Nitrate-Dependent, Neutral pH Bioleaching of Ni from an Ultramafic Concentrate

by

Han Zhou

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Chemical Engineering and Applied Chemistry University of Toronto

© Copyright by Han Zhou 2014 ii

Nitrate-Dependent, Neutral pH Bioleaching of Ni from an Ultramafic Concentrate

Han Zhou

Master of Applied Science

Chemical Engineering and Applied Chemistry University of Toronto

2014 Abstract

This study explores the possibility of utilizing bioleaching techniques for nickel extraction from a mixed sulfide ore deposit with high magnesium content. Due to the ultramafic nature of this material, well-studied bioleaching technologies, which rely on acidophilic bacteria, will lead to undesirable processing conditions. This is the first work that incorporates nitrate-dependent bacteria under pH 6.5 environments for bioleaching of base metals. Experiments with both defined bacterial strains and indigenous mixed bacterial cultures were conducted with nitrate as the electron acceptor and sulfide minerals as electron donors in a series of microcosm studies.

Nitrate consumption, sulfate production, and Ni released into the aqueous phase were used to track the extent of oxidative sulfide mineral dissolution; taxonomic identification of the mixed culture community was performed using 16S rRNA gene sequencing. Nitrate-dependent microcosms that contained indigenous sulfur- and/or iron-oxidizing microorganisms were cultured, characterized, and provided a proof-of-concept basis for further bioleaching studies.

iii

Acknowledgments

I would like to extend my most sincere gratitude toward both of my supervisors Dr. Vladimiros Papangelakis and Dr. Elizabeth Edwards. This work could not have been completed without your brilliant and patient guidance. Your enthusiasm and curiosity in scientific pursuit and teaching is a constant source of motivation in my work; your energy and optimistic take on life will forever guide me and I will always look upon you two as role models in life.

NSERC and our industrial sponsor Vale deserve recognition for providing funding and the ore material for my research.

I would like to thank Susie (Endang Susilawati) and Line Lomheim in Biozone in teaching me how to work in a large microbiology lab on a daily basis and always being there to offer their help with lab related issues and where to find stuff. (It’s surprising how big a difference it makes!) I would also like to recognize Paul Jowlabar for his help in earlier stages of my project with building my first set of reactors, and George Kretschemann for his expertise in mineralogy analysis.

I want to thank all my lab mates and colleagues, Georgiana, Doug, Srinath, Fei, Cheryl, Shuiquan, Chris, and everyone else in both APEC lab and Edlab, your friendship, guidance, and generosity have made the past two and a half years a memorable and rewarding experience in my life.

The most sincere gratitude and love goes to my parents, Bing Zhou and Jiang Gao, for their unconditional love and support in my education and all other aspects in life. Without them, I could not have become the person I am today.

Finally, I would like to dedicate this work to my grandfather, Ruju Zhou.

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Table of Contents

Acknowledgments ...... iii

Table of Contents ...... iv

List of Tables ...... vii

List of Figures ...... ix

List of Appendices ...... xi

1 Introduction ...... 1 1.1 Overview ...... 1 1.2 Objectives ...... 2 1.3 Outline of Thesis ...... 3

2 The Thompson Concentrate ...... 4 2.1 Thompson Deposit History ...... 4 2.2 Mineralogy ...... 4 2.3 Processing Concerns ...... 9 2.4 Incentives for the current study ...... 10

3 Literature Review on Biohydrometallurgy ...... 11 3.1 Introduction ...... 11 3.2 Acid Bioleaching ...... 11 3.3 Bioleaching at Elevated pH ...... 12 3.4 Nitrate-dependent Bioleaching System ...... 13 3.4.1 Thiobacillus denitrificans ...... 13 3.4.2 S oxidation by T. denitrificans ...... 14 3.4.3 Fe oxidation by T. denitrificans ...... 14 3.4.4 Nitrite reduction inhibition in T. denitrificans...... 15 3.4.5 Attachment to solid surface ...... 15 3.4.6 Other nitrate-dependent iron- and sulfur-oxidizing bacteria ...... 15

4 Methodology ...... 17 4.1 Ultramafic concentrate ...... 17 4.1.1 Material Preparation ...... 17 4.1.2 UMFC Preservation ...... 17 v

4.1.3 Acid pre-leach ...... 18 4.2 Analytical techniques ...... 18 4.2.1 Solid material characterization ...... 18 4.2.2 Aqueous Samples ...... 20 4.3 Experiments with Defined Culture – Thiobacillus denitrificans (ATCC 25259) ...... 21 4.3.1 Revitalization, Growth, and Maintenance ...... 21 4.3.2 Experimental Setup ...... 22 4.3.3 Sample Analysis ...... 23 4.4 Experiments with Indigenous Cultures – First Enrichment ...... 23 4.4.1 Collection of Indigenous Sediment Samples from Thompson ...... 24 4.4.2 First Enrichment – Experimental Design ...... 24 4.4.3 Microcosm Setup ...... 25 4.4.4 Sample Analysis ...... 25 4.5 Second Enrichment ...... 26 4.5.1 Experimental Design ...... 26 4.5.2 Microcosm Setup ...... 28 4.5.3 Sample Analysis ...... 28 4.6 Microbiology Investigation ...... 29 4.6.1 DNA Extraction and Isolation ...... 29 4.6.2 16S rRNA Gene Amplification and Purification ...... 30 4.6.3 16S Data Processing ...... 31 4.6.4 Phylogenetic Analysis ...... 32

5 Results and Discussion ...... 33 5.1 Experiments with T. denitrificans ATCC 25259 ...... 33 5.1.1 Growth of ATCC 25259 ...... 33 5.1.2 Bioleaching of UMFC with ATCC 25259 ...... 34 5.2 First enrichment - Experiments with indigenous Thompson cultures ...... 36 5.2.1 Preliminary results ...... 36 5.2.2 Stoichiometric correlation - denitrifying ...... 38 5.2.3 Stoichiometric correlations - aerobic ...... 40 5.2.4 Denitrifying vs. Aerobic ...... 41 5.2.5 Ni re-precipitation ...... 42 5.2.6 Magnesium Dissolution ...... 42 5.2.7 Tails, Dam, and Exp ...... 43 vi

5.3 Bacterial community analysis (16S rRNA gene sequencing) ...... 45 5.3.1 Taxonomic diversity in active denitrifying microcosms (Tails, Dam, Exp) ...... 46 5.3.2 Taxonomic diversity in active aerobic microcosms (Tails, Dam, Exp) ...... 49 5.3.3 Taxonomic diversity in TSRA microcosms (aerobic and denitrifying) ...... 51 5.3.4 Phylogenetic analysis of Betaproteobacteria ...... 53 5.4 Second enrichment – acid leached UMFC ...... 56

6 Conclusions and Recommendations for Future Work ...... 58 6.1 Bioleaching with defined strain Thiobacillus denitrificans ...... 58 6.2 Microcosm study with environmental cultures ...... 58 6.3 Bacterial community analysis ...... 60 6.4 Second microcosms study with acid-leached UMFC ...... 61 6.5 Recommendations for Future Work ...... 61

7 References ...... 63

Appendices ...... 66 Appendix A: Edlab Multi Vitamins Stock Solution (MM7) ...... 66 Appendix B: Thiobacillus denitrificans Growth Medium ...... 67 Appendix C: Leaching medium ...... 68 Appendix D: Thompson Indigenous Samples Collection ...... 69 Appendix E: Protocol for setting up microcosms using anaerobic glovebag ...... 72 Appendix F: Leaching medium II ...... 74 Appendix G: Supplementary Data ...... 75

vii

List of Tables

Table 1 Chemical Assay of UMFC* (by Aqua Regia, in wt.% and standard deviation σ) ...... 4

Table 2 Atomic Composition of Selected Points in Figure 2 (atomic wt.%) ...... 6

Table 3: Atomic composition of selected points in Figure 3 (atomic wt.%) ...... 6

Table 4: Mineralogy of UMFC ...... 8

Table 5: Ion Chromatography calibration standards ...... 21

Table 6: Leaching experiments with T. denitrificans ...... 23

Table 7: Microcosm treatment table for each series ...... 24

Table 8: First enrichment startup compositions ...... 25

Table 9: Aerobic treatments for second enrichment (Exposure and TSRA) ...... 27

Table 10: Denitrifying treatments for second enrichment (Dam, Exposures, and TSRA) ...... 28

Table 11: PCR reagents ...... 30

Table 12: Stoichiometric comparison in active denitrifying microcosms (410 days) ...... 39

Table 13: Stoichiometric comparison in active aerobic microcosms (410 days) ...... 41

Table 14: Nitrate, sulfate, and dissolved Ni in Tails, Dam, and Exp series (220 days) ...... 48

Table 15: Nitrate, sulfate, and dissolved Ni in denitrifying TSRA microcosms (220 days) ...... 52

Table 16: Major OTUs, abundance, and closest GenBank matches ...... 53

Table 17: Chemical Assay of Acid-leached UMFC ...... 56

Table 18: Nitrate, sulfate in second enrichment Dam, Exp, TSRA and Thd series (100 days) ..... 57

Table 19: Tails first enrichment complete data - Sterile ...... 75 viii

Table 20: Dam first enrichment complete data - Sterile ...... 76

Table 21: Exp first enrichment complete data - Sterile ...... 77

Table 22: TSRA first enrichment complete data - Sterile ...... 78

Table 23: Tails first enrichment complete data - Aerobic ...... 79

Table 24: Dam first enrichment complete data - Aerobic ...... 80

Table 25: Exp first enrichment complete data - Aerobic ...... 81

Table 26: TSRA first enrichment complete data - Aerobic ...... 82

Table 27: Tails first enrichment complete data - Denitrifying ...... 83

Table 28: Dam first enrichment complete data - Denitrifying ...... 85

Table 29: Exp first enrichment complete data - Denitrifying ...... 87

Table 30: TSRA first enrichment complete data - Denitrifying ...... 89

Table 31: Taxonomic assignment of denitrifying microcosms – Tails, Dam, and Exp ...... 91

Table 32: Taxonomic assignment of aerobic microcosms – Tails, Dam, and Exp ...... 92

Table 33: Taxonomic assignment of TSRA microcosms ...... 93

Table 32: Dam second enrichment partial data - Denitrifying ...... 94

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List of Figures

Figure 1 Backscattered electron image of polished mount embedded in epoxy (200x) ...... 5

Figure 2 Backscattered electron image with point analysis using EDX (500x) ...... 6

Figure 3: Backscattered electron image with point analysis using EDX (300x) ...... 6

Figure 4: X-ray diffraction (XRD) of UMFC ...... 7

Figure 5: Particle size distribution of UMFC ...... 9

Figure 6: Acid pre-leach, % leached ...... 18

Figure 7: Sealed anaerobic leaching setup ...... 22

Figure 8: Second enrichment transfer layout ...... 29

Figure 9: Number of sequences used for taxonomic and phylogenetic analysis per sample ...... 31

Figure 10: Sequence length distribution ...... 32

Figure 11: Substrate consumption for ATCC 25259 growth on thiosulfate (batch) ...... 33

Figure 12: Substrate consumption for ATCC 25259 during UMFC bioleaching ...... 35

Figure 13: TSRA microcosm study ...... 37

Figure 14: Magnesium dissolution in Exposures series ...... 43

Figure 15: Tails microcosm study ...... 44

Figure 16: Dam microcosm study ...... 44

Figure 17: Exposures microcosm study ...... 45

Figure 18: Taxonomic assignment of Tails, Dam, and Exp denitrifying microcosms ...... 47

Figure 19: Taxonomic assignment of aerobic Tails, Dam, and Exp microcosms ...... 50 x

Figure 20: Taxonomic assignment of TSRA microcosms ...... 52

Figure 21: Maximum likelihood tree of Betaproteobacteria in all 23 sequenced microcosms ..... 55

Figure 22: Second enrichment - Dam denitrifying ...... 56

xi

List of Appendices

Appendix A: Edlab Multi Vitamins Stock Solution (MM7) ...... 66 Appendix B: Thiobacillus denitrificans Growth Medium ...... 67 Appendix C: Leaching medium ...... 68 Appendix D: Thompson Indigenous Samples Collection ...... 69 Appendix E: Protocol for setting up microcosms using anaerobic glovebag ...... 72 Appendix F: Leaching medium II ...... 74 Appendix G: Supplementary Data ...... 75

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1 Introduction 1.1 Overview

This work presents a proof-of-concept study for biologically mediated nickel extraction from low-grade ultramafic deposit characterized by high magnesium contents. Nickel exists as sulfide, and magnesium is present in the deposit primarily as serpentine group minerals, which make up the bulk of the host materials. In processing an ore material with low contents of the desired metal, conventional pyro and hydro metallurgical processes usually lead to uneconomical scenarios due to high energy costs, consumption of large quantities of chemicals for neutralization, and expensive waste management.

In the case of a conventional process, or in other words, the mine-mill-smelter route, unaltered ore material is first pretreated using methods including grinding and flotation. Loss of nickel to rejected stream of the flotation circuit could be significant, depending on the natural associations of the nickel bearing phases. In addition, the amount of gangue materials remaining in the concentrate stream could reduce the energy efficiency of the smelter. These drawbacks are especially impeding in processing ultramafic deposits, as some gangue materials are naturally flotable, which will lead to high-MgO concentrates that (1) increase the total volume for handling, (2) require higher smelting temperatures which resulting in reduced furnace life [1], and (3) interfere with the flotation of the sulfide minerals, reducing flotation rates [2].

Conventional metallurgy for extraction of nickel from sulfide concentrates employs a combination of pyro and hydro metallurgical techniques. The recovery process usually starts with grinding the concentrate to desired size range, followed by oxidative leaching, which usually takes place in an autoclave or and agitated reactor. After several stages of impurity removal, nickel and other sellable metals are recovered by electrowinning. Operating costs and raw material costs associated with these techniques are high, and can only be recovered by the metal values from a high-grade ore. Compared to most industrial processes, the grade of the ultramafic material in this study is too low for a viable nickel recovery process.

Bioleaching is usually studied and practiced as an alternative to conventional mineral processing techniques that could potentially allow economical development of currently unfeasible deposits, either due to low grade of the minerals of interest or difficulties with processing. The most well 2 studied bioleaching system stems from the widely observed acid mine drainage (AMD) phenomenon, where acidophilic sulfur- and iron-oxidizing bacteria thrive under pH 1.5-2.5 and moderate temperature environments to enable oxidative dissolution of exposed sulfide minerals. The study of this category of bioleaching under low pH conditions has been on going since 1977 [3] for copper sulfides; more recently, the first commercial application of nickel sulfide heap bioleaching began at Talvivaara, Finland [4].

These studies and commercial practices have shown that acidic bioleaching could be suitable for low-grade sulfide deposits or mining wastes (e.g. pyrrhotite tailings), where processing costs, reagent costs, and waste management costs can be justified by the metal value embedded in the source material. However, the staggering acid requirements for MgO dissolution eliminate acidic bioleaching as a viable option for nickel recovery from a low-grade ultramafic deposit. Given that magnesium solubility decreases as pH increases, a different bioleaching system, possibly one that operates under neutral or near-neutral pH conditions, may offer oxidation of sulfide minerals while limit the extent of magnesium dissolution. Bioleaching at neutral pH could also potentially lead to a more environmentally friendly and cost effective process.

1.2 Objectives

This study explores bioleaching under neutral pH environments as an alternative method for the initial nickel mobilization in a hydrometallurgical recovery process for a low-grade ultramafic nickel deposit. Batch-type bioleaching experiments were set up with both defined bacterial strain and indigenous cultures from the local environment to study oxidative nickel dissolution, and an investigation into the bacteria community study was performed using DNA sequencing and taxonomy assignment based on sequence similarity to obtain insights into the community structure and to identify the main players in the bioleaching process.

Objective 1: To evaluate bioleaching capabilities of a pure strain nitrate-reducing bacterium in catalyzing oxidative dissolution of mixed sulfides, a well-known denitrifying bacterium, Thiobacillus denitrificans, was grown and applied under bioleaching conditions with an ultramafic concentrate material containing 8.3 wt.% Ni and 9.6 wt.% Mg obtained from the “middlings” stream of a flotation circuit. 3

Objective 2: To examine the capabilities of mixed indigenous bacterial cultures in carrying out oxidative dissolution of the same material as Objective 1, a number of indigenous mixed bacterial cultures were collected from several mine-related local environments, and were enriched on the same ultramafic concentrate for studying nickel mobilization under nitrate-reducing conditions.

Objective 3: To evaluate the bacterial community and provide a basis for inferring correlations between bioleaching performance and the community structure, microbiology techniques were used to identify bacteria in active microcosms after a period of microcosm enrichment on the ultramafic concentrate. The techniques used include DNA extraction, pyrotag sequencing, and taxonomic identification against a large database of known bacteria.

1.3 Outline of Thesis

This document begins with an overview of the history of the Thompson deposit and characterization of the ultramafic mixed sulfide material, outlining potential processing challenges for conventional extractive metallurgical methods and foreseeable limitations of acidic bioleaching due to the ultramafic nature of this particular material (Chapter 2). Chapter 3 first briefly reviews some well-studied bioleaching systems under acidic pH regime, and then provides a more detailed literature review on known bacterial species that have been found to possess iron-oxidizing and sulfur-oxidizing abilities. Chapter 4 details all the experimental methodology and designs involved in answering the three objectives of this study. Chapter 5 contains results, analysis, and discussions on experimental findings, which is followed by key conclusions and recommendations for future work in Chapter 6.

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2 The Thompson Concentrate 2.1 Thompson Deposit History

Thompson Nickel Belt in Manitoba, Canada is a large ore body that includes 125 metric tons of ultramafic material at 0.55 wt.% Ni and 600 metric tons at 0.5-0.7 wt.% Ni, among which 300 metric tons are amenable for surface mining. Since its first discovery in the 1950s, the Thompson Nickel Belt has produced more than 4 billion pounds of nickel from several mines. [5]

2.2 Mineralogy

The ultramafic concentrate (UMFC) studied is a highly heterogeneous material with serpentine group minerals as the gangue phase and a mix of metal sulfides. The main nickel-containing mineral is pentlandite, where a small fraction of nickel is dissolved in pyrrhotite. Other sulfide minerals include pyrite and chalcopyrite. Although the sulfide content has been significantly increased through flotation, the magnesium containing serpentine minerals, hematite, and carbonates still make up approximately 65% of the concentrate.

Chemical assay by Aqua Regia digestion (Table 1), combined with mineralogical analysis including scanning electron microscopy (Figure 1, Figure 2, Figure 3), energy dispersive x-ray spectroscopy (EDX) (Table 2, Table 3), x-ray diffraction (Figure 1), and x-ray fluorescence are used to fully characterize the ultramafic concentrate. The results from each method are shown below individually, and the overall mineralogy of the material is displayed in Table 4.

Table 1 Chemical Assay of UMFC* (by Aqua Regia, in wt.% and standard deviation σ)

Ni Mg Fe S Al Ca Mn Co Cu Zn wt.% 8.23 9.47 25.02 18.47 0.16 0.69 0.08 0.24 0.35 0.07

σ 0.16 0.33 0.43 0.48 0.10 0.03 0.002 0.006 0.008 0.002

* Average of 3 replicate measurements and standard deviation.

Scanning electron microscopy (SEM) images of epoxy embedded polished mounts illustrate the various mineral phases and the range of size of particles of the concentrate (Figure 1). The 5 brightest particles correspond to the heaviest mean atomic weight minerals, which in this case are pentlandite ((Fe,Ni)9S8) and pyrrhotite (Fe(1-0.8)S). pyrite particles appear to be slightly darker than the brightest phases, but still very bright in color to closely resemble pentlandite and pyrrhotite. Point analysis using energy dispersive x-ray spectroscopy (EDX) is used to differentiate among pentlandite, pyrrhotite, and pyrite based on their atomic composition. The dark gray particles in the image correspond to Mg silicates, which posses the lowest mean atomic weights.

Mg silicates

Pyrite

Pentlandite/Pyrrhotite

Figure 1 Backscattered electron image of polished mount embedded in epoxy (200x)

6

Figure 2 Backscattered electron image with point analysis using EDX (500x)

Table 2 Atomic Composition of Selected Points in Figure 2 (atomic wt.%)

Point of Closest O Mg Si S Fe Ni Mineral interest formula 1 70.61 29.39 FeS pyrite 2 2 50.98 21.95 27.07 (Fe,Ni) S pentlandite 9 8 3 62.3 21.95 14.71 0.1 0.69 Mg silicate serpentine type

Figure 3: Backscattered electron image with point analysis using EDX (300x)

Table 3: Atomic composition of selected points in Figure 3 (atomic wt.%)

Point of Closest O Mg Si S Fe Ni Mineral interest formula 1 57.92 41.59 0.49 Fe S pyrrhotite (1-0.8) 2 61.25 22.6 14.94 0.18 0.73 Mg silicate serpentine type

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X-ray diffraction analysis on dried UMFC samples suggests the presence of pyrrhotite, pyrite, pentlandite, and possibly various forms of magnesium silicates. The spectrum shown in Figure 4 has a high level of noise and interference, which indicates the degree of heterogeneity of the material.

