug-18 1490 5.34E+05 50 1 1.79E+04 Rep.1 Sampler 45 16-Aug-18 1530 9.33E+04 50 1 3.05E+03 Rep. 2 Sampler 45 16-Aug-18 1510 1.94E+05 50 1 6.44E+03 Rep. 1 Pad 7 North 16-Aug-18 1675 4.88E+06 50 1 1.46E+05 Rep. 2 Pad 7 North 16-Aug-18 1505 1.05E+07 50 1 3.50E+05 Rep. 1 Pad 7 South 16-Aug-18 1500 1.23E+07 50 1 4.11E+05 Rep. 2 Pad 7 South 16-Aug-18 1510 1.21E+07 50 1 3.99E+05 Rep. 1 Before CIC's 16-Aug-18 1560 5.44E+06 50 1 1.74E+05 Rep. 2 Before CIC's 16-Aug-18 1510 1.06E+07 50 1 3.52E+05 Rep. 1 After New CIC's 16-Aug-18 1490 9.14E+06 50 1 3.07E+05 Rep. 2 After New CIC's 16-Aug-18 1550 1.17E+07 50 1 3.78E+05 Rep.2 After Old CIC's 16-Aug-18 1545 1.31E+07 50 1 4.24E+05

85 Appendix O: Anions and nutrients

Alkalinity Ammonia Nitrite Sulfate Sample Nitrate (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Raw Water 69.80 0.01 1.01 <0.010 273 Rep. 1 Barren Solution 67.1 28.5 592 68.1 329 Rep. 2 Barren Solution 73.1 32.9 596 68.4 333 Rep. 1 Sampler 3 702.0 2.5 588 73.5 251 Rep. 2 Sampler 3 669.0 2.3 593 74.9 258 Rep.1 Sampler 45 339.0 51.9 539 60.7 402 Rep. 2 Sampler 45 317.0 52.8 87 9.8 59 Rep. 1 Pad 7 North 50.0 20.5 616 69.7 330 Rep. 2 Pad 7 North 55.7 21.8 639 72.0 345 Rep. 1 Pad 7 South 55.1 20.1 759 77.9 403 Rep. 2 Pad 7 South 54.6 24.6 625 63.7 330 Rep. 1 Before CIC's 64.1 21.2 701 81.4 379 Rep. 2 Before CIC's 64.9 20.7 613 69.4 329 Rep. 1 After New CIC's 67.5 21.7 608 70.5 327 Rep. 2 After New CIC's 65.4 19.9 692 79.8 373 Rep.2 After Old CIC's 67.7 21.0 593 68.1 318

Appendix P: Cyanide Species

Cyanide Total Cyanide Cyanate Thiocyanate Free Cyanide Sample (WAD) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Raw Water <0.010 0.01 <0.20 <0.50 0.01 Rep. 1 Barren Solution 93.20 91.30 <2.0 <2.5 89.80 Rep. 2 Barren Solution 120.00 117.00 5.40 <2.5 115.00 Rep. 1 Sampler 3 1.84 2.65 3.21 0.58 1.72 Rep. 2 Sampler 3 0.92 2.07 5.01 <0.50 0.85 Rep.1 Sampler 45 7.25 9.41 20.40 <0.5 6.76 Rep. 2 Sampler 45 4.33 7.30 9.60 <0.50 4.02 Rep. 1 Pad 7 North 5.44 8.48 13.80 <0.5 5.05 Rep. 2 Pad 7 North 4.67 7.49 <2.0 <0.50 4.33 Rep. 1 Pad 7 South 11.80 14.70 19.50 <0.5 11.10 Rep. 2 Pad 7 South 9.53 11.90 <2.0 <0.50 8.63 Rep. 1 Before CIC's 3.12 5.92 3.90 <0.5 2.92 Rep. 2 Before CIC's 3.51 5.88 <2.0 <0.50 3.22 Rep. 1 After New CIC's 2.04 4.81 7.50 <0.5 1.96 Rep. 2 After New CIC's 1.64 4.41 <2.0 <0.50 1.50 Rep.2 After Old CIC's 2.07 3.73 2.10 <0.50 1.92

87 Appendix Q: Total dissolved metals

Antimony Arsenic Barium Beryllium Bismuth Boron (B)- Cadmium Calcium Cesium Chromium Copper Gold (Au)- Aluminum (Al)- Cobalt (Co)- Sample (Sb)-Total (As)-Total (Ba)-Total (Be)-Total (Bi)-Total Total (Cd)-Total (Ca)-Total (Cs)-Total (Cr)-Total (Cu)-Total Total Total (mg/L) Total (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Raw Water <0.0030 0.00 0.00 0.03 <0.00010 <0.000050 0.85 <0.0000050 43.00 0.02 0.01 <0.00010 <0.00050 <0.0010