Pyrrhotite

Pyrite Pentlandite Magnesium iron (II) oxide Magnesium Aluminum Silicate

Figure 4: X-ray diffraction (XRD) of UMFC

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Table 4: Mineralogy of UMFC

Mass % Mineral Phases

18.0 Pentlandite (Fe,Ni)9S8

6.4 Pyrrhotite Fe(1-0.8)S

10.0 Pyrite FeS2

1.0 Chalcopyrite CuFeS2 Olivine/serpentine:

Antigorite (Mg,Fe)3Si2O5(OH)4

Clinochrysotile Mg3Si2O5(OH)4 55.3 Lizardite Mg3Si2O5(OH)4

Orthochrysotile Mg3Si2O5(OH)4

Parachrysotile (Mg,Fe)3Si2O5(OH)4

9.6 Hematite Fe2O3 100.3 Total

The ultramafic concentrate was a dark gray, fine, and sticky settled bed of material when arrived at the laboratory. When dried, the material is a powder of fine particles with a range of sizes. The average particle size is measured to be 45µm by a laser diffraction particle size analyzer with a d50 of 27.7µm and a d90 of 90.3µm. The size distribution in Figure 5 shows a tail on the left, indicating the presence of some extremely fine particles. The right hand side of the distribution shows the presence of some large particles with size up to 1µm; however, this is suspected to be a false measurement due to agglomeration of sulfide particles in water, as no such particles shows up in any of the SEM images. 9

UMFC Particle size distribution 7 6 5 4

3 % 2 1 0 0.01 0.1 1 10 100 1000 µm

Figure 5: Particle size distribution of UMFC

2.3 Processing Concerns

Ultramafic concentrate of this nature poses difficulties for extractive metal recovery using conventional pyrometallurgical and hydrometallurgical techniques. Although the sulfide mineral content has been significantly upgraded after flotation, as much as 55 wt.% of the concentrate is still gangue material, which is a collection of various hydrated Mg silicates. Firstly, the gangue material challenges the performance of smelting by reducing furnace life [1] and requiring a higher smelting temperature; secondly, serpentine group minerals and various carbonates in the gangue will consume large amounts of sulfuric acid during dissolution; and lastly, disposal of the depleted pregnant liquor after nickel removal containing dissolved magnesium is a significant logistic challenge. All of these difficulties will be reflected as increased costs and reduced economic attractiveness for processing through conventional routes.

Acidic heap bioleaching has been studied and practiced both on pilot and industrial scales for low-grade sulfide ores, such as the copper bioleaching heap at Talvivarra [4]. However, a typical acidic bioleaching process cannot avoid gangue dissolution in this material; therefore, the same difficulties associated with high acid requirements and expensive waste disposal for conventional methods still exist. R.A. Cameron [6] bioleached an ultramafic ore from the same ore body at elevated pH conditions ranging from pH 2 to pH 5 using an indigenous mixed culture of microorganisms that normally thrive under the acidic bioleaching conditions (pH 1.5-2.5). In the 10 most favorable scenario, which offered a %Ni:%Mg dissolution ratio of 6.65, 68% Ni dissolution was achieved at pH 5 and 30 °C in continuously stirred tank reactors for 35 days, and only 10% Mg dissolution was measured with a sulfuric acid consumption of 72 g/kg ore.

2.4 Incentives for the current study

To further reduce magnesium dissolution and acid requirements, this study explores bioleaching under near neutral pH (pH 6.5) conditions where nitrate is the ultimate oxidant.

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3 Literature Review on Biohydrometallurgy 3.1 Introduction

Biohydrometallurgy has become an increasingly attractive alternative for the mining industry for recovery of metal values from low-grade ores, as high-grade and easy-to-process deposits are becoming depleted worldwide. Nowadays, biohydrometallurgy is being employed extensively for recovery of copper, nickel, silver, and gold from sulfide ores; in particular, more than 15% of the world copper production comes from heap bioleaching [7].

The term “biohydrometallurgy” encompasses two different concepts: (a) bioleaching, which refers to dissolution of base metals from their host minerals, such as nickel dissolution from pentlandite ((Fe,Ni)9S8) when sulfur and iron are converted to soluble forms under the catalytic effects of microorganisms; (b) mineral biooxidation, which describes freeing or exposing precious metal units, such as gold, from sulfide minerals [7]

3.2 Acid Bioleaching

Research in biohydrometallurgy so far has largely focused on acidophilic bacteria that catalyze iron and/or sulfur oxidation when oxygen is the ultimate electron accepter. Often found at acid mine drainage (AMD) sites, these bacteria thrive under acidic conditions with pH values lower than 3 and derive their energy from sulfide minerals at exposed mine sites or tailings ponds.

The diversity of acidophilic iron- and/or sulfur-oxidizing bacteria, although thought to be low due to the general lack of nutrients at AMD sites has proven to be quite high. A range of mesophilic, moderately thermophilic, and extremely thermophilic bacteria have been found to be able to play a role in carrying out iron and/or sulfur oxidation [8].

Acidithiobacillus ferrooxidans (At. ferooxxidans), a mesophilic bioleaching microorganism was first identified and categorized under the genus Thiobacillus, is a colorless, rod-shaped, Gram- negative betaproteobacterium that utilizes a cytochrome oxidase as the terminal electron acceptor that enables it to metabolize metal ions such as Fe2+ [9]. In 2000, Kelly and Wood reclassified this acidophilic bioleaching bacterium and assigned it to the newly created genus of 12

Acidithiobacillus, a gamaproteobacterium [10]. Many other mesophilic and thermophilic bacteria have now been identified to form a consortium along with At. ferrooxidans to carry out iron and/or sulfur oxidation of sulfide minerals. These include, but are not limited to, Acidithiobacillus thiooxidans (At. thiooxxidans), Leptospirillum ferrooxidans (L. ferrooxidans),

Acidithiobacillus caldus (At. caldus), and Sulfobacillus thermosulfidooxidans [11].

The Talvivarra deposit in northern Finland is the first to have demonstrated commercial success of heap bioleaching in 2009 for nickel, copper, zinc, and cobalt. The demonstration-scale heap that operated from 2005 to 2006 bioleached 17000 tons of low-grade multi-metal black schist sulfide deposit containing 0.27% Ni, 0.56% Zn, 0.14% Cu, and 0.02% Co with 50% of SiO2 as the gangue material. About 70% of the nickel value resides in pentlandite, 20% as pyrrhotite, and the rest in pyrite. The heap was built with crushed ore particles stacking 8m high, 30m wide, and 60m long, operated at pH 1.5 with conditioned bacterial consortium containing At. ferrooxidans, At. caldus, L. ferrooxidans, and S. thermosulfodooxidans. Different strains of bacteria dominated the consortium at different periods due to temperature and other operating conditions. After 400 days of metal recovery, Ni extraction reached 80%. Sulfuric acid consumption overall averaged to 16 kg/t. [12]

The Talvivaara demonstration plant has shown that bioheapleaching is a feasible process for metal recovery from low-grade sulfide deposit of similar constitution, and offered valuable operational control knowledge regarding temperature gradient in heap and shifting of dominant species in microbial community due to different operating conditions.

3.3 Bioleaching at Elevated pH

Among the low-grade nickel sulfide resources that remain available for future exploration, a major type of deposit is of ultramafic nature, meaning that the gangue material has high magnesium content. Magnesium silicates and carbonates in the gangue of this type of deposit make acidic bioleaching at pH values 1.5-2.5 not feasible due to high acid requirement and challenges in the logistic and economic aspects of waste disposal.

Cameron [6] studied an ultramafic sulfide nickel deposit with 0.3% Ni, and 21% Mg. To reduce magnesium mobilization, Cameron conducted bioleaching experiments in batch reactors to study 13 the effect of pH (2 to 6) on nickel and magnesium extraction. The results showed that at pH 5, the growth of microbial consortium was able to survive at elevated pH conditions, and the ratio of %Ni extraction to %Mg extraction was the highest at 6.65. The preferential leaching of nickel compared to magnesium was attributed to nickel extraction being relatively insensitive to reduced acidity up to pH 5, whereas magnesium extraction dropped significantly from 70% to 20% when the acidity was decreased from pH 2 to pH 5.

The origin of the Cameron’s bacterial consortium is from mining-related locations in Sudbury, Ontario, and the sediment samples were enriched on ferrous iron and thiosulfate media for iron- and sulfur-oxidizing bacteria under pH 2.2. Therefore, it is not surprising that molecular biology investigation of the bacterial consortium revealed the prevailing species to be At. ferrooxidans, At. thiooxidans, L. ferrooxidans, and at least one species of Acidiphilium. The bacteria consortium present in Cameron’s work closely resembles those found in bioleaching heaps at commercial practices such as Talvivaara with an operating pH range of 1.5 to 3 [12].

Ni extraction over 35 days in stirred tank reactors showed satisfactory nickel extraction for pH 2 to 5, with %Ni extracted ranging from 70% to 90%. However at pH 6, the final %Ni extraction was significantly lower (~30%) than that of abiotic leaching experiments at pH 3 and pH 5, even though the presence of bacteria was initially beneficial. The author attributed this finding to reduced mass transfer rate at high pH condition due to diffusion limited kinetic behavior after the first two weeks of bioleaching.

3.4 Nitrate-dependent Bioleaching System

3.4.1 Thiobacillus denitrificans

Denitrification is the biochemical reduction of nitrate to nitrogen gas (N2). Weissenberg [13] [14] was the first to propose that under the influence of certain microorganisms, nitrate can act as the ultimate electron accepter, just as oxygen does under aerobic oxidation, when coupled with oxidation of organic substances. Ishaque and Aleem [15] identified nitrite, nitric oxide, and nitrous oxide as denitrification intermediates, under anaerobic conditions by Thiobacillus denitrificans (T. denitrificans). Since then, T. denitrificans has been the one of the most well- studied chemolithotrophic denitrifying bacteria. 14

3.4.2 S oxidation by T. denitrificans

T. denitrificans can derive its energy from coupling the reduction of nitrate to the oxidation of Fe(II) and reduced sulfur compounds. Schedel [16] suggested sulfide being a possible substrate for T. denitrificans based on high specific sulfite reductase activities found in T. denitrificans extracts. In 1980, the same author deciphered the metabolic pathway of anaerobic oxidation of thiosulfate by T. denitrificans. Thiosulfate is split to produce sulfite and elemental sulfur, which is then transiently accumulated in the cells in a reactive form until sulfite is fully oxidized to sulfate [17]. Dalsgaard and Bak [18] in a study of acetylene inhibition technique for denitrification quantification demonstrated anaerobic oxidation of soluble sulfide (H2S) to sulfate with N2O as the ultimate electron acceptor.

3.4.3 Fe oxidation by T. denitrificans

Nitrate-dependent Thiobacillus-like cultures are collected and enriched from a wide range of subsurface environments for their ability to catalyze Fe(II) oxidation. From town ditches and brackish water lagoon in Germany, T. denitrificans was found capable of coupling anaerobic oxidation of ferrous iron, both from soluble salts such as FeSO4 and solid sulfide “black FeS”, to denitrification [19]. The same enrichment culture was able to catalyze iron oxidation from biogenic solid Fe(II) compounds, such as chemically precipitated FeCO3, and Fe(III) compounds including goethite, hydrous ferric oxides and Fe(III) oxide-rich subsoils [20]. Microbial nitrate- dependent oxidation of pyrite (FeS2) was not supported by enrichment assays inoculated by marine sediments [21], or soil enrichment cultures from a high iron sulfide mineral environment in the south of the Netherlands containing Thiobacillus-like bacteria [22].

Incubation of sediments collected from various depths from an agricultural area with potato production in Denmark showed increased microbially catalyzed denitrification in the presence of pyrite under anoxic conditions [23]. Torrento et al. [24] studied the potential of pyrite addition to stimulate activity of denitrifying bacteria for treatment of nitrate-contaminated groundwater and sediment. T. denitrificans demonstrated continuous and complete nitrate removal in flow-through experiments driven by pyrite addition; moreover, nitrate reduction was dependent on pyrite grain 15 size, nitrate loading and pH. T. denitrificans is also found to carry out anaerobic nitrate- dependent oxidation of pyrite nanoparticles [25]. The reaction rates observed with pyrite nanoparticles are orders of magnitude higher than those of larger particles. Molecular studies of nitrate-dependent Fe(II) oxidation have been carried out recently by Beller et. al [26].

3.4.4 Nitrite reduction inhibition in T. denitrificans.

The complete denitrification of nitrate to nitrogen gas occurs in four enzyme-catalyzed steps: - - NO3 à NO2 à NO à N2O à N2. The T. denitrificans genome includes all the necessary genes for the enzymes involved in denitrification [27]. Transient nitrite accumulation is observed in many denitrification studies. Torrento et al. concluded that based on transient behavior of nitrate and nitrite concentrations in both batch and flow-through experiments, nitrite accumulation is due to competition between nitrate and nitrite reductases for the available electron donor pyrite [24]. As long as nitrate is still available, nitrite reductase appears to be inhibited, resulting in nitrite accumulation.

3.4.5 Attachment to solid surface

T. denitrificans has a polar flagellum that allows it to be motile. However, attachment on pyrite surfaces occurs to a limited extent; about 0.2% of total cells were estimated to grow attached to pyrite mineral surfaces with a total 2% of the pyrite surfaces being covered [28].

3.4.6 Other nitrate-dependent iron- and sulfur-oxidizing bacteria

The range of denitrifying bacteria capable of iron and sulfur oxidation is diverse. A groundwater denitrification study with pyrite addition using unsterilized core fragments resulted in mixed bacterial consortia with wild strains of both autotrophic and heterotrophic denitrifying bacteria, including Xanthomonadaceae, Sphingomonas, Chitinophagaceae, and Methylophilaceae [29]. Although the study showed growth and diversity of the consortia with pyrite addition, further research is required to confirm the roles each member plays in the denitrification process. 16

Gallionella ferruginea, a betaproteobacterium, is an iron-oxidizing chemolithotroph that lives under low-oxygen conditions. It is found in many types of habitats, such as freshwater ferruginous mineral springs, brackish waters, marine environments, and soil associated with iron [30]. It has a single elongated stalk made from mineralized fibrils that tend to form biofilms. Multiple species of the genus have been shown to grow in fully aerated environments catalyzing iron oxidation [31].

Chromatiales is an order of gram-negative anoxygenic phototrophic bacteria, collectively known as purple sulfur bacteria. They are phototrophic and are capable to performing photosynthesis under anoxic conditions without oxygen production [32]. 17

4 Methodology 4.1 Ultramafic concentrate

4.1.1 Material Preparation

The ultramafic concentrate (abbreviated as UMFC) material used in this work was obtained from Vale’s Base Metal Research Centre in March of 2012. Drill cores from the “Pipe exposure” at Thompson nickel belt were subjected to crushing, beneficiation, and flotation to produce a “middlings” stream containing approximately 8 wt.% Ni and 10 wt.% Mg. The “middlings” product was then passed through a few stages of cleaning columns for asbestos removal to ensure the safe handling of the material. The overflow and the tails from the last stage of cleaning were allowed to settle into two dense beds, which were then decanted, combined, and homogenized to produce three identical pails of 20 L ultramafic concentrate with approximately 45 vol.% solids.

To homogenize the ultramafic concentrate, a heavy-duty impeller driven by a hand drill was submerged into the pail, at a low speed first, to gradually re-suspend the dense material. The hand drill was then kept running on a higher speed with the impeller as close to the bottom of the pail as possible to produce a constant slurry, which was subsequently vacuum filtered into nineteen 1- kg UMFC cakes with P8-creped qualitative filter paper (Fisherbrand®).

4.1.2 UMFC Preservation

The cakes are kept in double sealed bags in -20 °C freezer to minimize any oxidation of the sulfides. The average moisture content of the cakes was determined to be approximately 18 wt.%. The moisture content is determined by keeping a pre-weighed wet sample in a vacuum chamber (-750 mm Hg) with desiccant for 2 days then re-measured again. The moisture content of each cake used for any experiment was determined individually at the same time as the experiment was set up.

18

4.1.3 Acid pre-leach

To better elucidate the second objective of this study - microbial catalyzed oxidation of nickel containing sulfide minerals in UMFC, it is desirable to conduct a set of experiments with acid- leached UMFC where any acid soluble materials, such as carbonates and MgO hydrates, are removed ahead of time. A heated acid pre-leach was performed at 80 °C where 1.4 L of 2N sulfuric acid was used for 169.4 g of wet UMFC (18.02 wt.% moisture). The leaching process took 46 hours in total in a jacketed reactor equipped with a mechanical stirrer to keep the UMFC well suspended (Figure 6).

100%

80% Ni Fe 60% Mg

40% %extracted

20%

0% 0 1 2 3 4 5 6 7 8 46 hours

Figure 6: Acid pre-leach, % leached

4.2 Analytical techniques

4.2.1 Solid material characterization

4.2.1.1 Chemical assay (Aqua Regia)

Characterization of fresh UMFC, acid-washed UMFC (Section 4.1.3), and leach residues includes determining elemental composition by acid digestion with aqua regia. The aqua regia solution (70 wt.% HNO3 : 35 wt.% HCl = 1:3) and the target material are combined and heated in a sealed vessel with microwave in a microwave digester (Ethos EZ Microwave Digestion System) following a predetermined temperature profile. Digestion starts with rapid heating to 200 19

°C in 10 minutes, followed by 30 minutes digestion, before cooling down to 40 °C. For each digestion, approximately 0.5 g of material is dissolved in 15mL aqua regia solution to make sure that the acid is in excess.

The digestion product is usually a dark brownish green solution with a small amount of gray sandy material (SiO2). The mixture is filtered with pre-weighed Q5 quantitative filter paper (Fisherbrand®) on which the solids are dried in oven at 80 °C. The filtrated is diluted with 5 wt.%

HNO3 to 100 mL in volumetric flask. Elemental composition of the filtrate is measured using Inductive Coupled Plasma equipped with an Optical Emission Spectrophotometer (ICP-OES). The operating parameters and techniques used for elemental analysis for liquid samples are detailed in Section 4.2.2.1.

4.2.1.2 X-ray Diffraction (XRD)

UMFC samples were characterized for predominate crystalline mineral phases by semi- quantitative x-ray diffraction (XRD) with signal detection covering 15° to 60° theta range. Only the best matching mineral phases were accepted and used as supporting information for XRF analysis.

4.2.1.3 Scanning electron microscopy (SEM) & Energy-dispersive x-ray (EDX)

UMFC particles cross-sections were visualized using a scanning electron microscope (SEM) on polished epoxy mounts. EDX point analysis or element mapping enabled characterization of specific mineral phases to the atomic composition level. Each solid samples was gently dispersed in epoxy resin immediately after hardener addition and degassed in a vacuum chamber for 5 minutes at -25 mm Hg; the resin was allowed to cure for at least 24 hours before polishing and characterization. Backscattered electron (BSE) images were able to distinguish various mineral phases by different gray scale. EDX point analysis of atomic composition further solidified the identity of present minerals.

20

4.2.1.4 X-ray Fluorescence (XRF)

With all the information gathered from chemical assay by acid digestion, XRD, and SEM/EDX, it is possible to (semi)-quantify the amount of each mineral phase by X-ray fluorescence. In a standard quantitative XRF analysis, the interferences of signals from various elements present in the material are compensated by mathematical corrections. However, the correction methods applied are dependent on the material background, making analysis for highly heterogeneous mixtures only a semi-quantitative technique. Therefore, the mineralogy results for UMFC and residues are often “best estimates” of the reality.

4.2.2 Aqueous Samples

4.2.2.1 Elemental Composition Analysis (ICP-OES)

Aqueous samples are diluted using an auto-diluter (Hamilton Microlab® 500) to concentrations within the calibrated range of an Inductively Coupled Plasma equipped with optical emission spectroscopy (ICP-OES, Agilent Technologies 700). Two sets of multi-element standards, including Ni, Mg, Fe, S, and a list of other elements at specified concentrations were used for equipment baseline and sample analysis calibration. Preprogrammed methods are routinely applied to check for alignments, baseline to ensure the normal startup and operation of the machine. For every analysis, the Argon plasma (1.2 kV) is allowed 15 minutes to stabilize prior to the calibration standards, followed by samples. The calibration is only accepted when the %error for a specific element is below 10% and the r2 value is greater than 0.9999.

4.2.2.2 Anion Analysis with Ion Chromatography (IC)

The concentrations of nitrate, nitrite, sulfate, and phosphate were routinely monitored by diluting (1:50) a small volume of filtered liquid sample with deionized (DI) water and injecting the sample into an ion chromatography (IC, Dionex Integrated Regenerative Ion Chromatography System 2100) equipped with an AS18 4×250 mm anion column and electrical conductivity detector. The analysis was carried out using 23 mM KOH eluent with baseline suppression with 21

57 mA at 30 °C. The calibration of the IC was performed using house standards at the following composition in Table 5.

Table 5: Ion Chromatography calibration standards

Anion Low (mM) Medium (mM) High (mM)

Cl- 0.01 0.1 0.5

- NO2 0.02 0.2 1

- NO3 0.02 0.2 1

2- SO4 0.04 0.4 2

3- PO4 0.01 0.1 0.5

4.3 Experiments with Defined Culture – Thiobacillus denitrificans (ATCC 25259)

Thiobacillus denitrificans (T. denitrificans) is a well-studied facultative chemolithoautotroph that is capable of iron and sulfur oxidation when nitrate is the ultimate electron acceptor. As a first step into this study, a defined strain of Thiobacillus denitrificans ATCC 25259 from the American Type Culture Collection (ATCC) was grown and tested for bioleaching of UMFC.

4.3.1 Revitalization, Growth, and Maintenance

A freeze-dried pellet containing the bacteria strain was revitalized according to manufacture’s procedure. The initial culture turned cloudy after 2 weeks, suggesting successful growth, and was transferred to a 160-mL serum bottle sealed with a butyl rubber stopper and aluminum crimp top in an anaerobic glovebox with an 80%N2-10%H2-10%CO2 atmosphere. The growth medium used contained: Na2S2O3Ÿ5H2O (20 mM), KNO3 (20 mM), KH2PO4 (14.7 mM), NH4Cl (18.7 mM), MgSO4Ÿ7H2O (3.25 mM), CaCl2Ÿ2H2O (0.05 mM), and 2.52 g of NaHCO3 (30 mM) as buffer [33], and anaerobic sterile trace element and selenite-tungstate solutions prepared as described by Widdel and Bak [34], and anaerobic sterile multivitamin solutions used in the lab (Appendix A: Edlab Multi Vitamins Stock Solution (MM7)) in 1 L DI water. The procedure for 22 medium preparation follows [33], and the complete growth medium composition is in Appendix B: Thiobacillus denitrificans Growth Medium adapted from .

T. denitrificans ATCC 25259 was cultured without pH control and aseptically transferred by inoculating 5 mL of a stationary phase culture to 100 mL fresh medium in a sterilized serum bottle. Concentrations of thiosulfate, sulfate, nitrate, and nitrite were monitored by IC every 2 to 3 days for one transfer; it was determined that a 7-day interval was appropriate for each subsequent transfer. Growth under pH 6.0 and pH 7.5 were also attempted with pH adjustment using 2N HCl and 2N NaOH when a sample was taken.

4.3.2 Experimental Setup

Batch leaching experiments were set up in 160-mL serum bottles with anaerobic headspace sealed using a butyl rubber stopper and aluminum crimp as shown below in Figure 7.

Figure 7: Sealed anaerobic leaching setup

Three leaching experiments with 1 wt.%, 4 wt.%, and 20 wt.% solid loadings of UMFC were carried out in parallel. For each experiment, 1 g, 4 g, or 20 g of wet UMFC was added to 100mL leaching medium (composition detailed in Appendix C), inoculated with a T. denitrificans cell pellet centrifuged from a 100mL maintenance culture that had been growing on growth medium for 7 days. Duplicates and a sterile control with no bacteria were also created for each experiment. 23

Table 6: Leaching experiments with T. denitrificans

Experiment Medium e- donor e- acceptor Inoculum Replicates Comments

T. denitrificans Maintenance Growth Na S O (20 KNO Keep active 2 2 3 3 N/A culture medium mM) (20mM) 5 vol.% from at all times previous transfer

1 wt.% solids 1 g UMFC T. denitrificans 100 vol.% Working 4 wt.% solids Leaching 4 g UMFC KNO3 maintenance volume: 2 medium (5mM) culture washed 100mL 20 wt.% solids 20 g UMFC

4.3.3 Sample Analysis

Concentrations of nitrate, nitrite, sulfate, as well as metal ions including Ni2+, Mg2+, and Fe2+/Fe3+ were determined by taking liquid samples from leaching experiments at appropriate intervals. Samples were collected using a 1mL sterile syringe (Becton Dickenson & Co.) and a sterile 22-gauge needle through the rubber stopper in an anaerobic glovebox. A 0.7 mL liquid sample was filtered with a 0.2 µm syringe filter (Pall Co. Acrodisc® Syringe Filters) and brought out of the glovebox, immediately diluted with DI water 1:50 times with pipettes for anion analysis with IC, and with 5 wt.% HNO3 for ICP elemental analysis.

4.4 Experiments with Indigenous Cultures – First Enrichment

Indigenous environmental samples collected from mining related locations in Thompson, Manitoba were enriched on UMFC under both aerobic and denitrifying conditions in this microcosm study. Four series of microcosms were created; each series contained environmental sample(s) from a different site. A total of 48 microcosms were started in August of 2012 and monitored for 450 days.

24

4.4.1 Collection of Indigenous Sediment Samples from Thompson

Environmental water and sediment samples were collected from surface waters around the Thompson refinery that may have been exposed to mining related activities, such as tailings ponds and open pit mines. Samples from 6 locations were collected (with the help of our industrial partner) in July 2012, with 1-liter sediment/water slurry from each location. The samples were kept in airtight plastic containers and stored at 4 °C during transportation to the university. The sample collection information table in Appendix D provides more background on the location and conditions of sample origin.

4.4.2 First Enrichment – Experimental Design

Samples from two tailings ponds (Cell B, Area 1), and samples from inactive mine exposures (Station B, Birch Tree) were combined to reduce the total number of environmental samples to four unique indigenous consortia (also referred to as “series” or “site”). For each series, 3 replicates were created with anaerobic headspace where nitrate was added periodically in 5 mM doses, 3 replicates were created with atmospheric headspace for enrichment under aerobic conditions, 3 sterile controls were created to examine abiotic denitrification at the presence of UMFC, and 3 no oxidant (or no electron acceptor) controls were created without oxygen nor nitrate to determine background activities of the environmental samples. With 12 microcosms for each series, a total of 48 microcosms were maintained and monitored for the first enrichment on UMFC, illustrated in Table 7 below.

Table 7: Microcosm treatment table for each series

Treatment Bottle No. Replicates Volume e- donor e- acceptor Headspace Sterilization Sterile 1-3 3 5mM KNO Anaerobic Gamma ray control 3 No acceptor 4-6 3 --- Anaerobic 5g 100 mL UMFC Aerobic 7-9 3 air Aerobic ---

Nitrate 10-12 3 5mM KNO3 Anaerobic

Number of Treatments: 4 Number of Microcosms: 12

Number of Total Microcosms: 48 25

4.4.3 Microcosm Setup

Sterile control, No-acceptor control, and Nitrate-amended bottles were set up in an anaerobic glovebag purged with N2. A glovebag was used to create a temporary anaerobic environment to avoid contamination to other experiments and tampering with the anoxic atmosphere in glovebox. Glassware, plastic containers, and tools were brought into the glovebag the day before to degas overnight; sediment/water samples from Thompson were stored at 4 °C until introduced into the glovebag. The step-by-step protocol followed for setting up the microcosms is presented in Appendix E. Aerobic microcosms were set up in normal atmosphere.

Table 8: First enrichment startup compositions (*Approximate volumes due to presence of sediment)

Series/Sites Indigenous sediment/water* Medium Total volume*

Tails 50 mL Cell B + 50 mL Area 1 ---

Dam 50 mL Dam B 50 mL 100 mL Exposures 50 mL Birch tree + 50 mL Station B ---

TSRA 50 mL TSRA 50 mL

5 mL of sterile 1M KNO3 stock was added to each nitrate and sterile control microcosms for all series at the same time when the bottles were setup. As an attempt to overcome the stress of anaerobic transfer, 0.2 mL D-glucose from a sterile 0.1M stock was added to boost growth of bacteria in these bottles. The same was done for no acceptor controls. All of the microcosms were sampled twice on the 7th day and the 11th day as time zero, and incubated for 70 days until regular weekly nitrate addition and sampling took place.

4.4.4 Sample Analysis

Progress of leaching and growth of bacteria consortia were monitored on a weekly basis by analyzing an aqueous sample from the supernatant phase. Elemental metal concentrations, including Ni, Mg, and Fe, were measured by ICP-OES, and substrate and product concentrations, such as nitrate and sulfate by IC. The pH was measured and adjusted with HCl or NaOH to always remain within pH 6.5±0.1 at the same time as sampling took place. DNA was extracted 26 from selected microcosms for 16S rRNA gene sequencing and microbial community analysis. Section 4.6 Microbiology Analysis is dedicated to this topic.

Nitrate concentration was monitored by IC for every aqueous sample (and just before DNA extraction), or occasionally measured by nitrate strip test (EMD Millipore Co.) for a quick estimate. To avoid nitrite buildup, KNO3 was added in small doses to maintain between 0.5mM to 5mM (except a few scenarios due to errors).

4.5 Second Enrichment

To obtain a better mass balance of sulfide mineral oxidation and track the fate of Ni mobilized from pentlandite, a second round of enrichment using the existing microbial consortia were created with acid-leached UMFC. Microbial consortia from selected series: Dam (nitrate amended), Exposures (aerobic and nitrate amended), and TSRA (aerobic and nitrate amended), were transferred to new microcosms where acid-leached UMFC was the electron donor in a modified leaching medium (Appendix F).

4.5.1 Experimental Design

For each treatment selected for further enrichment, the microbial consortia from triplicate microcosms were combined and divided to form new microcosms, as illustrated in the layout below. For example, Dam 10, 11, and 12 are triplicates of denitrifying treatment from the Dam series. The microbial consortia from these microcosms were mixed and evenly distributed among No donor control and Nitrate active triplicates. 27

Dam 10 Dam 11 Dam 12

Master 90 mL

No Donor Control Active Sterile Control 15 mL mother culture 15 mL mother culture 50 mL medium 35 mL medium 35 mL medium Acid-leached UMFC 3 g Maintain 0.5 – 5 mM nitrate Acid-leached UMFC 3 g Maintain 0.5 – 5 mM nitrate Or aerobic headspace Maintain 0.5 – 5 mM nitrate Or aerobic headspace Or aerobic headspace

Figure 8: Second enrichment transfer layout

Treatment tables for aerobic and denitrifying microcosms are shown below (Table 9 and Table 10). 5 series of microcosms, including 18 aerobic microcosms and 27 denitrifying microcosms were created for the second enrichment with acid-leached UMFC.

Table 9: Aerobic treatments for second enrichment (Exposure and TSRA) Treatment Bottle No. Replicates e- donor e- acceptor Inoculum Sterilization 3 g Acid-leached Sterile control 1-3 3 --- Gamma ray UMFC No donor 4-6 3 --- air 20 vol.% 3 g Acid-leached mother --- Aerobic active 7-9 3 UMFC culture

Number of Treatments: 3 Number of Microcosms: 9

Number of total aerobic microcosms: 18

28

Table 10: Denitrifying treatments for second enrichment (Dam, Exposures, and TSRA) Treatment Bottle No. Replicates e- donor e- acceptor Inoculum Sterilization 3 g Acid-leached Sterile control 1-3 3 --- Gamma ray UMFC KNO No donor 4-6 3 --- 3 20 vol.% 5 mM 3 g Acid-leached mother --- Nitrate active 7-9 3 UMFC culture

Number of Treatments: 3 Number of Microcosms: 9

Number of total denitrifying microcosms: 27

4.5.2 Microcosm Setup

One of the major incentives for conducting the second enrichment was to allow better mineralogy characterization of leach residue and hence obtaining a clearer mass balance for Ni. Therefore, supernatants from the first enrichment were centrifuged and only the cell pellets were transferred to start the new bottles.

In the glovebox, 30 mL of supernatant from each triplicate microcosm was extracted using a 50mL sterile syringe and combined in a centrifuge bottle with O-ring fitted screw cap and sealed with anaerobic tape. The bottle was brought out of the glovebox and centrifuged for 20-40 minutes at 5000 g until a pellet formed on the bottom. The supernatant was then discarded in the glovebox and the pellet resuspended with equal volume of Leaching medium 2. 15 mL of culture and 35 mL of extra medium were transferred to each 120-mL sterilized serum bottle, along with ~3 g of acid-leached UMFC. The serum bottle was capped with butyl rubber stopper and sealed with an aluminum crimp top.

th The first dose of 5 mM KNO3 was added to denitrifying bottles on the 15 day after the beginning of the enrichment. Aerobic bottles were kept open to the atmosphere with a needle attached to a syringe filter through the rubber stoppers.

4.5.3 Sample Analysis

Sampling and pH adjustments were performed in the same manner as in the first enrichment. The pH was adjusted using 2 N NaOH and 2 N HCl solutions and maintained around pH 6.5; liquid 29 samples were collected every 2 weeks for aqueous Ni and Mg levels, as well as sulfate production; nitrate concentrations were kept between the 0.5 mM and 5 mM range to avoid accumulation of nitrite.

4.6 Microbiology Investigation

To investigate if enrichment of any organisms occurred over the first 220 days of the study, variable regions (V6-V8) of 16S rDNA of all (but one) microcosms was sequenced to identify the organisms existing in these communities. This section explains (a) the microbiology techniques used for DNA extraction, purification, and 16S rDNA amplification; (b) 16S sequence data analysis and taxonomic assignment; and (c) phylogenetic inference by comparing unknown sequences to cultured isolates or environmental sequences found in the literature.

4.6.1 DNA Extraction and Isolation

In an anaerobic glovebox, a denitrifying culture was shaken to keep the sediment suspended while a 10-mL culture and sediment mixture was extracted using a 10-mL sterile syringe (Becton Dickenson & Co.). The microcosm was actively coupling denitrification with sulfur oxidation, as approximately 1/2 of KNO3 fed was consumed and sulfate evolution was continuing at the time DNA was extracted. The extracted volume was transferred to a 15-mL sterile polypropylene tube (BD Falcon™) capped with a screw lid and sealed with anaerobic tape. The tube was brought out of the glovebox and centrifuged at 3600 rpm and 4 °C for 30 minutes (JE Avanti®, Beckman Coulter). The supernatant was removed and discarded with a pipette without disturbing the pellet and all material remaining in the tube was transferred to a PowerSoil® DNA Isolation Kit PowerBead Tube (Mo Bio Laboratories, Inc., Solana Beach, CA). The protocol provided by the manufacturer was followed to lyse cells and purify extracted DNA. Final DNA product was eluted using 50 µl C6 solution and stored at -20 °C. The concentration of purified DNA was checked with an UV spectrophotometer (NanoDrop® ND-1000).

30

4.6.2 16S rRNA Gene Amplification and Purification

16S rDNA was amplified from purified DNA by PCR using forward adapter (5’-CTA TGC GCC TTG CCA GCC CGC TCAG-3’) + forward primer 926f (5’-AAA CTY AAA KGA ATT GAC GG-3’), and reverse adapter (5’-CGT ATC GCC TCC CTG CGC CAT CAG-3’) + barcode + reverse primer 1392r (5’-ACG GGC GGT GTG TRC-3’). A unidirectional 16S sequencing method (pyrotag sequencing) was used, which performs sequencing of multiple samples in one batch simultaneously. Therefore, a unique barcode (8 base pair) was assigned and attached to DNA amplicons of a particular microcosm via PCR, therefore allowing grouping of sequences in downstream data analysis. 100 µL PCR mixtures included the following reagents listed in Table 11. The selective amplification of 16S rDNA during PCR followed these steps: (1) initial denaturation at 95 °C for 3 minutes, (2) denaturation at 95 °C for 30 seconds, (3) primer annealing at 54 °C for 45 seconds, (4) chain extension at 72 °C for 1.5 minutes, and 25 cycles of steps (2) to (4), and followed by a final extension at 72 °C for 10 minutes. An MJ Research PTC- 200 PCR machine was used in this study.

Table 11: PCR reagents

Reagent Volume Concentration Amount 2x PCR master mix (Fermentas): As 50 µL --- dNTP manufactured Taq polymerase Forward primer 2 µL 200 nM 0.4 pmol Reverse primer 2 µL 200 nM 0.4 pmol

DNA template 4 µL 12 ~ 113 ng/µL 48 ~ 452 ng

UV treated ddH2O 42 µL ------Total 100 µL ------

The PCR products were separated on a 2% agarose gel containing SYBR® Safe DNA Gel Stain for 30 minutes at 120 V, and visualized under UV light in GelDoc chamber. PCR products that showed a strong band at the expected length were further purified using Fermentas GeneJET® PCR Purification kit. Concentration of purified PCR products was determined by image analysis of agarose gel images using 100 bp (base pair) ladder (GeneRuler™, Fermentas) of known 31

concentrations as calibration. Purified PCR products were kept in ddH2O at -20°C until shipment to Genome Quebec for pyrotag sequencing.

4.6.3 16S Data Processing

Pyrotag sequencing gave two output files: a fasta file that contained all raw reads and a second file with quality control scores for each read. Quantitative Insights Into Microbial Ecology (or Qiime) software package was used to analyze sequencing results. Using the Qiime platform, raw reads were first de-multiplexed as follows: (1) reads were grouped according to source microcosms, (2) reads with low quality score (<25 out of 40) and shorter than 220 bp were discarded, (3) reads with mismatches in primer sequence and 8 or more homopolymers were removed, and (4) adapter sequence and primer sequence were chopped from raw reads. Sequences that passed the above quality control qualifiers were assigned to operational taxonomic units (OTUs) based on 97% sequence similarity. One representative sequence was randomly selected from each OTU and used for taxonomic identification and phylogenetic alignment using the RDP classifier against the Greengenes database.

10000 9000 8000 7000 6000 5000 4000 3000 2000

Sequences/sample 1000 0

samples

Figure 9: Number of sequences used for taxonomic and phylogenetic analysis per sample 32

60000

50000

40000

30000

20000

No. of sequences of No. 10000

0 220 260 300 340 380 420 460 500 540 580 620 660 sequence length (bp) Figure 10: Sequence length distribution

The majority of the sequences used for further processing center around 460-520 bp long. The amount of variability is expected for V6-V8 regions of 16S rDNA.

4.6.4 Phylogenetic Analysis

Representative sequences (with lengths between 440 bp to 473 bp) from OTUs that make up more than 2% of each consortium were entered into the NCBI GenBank BLASTn suite search engine (http://blast.ncbi.nlm.nih.gov/Blast) to obtain most similar isolates and most similar sequences. Other relevant identified bacteria species found on the SILVA rRNA database’s All- Species Living Tree (LTPs111 release), along with NCBI blast results and OTUs >2% were used to produce a phylogenetic tree illustrating relative distances and lineage between identified species and major OTUs.

33

5 Results and Discussion 5.1 Experiments with T. denitrificans ATCC 25259

5.1.1 Growth of ATCC 25259

Strain ATCC 25259 was revitalized from freeze dried pellet in the lab on growth medium specified in Section 4.3.1, where thiosulfate is the sole electron donor and nitrate is the sole electron accepter. Assuming that sulfate is the stable sulfur product and dinitrogen gas is the final form of nitrogen, the theoretical balanced redox reaction should follow Equation 1.

!! ! !! ! !!!!! + !"!! + !!! → !"#!! + !!! + !! Equation 1

The sulfate concentration measured in one growth test matched stoichiometrically to thiosulfate consumption over the whole course of growth. From Day 0 to Day 7, ~11 mM of thiosulfate was consumed; this corresponded to an increase of 22 mM of sulfate (thiosulfate:sulfate = 5:10). The same adherence to stoichiometry is demonstrated by thiosulfate to nitrate ratio; over the same period of time, a decrease of 16 mM nitrate corresponded to a 20 mM increase in sulfate concentration (nitrate:sulfate = 8:10).

Growth on thiosulfate 30 NO2(-) NO3(-) SO4(2-) 20 S2O3(2-) mM

10

0 0 2 4 6 8 10 12 day

Figure 11: Substrate consumption for ATCC 25259 growth on thiosulfate (batch)

34

Nitrite was transiently measured over the first few days of growth until Day 5 when nitrite concentration decreased to below the detection limit. No inhibition to denitrification was observed as nitrate continued to decrease and sulfate continued to increase at steady rates.

Positive pressure in the headspace of serum bottles (observed during sampling) and increasing turbidity were direct evidence of nitrogen gas production and cell growth that indicated successful growth.

5.1.2 Bioleaching of UMFC with ATCC 25259

To test if pure strain T. denitrificans can carry out oxidative bioleaching of UMFC and provide a benchmark for indigenous culture bioleaching under denitrifying conditions, cultures of ATCC 25259 were amended with UMFC in leaching medium specified in Section 4.3.2 without any sulfur source except sulfide minerals in UMFC. As nitrite accumulation during groundwater denitrification with pyrite was previously documented [24] in the presence of excessive nitrate, nitrate was added in small doses of KNO3 to the batch experiments to not exceed 5 mM.

For each test, 1 g, 4 g, or 20 g of wet UMFC was added to a serum bottle with 100 mL leaching medium and an anaerobic headspace. The UMFC-medium slurry was kept suspended by securing the bottle to an orbital shaker at 160 rpm as an attempt to avoid mass transfer limitations. Nitrate was added to the bottles in doses, when the concentration dropped to below 0.5 mM.

The experiment ran for 52 days and was terminated due to the lack of denitrifying activity. The results (as shown in Figure 12 below) showed no significant difference between the active tests and the controls at all three levels of solid loading. An initial decrease in nitrate concentration was observed in all cases, with the magnitude of decrease positively correlated with high solid loading. The 20% solid loading test was able to consume the first dose of nitrate but denitrification stopped after the second 5 mM dose was added. 1% and 4% solid loading tests did not consume more than 2 mM KNO3. Nitrite temporarily accumulated in all active tests.

By the end of the experiment, it became apparent that nitrate reduction ceased after approximately 15 days; the microorganisms were suspected to have gone into a resting state due to absence of easily accessible electron donor or low tolerance toward high Mg levels brought 35 upon by MgO contents in the UMFC. A small volume from each level of solid loading was inoculated into growth medium with thiosulfate; growth was observed and denitrification resumed.

1 wt.% UMFC solids 4 wt.% UMFC solids 4 4

3 3

2

mM 2 mM

1 1

0 0 0 10 20 30 40 50 60 day 0 10 20 day30 40 50 60

20 wt.% UMFC solids Controls 4 4 No donor 1 wt.% solids sterile 3 3 NO2(-) 4 wt.% solids sterile NO3(-) 20 wt.% solids sterile 2 SO4(2-) 2 mM mM

1 1

0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 day day

Figure 12: Substrate consumption for ATCC 25259 during UMFC bioleaching

The findings from this experiment are in agreement with similar studies in the literature. Nitrate- dependent pyrite oxidation in the presence of T. denitrificans in small reaction volumes demonstrated the existence of such process, but the reaction rate was on the order of 0.02-0.69 - mM NO3 /day [25]. As the current experiment showed unsustainable nitrate-dependent UMFC bioleaching, pure strain T. denitrificans ATCC 25259 was deemed not suitable for further study.

36

5.2 First enrichment - Experiments with indigenous Thompson cultures

As bioleaching with pure strain T. denitrificans did not demonstrate sustainable denitrification when amended with UMFC, it became increasingly attractive to experiment with mixed microorganism communities for bioleaching. This approach was motivated by the general observation that mixed communities tend to be more versatile and resilient in performing certain biological processes after enrichment for a specific metabolic condition. For this study, it was proposed that sediment samples collected from mine related sources could be enriched on UMFC in well-controlled environments and evolved into a viable microbial community that is capable of nitrate-dependent bioleaching of Ni.

Sediment and water samples containing indigenous microorganisms from mining and refining activities related locations were enriched on UMFC in this microcosms study. Four distinct series of tests with different indigenous source were cultured under denitrifying and aerobic conditions. For each series, 3 denitrifying microcosms were amended with nitrate under anaerobic headspace (No. 10, 11, 12), 3 aerobic microcosms were maintained under atmospheric headspace (No. 7, 8, 9), 3 were free of any additional electron donor (No. 4, 5, 6), and 3 were sterile denitrifying controls (No. 1, 2, 3). The denitrifying microcosms were amended with KNO3 within the range of 0.5 mM to 5 mM. The pH in all microcosms was adjusted regularly with HCl and NaOH to be 6.5±0.2 whenever samples were collected.