Rep. 1 Barren Solution <0.030 <0.0010 0.04 0.08 <0.0010 <0.00050 0.22 <0.00012 352.00 0.00 0.01 0.52 0.13 <0.010 Rep. 2 Barren Solution <0.030 <0.0010 0.04 0.07 <0.0010 <0.00050 0.24 <0.00013 349.00 0.00 0.01 0.51 0.15 <0.010 Rep. 1 Sampler 3 0.13 <0.0010 0.00 0.11 <0.0010 <0.00050 0.20 <0.000080 706.00 0.00 0.01 0.48 0.10 0.04 Rep. 2 Sampler 3 0.09 <0.0010 0.00 0.11 <0.0010 <0.00050 0.19 <0.000065 704.00 0.00 0.01 0.50 0.06 0.03 Rep.1 Sampler 45 <0.015 0.00 0.00 0.09 <0.00050 <0.00025 0.27 <0.00015 351.00 0.00 0.01 0.62 0.50 0.47 Rep. 2 Sampler 45 <0.015 0.00 0.00 0.08 <0.00050 <0.00025 0.27 <0.00011 356.00 0.00 0.01 0.63 0.48 0.50 Rep. 1 Pad 7 North <0.015 0.00 0.01 0.09 <0.00050 <0.00025 0.21 <0.00035 319.00 0.00 0.01 0.50 0.63 0.16 Rep. 2 Pad 7 North <0.015 0.00 0.01 0.09 <0.00050 <0.00025 0.20 <0.00031 323.00 0.00 0.00 0.54 0.52 0.16 Rep. 1 Pad 7 South <0.015 0.00 0.01 0.08 <0.00050 <0.00025 0.18 <0.00060 278.00 0.00 0.01 0.52 1.07 0.23 Rep. 2 Pad 7 South <0.015 0.00 0.01 0.08 <0.00050 <0.00025 0.18 <0.00059 288.00 0.00 0.00 0.54 0.94 0.20 Rep. 1 Before CIC's <0.015 0.00 0.04 0.08 <0.00050 <0.00025 0.21 <0.00021 346.00 0.00 0.01 0.53 0.18 0.13 Rep. 2 Before CIC's <0.015 0.00 0.04 0.07 <0.00050 <0.00025 0.21 <0.00018 351.00 0.00 0.01 0.49 0.14 0.12 Rep. 1 After New CIC's <0.015 0.00 0.04 0.08 <0.00050 <0.00025 0.21 <0.00017 345.00 0.00 0.01 0.51 0.17 <0.0050 Rep. 2 After New CIC's <0.015 0.00 0.04 0.07 <0.00050 <0.00025 0.21 <0.00013 352.00 0.00 0.01 0.50 0.21 <0.0050 Rep.2 After Old CIC's <0.015 0.00 0.04 0.07 <0.00050 <0.00025 0.24 <0.00015 359.00 0.00 0.01 0.53 0.19 0.01

Magnesium Manganese Molybdenum Phosphorus Rubidium Selenium Silicon (Si)- Silver (Ag)- Iron (Fe)- Lead (Pb)- Lithium (Li)- Nickel (Ni)- Potassium (K)- Sample (Mg)-Total (Mn)-Total (Mo)-Total (P)-Total (Rb)-Total (Se)-Total Total Total Total (mg/L) Total (mg/L) Total (mg/L) Total (mg/L) Total (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Raw Water 0.26 <0.000050 0.19 3.83 0.01 0.02 0.00 <0.050 6.44 0.04 0.01 11.90 <0.000010

Rep. 1 Barren Solution 1.32 <0.00050 0.02 11.50 0.00 0.38 0.01 <0.50 20.90 0.01 0.16 4.70 0.01 Rep. 2 Barren Solution 1.28 <0.00050 0.02 11.40 0.00 0.37 0.01 <0.50 20.60 0.01 0.16 4.90 0.01 Rep. 1 Sampler 3 0.45 <0.00050 0.02 0.15 <0.0010 0.40 0.01 <0.50 19.60 0.01 0.14 1.30 0.00 Rep. 2 Sampler 3 0.48 <0.00050 0.02 0.27 <0.0010 0.40 0.01 <0.50 18.70 0.01 0.14 1.30 0.00 Rep.1 Sampler 45 1.42 <0.00025 0.02 0.99 <0.00050 0.40 0.04 <0.25 23.00 0.01 0.22 2.02 0.06 Rep. 2 Sampler 45 1.43 <0.00025 0.02 0.86 <0.00050 0.40 0.04 <0.25 23.60 0.01 0.20 1.97 0.05 Rep. 1 Pad 7 North 1.54 <0.00025 0.02 19.70 0.00 0.38 0.02 <0.25 20.80 0.01 0.17 4.34 0.05 Rep. 2 Pad 7 North 1.51 <0.00025 0.02 19.90 0.00 0.39 0.02 <0.25 22.30 0.01 0.17 4.47 0.04 Rep. 1 Pad 7 South 1.52 <0.00025 0.02 28.40 0.02 0.39 0.04 <0.25 21.60 0.01 0.18 5.44 0.06 Rep. 2 Pad 7 South 1.52 <0.00025 0.02 28.80 0.02 0.40 0.04 <0.25 22.20 0.02 0.19 5.51 0.06 Rep. 1 Before CIC's 1.36 <0.00025 0.01 12.00 0.00 0.37 0.01 <0.25 21.20 0.01 0.16 4.43 0.02 Rep. 2 Before CIC's 1.28 <0.00025 0.01 11.90 0.00 0.36 0.01 <0.25 20.90 0.01 0.15 4.36 0.02 Rep. 1 After New CIC's 1.36 <0.00025 0.01 12.00 0.00 0.38 0.01 <0.25 21.60 0.01 0.16 4.51 0.01 Rep. 2 After New CIC's 1.37 <0.00025 0.01 11.70 0.00 0.37 0.01 <0.25 20.60 0.01 0.15 4.51 0.01 Rep.2 After Old CIC's 1.38 <0.00025 0.01 12.40 0.00 0.38 0.01 <0.25 20.80 0.01 0.16 4.50 0.01