5.2.1 Preliminary results

The results suggest that indigenous sediment cultures grew both aerobically and under denitrifying conditions, deriving energy from sulfur oxidation, evidenced by increasing sulfate concentrations in all active microcosms over the course of the experiment. Any or all of the sulfide minerals, including pyrite, pentlandite, pyrrhotite, and trace amount of Chalcopyrite, could be the S source. Oxidation and dissolution of pentlandite, although not demonstrated by mineralogical analysis yet, occurred due to the continuous Ni evolution in most of the denitrifying microcosms and a few aerobic microcosms. 37

TSRA Denitrifying TSRA Denitrifying 40 120 7.2 TSRA 10 35 TSRA 11 100 7.0 TSRA 12 30 Sterile ave. 25 Nitrate addition 80 6.8 20 60 6.6 pH 15 ppm TSRA10

sulfate(mM) TSRA11 10 40 TSRA12 6.4 Ni sterile ave. 5 20 Mg ave. 6.2 pH ave. 0 0 100 200 300 400 0 6.0 days 0 100 200 days 300 400

TSRA Aerobic TSRA Aerobic 200 7.2 25 TSRA 7 TSRA 8 7.0 20 TSRA 9 150 6.8 15 ppm TSRA 7 100 6.6 10 TSRA 8 pH TSRA 9 sulfate(mM) Mg ave. 6.4 5 50 pH ave. 6.2 0 0 100 200 300 400 0 6.0 0 100 200 300 400 days days

Figure 13: TSRA microcosm study. (a) TSRA denitrifying sulfate production and nitrate additions; (b) TSRA denitrifying Ni (green), Mg (maroon), and pH (blue cross); (c) TSRA aerobic sulfate production; (d) TSRA aerobic Ni (green), Mg (maroon), pH (blue cross) (different shades correspond to triplicates)

In the denitrifying TSRA microcosms (Figure 13 (a) and (b)), sulfate concentrations in all three active bottles are significantly higher than the average of sterile controls, and Ni concentrations in two out of three active bottles were significantly higher than that in the sterile controls. While the KNO3 concentrations in both active and sterile microcosms were kept between 0.5 mM and 5 mM at all time, the most active bottle, TSRA 10, overall received 18 doses of KNO3 (9 mmol) and the sterile bottles received 2 doses (1 mmol). All three active microcosms demonstrated microbial sulfur oxidation coupled to denitrification under anaerobic conditions, in which the (sulfide minerals from) UMFC was the major electron donor. Other possible donors were 38 substances in the original sediment, including humic acids; after the first 70 days of incubation, these donors were likely to be depleted and no longer available as major energy sources for the microorganisms.

Two active microcosms, TSRA 10 and TSRA 11, showed significantly higher dissolved Ni levels that are indicative of microbial mediated Ni dissolution from pentlandite and/or pyrrhotite. Although TSRA 12 has a dissolved Ni concentration similar to the controls, its sulfate production still showed microbial sulfur oxidation coupled to denitrification. The low Ni level in TSRA 12 could be a result of either low pentlandite oxidation due to preferential leaching of less noble sulfides (pyrrhotite for example), or no pentlandite oxidation due to a difference in the microbial community that was enriched in this microcosm.

5.2.2 Stoichiometric correlation - denitrifying

Microbial mediated oxidative dissolution of sulfide minerals in this study (in increasing nobility) with nitrate as electron acceptor can be described using the following equations:

! !! ! Pyrrhotite: !"!(!) + 1.8!!! + 1.6!!! → !"(!")! ! + !!! + 0.9!! ! + 0.2!

Equation 2

! !! ! Pyrite: !"!!(!) + 3!!! + 2!!! → !"(!")! ! + 2!!! + 1.5!! ! + !

Equation 3

! ! !! Pentlandite: !"#$!! + 3.4!!! + 1.4! + 0.8!!! → !"(!")! ! + 2!!! + 1.7!! ! + !!!!

Equation 4

Sulfate production and Ni dissolution observed in active microcosms are likely a combination of more than one of the listed redox reactions for different electron donors. The total amounts of

KNO3 consumed and sulfate produced in all denitrifying microcosms at the end of the first enrichment (410 days) are listed in Table 12. The ratio of nitrate to sulfate ranges from 1.86 for Dam 11 to 3.66 for TSRA 11, all of which are higher than any of the theoretical stoichiometric ratios for pyrrhotite (1.8), pyrite (1.5), and pentlandite (1.7). Although nitrate utilization for sulfide mineral oxidation is always below 100%, the number gets close to the theoretical value in 39 a few microcosms, such as the Tails series and the Dam series. A number of reasons could contribute to this observation, including presence of other non-sulfur electron donors in the sediment or other nitrate-dependent microorganisms.

Under denitrifying conditions, both pyrrhotite and pyrite have been found to be an electron donor for nitrate-dependent microbially mediated sulfide oxidation. Sulfate is assumed to be the stable and final product of sulfur compounds in the oxidative dissolution of UMFC, therefore aqueous sulfate concentration is used as the measurement of the overall extent of sulfide mineral oxidation. In the absence of any mineralogical analysis of leach residue, it is difficult to evaluate the extent of oxidation for each sulfide mineral alone. However, the ratio of sulfate:dissolved Ni (Table 12) ranges from 15.4 in Exp 12 to 46.6 in Dam 10, all of which are significantly higher 2- 2+ than the theoretical stoichiometric ratio of 2.0 (2SO4 :1Ni ) in Equation 4. Therefore, it is certain that at least one of the non-Ni-bearing iron sulfides consumed a significant amount of nitrate and contributed largely to the levels of sulfate measured.

Table 12: Stoichiometric comparison in active denitrifying microcosms (410 days)

- 2- Aqueous Ni Denitrifying NO3 SO4 - 2- 2- Acid consumption NO3 : SO4 SO4 : Aq. Ni microcosms (mmol) (mmol) (mmol) (2 N HCl, mL) Tails 10 8 3.75 2.13 0.11 35.1 1.34 Tails 11 8 3.62 2.21 0.12 29.2 1.41 Tails 12 7 3.39 2.07 0.12 27.3 1.26 Dam 10 12 5.91 2.03 0.13 46.6 1.36 Dam 11 9.5 5.11 1.86 0.15 34.7 1.15 Dam 12 10 5.31 1.88 0.13 40 1.02 Exp 10 8.5 3.63 2.34 0.14 26 1 Exp 11 8.5 3.86 2.2 0.16 24.6 1.24 Exp 12 8 3.26 2.45 0.21 15.4 1.17 TSRA 10 8 3.46 2.31 0.13 26.9 0.35 TSRA 11 5 2.01 2.48 0.12 16.4 0.5 TSRA 12 4 1.38 2.9 0.04 37.5 0.55

Although pentlandite nitrate-dependent oxidation is the only acid-consuming process (Equation 4) compared to pyrrhotite and pyrite (Equation 2 and Equation 3), all of the microcosms required addition of HCl solution to maintain a pH of 6.4 – 6.6. Tails, Dam, and Exp series consumed 40 similar amounts of acid solution, while TSRA series required much less (Table 12, last column “Acid consumption”). The amount acid used does not appear to be correlated with dissolved Ni or sulfate production, but with source of the original sediments. It is believed that since the sediments in this study were directly enriched on UMFC without any washing or manipulation, the difference in acid consumption is due to the nature of the original environmental sediment. For Tails and Dam, the sediments were collected from tailings ponds in which neutralizing agents were regularly added to maintain a slightly alkaline pH to avoid acidification. It is likely that the sediments carried some neutralizing capacity and contributed to the overall acid consumption in these denitrifying microcosms.

5.2.3 Stoichiometric correlations - aerobic

Under aerobic conditions in which oxygen is the electron acceptor, the oxidation of main sulfide minerals in the UMFC proceed as follows in increasing nobility:

!! ! pyrrhotite: !"!(!) + 2.25!! ! + 2.5!!! → !"(!")! ! + !!! + 2!

Equation 5

!! ! pyrite: !"!!(!) + 3.75!! ! + 3.5!!! → !"(!")! ! + 2!!! + 4!

Equation 6

!! ! !! pentlandite: (!!)!!!! + 4.25!! ! + 2.5!!! → !"(!")! ! + 2!!! + 2! + (!" )

Equation 7

Similar to the denitrifying microcosms, sulfate production observed in the aerobic microcosms is most likely a combination of at least two or all of the redox reactions above. Since pentlandite is the only major Ni-bearing sulfide, any sulfate-to-Ni ratio (listed in Table 13) higher than 2.0 (or 2- 2+ 2SO4 :1Ni ) suggests oxidation of pyrrhotite and/or pyrite, both of which are well-characterized processes.

According to Equation 5 to Equation 7, all of the oxidation processes under aerobic condition are theoretically acid producing; however, only the TSRA series required addition of a base 41 solution, 2N NaOH. This observation is in agreement with denitrifying microcosms in the previous section, and reaffirms the background neutralizing capacity of environmental sediments in Tails, Dam, and Exp series.

Table 13: Stoichiometric comparison in active aerobic microcosms (410 days)

2- 2- Aerobic SO4 Aqueous Ni SO4 : Aq. Acid consumption microcosms (mmol) (mmol) Ni (2N HCl, mL) Tails 7 1.17 0.002 604.7 1.07 Tails 8 1.06 0.008 126.1 1.08 Tails 9 1.38 0.022 62 0.92 Dam 7 1.68 0.048 34.8 0.41 Dam 8 1.82 0.053 34.7 0.66 Dam 9 1.9 0.072 26.3 0.65 Exp 7 1.44 0.14 10.3 0.6 Exp 8 1.33 0.072 18.6 0.57 Exp 9 1.24 0.043 28.6 0.56 TSRA 7 1.54 0.2 7.69 -0.2 TSRA 8 1.5 0.2 7.51 -0.11 TSRA 9 1.27 0.219 5.82 -0.18

5.2.4 Denitrifying vs. Aerobic

Overall, TSRA aerobic microcosms (TSRA 7 to 9) have the highest dissolved Ni (Table 13). As they are the only active bottles that required NaOH addition, it is possible that the measurement is a result of lower pH levels at every sampling point. The extents of oxidation in TSRA 7 to 9 by sulfate analysis are not significantly higher than other active aerobic microcosms. This observation led to the belief that some Ni units that were mobilized from their sulfide crystalline host might have re-precipitated as insoluble salt(s); Ni measured by aqueous phases analysis is only representative of a fraction of the total amount of Ni mobilized by oxidation. Therefore, the accuracy of aqueous Ni analysis is highly dependent on solution pH. This issue is elaborated in the next Section 5.2.5 Ni re-precipitation.

All other series, Tails, Dam, and Exp, exhibit a higher degree of oxidation and Ni dissolution at the end of this enrichment study. This could be the result of a number of factors: (1) higher concentration of KNO3 as electron acceptor in the denitrifying scenario, versus low oxygen 42 solubility in water; (2) difference in thermodynamic driving forces for different oxidants; (3) differences in the microbial communities in catalyzing oxidative sulfide dissolution. All of these subjects are worth looking into to better understand microbial mediated bioleaching process.

5.2.5 Ni re-precipitation

The amount of Ni measured in the aqueous phase in both aerobic and denitrifying microcosms is possibly a fraction of the total amount of Ni mobilized by oxidation, due to re-precipitation as a less soluble solid. At pH 6.5, Ni is very close to becoming insoluble (pH 7) and precipitate as

Ni(OH)2. The phosphate buffer system in the leaching medium can also form insoluble salts with Ni2+ at pH 6.5. A mineralogy analysis of the leach residue could answer this question, however this is not feasible for this particular enrichment due to the high amount of sediment still present in the system. It is of great interest to track the fate of possibly re-precipitated Ni, as this can shed light on potential processing techniques such as a mild acidification step to mobilize Ni after pH neutral bioleaching.

5.2.6 Magnesium Dissolution

Dissolved magnesium (Figure 14) in aerobic, denitrifying, and both types of control microcosms did not show a significant difference in either trend or magnitude, suggesting that Mg dissolution is an abiotic chemical process. The time zero data point was measured 7 days after the start of the experiment; the initial magnesium dissolution can be attributed to dilution of the water content in the wet UMFC and dissolution of easily soluble MgO units on the surface of UMFC particles. The amount of dissolved Mg measured at the beginning corresponds to 30% (in no acceptor control) to 49% (in sterile control) of the final magnesium level after 410 days of enrichment. Up to approximately 170 days, Mg concentrations in the aqueous phase continued to increase at a steady rate, which suggests that a diffusion-controlled process was in play as Mg is soluble at pH 6.5. After which, the dissolved Mg begins to approach a constant concentration, reaching an equilibrium regardless of the treatment conditions, sterile or active, aerobic or denitrifying. 43

EXP - Mg 200 180 160 140 120 100 ppm 80 Sterile control 60 No acceptor control 40 Aerobic active 20 Denitrifying active 0 0 100 200 300 400 days

Figure 14: Magnesium dissolution in Exposures series

5.2.7 Tails, Dam, and Exp

The enrichment results for Tails, Dam, and Exp series are shown in Figure 15, Figure 16, and Figure 17 below. (Complete raw data can be found in Appendix G: Supplementary Data, Table 19 to Table 30.) They demonstrated similar behaviors as TSRA microcosms in sulfate production, nitrate consumption, and Ni and Mg dissolution. The figures are presented here for the convenience of discussion in the next section, Bacterial community analysis.

Tails - Denitrifying Tails - Denitrifying 45 250 7.2 40 Tails 10 Tails 11 7.0 35 200 Tails 12 30 Sterile 6.8 150 25 KNO3 add. 6.6 pH

20 ppm 100 Tails 10 Tails 11 sulfate(mM) 15 6.4 Tails 12 10 50 Sterile Mg ave. 6.2 5 pH 0 0 6.0 0 100 200days 300 400 0 100 200 days 300 400 44

Tails - Aerobic Tails - Aerobic 20 350 7.2 Tails 7 Tails 8 300 7.0 15 Tails 9 250 6.8 200 10 6.6 pH 150ppm Tails7 Tails8 6.4

sulfate(mM) Tails9 5 100 Mg ave. 50 pH ave. 6.2

0 0 6.0 0 100 200 300 400 days 0 100 200days 300 400 Figure 15: Tails microcosm study. (a) Tails denitrifying sulfate production and nitrate additions; (b) Tails denitrifying Ni (green, different shades correspond to triplicates), Mg (maroon), and pH (blue cross); (c) Tails aerobic sulfate production; (d) Tails aerobic Ni (green), Mg (maroon), pH (blue cross)

DAM - Denitrifying DAM - Denitrifying 60 7.2 DAM 10 120 NO3 addition 50 100 7.0 DAM 11 40 DAM 12 80 6.8 Sterile ave. 30 6.6 60 pH ppm DAM10 20 40 Sterile ave. 6.4 sulfate(mM) DAM 11 DAM 12 10 6.2 20 Mg ave. pH ave. 0 0 6.0 0 100 200days 300 400 0 100 200 days 300 400 DAM - Aerobic DAM - Aerobic 25 160 7.2 DAM 7 140 DAM 8 7.0 20 DAM 9 120 6.8 15 100 80 6.6 pH ppm 10 60 Dam 7 Mg ave. 6.4 40 sulfate(mM) Dam 8 5 6.2 20 DAM 9 pH ave. 0 0 6.0 days 0 100 200 300 400 0 100 200 300 400 days Figure 16: Dam microcosm study. (a) Dam denitrifying sulfate production and nitrate additions; (b) Dam denitrifying Ni (green, different shades correspond to triplicates), Mg (maroon), and pH (blue cross); (c) Dam aerobic sulfate production; (d) Dam aerobic Ni (green), Mg (maroon), pH (blue cross) 45

EXP - Denitrifying EXP - Denitrifying 40 180 7.2 EXP 10 35 KNO3 add. 160 EXP 11 7.0 140 30 EXP 12 25 Sterile 120 6.8 100 20 6.6 EXP10 pH ppm 80 15 EXP 11 sulfate(mM) 60 EXP 12 6.4 10 40 Sterile control 5 Mg ave. 6.2 20 pH 0 0 6.0 0 100 200 300 400 days days 0 100 200 300 400 EXP - Aerobic EXP - Aerobic 20 7.2 EXP 7 160 EXP 8 140 7.0 15 120 EXP 9 6.8 100 10 6.6 80 pH ppm EXP7 60 EXP 8 EXP 9 6.4 sulfate(mM) 5 40 Mg ave. pH ave. 6.2 20 0 0 6.0 0 100 200 days 300 400 0 100 200 days 300 400 Figure 17: Exposures microcosm study. (a) Exposures denitrifying sulfate production and nitrate additions; (b) Exposures denitrifying Ni (green, different shades correspond to triplicates), Mg (maroon), and pH (blue cross); (c) Exposures aerobic sulfate production; (d) Exposures aerobic Ni (green), Mg (maroon), pH (blue cross)

5.3 Bacterial community analysis (16S rRNA gene sequencing)

As described in Section 4.6 Microbiology Investigation, after 220 days of enrichment on UMFC, DNA from each active aerobic and denitrifying microcosm was extracted, amplified, and sequenced for bacterial community analysis. Only sequences that passed a set of quality control parameters for sequencing quality score, length, and homopolymers were used for OTU grouping (based on 97% sequence similarity), taxonomic assignment, and building a phylogenetic tree. 16S rRNA sequences and taxonomic identification files are deposited in an online server (smb://files.biozone.utoronto.ca/syntrophy). The primers used for this study were general to both bacteria and archaea; hence any archaea present in the microcosms would have been amplified. 46

However, as this analysis focuses on taxonomic groups that have >2% relative abundance in each microcosm and no archaea was found to reach this level of abundance, the discussion is focused on bacterial species.

Bacterial communities in all active microcosms were highly diverse at the time of DNA extraction; this is not unexpected since the enrichment started directly from environmental sources and had only been incubating for 220 days. However, enrichment is observed in certain types of microorganisms, and their taxonomic relations point toward sulfur oxidation, iron oxidation, and/or denitrification. The taxonomic assignment is performed using RDP classifier against the Greengenes database.

5.3.1 Taxonomic diversity in active denitrifying microcosms (Tails, Dam, Exp)

In Figure 18 below, denitrifying microcosms from Tails, Dam, and Exp series appear to have a similar community structure where a significant proportion is consistently made of a mix of Chromatiales (also collectively referred to as purple sulfur bacteria), OTU7494 (assigned as Gallionellaceae), envOPS12, OTU3487 (Thiobacillus), and OTU6475 (unspecific Betaproteobacteria). Taxonomic abundance can be found in Table 31 in Appendix G: Supplementary Data.

It should be noted that in the bar figures below for community diversity, the convention for showing certainty and goodness of the match is by one of the three ways: 1) when a representative sequence of a particular OTU matches well with a defined sequence, the OTU can be identified to the genus level (Example: OTU3487 is assigned to the genus level as Thiobacillus); 2) when ambiguity between distinct taxa arises during classification by the RDP classifier, the assignment below a certain level is designated as “Other” (Example Chromatiales;Other;Other); 3) when the representative sequence matches to a poorly defined, or a not previously named reference sequence in the database, the taxonomic assignment for that level will be left blank (Example: Gallionellaceae).

47

100% Uncategorized Less than 2% 90% Cyanobacteria;c_Chloroplast;o_Stramenopiles;f_;g_ Gammaproteobacteria;Other;Other;Other 80% Betaproteobacteria;o_Rhodocyclales;f_Rhodocyclaceae;g_Methyloversatilis Proteobacteria;Other;Other;Other;Other 70% OP11;c_OP11-3;o_;f_;g_

60% Chlorobi;c_Ignavibacteria;o_Ignavibacteriales;f_Ignavibacteriaceae;g_ Chloroflexi;c_Anaerolineae;Other;Other;Other

50% Betaproteobacteria;o__Rhodocyclales;f__Rhodocyclaceae;Other Betaproteobacteria;o_;f_;g_ (OUT594) 40% Planctomycetes;c_Phycisphaerae;o_Phycisphaerales;f_;g_ Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;Other (OUT7062) 30% Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;g_(OUT3261) Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_ 20% Betaproteobacteria;Other;Other;Other (OTU6475) Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;g_Thiobacillus (OUT3487) 10% Gammaproteobacteria;o_Chromatiales;f_;g_

0% Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;g_(OTU7494) Gammaproteobacteria;o_Chromatiales;Other;Other Figure 18: Taxonomic assignment of Tails, Dam, and Exp denitrifying microcosms

The order Chromatiales contains unicellular Gram-negative anoxygenic phototrophic bacteria, known as phototrophic purple sulfur bacteria, able to perform photosynthesis under anoxic conditions without oxygen production. In addition, the order contains nonphototrophic purely chemotrophic relatives [32].

The family Gallionellaceae consists Gram-negative, bean-shaped cells that secrete an extracellular twisted stalk that is composed of numerous fibers under microaerophilic conditions.

Chemolithotrophic growth can be obtained in vitro using oxygen and ferrous iron with CO2 as a sole carbon source [35]. The type species Gallionella ferruginea is an iron-oxidizing chemolithotrophic bacterium that lives in low-oxygen conditions that contains dissolved reduced iron; it has been found in a variety of aquatic habitats, including freshwater, ferruginous mineral springs, as well as in soil environments associated with iron [30].

The genus Thiobacillus are rod-shaped Gram-negative bacteria with polar flagella. NCBI blast of the representative sequence of OTU3487 showed 100% identity to the defined species Thiobacillus denitrificans (NCBI Accession: NR_025358.1), a sulfur and iron oxidizer capable of coupling oxidation to dentrification. A strain of T. denitrificans (ATCC 25259) was used in pure culture bioleaching experiments in this study. 48

Except for envOPS12, the other five major taxa appear to be the result of enrichment on UMFC, as they all rely on sulfur or iron sources for growth under denitrifying or microaerobic conditions. Among all Betaproteobacteria present in these microcosms, Gallionellaceae (OTU7494) and

Thiobacillus (OTU3487) are both iron-oxidizing bacteria capable of using CO2 as a sole carbon source. Enrichment of OTU3487 (representative sequence NCBI blast as Thiobacillus denitrificans) offers an explanation for rapid nitrate consumption, and increased N2 concentration in the headspace of denitrifying microcosms when analysis was occasionally performed with a gas chromatography equipped with a thermal conductivity detector (GC-TCD).

Table 14: Nitrate, sulfate, and dissolved Ni in Tails, Dam, and Exp series (220 days)

- 2- - 2- NO3 SO4 - 2- Aqueous Ni NO3 : SO4 NO3 : SO4 (mmol) (mmol) (mmol) (410 days) Tails 10 5.5 2.21 2.49 0.021 2.13 Tails 11 5.5 2.34 2.35 0.019 2.21 Tails 12 5 2.4 2.08 0.029 2.07 Dam 10 5.5 2.41 2.28 0.023 2.03 Dam 11 3.5 1.09 3.21 0.025 1.86 Dam 12 5.5 2.28 2.41 0.027 1.88 Exp 10 5.5 1.89 2.9 0.02 2.34 Exp 11 5.5 2.07 2.65 0.024 2.2 Exp 12 5.5 2.09 2.63 0.029 2.45

Although nitrate consumption and sulfate production by Day 220 in these microcosms are very similar, and no obvious correlation between sulfate production and relative abundance of a particular taxa can be drawn, the stoichiometry (Table 14) still offers insights into the dynamics of the redox system. 1) Compared to the rest, Dam 11 consumed the least nitrate, and also produced the least amount of sulfate; this correlation confirms the previous conclusion that mineral sulfides were the primary electron donors for denitrification in these microcosms. In addition, dissolved Ni concentration in Dam 11 is on par with the rest of the group, suggesting - again that dissolved Ni is not an accurate representation of pentlandite oxidation. 2) The NO3 : 2- SO4 ratio for each microcosm decreased from Day 220 to Day 410, approaching the theoretical 49 values of 1.8, 1.5, 1.7 for pyrrhotite, pyrite, and pentlandite, respectively, as the enrichment continued (Table 14, column 4 and 6).

5.3.2 Taxonomic diversity in active aerobic microcosms (Tails, Dam, Exp)

In Figure 19 below, aerobic microcosms from Tails, Dam, and Exp series appear to be enriched in one or two taxa, instead of six in their denitrifying counterparts, while consisting of many less abundant taxa that are <2% of the community. Hydrogenophilaceae, consisting OTU3261, OTU3800, and OTU6684, is the most abundant taxon in all aerobic microcosms, ranging from 18% in Tails 9 to 64% in Exp 8. Unspecified Betaproteobacteria (OTU4252) is also present in all microcosms, but to a much smaller extent. Chloroflexi, Bacteroidetes, and Cyanobacteria that are not specified below the order level are often found in a number of microcosms, as well as some well defined taxa including Methyloversatilis (OTU408), Gallionellaceae (OTU6467, OTU7494), and Thiobacillus (OTU3487). Taxonomic abundance can be found in Table 32 in Appendix G: Supplementary Data.

Hydrogenophilaceae are a family of Gram-negative bacteria that obtain their energy from oxidizing hydrogen. Two genuses are identified under this family: Hydrogenophilus, and Thiobacillus. Representative sequences of the three OTUs that belong to this taxon blast similar to (1) Thiobacillus species found in a study of carbon and sulfur cycling by microbial communities in oil sands tailings pond (99% similarity, NCBI Accession: HQ086221.1), (2) Thiobacillus aquaesulis isolate (96% similarity, NCBI Accession: NR_044793.1), and (3) Limnobacter thiooxidans isolate (97% similarity, NCBI Accession: NR_025421.1). Sequence similarity to these Gram-negative, thiosulfate-oxidizing isolates [36] [37] suggests this highly abundant group of bacteria consisting three OTUs are strongly associated with sulfur oxidation in the microcosms.

OTU4252 is found in all aerobic microcosms in the Tails, Dam, and Exp series. It is poorly identified to the phylum level as Betaproteobacteria, but its representative sequence blast 100% identity with microbial communities in karst groundwater environments (NCBI Accession: AM991243.1). Karst is a landscape formed by underground erosion of rocks such as limestone and marble that dissolve in water. 50

OTU6467 (and OTU7494, discussed in Section 5.3.1) is classified as Gallionellaceae, and found in six out of nine microcosms discussed here. Although it is assigned to the family of Gallionellacea, it is found to be 99% similar to a Sideroxydans species (NCBI Accession: EU809885.1) based on a NCBI blast of the representative sequence. Nonetheless, both OTUs contain Gram-negative iron- oxidizing bacteria with properties that suggest a correlation to the enrichment conditions with UMFC.

100% Root;Other;Other;Other;Other;Other

Less than 2% 90% Deltaproteobacteria;o_;f_;g_

80% Alphaproteobacteria;Other;Other;Other

Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;Other (OTU6263) 70% Gammaproteobacteria;o_Chromatiales;f_;g_

60% Gammaproteobacteria;o_Chromatiales;Other;Other Gammaproteobacteria;Other;Other;Other

50% Nitrospirae;c_Nitrospira;o_Nitrospirales;f_Thermodesulfovibrionaceae;g_GOUTA19

Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;g_Thiobacillus (OTU3487) 40% Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;g_(OTU6467,7494)

30% Betaproteobacteria;o_Rhodocyclales;f_Rhodocyclaceae;g_Methyloversatilis (OTU408) Cyanobacteria;c_Chloroplast;o_Stramenopiles;f_;g_ 20% Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_

Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ 10% Betaproteobacteria;Other;Other;Other (OTU4252)

0% Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;g_(OTU3261,3800,6684)

Figure 19: Taxonomic assignment of aerobic Tails, Dam, and Exp microcosms

Aerobic microcosms from Tails, Dam, and Exp series share many similarities in microbial community structure. The most abundant group of bacteria in all cases is the family of Hydrogenophilacea. OTUs belonging to this taxonomic assignment showed strong similarities to hydrogen-oxidizing and sulfur-oxidizing bacteria isolates, which correlate well to the enrichment conditions. At least one of iron-oxidizing bacteria Gallionellacea (OTU6467 and OTU7494) and Thiobacillus (OTU3487) is present in all aerobic microcosms discussed here, except for Dam 7.

Unlike the rest of the aerobic microcosms, Dam 7 seems to have a bacterial community similar to denitrifying microcosms in Section 5.3.1. As much as 35% of the Dam 7 community is composed of purple sulfur bacteria Chromatiales. It is uncertain what caused this disparity, but another 51 microbial community analysis of DNA extracted at a later date may shed light on whether this is a result of random errors in the analytical process.

5.3.3 Taxonomic diversity in TSRA microcosms (aerobic and denitrifying)

Microbial communities in the TSRA series showed higher variability among triplicate microcosms than other three series (Figure 20). Among TSRA aerobic microcosms, TSRA7 and TSRA8 consist similar types of bacteria, such as Thiobacillus (OTU3487), Gallionellaceae (OTU7494), and Betaproteobacteria (OTU1225). On the other hand, TSRA9 shares only the first most abundant OTU with its replicates and the remainder of the community is made up of dramatically different phyla such as Bacteriodetes and Nitrospirae. Abundance of Thiobacillus (OTU3487) also varies over a wide range from 8% to 48%. The second most abundant taxon, Gallionellaceae (OTU7494), in TSRA 7 and TSRA8 is absent from TSRA9. Taxonomic abundance can be found in Table 33 in Appendix G: Supplementary Data.

Among TSRA denitrifying microcosms, the first four major taxa, namely Thiobacillus (OTU3487), Gallionellaceae (OTU7494), Betaproteobacteria (OTU1225), and Methyloversatilis (OTU408 and OTU2838), are shared by all triplicates, but the abundance of Thiobacillus is much lower in TSRA12. There is another taxon of Gallionellacea, designated as “Gallionellaceae;Other”, as the OTUs match poorly to any reference sequence in the Greengenes database below the family level. However, a blast search against the NCBI database showed OTU343 to be similar to bacteria found in a bioremediation study by iron oxide and sulfide, and OTU7062 to be similar to those found in denitrification coupled to pyrite oxidation. It is at least possible to speculate that OTU343 and OTU7062 are similar to OTU7494 and OTU3487 in terms of their functions in oxidation of iron and sulfur. A large percentage of TSRA12 is made from Chromatiales (19%), which is a major taxon in the denitrifying microcosms from Tails, Dam, and Exp series but not present in any other TSRA bottles. 52

100% Uncategorized

90% Less than 2% Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_ 80% Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;g_Gallionella Betaproteobacteria;o_Rhodocyclales;f_Rhodocyclaceae;Other 70% Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ Chloroflexi;c__Anaerolineae;o_GCA004;f_;g_ 60% Gammaproteobacteria;o_Chromatiales;f_;g_

50% Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;Other (OTU343,7062) Bacteroidetes;c_Sphingobacteria;o_Sphingobacteriales;f_;g_ 40% Nitrospirae;c_Nitrospira;o_Nitrospirales;f_Nitrospiraceae;g_Nitrospira Bacteroidetes;c_Sphingobacteria;o_Sphingobacteriales;f_Sphingobacteriaceae;g_ 30% Deltaproteobacteria;o_Myxococcales;f_;g_ Betaproteobacteria;o_Rhodocyclales;f_;g_(OTU1041,5214) 20% Betaproteobacteria;o_Methylophilales;f_;g_(OTU4411) 10% Betaproteobacteria;o_Rhodocyclales;f_Rhodocyclaceae;g_Methyloversatilis (OTU408,2838) Betaproteobacteria;Other;Other;Other (OTU1225) 0% Betaproteobacteria;o_Gallionellales;f_Gallionellaceae;g_(OTU7494) Betaproteobacteria;o_Hydrogenophilales;f_Hydrogenophilaceae;g_Thiobacillus (OTU3487)

Figure 20: Taxonomic assignment of TSRA microcosms

After 220 days of enrichment on UMFC, the amounts of nitrate consumed and sulfate produced (Table 15 below) may be correlated with the total abundance of Thiobacillus, Gallionellaceae, and OTU1225. TSRA10 produced the highest amount of sulfate, and the total abundance of the abovementioned taxa is as high as 75%; TSRA12 produced the least amount of sulfate, and the corresponding taxa abundance is just below 50%; TSRA11’s sulfate production as well as corresponding taxa abundance is the mid performer in both aspects.

Table 15: Nitrate, sulfate, and dissolved Ni in denitrifying TSRA microcosms (220 days) NO - SO 2- NO - : SO 2- Aqueous Ni NO - : SO 2- 3 4 3 4 3 4 (mmol) (mmol) (up to 220 days) (mmol) (up to 410 days) TSRA 10 5.5 2.07 2.66 0.063 2.31 TSRA 11 3.5 0.78 4.51 0.043 2.48 TSRA 12 3 0.57 5.3 0.012 2.9

Similar to other denitrifying series, nitrate to sulfate ratio also improved in TSRA denitrifying microcosms from Day 220 to Day 410. For the amount of nitrate consumed, a higher percentage 53 of which is used toward coupling sulfur oxidation, indicating continuous enrichment for sulfur- oxidizing bacteria.

5.3.4 Phylogenetic analysis of Betaproteobacteria

All OTUs >2% abundant in each microbial community and identified as Betaproteobacteria in the taxonomic analysis were compared with nucleotide sequences in the GenBank database using NCBI BLAST search with default settings; similar 16S rDNA sequences were identified and used for phylogenetic comparison for the OTUs. Major OTUs that are abundant or appear consistently in a number of microcosms and their closest match on the GenBank database are listed in Table 16.

Table 16: Major OTUs, abundance, and closest GenBank matches

OTUs Description of abundance GenBank closest match (% similarity)

10.3% in TSRA12; Unidentified bacterium from bioremediation site 343 <2% in other series by iron oxides and sulfides (99%) Moderately abundant in TSRA: Unidentified bacterial sp. in iron-reducing 408 31.8% in TSRA11 ~ 3.8% in TSRA8; enrichment culture (99%) Also found in other series but <4% 5.6% in TSRA11; 4.8% in Tails12 2838 Uncultured Rhodocyclaceae bacterium (97%) ~2% or lower in other series Moderately abundant in Tails, Dam, Unidentified bacterial sp. in lake sediment 3261 Exp. Most abundant in Tails7 at (100%) 28.6% Thiobacillus denitrificans (97%)

Highly abundant in all microcosms. 3487 Thiobacillus denitrificans (100%) Most abundant in TSRA9 at 51.5% 7.8% in Exp9; 4.4% in Dam8; Unidentified bacterial sp. in Karst groundwater 4252 <2% in other (100%) Highly abundant in aerobic Dam and 6684 Exp; most abundant in Exp8 at Limnobacter thiooxidans (97%) 63.8% 11.9% in TSRA12, 6.4% in TSRA11 Unidentified bacterial sp. in denitrification 7062 coupled pyrite oxidation groundwater sediment ~2% or lower in other (99%) Moderately abundant in all 7494 microcosms. Most abundant in Unidentified bacterial sp. (100%) TSRA10 at 34.5%

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A phylogenetic tree is a graphical illustration of evolutionary relationships between organisms. The partial 16S rDNA sequences of major OTUs in this study and closest matches on the GenBank database were entered into Geneious R6 software and a maximum likelihood phylogenetic tree was generated (Figure 21). Distances between a known organism and an organism found in the microcosms may suggest functional similarities/dissimilarities, and allow inferences for the roles certain organisms might play in the bioleaching microcosm environments.

The blue clade (C1) in Figure 21 consists OTUs with relatively high sequencing depth and was identified to the genus level as Thiobacillus. OTU3487, a highly abundant OTU in all microcosms, was found to be 100% identical to Thiobacillus denitrificans, the nitrate-dependent iron/sulfur-oxidizing organism that inspired this project. Also under the blue clade is OTU6684, which is highly abundant in aerobic microcosms and positioned closely to Limnobacter thiooxidans with 97% sequence similarity. The red clade (C2) consists a large number of OTUs and known species that are capable of using iron and/or sulfur as energy sources; a few matches to pyrite oxidation environments under denitrifying conditions. The green clade (C3) includes a few moderately abundant OTUs and bacterial species found in iron-related environments, such as Iron Snow. 55

desulfobacterium anilini (EU020016.1) 56.2 1041 Polaromonas sp. strain JS666 (NR_074725.1) 100 4411 Pyrite mine AMD 16S clone (KC620646.1) 90.7 343 38.4 Bioremediation by iron oxides and sulfides site 16S clone (JQ976370.1) 99.7 6467 56.7 Siderooxidans sp. (EU809885.1) 4471 92.5 90.2 17.3 2280 97.1 Rhodocyclaceae 16S clone from CAHs contaminated groundwater (JQ279055.1) Sideroxydans lithotrophicus (NR_074731.1) 17.8 93.2 7062 Denitrification coupled to pyrite oxidation 16S clone (HM641565.1) 28.6 99.8 6475 6.7 PAH degrading community 16S clone (FQ659310.1) 7494 21 2.3 98.9 Azospira oryzae (NR_024852.1) Dechlorosoma suillum (NR_074103.1) 21 Zoogloea oryzae (NR_041286.1) Denitratisoma oestradiolicum (NR_043249.1) 73.8 3800 2.8 61.2 6263 99.4 Thiobacillus aquaesulis (NR_044793.1) 85.1 Thiobacillus sp. from gypsum-treated oil sands tailings pond (HQ086221.1) 78.8 3261 99.2 3487 6.9 50.2 99 Thiobacillus denitrificans (NR_025358.1) Thiobacillus denitrificans (NR_074417.1) C2 61.1 6684 Limnobacter thiooxidans (NR_025421.1) C1 100 4252 15.1 Karst groundwater microbial community 16S clone (AM991243.1) 100 Nitrosomonadaceae 16S clone from gypsum-treated oil sands tailings pond (HQ043799.1) 67.1 594 Dechloromonas agitata (NR_024884.1) 79.9 408 9.1 100 Iron-reducing bacterium enrichment 16S clone (FJ802319.1) 75.1 Rhodocyclaceae 16S clone from gypsum-treated oil sands tailings pond (HQ042342.1) 22.6 2838 77.6 Castellaniella denitrificans (NR_044802.1) 17.3 Accumulibacter phosphatis strain UW-1 (NR_074763.1) 100 5214 Iron-rich particles (Iron Snow) 16S clone (FR667826.1) C3

0.03

Figure 21: Maximum likelihood phylogenetic tree of Betaproteobacteria OTUs (>2%) in all 23 sequenced microcosms, rooted to desulfobacterium anilini (EU020016.1). Generated using Geneious R6. The scale bar represents 3 substitutions per 100 positions.

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5.4 Second enrichment – acid leached UMFC

Bacteria cultures from the first enrichment study were collected and applied to acid leached UMFC (Table 17) to (1) test if the cells in the supernatants can be transferred successfully, and (2) be able to analyze post leach residues to better track the fate of Ni during bioleaching.

Table 17: Chemical Assay of Acid-leached UMFC* (by Aqua Regia, in wt.% and standard deviation, σ)

Ni Mg Fe S Al Ca Mn Co Cu Zn Wt.% 11.79% 0.11% 25.20% 26.83% 0.07% 0.05% 0.03% 0.38% 0.42% 0.03% σ 0.18% 0.15% 0.43% 0.68% 0.001% 0.003% 0.001% 0.002% 0.002% 0.001%

* Average of five replicates and standard deviation.

After 90 days of enrichment on acid-leached UMFC, bacterial cultures collected from the all of the denitrifying microcosms in the first enrichment study showed denitrification activity and sulfur oxidation. As shown in Figure 22 below, sulfur oxidation coupled to denitrification started as soon as the first dose of 5 mM KNO3 was added on Day 17, and continues to increase at a steady rate. pentlandite dissolution was also detected, as Ni in the aqueous phase increases with the sulfate profile. (Partial raw data can be found in Table 34 in Appendix G: Supplementary Data.)

Dam_2 denitrifying Dam_2 denitrifying 35 10 7.0 Dam N7 30 Dam N8 8 6.8 25 Dam N9 killed ave. 6.6 20 NO3 add 6 6.4 Dam N7 pH 15 ppm 4 Dam N8

sulfate(mM) 6.2 10 Dam N9 Mg 2 6.0 5 pH ave 0 0 5.8 0 20 40 60 80 100 120 0 20 40 60 80 100 120 days days Figure 22: Second enrichment – Dam_2 denitrifying. (a) Dam denitrifying sulfate production and nitrate additions; (b) Dam_2 denitrifying Ni (green, different shades correspond to triplicates), Mg (maroon), and pH (blue cross).

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Cultures from Exp, TSRA, and Thiobacillus denitrificans (ATCC 25259) (Thd series) were also enriched on acid leached UMFC, and have shown the same trends for sulfur oxidation, denitrification, and Ni dissolution.

Interestingly, nitrate to sulfate ratios in all denitrifying microcosms in this enrichment are below the stoichiometric values for pyrrhotite, pyrite, and pentlandite according to Equation 2, Equation 3, and Equation 4. It is unclear why this behavior is observed; further analysis of the microbial communities may offer some explanation in terms of alternative electron accepters.

Table 18: Nitrate, sulfate in second enrichment Dam, Exp, TSRA and Thd series (100 days) NO - SO 2- 3 4 NO - : SO 2- (mmol) (mmol) 3 4 Dam 7 1.50 1.42 1.06 Dam 8 1.50 1.44 1.04 Dam 9 1.50 1.46 1.03 Exp 7 1.50 1.45 1.03 Exp 8 1.50 1.45 1.03 Exp 9 1.50 1.42 1.06 TSRA 7 1.25 1.16 1.08 TSRA 8 1.25 1.21 1.03 TSRA 9 1.25 1.13 1.11 Thd 7 1.00 1.07 0.93 Thd 8 1.00 1.09 0.92 Thd 9 1.00 1.07 0.93

This part of the study will be continued until an appreciable amount of pentlandite is oxidized (based on estimation of nitrate consumption), and mineralogy analysis of the leach residue will answer some earlier questions, such as whether Ni re-precipitation occurred at pH 6.5 and in what form, and shed light on the relative extents of oxidative dissolution among pyrrhotite, pyrite, and pentlandite.

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6 Conclusions and Recommendations for Future Work

This study aimed to provide a proof-of-concept understanding of nitrate-dependent bioleaching of nickel from ultramafic sulfide ore under neutral pH conditions. Although bioleaching has been studied for more than half a century and applied to base and precious metal extraction from low- grade ores in the past, the majority of these applications rely on bacteria that thrive under acidic environments, typically pH 1.5-2.5. Due to the ultramafic nature of the host material, it was expected that bioleaching under neutral pH environments (pH 6-8) may circumvent processing challenges associated with high MgO content, such as increased acid consumption, costly waste management, and high operating costs.

6.1 Bioleaching with defined strain Thiobacillus denitrificans

Objective 1 of this study was to examine whether defined single strain bacterium Thiobacillus denitrificans could carry out oxidative sulfide dissolution of UMFC. Bioleaching experiments were conducted with an axenic culture of Thiobacillus denitrificans (ATCC 25259) under neutral pH conditions, in which ultramafic concentrate (UMFC) is the electron donor and nitrate is the electron acceptor. The experiments were carried out in sealed serum bottles at pH 6.5 and kept under 30 °C and 160 rpm in an orbital shaker with an anaerobic headspace for 52 days.

In 1 wt.% and 4 wt.% solid loading tests, the initial dose of nitrate added at 5 mM was only partially consumed over the first 14 days before denitrification came to a halt. In 20 wt.% solid loading test, denitrification stopped after the addition of the second dose of 5 mM nitrate. These results suggest that nitrate-dependent oxidation of sulfide minerals in UMFC could not be sustained under the experimental conditions. No further experiment was carried out in the same configuration with defined culture.

6.2 Microcosm study with environmental cultures

Objective 2 of this study was to collect and enrich indigenous mixed culture bacteria for the same purpose as Objective 1. Microcosm studies were conducted with environmental sediment cultures 59 under pH 6.5 bioleaching conditions, in which ultramafic concentrate (UMFC) was the electron donor and nitrate was the electron acceptor. The study involved enrichment of 4 series of microcosms with sediments collected from 6 different mine-related sites. Over the course of 410 days, all microcosms under denitrifying conditions demonstrated microbially mediated, nitrate- dependent oxidative dissolution of sulfide minerals in UMFC.