Sodium Strontium Tellurium Thallium Thorium Tungsten Uranium Vanadium Zirconium Sulfur (S)- Tin (Sn)- Titanium (Ti)- Zinc (Zn)- Sample (Na)-Total (Sr)-Total (Te)-Total (Tl)-Total (Th)-Total (W)-Total (U)-Total (V)-Total (Zr)-Total Total (mg/L) Total (mg/L) Total (mg/L) Total (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Raw Water 314.00 1.33 81.30 <0.00020 0.00 <0.00010 <0.00010 <0.00030 0.02 0.00 0.00 <0.0030 0.00

Rep. 1 Barren Solution 1420.00 5.80 109.00 <0.0020 <0.00010 <0.0010 <0.0010 <0.0030 0.00 0.00 0.01 <0.030 <0.00060 Rep. 2 Barren Solution 1400.00 5.75 109.00 <0.0020 <0.00010 <0.0010 <0.0010 <0.0030 0.00 0.00 0.01 <0.030 <0.00060 Rep. 1 Sampler 3 1170.00 7.25 86.30 <0.0020 <0.00010 <0.0010 <0.0010 <0.0030 <0.0010 <0.00010 0.01 <0.030 <0.00060 Rep. 2 Sampler 3 1160.00 7.06 82.40 <0.0020 <0.00010 <0.0010 <0.0010 <0.0030 <0.0010 <0.00010 <0.0050 <0.030 <0.00060 Rep.1 Sampler 45 1340.00 4.17 132.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 <0.000050 0.01 <0.015 <0.00030 Rep. 2 Sampler 45 1320.00 4.23 133.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 <0.000050 0.01 <0.015 <0.00030 Rep. 1 Pad 7 North 1330.00 5.66 112.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.00 <0.015 <0.00030 Rep. 2 Pad 7 North 1340.00 5.72 114.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.00 <0.015 <0.00030 Rep. 1 Pad 7 South 1380.00 5.73 110.00 <0.0010 0.00 <0.00050 <0.00050 <0.0015 0.00 0.01 0.00 0.02 <0.00030 Rep. 2 Pad 7 South 1360.00 5.75 114.00 <0.0010 0.00 <0.00050 <0.00050 <0.0015 0.00 0.01 0.00 <0.015 <0.00030 Rep. 1 Before CIC's 1240.00 5.73 109.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.01 0.11 <0.00030 Rep. 2 Before CIC's 1230.00 5.76 106.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.01 <0.015 <0.00030 Rep. 1 After New CIC's 1280.00 5.88 108.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.01 <0.015 <0.00030 Rep. 2 After New CIC's 1240.00 5.60 110.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.01 <0.015 <0.00030 Rep.2 After Old CIC's 1250.00 5.89 111.00 <0.0010 <0.000050 <0.00050 <0.00050 <0.0015 0.00 0.00 0.01 <0.015 <0.00030

90 Appendix R: Comparison between bacterial cell count, filtered volume and Hydrogenophaga relative abundance per sample

Bacterial Volume Hydrogenophaga Sample copies Filtered (mL) Relative (copies/uL) Abundance (%)

Raw Water 1.14E+06 1500 0.11 Rep. 1 Barren Solution 1.27E+07 1480 89.10 Rep. 2 Barren Solution 1.14E+07 1480 90.72 Rep. 1 Sampler 3 8.45E+04 1530 99.33 Rep. 2 Sampler 3 5.34E+05 1490 99.64 Rep.1 Sampler 45 9.33E+04 1530 96.11 Rep. 2 Sampler 45 1.94E+05 1510 94.78 Rep. 1 Pad 7 North 4.88E+06 1675 85.52 Rep. 2 Pad 7 North 1.05E+07 1505 87.07 Rep. 1 Pad 7 South 1.23E+07 1500 88.15 Rep. 2 Pad 7 South 1.21E+07 1510 85.53 Rep. 1 Before CIC's 5.44E+06 1560 71.67 Rep. 2 Before CIC's 1.06E+07 1510 75.72 Rep. 1 After New CIC's 9.14E+06 1490 81.15 Rep. 2 After New CIC's 1.17E+07 1550 80.74 Rep.2 After Old CIC's 1.31E+07 1545 81.64

*Raw sequence reads can be found in supplementary file: Raw-DNA-Sequence_Chapter3.xlsx