Parallel to the denitrifying microcosms, the same environmental sediments were enriched on UMFC aerobically. Oxidative sulfide dissolution was also observed in all aerobic microcosms. Although no aerobic sterile control experiments was conducted to offer an abiotic baseline for dissolution, the abundance of iron- and sulfur-oxidizing bacteria in aerobic microcosms strongly suggests the presence of microbially-catalyzed aerobic sulfide dissolution process.

In denitrifying scenarios, the stoichiometric ratios of nitrate consumption and sulfate production indicate that nitrate utilization was always below 100%, but the ratio approached theoretical values toward the end of the study in a number of bottles. While the theoretical stoichiometric ratios for pyrrhotite, pyrite, and pentlandite are 1.8, 1.5, and 1.7, respectively, the overall measured nitrate:sulfate ratio in Dam 11 reached 1.88. As the enrichment progresses, the indigenous cultures appeared to have become more robust at coupling sulfide oxidation to denitrification under the conditions tested.

Dissolved Ni in the aqueous phase is believed to be a poor indicator for the extent of pentlandite dissolution, due to limited solubility of Ni at pH 6.5. Some Ni units that were mobilized from their sulfide host could have re-precipitated as insoluble salts; therefore Ni measured in aqueous phase represent only a fraction of the total amount of Ni associated with pentlandite dissolution. It is of great interest to track the fate of Ni by mineralogy analysis or gentle acidification of post leach residues in the second microcosm study.

Magnesium dissolution appeared to be an abiotic process, as after approximately 170 days dissolved Mg began to approach a constant concentration regardless of treatment conditions, such as sterile or active, aerobic or denitrifying. The initial Mg dissolution, which accounted for approximately 30% to 50% of the final Mg concentration, was attributed to dilution of water content in wet UMFC and immediately dissolution of soluble MgO units on the surface of UMFC particles. 60

6.3 Bacterial community analysis

As the microcosm study for Objective 2 provided active cultures for sulfide mineral oxidation, it offered us the opportunity to study the microbial community involved in the neutral pH bioleaching of Ni from UMFC. Objective 3 of the study was to perform 16S rDNA sequencing of both denitrifying and aerobic microcosms in Objective 2 to identify community members present after 220 days of enrichment. The results will also serve as a benchmark for future community analysis of the same cultures for the evaluation of the community upon further enrichment and treatment.

The sequencing results showed high diversity in all microcosms with as high as 35% of the entire community being low abundance bacteria (<2%) in denitrifying microcosms, and 44% in aerobic microcosms. Nonetheless, enrichment in OTUs whose representative sequences are similar (>97%) to known iron-oxidizing and/or sulfur-oxidizing bacteria is also observed across all microcosms.

Among denitrifying microcosms, the communities consistently contain a mix of Chromatiales, Gallionellaceae, envOPS12, and Thiobacillus, in which Chromatiales, collectively referred to as purple sulfur bacteria, is the predominant group. The representative sequence of OTU3487 (5% - 7% abundance in each community) gave 100% sequence similarity to the defined species Thiobacillus denitrificans. Enrichment in these taxa of bacteria correlates well to sulfur oxidation measured in these microcosms and increased N2 concentration in the headspace of the microcosms.

Among aerobic microcosms, the most predominant OTUs were OTU3261, OTU3800, and OTU6684. OTU3261 is 99% similar to a Thiobacillus species found in a study of carbon and sulfur cycling microbial community in oil sands tailings pond; OTU3800 is 96% similar to a Thiobacillus aquaesulis isolate; OTU6684 is 97% similar to a Limnobactoer thiooxidans isolate. When grouped together, these 3 OTUs make up 18% - 64% of each community. Similarity to relevant bacterium or identified species that are sulfur-oxidizing suggests these 3 highly enriched OTUs are associated with sulfur oxidation in the aerobic microcosms. OTU6467 was also present in more than half of the aerobic microcosms, and was found to be 99% similar to a Sideroxydans 61 species, which is an iron-oxidzing bacterium with properties that suggest a correlation to the presence of UMFC during enrichment. OTU3487, which made up 2% - 6% of 5 out of 8 aerobic communities, blasts 100% similar to Thiobacillus denitrificans. At least one of iron-oxidizing bacteria Sideroxydans (OTU6467) and Thiobacillus (OTU3487) is present in aerobic microcosms.

6.4 Second microcosms study with acid-leached UMFC

Following the findings from Objective 2 and Objective 3, it became apparent that a second round of enrichment was necessary to provide a better understanding of the effects of bioleaching on different mineral sulfides in UMFC. Objective 4 was a new microcosm study with acid-leached UMFC using bacterial cultures already acclimatized to UMFC. Preliminary results showed that the bacterial culture was successfully transferred to new conditions where acid-leached UMFC is the electron donor, and oxidative sulfide dissolution is continuously carried out by the bacterial communities.

6.5 Recommendations for Future Work

In this study so far, there is strong experimental evidence for microbially mediated oxidative dissolution of sulfide minerals in UMFC under nitrate-dependent and neutral pH conditions. To further investigate the effects of bioleaching on different sulfide phases and optimize Ni dissolution from pentlandite, it is recommended to continue the microcosm enrichment described in Objective 4. With the removal of environmental sediment, it is now possible to perform mineralogy analysis on leach residues to further our understanding of the degree of leaching and interactions among different sulfides: pyrrhotite, pyrite, and pentlandite. These microcosms were also set up to separate the biotic and abiotic effects of aerobic bioleaching, which will offer insights on the relative effectiveness of the working bacteria and abiotic oxidative leach.

The kinetics of this study did not offer a comparative alternative to current industrial methods for Ni extraction from sulfide sources. Extensive studies for scale-up or optimization for industrial application appear to be premature in the current global mining economy and is not 62 recommended as an immediate step in the pursuit of viable mining technology. However, as cost of material and energy increases in the future, as higher penalties for carbon emission become a limiting factor, the mining industry and our economic environment may gain more acceptance toward longer processing time, and this method could attract further interest as a feasible mining technology for extraction from low-grade ore or waste material recovery. As a recommended next step to bring this system to a larger scale, a packed column with 2-5 kg of UMFC and continuous circulation of leach solution containing bacteria, minimum nutrients, and electron acceptor is desirable to evaluate the interactions and parameters of bioleaching in a regime similar to heap leaching. In addition, it would be beneficial to isolate dominant cultures identified by the 16S rRNA gene analysis and test them for bioleaching capability, including the T. denitrificans strains that differ from ATCC 25259 with respect to the ability in using solid-phase sulfide minerals as electron donors.

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Appendices Appendix A: Edlab Multi Vitamins Stock Solution (MM7)

MM7: Vitamins (10,000x and 100x)

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

Adjust pH to 7.0 with 2 N NaOH. Make up to 1 Liter. Store in one or two mL aliquots frozen. Dilute the stock 1/100 to get 100x stock. Filter-sterilize 100x stock into sterile 160-mL serum bottle and sparge with sterile O2-free N2 for 15 minutes. Seal with sterile black butyl rubber stopper and crimp.

67

Appendix B: Thiobacillus denitrificans Growth Medium adapted from [33] Chemical List M.W. mg/L mM Na2S2O3Ÿ5H2O 248.19 5000 20.15 KNO3 101.10 2000 19.78 KH2PO4 136.09 2000 14.70 NH4Cl 53.49 1000 18.69 MgSO4Ÿ7H2O 246.48 800 3.25 MgCl2Ÿ6H2O 203.30 0 0.00 CaCl2Ÿ2H2O 147.02 735 5.00 NaHCO3 84.01 2520 30.00 mL/L Multi Vitamins Solution (Edlab MM7, Appendix A) 50 Trace element (#1 in [34]) 1 Selenite-tungstate (#4 in [34]) 1

68

Appendix C: Leaching medium Chemical List M.W. mg/L mM

KH2PO4 136.09 2000 14.70 NH4Cl 53.49 1000 18.69 MgCl2-6H2O 203.30 660 3.25 CaCl2Ÿ2H2O 147.02 735 5.00 NaHCO3 84.01 2520 30.00 mL/L Multi Vitamins Solution (Edlab MM7, Appendix A) 50 Trace element (#1 in [34]) 1 Selenite-tungstate (#4 in [34]) 1

69

Appendix D: Thompson Indigenous Samples Collection

D.1 Step-by-Step Sampling Procedures 1. Submerge the plastic bottles under water. (Use bottles sent from U of T.) 2. Scoop to collect a mixture of soil/sediment and water. Do NOT stir up the soil ahead of time; try to get a good chunk of the sediment. Fill the bottle full without any headspace. 3. Close the lids tight. Check for any leaks. 4. Fill the sample information form; please be as specific as possible. 5. Keep in a cooler with ice packs at 4 °C – 25 °C after sampling. Do not allow samples to freeze or go above 25 °C.

D.2 Transportation

Keep in a cooler with ice packs at 4 °C – 25 °C during transportation.

70

D.3 Sample Information

71

72

Appendix E: Protocol for setting up microcosms using anaerobic glovebag

The microcosms are prepared under the fumehood in a glove bag to develop an anaerobic environment within the microcosm bottles. The anaerobic environment is made of 80% nitrogen and 20% carbon dioxide. Here are the steps undertaken to set-up the microcosms [SIREM]:

E.1 Preparations:

• Prepare approximately 100 mL of media per bottle (60 bottles * 100 mL = 6 L) • Purge the glove bag twice with an 80% nitrogen 20% carbon dioxide mixture. o Attach one end of the hose to the gas cylinder and attach the other end to a hole created in the glovebag sealed with tape o After the first purge, discard of the gas (temporarily untape the seal) and re-fill the glovebag with the gas mixture • Sterilize all items entering the glove bag. o Autoclave funnel, measuring spoon, mixing utensils, tray, Erlenmeyer flask, graduated cylinder (100 mL), measuring spoon (15 or 30 mL) o Clean mininert caps with soap and water, followed by wiping with 70% ethanol o Collect latex gloves, paper towels, sterile wipes, permanent markets, scissors, crimper o Wash all bottles with soap and water, then rinse until no visible residue remains with tap water, then rinse three times with distilled water. Obtain one autoclavable bin for each set of 20 bottles to be set up and line with aluminum foil. Place each set of 20 bottles in an autoclave bag, seal with tape, place in an autoclavable bin and autoclave. • Place autoclaved items in glovebag as soon as possible. • Add the medium, substrates and inocula to the glove bag, and then seal the bag with tape.

E.2 Setup:

• Setup one stage at a time (medium, substrate, inoculum)

Medium:

• Pour ~500 mL of media into a large beaker, topping up as necessary, attempting to keep the solution as homogeneous as possible to avoid large amounts of FeS. • Add 60 mL of media into each bottle using a graduated cylinder (or a 60 mL syringe) 73

• Remove empty media bottles and beaker carefully from the bag

Inoculum:

• Mix inoculum thoroughly in a sterile autoclavable bin. • Start adding inoculum 15 mL at a time to each bottle (or 30 mL if enough inoculum available) to each bottle. • Cap and crimp each bottle.

Substrate:

• Add prepared samples of substrate to the designated bottles after being removed from glovebag.

E.3 Cleanup:

• Re-seal, remove and refrigerate remaining inoculum. • Remove all bottles and rinse surface ensuring substrate containing samples are still labelled. • Remove all remaining reusable materials, place in autoclave bin to be sterilized and cleaned. • Pack up portable glove bag with all disposable components and dispose in orange wire garbage (to be autoclaved).

74

Appendix F: Leaching medium II Chemical List M.W. mg/L mM

KH2PO4 136.09 272 2.00 K2HPO4 174.18 348 2.00 NH4Cl 53.49 1000 18.69 CaCl2Ÿ2H2O 147.02 735 5.00 NaHCO3 84.01 2520 30.00 mL/L Multi Vitamins Solution (Edlab MM7, Appendix A) 50 Trace element (#1 in [34]) 1 Selenite-tungstate (#4 in [34]) 1

75

Appendix G: Supplementary Data

Table 19: Tails first enrichment complete data - Sterile

Tails Sterile Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate (mM)

Tails Tails Tails Ni std Tails Tails Tails Tails Tails Tails Mg std Tails Tails Tails Tails Tails Tails Sample days 1 2 3 ave dev 1 2 3 1 2 3 ave dev 1 2 3 1 2 3 Tails-1 7 1.39 1.38 1.19 1.32 0.11 6.67 6.71 6.67 113.77 118.47 110.32 114.19 4.09

Tails-2 11 1.40 1.33 1.25 1.33 0.08 6.65 6.65 6.71 116.87 115.92 114.37 115.72 1.26 4.17 4.17 4.17 1.26 1.26 1.26

Tails-3 70 0.49 0.39 0.17 0.35 0.16 7.12 7.07 7.06 144.52 140.78 144.42 143.24 2.13 5.00 5.92 6.78 0.01 0.01 0.01

Tails-4 75 0.45 0.36 0.18 0.33 0.14 7.12 7.15 7.16 140.74 140.54 146.29 142.52 3.26 5.29 6.21 6.03 0.03 0.04 0.07 Tails-6 92 0.46 0.34 0.22 0.34 0.12 6.93 6.95 6.82 151.35 151.15 155.95 152.81 2.72 5.10 6.20 7.37 0.14 0.17 0.13 Tails-7 98 0.57 0.43 0.25 0.42 0.16 6.76 6.81 6.80 139.43 137.28 143.73 140.14 3.28 5.00 6.33 8.32 0.03 0.04 0.03 Tails-8 109 0.85 0.72 0.48 0.68 0.19 6.66 6.69 6.69 166.86 182.41 172.10 173.79 7.91 6.62 6.36 8.08 0.05 0.01 0.04 Tails-10 147 0.65 0.49 0.40 0.51 0.13 6.68 6.64 6.58 194.50 197.75 208.80 200.35 7.50 5.34 4.83 6.68 0.06 0.03 0.04 Tails-11 162 0.60 0.48 0.36 0.48 0.12 6.68 6.67 6.67 198.22 200.58 99.61 166.14 57.62 4.47 4.89 6.00 0.03 0.03 0.03 Tails-15 172 0.68 0.59 0.45 0.57 0.12 6.64 6.65 6.62 203.36 221.26 204.71 209.77 9.97 4.80 5.57 5.59 0.04 0.03 0.02 Tails-16 178 1.17 0.96 1.26 1.13 0.15 6.38 6.36 6.37 213.26 218.21 211.26 214.24 3.58 4.67 4.54 5.81 2.97 3.73 2.18 Tails-19 216 2.54 1.85 1.87 2.09 0.39 6.51 6.53 6.56 225.88 224.53 222.48 224.30 1.71 5.08 5.44 6.14 2.25 3.37 0.48 Tails-21 239 2.63 1.80 1.91 2.11 0.45 6.45 6.54 6.56 233.26 238.41 235.96 235.88 2.58 4.68 5.23 6.36 2.03 2.72 4.98 Tails-32 305 3.03 2.06 2.46 2.52 0.49 6.63 6.74 6.68 243.21 269.31 243.46 251.99 15.00 4.91 7.07 6.53 6.19 5.28 3.71

Tails-43 409 1.29 0.33 1.50 1.04 0.62 6.84 6.90 6.84 238.25 251.81 235.84 241.97 8.61 6.37 6.70 8.12 7.40 8.13 3.20

76

Table 20: Dam first enrichment complete data - Sterile

DAM Sterile

Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate (mM)

DAM DAM DAM Ni std DAM DAM DAM DAM DAM DAM Mg std DAM DAM DAM DAM DAM DAM Sample days 1 2 3 ave dev 1 2 3 1 2 3 ave dev 1 2 3 1 2 3 DAM-1 7 0.62 0.52 0.64 0.59 0.06 6.88 6.89 6.94 54.46 46.34 48.50 49.77 4.21

DAM-2 11 0.62 0.54 0.66 0.61 0.06 6.96 6.91 6.94 54.50 45.66 50.33 50.16 4.42 2.73 2.96 3.42 1.61 1.03 0.85 DAM-3 70 0.90 0.47 0.19 0.52 0.36 7.09 7.07 7.03 64.90 51.70 69.34 61.98 9.18

DAM-4 75 0.88 0.42 0.18 0.49 0.36 7.11 7.08 7.03 64.97 51.71 69.33 62.00 9.18 2.98 3.11 3.80 0.39 0.03 0.04 DAM-5 82 0.81 0.30 0.10 0.40 0.37 7.10 7.08 7.03 63.46 49.71 71.56 61.57 11.05 3.90 4.07 4.73 0.18 0.14 0.11 DAM-6 93 1.14 0.38 0.18 0.57 0.51 6.80 6.77 6.79 66.42 55.65 75.19 65.75 9.78 5.16 5.04 5.69 0.09 0.05 0.03 DAM-7 99 1.55 0.46 0.27 0.76 0.69 6.72 6.66 6.68 75.66 61.09 80.22 72.32 9.99 2.45 4.15 4.75 0.02 0.03 0.04 DAM-8 109 1.32 0.65 0.52 0.83 0.43 6.69 6.58 6.56 76.20 69.39 89.23 78.27 10.08 2.21 3.99 4.49 0.04 0.03 0.03 DAM-10 148 1.28 0.89 0.78 0.98 0.26 6.70 6.60 6.58 102.43 85.29 102.80 96.84 10.00 2.03 3.49 3.90 0.03 0.03 0.03 DAM-11 163 1.56 1.02 0.77 1.12 0.40 6.57 6.55 6.52 110.17 90.59 104.77 101.84 10.11 2.02 3.39 4.05 0.03 0.02 0.05 DAM-15 173 1.78 1.16 0.87 1.27 0.46 6.56 6.52 6.64 117.61 94.17 106.51 106.09 11.73 2.06 3.53 4.00 3.47 0.02 0.02 DAM-16 179 2.56 1.17 1.03 1.59 0.85 6.53 6.55 6.53 114.91 96.67 111.66 107.74 9.73 2.04 3.64 4.26 3.50 3.44 2.49 DAM-19 217 4.72 2.23 2.76 3.24 1.31 6.48 6.53 6.59 125.48 107.23 121.58 118.10 9.61 2.17 3.64 2.95 3.54 3.29 2.16 DAM-21 239 5.02 2.48 3.26 3.59 1.30 6.49 6.50 6.57 127.11 111.41 125.06 121.19 8.53 2.21 3.88 3.18 3.53 3.35 1.93 DAM-32 305 6.29 3.55 5.14 4.99 1.38 6.58 6.59 6.61 128.46 117.51 129.21 125.06 6.55 2.19 3.74 4.01 3.13 2.88 5.66

DAM-43 411 5.87 2.87 4.85 4.53 1.53 6.57 6.58 6.63 117.72 107.15 121.12 115.33 7.28 2.67 4.68 5.23 3.70 3.31 6.86

77

Table 21: Exp first enrichment complete data - Sterile

EXP Sterile Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate (mM)

std Mg std Sample days EXP1 EXP2 EXP3 Ni ave EXP1 EXP2 EXP3 EXP1 EXP2 EXP3 EXP1 EXP2 EXP3 EXP1 EXP2 EXP3 dev ave dev EXP-1 7 0.78 0.86 0.98 0.87 0.10 6.71 6.67 6.75 72.74 62.08 74.94 69.92 6.88

EXP-2 11 0.79 0.88 0.97 0.88 0.09 6.83 6.82 6.83 69.24 63.32 71.38 67.98 4.18 0.62 0.60 0.76 2.44 2.20 2.24 EXP-3 70 1.00 1.05 0.14 0.73 0.51 7.17 7.12 7.27 80.74 77.05 112.96 90.25 19.75 0.96 0.73 1.88 0.66 0.79 0.01 EXP-4 75 0.99 1.02 0.14 0.72 0.50 7.17 7.13 7.17 80.79 76.77 114.69 90.75 20.83 0.81 0.66 1.56 0.55 0.70 0.04

EXP-6 93 1.17 1.16 0.08 0.80 0.63 6.89 6.84 6.82 81.56 74.98 113.30 89.94 20.49 0.87 0.86 2.06 0.55 0.81 0.00

EXP-7 99 1.54 1.48 0.09 1.04 0.82 6.62 6.64 6.62 89.11 82.19 123.43 98.24 22.08 1.03 0.89 1.77 0.60 0.78 0.03 EXP-8 109 1.75 1.66 0.11 1.17 0.92 6.68 6.64 6.62 99.30 87.07 127.67 104.68 20.83 1.33 1.09 2.38 0.36 0.63 0.07 EXP-10 148 1.92 2.07 0.17 1.39 1.06 6.68 6.64 6.58 115.55 100.69 138.65 118.30 19.13 1.05 0.84 1.80 0.03 0.03 0.02 EXP-11 163 2.21 2.35 0.09 1.55 1.27 6.55 6.54 6.48 113.29 101.31 134.58 116.39 16.85 0.89 0.76 1.56 0.03 0.03 0.02 EXP-15 173 2.67 2.61 0.16 1.81 1.43 6.57 6.55 6.52 124.01 104.21 143.51 123.91 19.65 0.94 0.82 1.72 0.03 0.07 0.04 EXP-19 216 5.23 5.13 2.93 4.43 1.30 6.55 6.51 6.55 136.33 117.58 152.73 135.55 17.59 1.34 1.07 2.16 3.88 3.23 2.21 EXP-21 239 5.42 5.46 3.40 4.76 1.18 6.47 6.48 6.52 139.16 120.36 155.56 138.36 17.61 1.42 1.21 2.23 3.91 3.38 2.19 EXP-32 305 5.90 6.15 4.85 5.63 0.69 6.53 6.53 6.61 145.51 125.01 158.86 143.13 17.05 1.39 1.12 2.07 3.45 2.78 5.71

EXP-43 411 4.17 4.30 5.14 4.54 0.53 6.62 6.65 6.57 141.83 121.09 153.90 138.94 16.60 1.63 1.35 2.62 4.00 3.15 7.09

78

Table 22: TSRA first enrichment complete data - Sterile

TSRA Sterile Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate (mM)

TSRA TSRA TSRA std TSRA TSRA TSRA TSRA TSRA TSRA Mg std TSRA TSRA TSRA TSRA TSRA TSRA Sample days Ni ave 1 2 3 dev 1 2 3 1 2 3 ave dev 1 2 3 1 2 3 TSRA-1 7 0.84 0.85 1.14 0.94 0.17 6.78 6.82 6.89 64.52 69.69 65.59 66.60 2.73

TSRA-2 11 0.89 0.86 1.20 0.98 0.19 6.88 6.90 6.90 66.15 67.90 69.57 67.87 1.71 0.43 0.46 0.57 2.58 2.62 3.18 TSRA-3 70 1.76 1.65 1.69 1.70 0.05 7.08 7.09 7.04 81.83 86.39 78.05 82.09 4.17

TSRA-4 75 1.73 1.59 1.37 1.56 0.18 7.14 7.12 7.09 83.68 85.30 75.76 81.58 5.10 0.50 0.53 2.16 1.34 1.41 0.06 TSRA-6 93 2.26 2.09 1.58 1.98 0.35 6.87 6.88 6.82 82.59 84.93 79.75 82.42 2.60 0.79 0.78 2.96 1.71 1.72 0.05 TSRA-7 99 3.12 3.02 2.85 3.00 0.14 6.68 6.69 6.62 90.20 93.39 86.64 90.07 3.38 0.72 0.77 2.92 1.68 1.70 0.04 TSRA-8 109 2.30 3.90 5.00 3.73 1.36 6.77 6.64 6.57 87.93 97.40 98.53 94.62 5.82 0.46 0.70 2.50 0.46 1.23 0.02 TSRA-10 148 2.85 5.98 6.58 5.14 2.00 6.71 6.61 6.62 113.70 121.80 116.30 117.27 4.14 0.45 0.69 2.35 0.42 1.00 0.02 TSRA-11 163 3.22 6.49 7.03 5.58 2.06 6.60 6.56 6.56 115.54 117.31 113.31 115.39 2.00 0.43 0.74 2.44 0.40 0.98 0.04 TSRA-15 173 3.42 7.68 7.63 6.24 2.45 6.64 6.57 6.61 119.46 125.71 120.26 121.81 3.40 0.67 1.03 2.95 3.82 4.85 3.55 TSRA-19 216 9.27 14.83 17.15 13.75 4.05 6.53 6.53 6.54 125.03 130.38 127.28 127.56 2.69 0.66 0.86 2.86 3.66 4.42 3.34 TSRA-21 239 9.66 15.76 19.27 14.90 4.86 6.53 6.51 6.52 129.86 135.81 134.86 133.51 3.20 0.70 1.05 2.93 3.75 4.72 3.28 TSRA-32 305 11.26 18.48 22.59 17.44 5.74 6.58 6.57 6.58 133.26 137.21 130.56 133.68 3.34 0.66 1.01 2.86 3.47 4.31 2.75

TSRA-43 411 8.84 19.52 22.99 17.12 7.38 6.55 6.59 6.60 121.02 130.10 119.65 123.59 5.68 0.79 1.26 3.71 4.29 5.36 2.87

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Table 23: Tails first enrichment complete data - Aerobic

Tails Aerobic Ni (ppm) pH Mg (ppm) sulfate (mM)

pH std Mg std Sample days Tails7 Tails8 Tails9 Tails7 Tails8 Tails9 Tails7 Tails8 Tails9 Tails7 Tails8 Tails9 ave. dev ave. dev Tails-1 7 0.56 0.55 1.17 6.86 6.73 6.71 6.77 0.08 100.99 99.31 99.54 99.94 0.91 4.21 4.55 4.78 Tails-2 11 1.14 0.35 1.52 6.92 6.97 6.87 6.92 0.05 111.17 109.07 108.22 109.49 1.52 5.97 4.93

Tails-3 70 0.17 0.13 0.18 7.33 7.39 7.30 7.34 0.05 163.96 143.27 146.37 151.20 11.16 5.75 3.75 3.73 Tails-4 75 0.22 0.17 0.33 7.39 7.51 7.42 7.44 0.06 163.54 144.29 148.34 152.06 10.15

Tails-6 93 0.36 0.30 0.78 7.00 6.91 6.98 6.96 0.05 182.75 154.50 154.35 163.86 16.35 6.41 3.73 6.10 Tails-7 99 0.42 0.27 0.94 6.82 6.76 6.62 6.73 0.10 203.03 165.83 171.18 180.01 20.11 6.64 3.77 6.11 Tails-8 109 0.33 0.24 0.89 6.77 6.71 6.57 6.68 0.10 229.27 178.49 183.48 197.08 27.99 6.24 3.41 5.92 Tails-10 148 5.14 4.93 9.63 6.83 6.79 6.80 6.81 0.02 266.20 203.35 204.20 224.58 36.04 7.98 4.97 6.93 Tails-11 163 4.47 5.81 8.54 6.88 6.83 6.79 6.83 0.05 280.76 207.16 206.24 231.39 42.76 8.60 5.57 6.66 Tails-15 173 3.63 3.86 6.94 6.85 6.82 6.77 6.81 0.04 305.11 225.16 220.36 250.21 47.61 8.13 5.39 7.68 Tails-16 179 4.31 4.45 9.24 6.75 6.66 6.65 6.69 0.06 314.21 233.31 230.51 259.34 47.54 8.92 5.90 8.07 Tails-19 216 5.05 7.37 11.94 6.86 6.88 6.84 6.86 0.02 341.48 244.63 242.18 276.10 56.64 11.64 7.96 10.39 Tails-25 273 1.97 4.96 11.99 6.48 6.43 6.45 6.45 0.03 418.12 307.12 292.17 339.14 68.81 10.27 5.06 10.08 Tails-34 307 4.76 11.23 22.88 6.78 6.67 6.59 6.68 0.10 396.06 281.26 269.51 315.61 69.92 8.95 7.54 9.28 Tails-42 390 1.54 6.64 17.91 6.76 6.77 6.71 6.75 0.03 417.39 285.26 285.67 329.44 76.17 18.60 14.57 19.00

Tails-43 409 2.15 8.93 21.73 6.88 6.87 6.84 6.86 0.02 409.61 276.80 275.78 320.73 76.98 19.34 16.00 19.52

80

Table 24: Dam first enrichment complete data - Aerobic

DAM Aerobic Ni (ppm) pH Mg (ppm) sulfate (mM)

Sample days DAM7 DAM8 DAM9 DAM7 DAM8 DAM9 pH ave. std dev DAM7 DAM8 DAM9 Mg ave. std dev DAM7 DAM8 DAM9 DAM-1 7 0.37 0.34 0.45 6.88 6.91 6.92 6.90 0.02 43.86 43.16 46.15 44.39 1.56 1.94 1.98

DAM-2 11 0.58 0.45 0.65 7.00 7.03 7.00 7.01 0.02 46.65 47.54 49.26 47.82 1.33 2.23 2.00 2.05 DAM-3 70 0.19 0.14 0.18 7.16 7.22 7.15 7.18 0.04 69.87 79.55 75.67 71.32 4.87

DAM-4 75 0.20 0.14 0.32 7.42 7.48 7.50 7.47 0.04 63.98 70.29 68.10 64.86 3.20 2.34 2.02 1.40 DAM-5 82 0.43 0.37 0.77 7.00 6.96 6.88 6.95 0.06 65.10 76.54 72.54 68.24 5.80

DAM-6 92 0.61 0.53 0.92 6.63 6.68 6.69 6.67 0.03 73.69 86.31 79.31 75.39 6.32 3.88 3.70 3.99 DAM-7 98 0.91 0.59 1.15 6.64 6.64 6.65 6.64 0.01 81.19 91.03 83.86 80.12 5.09 5.21 4.79 5.77 DAM-8 109 0.77 0.62 1.34 6.68 6.67 6.65 6.67 0.02 75.17 104.38 94.01 85.16 14.81 2.65 4.25 4.68 DAM-10 147 6.11 10.06 13.27 6.52 6.62 6.61 6.58 0.06 98.27 129.15 116.95 105.23 15.55 4.53 6.71 7.30 DAM-11 162 10.40 14.26 18.48 6.53 6.63 6.63 6.60 0.06 103.42 132.86 122.83 109.38 14.97 5.15 7.32 7.75 DAM-15 172 15.46 12.87 18.05 6.78 6.73 6.71 6.74 0.04 107.71 141.56 124.91 113.72 16.93 6.08 7.82 7.98 DAM-16 178 18.38 16.21 23.50 6.70 6.71 6.69 6.70 0.01 111.81 148.96 131.86 118.95 18.59 6.60 8.35 8.58 DAM-19 217 23.64 24.54 30.77 6.69 6.86 6.85 6.80 0.10 115.98 156.63 135.88 123.49 20.33 9.15 11.37 11.04 DAM-25 273 30.79 35.90 46.76 6.60 6.56 6.56 6.57 0.02 139.07 185.92 164.42 146.43 23.45 9.89 12.41 11.94 DAM-34 307 37.19 45.20 55.45 6.55 6.63 6.62 6.60 0.04 126.56 172.11 145.71 143.46 22.87 9.58 13.03 12.66 DAM-42 390 29.51 36.51 46.81 6.72 6.76 6.71 6.73 0.03 127.09 172.37 149.37 149.61 22.65 17.33 21.27 20.70

DAM-43 409 35.09 40.29 52.17 6.63 6.76 6.71 6.70 0.07 120.44 163.24 139.66 141.12 21.44 17.01 20.77 19.06

81

Table 25: Exp first enrichment complete data - Aerobic

EXP Aerobic Ni (ppm) pH Mg (ppm) sulfate (mM)

pH std Mg std Sample days EXP7 EXP8 EXP9 EXP7 EXP8 EXP9 EXP7 EXP8 EXP9 EXP7 EXP8 EXP9 ave. dev ave. dev EXP-1 7 0.44 0.47 0.34 6.89 6.86 6.89 6.88 0.02 49.98 46.65 48.39 48.34 1.67 0.16 0.24 0.18

EXP-2 11 1.22 1.03 0.71 7.03 7.07 7.06 7.05 0.02 62.63 55.40 61.81 58.06 3.96 0.35 0.31 0.18 EXP-3 70 0.19 0.20 0.14 7.39 7.30 7.30 7.33 0.05 107.69 89.39 105.84 91.34 10.07 0.18 0.98 0.01 EXP-4 75 0.37 0.46 0.20 7.55 7.52 7.52 7.53 0.02 106.89 87.37 104.99 90.34 10.77 0.21 0.88 0.26 EXP-5 82 2.24 1.94 0.84 7.07 6.95 6.89 6.97 0.09 100.88 88.60 103.56 88.61 7.98

EXP-6 93 1.15 1.97 0.69 6.70 6.70 6.69 6.70 0.01 111.35 95.80 107.15 94.46 8.04 0.71 1.61 0.41 EXP-7 99 1.29 2.05 0.63 6.60 6.57 6.62 6.60 0.03 119.93 102.18 121.23 102.23 10.64 0.85 1.63 0.49 EXP-8 109 1.32 2.42 0.61 6.61 6.61 6.60 6.61 0.01 124.77 107.70 120.66 104.96 8.91 1.05 1.96 0.60 EXP-10 148 19.62 17.60 8.73 6.83 6.79 6.80 6.81 0.02 142.45 123.85 136.65 118.46 9.52 3.29 3.90 2.61 EXP-11 163 21.93 19.66 10.87 6.76 6.68 6.67 6.70 0.05 145.26 129.88 139.05 121.52 7.73 3.90 4.43 3.12 EXP-15 173 31.94 27.06 15.85 6.79 6.70 6.75 6.75 0.05 154.56 132.46 150.11 127.67 11.69 4.95 5.51 4.42 EXP-16 179 38.02 31.42 18.05 6.70 6.66 6.64 6.67 0.03 152.11 136.26 152.66 128.68 9.31 4.76 4.46

EXP-19 216 65.08 47.63 27.52 6.84 6.84 6.82 6.83 0.01 162.53 150.78 158.93 137.21 6.02 8.34 8.28 7.22 EXP-25 273 90.11 49.49 35.18 6.28 6.47 6.49 6.41 0.12 189.02 159.37 187.52 154.33 16.70 10.78 10.60 9.47 EXP-34 307 115.73 58.48 41.10 6.33 6.52 6.52 6.46 0.11 182.41 150.26 175.06 146.79 16.85 11.01 10.65 10.25 EXP-42 390 6.62 6.66 6.64 6.64 0.02 170.40 140.78 161.21 157.46 15.16 17.24 15.09 14.65

EXP-43 409 150.72 73.90 54.56 6.62 6.64 6.62 6.63 0.01 186.92 138.41 157.59 160.97 24.43 17.64 15.62 15.41

82

Table 26: TSRA first enrichment complete data - Aerobic

TSRA Aerobic Ni (ppm) pH Mg (ppm) sulfate (mM)

TSRA TSRA TSRA TSRA TSRA TSRA pH std TSRA TSRA TSRA Sample days TSRA7 TSRA8 TSRA9 Mg ave std dev 7 8 9 7 8 9 ave dev 7 8 9 TSRA-1 7 1.00 1.02 0.55 6.97 6.96 6.98 6.97 0.01 64.99 58.31 64.44 62.58 3.71 0.44 0.35 0.25 TSRA-2 11 1.78 3.15 1.58 7.01 6.98 7.02 7.00 0.02 77.41 64.69 72.49 71.53 6.41 0.51 0.35 0.31 TSRA-3 70 0.54 5.70 0.66 7.20 6.93 7.11 7.08 0.14 103.45 84.48 95.43 94.45 9.52 0.96 1.69 0.97 TSRA-4 75 1.25 4.52 1.28 7.53 7.28 7.42 7.35 0.13 102.89 81.81 95.19 93.30 10.67 0.91 1.46 0.89 TSRA-5 82 6.75 6.72 6.81 6.76 0.05

TSRA-6 93 7.27 18.27 10.78 6.55 6.56 6.61 6.57 0.03 107.80 82.73 99.47 96.66 12.77 2.12 3.16 2.45 TSRA-7 99 10.16 22.49 12.87 6.56 6.50 6.62 6.56 0.06 115.03 89.30 107.18 103.83 13.18 2.50 3.39 2.68 TSRA-8 109 12.05 27.07 7.72 6.47 6.50 6.51 6.49 0.02 115.23 89.43 82.60 95.75 17.21

TSRA-10 148 66.22 67.71 42.89 6.31 6.37 6.09 6.26 0.15 133.60 100.18 108.00 113.93 17.48 6.75 6.52 3.87 TSRA-11 163 70.86 74.70 54.91 6.29 6.39 6.22 6.30 0.09 133.00 99.40 109.07 113.82 17.29 7.20 7.05 4.59 TSRA-15 173 76.88 89.93 57.53 6.34 6.34 6.35 6.34 0.01 135.26 111.56 109.96 118.92 14.17 8.10 7.99 4.99 TSRA-16 179 82.26 90.02 59.73 6.44 6.52 6.45 6.47 0.04 140.76 107.61 112.76 120.37 17.84 8.86 8.53 5.31 TSRA-19 216 115.18 125.51 96.34 6.44 6.53 6.30 6.42 0.12 152.38 125.83 117.13 131.78 18.36 12.69 12.79 8.43 TSRA-25 273 158.41 154.71 165.40 6.34 6.34 6.30 6.33 0.02 201.87 176.67 156.87 178.47 22.55 15.37 16.02 11.09 TSRA-34 307 171.34 171.88 171.58 6.23 6.09 6.10 6.14 0.08 192.86 189.91 134.01 172.26 33.16 16.88 17.11 11.42

TSRA-42 390 170.08 172.57 161.27 6.65 6.74 6.70 6.70 0.05 174.15 163.27 125.13 154.18 25.74 21.84 22.43 16.32

83

Table 27: Tails first enrichment complete data - Denitrifying

Tails Denitrifying Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate additions

Tails Tails Tails Tails Tails Tails pH std Tails Tails Tails Mg std Tails Tails Tails Tails Tails Tails Sample days 10 11 12 10 11 12 ave. dev 10 11 12 ave. dev 10 11 12 10 11 12 Tails-1 7 1.90 1.33 1.22 6.76 6.69 6.65 6.70 0.06 101.89 112.19 101.83 95.19 5.97 5.17 4.88 4.88 + + + Tails-2 11 2.55 1.29 1.20 6.91 6.90 6.98 6.93 0.04 104.92 113.17 106.92 97.76 4.30 6.36 6.00 5.62

Tails-3 70 0.40 0.14 0.15 7.00 7.20 7.25 7.15 0.13 144.10 150.43 140.61 127.64 4.98 7.50 6.11 6.77

Tails-4 75 0.50 0.23 0.22 6.94 7.11 7.11 7.05 0.10 144.74 148.99 140.39 127.35 4.30 7.89 5.99 6.09 + + + Tails-6 92 1.49 0.85 0.99 6.75 6.76 6.79 6.77 0.02 147.60 152.15 142.40 129.51 4.88 10.93 8.80 8.67

Tails-7 98 1.68 0.94 1.14 6.66 6.68 6.66 6.67 0.01 164.83 161.93 150.88 138.95 7.36 13.15 10.55 10.45 + + + Tails-8 109 3.58 2.84 3.01 6.58 6.57 6.57 6.57 0.01 174.49 175.71 163.55 148.88 6.69 12.73 11.59 10.81

122 + + +

132 + + +

Tails-9 138 5.48 4.92 4.95 6.53 6.55 6.53 6.54 0.01 223.66 231.16 195.01 185.36 19.08 15.94 12.41 13.90

Tails-10 147 4.92 4.11 4.95 6.61 6.60 6.62 6.61 0.01 210.35 213.85 197.55 178.27 8.58 18.11 15.68 16.35 + + + 156 + + +

Tails-11 162 9.07 7.74 9.00 6.59 6.59 6.56 6.58 0.02 219.90 226.05 200.67 184.82 13.24 21.45 19.18 20.09 + + + Tails-12 164 10.61 9.14 10.39 6.63 6.62 6.58 6.61 0.03 224.77 230.10 209.71 189.87 10.58 20.53 17.09 16.95

Tails-13 166 12.82 11.06 12.31 6.72 6.66 6.60 6.66 0.06 256.60 257.52 239.41 214.10 10.20 17.60 18.76 19.55

Tails-14 168 13.23 11.68 12.39 6.62 6.65 6.63 6.63 0.02 254.95 260.49 236.16 213.48 12.75 19.90 16.48 18.45

Tails-15 172 12.53 9.92 11.65 6.58 6.63 6.62 6.61 0.03 233.51 231.06 216.81 194.45 9.02 25.07 22.59 21.58 + + + Tails-16 178 14.98 13.06 13.49 243.36 241.61 219.26 200.43 13.44 27.36 24.73 21.95 + +

Tails-18 215 6.43 6.42 6.42 6.42 0.01 272.68 257.33 225.48 213.74 24.08 26.47 26.59 26.18 + + +

Tails-19

Tails-20 225 14.49 12.41 20.38 6.68 6.69 6.64 6.67 0.03 258.13 260.93 228.18 211.94 18.15 28.94 27.71 27.99 + +

Tails-21 239 23.35 20.60 24.59 6.60 6.62 6.61 6.61 0.01 271.71 263.86 236.21 218.53 18.65 38.86 35.90 32.65 + + + Tails-22 251 25.25 24.52 25.93 6.63 6.64 6.62 6.63 0.01 238.30 248.10 216.80 200.15 16.01 32.46 29.70 26.90

Tails-23 262 30.99 30.80 30.45 6.63 6.66 6.62 6.64 0.02 249.30 235.80 224.15 201.86 12.59 29.43 26.93 26.76

Tails-24 269 33.30 32.89 31.55 6.53 6.58 6.60 6.57 0.04 247.35 244.90 214.45 200.81 18.33 29.15 30.22 27.30 + +

84

Tails-25 273 +

Tails-26 276 37.28 40.02 36.51 6.56 6.56 6.55 6.56 0.01 289.72 293.17 255.07 236.53 21.07 34.20 31.35 26.84

Tails-27 282 38.94 42.20 37.06 6.55 6.56 6.55 6.55 0.01 284.52 291.02 244.82 231.53 25.01 33.71 31.85 28.02

Tails-28 289 41.48 45.99 40.24 6.54 6.56 6.55 6.55 0.01 259.36 266.51 230.91 214.56 18.83 33.08 32.46 29.25

Tails-29 297 43.54 48.41 43.76 6.55 6.58 6.55 6.56 0.02 261.91 262.46 240.61 217.09 12.46 35.91 32.43 30.34

Tails-30 300

Tails-31 303 47.36 51.85 49.38 6.51 6.53 6.52 6.52 0.01 271.91 260.36 208.81 209.23 33.60 33.74 31.87 29.90 + +

Tails-32 305

Tails-35 312 50.88 55.44 51.66 6.67 6.63 6.62 6.64 0.03 30.62 30.62 27.16

Tails-36 318 56.53 60.78 55.99 6.61 6.59 6.56 6.59 0.03 217.83 221.20 200.02 213.02 11.38 35.38 31.29 28.17

Tails-37 323 56.91 61.67 56.76 6.55 6.56 6.50 6.54 0.03 223.70 227.85 204.32 218.62 12.56 34.32 31.31 28.50 +

Tails-38 331 57.74 64.35 55.82 6.59 6.58 6.58 6.58 0.01 221.96 227.91 202.35 217.41 13.38 35.89 31.78 29.61

Tails-39 349 62.53 69.51 62.52 6.69 6.62 6.63 6.65 0.04 229.31 233.22 208.33 223.62 13.39 35.75 31.61 29.07

Tails-40 359 6.62 6.62 6.61 6.62 0.01 34.84 36.78 + +

Tails-41 377 83.55 90.80 83.80 6.61 6.62 6.61 6.61 0.01 239.01 238.91 215.38 231.10 13.61 37.09 35.95 31.85

Tails-42 391 6.56 6.59 6.60 6.58 0.02 250.12 249.29 224.60 241.33 14.50 45.81 41.41 35.84

Tails-43 409 81.19 89.25 83.50 6.69 6.68 6.67 6.68 0.01 45.33 40.47 37.66 +

85

Table 28: Dam first enrichment complete data - Denitrifying

DAM Denitrifying Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate additions

DAM DAM DAM DAM DAM DAM pH std DAM DAM DAM Mg std DAM DAM DAM DAM DAM DAM Sample days 10 11 12 10 11 12 ave. dev 10 11 12 ave. dev 10 11 12 10 11 12 DAM-1 7 0.41 0.47 0.50 6.87 6.89 6.88 6.88 0.01 50.67 51.97 48.39 50.03 1.81 2.63 2.66 2.51 + + + DAM-2 11 0.66 0.66 0.60 7.09 7.03 6.99 7.04 0.05 50.59 53.09 51.13 51.07 1.31 3.39 3.11

DAM-3 70 0.23 0.14 0.14 7.09 7.30 7.26 7.22 0.11 75.32 91.70 86.35 74.31 8.36

DAM-4 75 0.18 0.11 0.10 7.11 7.36 7.34 7.27 0.14 70.77 86.63 81.32 70.81 8.08 3.63 2.05 1.84 + + + DAM-5 82 0.46 0.34 0.26 7.18 7.25 7.20 7.21 0.04 69.65 91.17 86.54 72.78 11.33

DAM-6 93 1.49 1.06 0.75 6.92 6.94 6.94 6.93 0.01 77.96 100.79 93.84 78.75 11.70 7.13 5.28 4.40

DAM-7 99 1.99 1.32 1.04 6.70 6.77 6.74 6.74 0.04 84.85 105.83 102.28 83.71 11.23 9.62 6.79 5.69 + + + DAM-8 109 4.50 2.37 2.83 6.58 6.78 6.65 6.67 0.10 98.69 84.20 111.96 85.38 13.88 11.43 1.58 7.29

112 + + +

132 + + +

DAM-9 138 8.52 5.83 6.52 121.11 98.75 138.61 100.95 19.98 18.09 5.39 13.09

DAM-10 148 8.13 7.54 5.95 6.48 6.55 6.56 6.53 0.04 119.15 99.13 133.65 99.25 17.33 16.89 5.88 12.90 + +

156 + +

DAM-11 163 12.12 9.39 9.54 6.53 6.67 6.62 6.61 0.07 124.86 101.11 137.53 102.13 18.49 21.13 6.39 16.41 + +

DAM-12 164 14.38 11.31 12.14 6.57 6.51 6.57 6.55 0.03 130.49 106.91 144.46 106.53 18.97 17.88 5.69 16.76

DAM-13 166 16.26 13.17 15.09 6.61 6.56 6.56 6.58 0.03 146.79 119.16 161.30 117.54 21.41 22.46 5.94 17.10

DAM-14 168 16.02 13.38 16.04 6.60 6.57 6.57 6.58 0.02 147.74 116.28 163.76 117.83 24.16 22.16 6.57 18.39

DAM-15 173 12.41 13.29 13.19 6.62 6.62 6.62 6.62 0.00 130.16 108.16 143.71 106.50 17.94 23.83 7.16 18.99 + +

DAM-16 179 16.25 16.55 16.54 6.57 6.58 6.60 6.58 0.02 136.81 115.36 152.41 111.91 18.60 27.20 8.26 22.11 + + + DAM-18 214 13.35 16.98 13.19 6.43 6.32 6.41 6.39 0.06 146.28 120.88 156.23 116.46 18.23 28.24 12.41 23.76 + + + DAM-19 217

DAM-20 224 19.34 17.49 16.54 6.62 6.58 6.62 6.61 0.02 145.83 120.43 147.43 114.00 15.15 31.09 15.38 27.76 + + + DAM-21 239 25.69 18.85 22.35 6.60 6.54 6.62 6.59 0.04 154.81 127.66 162.76 121.71 18.40 38.05 20.46 32.62 + + + DAM-22 251 31.42 19.26 29.02 6.60 6.52 6.50 6.54 0.05 142.10 118.65 149.00 113.07 15.91 19.36 28.50

261 + + +

86

DAM-23 262 30.92 18.44 30.13 6.60 6.61 6.56 6.59 0.03 143.65 121.40 155.40 115.67 17.27 35.24 20.90 31.21

DAM-24 269 37.81 22.68 36.14 6.59 6.51 6.55 6.55 0.04 140.85 116.95 148.05 112.16 16.28 36.46 24.58 33.28 + + + DAM-25 273

DAM-26 276 50.41 30.59 45.09 6.56 6.55 6.56 6.56 0.01 160.72 142.62 166.97 12.65 31.27 24.98 33.98 + + +

DAM-27 282 54.15 32.33 50.08 6.55 6.54 6.56 6.55 0.01 158.57 128.52 163.82 19.05 38.74 27.07 31.51 + +

285 +

DAM-28 289 58.83 41.16 53.46 6.54 6.50 6.52 6.52 0.02 144.06 123.31 150.06 114.80 14.04 42.42 31.87 36.91 + +

DAM-29 297 59.74 44.21 55.07 6.55 6.55 6.56 6.55 0.01 142.21 122.01 150.61 114.20 14.70 45.75 34.09 38.03

298 +

DAM-30 300 6.62 6.54 6.53 6.56 0.05 39.12 28.28 36.17 + +

DAM-31 303 62.36 44.74 55.59 6.56 6.57 6.53 6.55 0.02 134.01 112.81 141.96 108.00 15.07 45.83 32.00 37.14

DAM-32 305

DAM-35 312 58.06 46.35 55.59 6.56 6.53 6.58 6.56 0.03 96.85 41.48 33.60 36.62 + + +

DAM-36 318 62.36 49.80 56.47 6.58 6.59 6.56 6.58 0.02 116.75 99.70 122.13 112.86 11.71 41.34 37.64

DAM-37 323 57.82 51.75 57.12 6.56 6.54 6.51 6.54 0.03 116.78 100.48 124.86 114.04 12.42 43.80 35.29 37.52 + +

DAM-38 331 61.65 56.96 57.65 6.59 6.58 6.57 6.58 0.01 117.03 101.11 124.43 114.19 11.92 43.75 37.38 40.28 +

339 + + +

DAM-39 349 66.07 67.56 62.09 6.65 6.57 6.59 6.60 0.04 117.73 102.65 124.70 115.03 11.27 47.65 38.92 40.42

DAM-40 359 6.63 6.58 6.58 6.60 0.03 45.78 38.00 40.27 + +

DAM-41 377 79.33 86.96 76.76 6.62 6.56 6.56 6.58 0.03 117.32 101.20 124.40 114.30 11.89 51.61 43.23 44.51

DAM-42 391 65.31 75.88 65.41 6.59 6.56 6.53 6.56 0.03 123.48 107.54 131.34 120.79 12.12 60.64 50.32 50.00 + +

DAM-43 411 6.71 6.63 6.67 6.67 0.04

87

Table 29: Exp first enrichment complete data - Denitrifying

EXP Denitrifying Ni (ppm) pH Mg (ppm) sulfate (mM) nitrate additions

EXP EXP EXP EXP EXP EXP pH std EXP EXP EXP Mg std EXP EXP EXP EXP EXP EXP Sample days 10 11 12 10 11 12 ave. dev 10 11 12 ave. dev 10 11 12 10 11 12 EXP-1 7 0.49 0.51 0.53 6.78 6.72 6.76 6.75 0.03 54.09 55.31 56.86 54.23 1.39 0.43 0.42 0.41 + + + EXP-2 11 1.39 1.36 1.40 6.99 7.01 7.04 7.01 0.03 63.36 63.02 65.24 60.84 1.20 1.04 1.16

EXP-3 70 0.15 0.14 0.14 7.27 7.34 7.31 7.31 0.04 121.24 110.24 103.43 98.19 8.99 0.01 0.40 1.50

EXP-4 75 0.21 0.22 0.25 7.22 7.28 7.27 7.26 0.03 118.34 107.64 101.99 96.39 8.30 0.25 0.62 1.32 + + + EXP-5 82 1.09 0.96 1.21 7.08 7.15 7.11 7.11 0.04 113.86 104.56 100.08 93.91 7.03

EXP-6 93 0.67 0.65 1.21 6.87 6.90 6.84 6.87 0.03 117.90 112.25 105.20 98.28 6.36 2.28 3.04 3.91

EXP-7 99 0.63 0.67 1.39 6.71 6.79 6.71 6.74 0.05 124.73 121.33 115.38 105.06 4.73 2.04 3.00 3.86 + + + EXP-8 109 3.05 2.92 4.50 6.67 6.63 6.55 6.62 0.06 132.78 133.77 124.48 112.74 5.10

112 + + +

132 + + +

EXP-9 138 7.00 7.09 7.60 6.47 6.58 6.59 6.55 0.07 159.56 162.71 155.76 135.39 3.48 9.58 12.22 12.82

EXP-10 148 6.41 6.47 6.56 6.53 6.52 6.53 6.53 0.01 161.60 162.55 151.70 134.83 6.01 8.65 10.88 10.93 + + + 156 + + +

EXP-11 163 10.29 12.58 12.72 6.59 6.56 6.58 6.58 0.02 160.08 163.61 153.33 135.12 5.22 12.93 14.62 15.95

EXP-12 164 10.27 13.08 13.35 6.61 6.59 6.60 6.60 0.01 163.24 172.82 160.30 140.12 6.55 12.99 15.09 15.38 + + + EXP-13 166 13.58 16.02 16.45 6.60 6.57 6.61 6.59 0.02 183.67 189.37 176.29 153.93 6.56 14.02 16.50 16.44

EXP-14 168 14.52 17.17 17.97 6.58 6.62 6.63 6.61 0.03 184.47 188.63 177.45 154.26 5.65 15.39 17.26 17.26

EXP-15 173 11.22 13.71 15.24 6.63 6.63 6.63 6.63 0.00 161.61 169.46 157.21 138.02 6.21 16.14 18.58 18.38 + + + EXP-16 179 13.43 17.00 19.55 168.51 175.81 163.46 143.10 6.21 17.56 21.14 20.36 + + +

EXP-18 214 6.40 6.39 6.38 6.39 0.01 176.43 188.13 172.08 150.59 8.30 21.60 24.74 23.70 + + +

EXP-19

EXP-20 224 13.58 14.18 37.18 6.59 6.63 6.68 6.63 0.05 170.38 174.33 166.13 143.93 4.10 22.85 27.91 25.82 + + + 11.6 EXP-21 239 20.33 21.54 41.68 6.55 6.57 6.61 6.58 0.03 186.61 192.91 170.36 154.05 28.80 33.29 30.56 + + + 4 EXP-22 251 29.06 27.89 50.48 6.53 6.59 6.61 6.58 0.04 165.15 174.65 156.70 140.14 8.98 25.46 28.48 24.89

261 + + +

88

EXP-23 262 6.57 6.58 6.63 6.59 0.03 27.43 29.54 27.67

EXP-24 269 35.00 33.47 55.10 6.57 6.58 6.61 6.59 0.02 162.50 173.15 157.30 139.22 8.08 29.17 34.13 29.11

EXP-25 273 + +

11.9 EXP-26 276 38.00 40.42 62.45 6.55 6.58 6.58 6.57 0.02 182.22 195.07 171.27 153.67 26.55 31.12 26.78 1 278 +

12.1 EXP-27 282 38.72 41.80 64.42 6.53 6.53 6.55 6.54 0.01 177.52 189.17 164.97 149.28 27.29 30.48 26.03 0 EXP-28 289 41.88 45.48 73.32 6.51 6.55 6.56 6.54 0.03 164.81 172.76 160.36 140.54 6.28 28.88 31.88 27.21

14.4 EXP-29 297 42.89 50.55 75.44 6.56 6.56 6.57 6.56 0.01 162.46 181.21 152.71 140.05 27.47 32.36 26.64 8 EXP-30 300

15.2 EXP-31 303 45.66 54.99 82.27 6.52 6.53 6.52 6.52 0.01 161.71 182.31 152.46 140.06 28.55 32.70 27.74 + 8 EXP-32 305

EXP-35 312 52.02 60.26 87.95 6.56 6.58 6.60 6.58 0.02 28.81 32.23 28.45 + +

EXP-36 318 53.25 62.23 89.37 6.56 6.58 6.58 6.57 0.01 140.53 152.06 133.07 136.01 9.57 27.67 31.88

EXP-37 323 54.94 66.18 92.98 6.51 6.54 6.53 6.53 0.02 142.33 155.33 135.85 138.42 9.92 28.78 32.31 27.45

EXP-38 331 57.04 68.58 98.96 6.56 6.55 6.54 6.55 0.01 141.72 152.39 135.69 137.20 8.46 30.20 33.20 28.64

EXP-39 349 66.08 75.71 104.74 6.57 6.57 6.63 6.59 0.03 141.77 154.21 135.27 137.72 9.63 30.05 33.75 28.55 +

EXP-40 359 6.58 29.53 31.56 27.87 +

10.6 EXP-41 377 90.16 101.67 136.11 6.57 6.57 6.61 6.57 0.02 142.32 154.45 133.30 137.46 31.84 30.08 + 1 11.0 EXP-42 391 115.88 129.12 168.58 6.56 6.58 6.58 6.64 0.01 138.36 151.74 129.89 134.34 34.24 36.48 30.28 2 EXP-43 411 6.62 6.66 6.64 6.69 0.02 34.91 37.55 31.14 + +

89

Table 30: TSRA first enrichment complete data - Denitrifying

TSRA Denitrifying Ni (ppm) pH Mg (ppm) Sulfate (mM) nitrate additions

TSRA TSRA TSRA TSRA TSRA TSRA pH std TSRA TSRA TSRA Mg std TSRA TSRA TSRA TSRA TSRA TSRA Sample days 10 11 12 10 11 12 ave dev 10 11 12 ave dev 10 11 12 10 11 12 TSRA-1 7 0.65 0.20 0.61 6.86 6.88 6.80 6.85 0.04 61.80 66.07 60.31 60.49 2.99 0.41 0.07 0.44 + + + TSRA-2 11 0.88 0.31 0.92 7.09 6.97 7.06 7.04 0.06 63.69 80.11 59.89 65.04 10.75 0.51 0.53

TSRA-3 70 0.17 0.13 0.64 7.17 7.27 7.19 7.21 0.05 85.72 126.34 69.04 86.82 29.47 1.64 0.02 3.23

TSRA-4 75 0.58 0.15 0.65 7.25 7.24 7.16 7.22 0.05 83.97 127.79 67.88 86.49 31.01 0.82 2.58 + + +

TSRA-5 82 7.10 7.11 7.02 7.08 0.05 1.75 6.80

TSRA-6 93 7.03 3.49 3.94 6.84 6.92 6.79 6.85 0.07 86.88 130.15 69.58 88.38 31.20 4.76 2.09 6.73

TSRA-7 99 9.74 5.48 4.74 6.65 6.77 6.74 6.72 0.06 91.91 137.73 74.54 93.22 32.64 4.91 + + +

TSRA-8 109 18.26 11.72 3.97 6.57 6.58 6.76 6.64 0.11 99.18 140.52 63.70 93.08 38.45

112 + + +

132 + + +

TSRA-9 138 34.35 23.72 6.02 114.91 158.06 78.40 106.16 39.88 15.16 6.15 6.71

TSRA-10 148 32.16 26.65 6.23 6.46 6.54 6.65 6.55 0.10 110.40 157.05 77.46 104.46 39.99 13.67 5.96 5.63 +

156 6.64 5.74 +

TSRA-11 163 45.82 26.97 7.11 6.39 6.55 6.59 6.51 0.11 112.10 145.66 77.97 101.64 33.85 19.22 6.89 5.96

TSRA-12 164 47.70 28.55 7.73 6.43 6.61 6.58 6.54 0.10 119.50 153.22 82.57 106.99 35.34 19.13 6.76 5.70 +

TSRA-13 166 52.57 32.30 8.85 6.41 6.57 6.58 6.52 0.10 127.48 170.93 91.60 116.66 39.72 20.09 7.00 5.88

TSRA-14 168 55.34 32.10 9.02 6.39 6.59 6.60 6.53 0.12 130.06 168.32 91.78 116.59 38.27 21.43 7.43 5.99

TSRA-15 173 53.82 28.87 8.19 6.38 6.67 6.65 6.57 0.16 121.46 149.91 81.91 106.33 34.15 22.72 +

TSRA-16 179 57.00 31.52 9.13 6.41 6.59 6.59 6.53 0.10 122.81 154.96 84.97 108.98 35.03 25.66 8.26 6.17 +

TSRA-18 214 50.27 6.33 6.38 6.42 6.38 0.05 127.33 109.36 28.87 9.78 7.35 + +

TSRA-19 216

TSRA-20 224 54.37 35.17 14.74 6.54 6.55 6.61 6.57 0.04 128.58 155.63 86.54 111.07 34.82 32.90 11.17 8.06 +

TSRA-21 239 58.91 6.53 6.49 6.51 6.51 0.02 134.61 164.16 93.28 116.91 35.60 39.42 13.95 9.89 + +

TSRA-22 251 43.66 17.95 6.63 6.57 6.53 6.58 0.05 151.80 84.77 109.04 47.39 32.98 12.31 8.48

TSRA-23 262 68.62 48.57 16.62 6.52 6.55 6.55 6.54 0.02 125.15 150.15 84.94 108.14 32.90 36.40 14.99 9.72 + + + 90

TSRA-24 269 72.42 50.55 18.20 6.56 6.53 6.50 6.53 0.03 121.50 145.40 82.79 105.23 31.59 39.08 16.38 10.62

TSRA-25 273

TSRA-26 276 84.00 54.60 21.15 6.55 6.54 6.49 6.53 0.03 137.22 160.87 95.04 117.07 33.35 34.92 15.57 10.29

TSRA-27 282 84.60 54.79 19.19 6.52 6.51 6.48 6.50 0.02 129.42 155.32 86.89 111.28 34.55 34.50 15.26 10.01 + +

TSRA-28 289 86.77 57.12 21.07 6.57 6.49 6.49 6.52 0.05 119.06 145.86 83.01 104.81 31.54 35.57 16.76 11.19

TSRA-29 297 96.73 58.46 21.36 6.57 6.54 6.51 6.54 0.03 127.46 145.46 82.59 106.73 32.38 34.75 16.22 11.28

TSRA-30 300 +

TSRA-31 303 103.16 60.36 22.54 6.54 6.53 6.49 6.52 0.03 125.66 143.81 83.84 106.10 30.76 36.46 17.74 12.14

TSRA-32 305

TSRA-35 312 108.00 62.10 20.23 6.60 6.56 6.52 6.56 0.04 100.08 71.59 20.14 35.84 17.65 11.91

TSRA-36 318 108.89 61.79 19.74 6.53 6.55 6.54 6.54 0.01 102.44 128.64 76.01 102.36 26.32 35.48

TSRA-37 323 100.54 62.37 19.26 6.58 6.54 6.55 6.56 0.02 103.74 129.58 75.59 102.97 27.01 34.89 17.56 12.62

TSRA-38 331 100.55 63.51 20.18 6.59 6.57 6.51 6.56 0.04 104.70 129.08 76.19 103.33 26.47 36.16 18.47 13.43

339 +

TSRA-39 349 92.85 69.18 22.60 6.51 6.50 6.51 6.51 0.01 104.86 128.40 77.77 103.68 25.34 35.86 19.15 14.27 +

TSRA-40 359 6.64 6.57 6.58 6.60 0.04 34.42 18.72 13.80

TSRA-41 377 94.34 84.06 26.82 6.61 6.56 6.49 6.55 0.06 101.08 129.09 77.80 102.66 25.68 38.01 15.46 +

TSRA-42 391 63.99 62.76 19.72 6.76 6.65 6.63 6.68 0.07 95.88 123.46 71.26 96.87 26.11 37.18 21.22 15.10 + +

91

Table 31: Taxonomic assignment of denitrifying microcosms – Tails, Dam, and Exp

Taxon Tails10 Tails11 Tails12 Dam10 Dam11 Dam12 Exp10 Exp11 Exp12 Gammaproteobacteria;o_Chromatiales;Other;Other 15.99% 8.20% 17.37% 33.84% 22.02% 44.54% 29.57% 35.23% 21.37% OTU7494 f_Gallionellaceae;g_ 7.81% 10.34% 7.66% 5.79% 10.26% 5.36% 12.14% 10.13% 7.75% Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ 7.50% 9.17% 6.72% 3.18% 2.74% 5.87% 4.47% 7.01% Gammaproteobacteria;o_Chromatiales;f_;g_ 5.96% 7.54% 8.17% 5.17% 16.79% 3.16% 5.57% 6.44% 6.92% OTU3487 f_Hydrogenophilaceae;g_Thiobacillus 5.54% 7.74% 5.24% 4.82% 5.27% 6.30% 6.62% 5.25% 7.49% OTU6475 Betaproteobacteria;Other;Other;Other 4.86% 5.12% 4.53% 3.70% 3.98% 3.92% 3.16% 4.34% 3.37% Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_ 2.62% 2.38% 2.41% 2.79% OTU3261 f_Hydrogenophilaceae;g_ 2.51% 2.42% 2.76% 2.09% OTU7062 f_Gallionellaceae;Other 2.04% 2.13% 2.03% 2.38% Planctomycetes;c_Phycisphaerae;o_Phycisphaerales;f_;g_ 2.00% 2.03% 3.93% OTU594 Betaproteobacteria;o_;f_;g_ 2.70% f__Rhodocyclaceae;Other 2.22% 3.81% Chloroflexi;c_Anaerolineae;Other;Other;Other 2.12% 2.76% Chlorobi;c_Ignavibacteria;o_Ignavibacteriales;f_Ignavibacteriaceae;g_ 2.03% OP11;c_OP11-3;o_;f_;g_ 4.25% 3.75% 3.32% Proteobacteria;Other;Other;Other;Other 2.88% 3.20% 3.48% 2.25% 4.41% f_Rhodocyclaceae;g_Methyloversatilis 2.19% 2.77% 3.25% Gammaproteobacteria;Other;Other;Other 2.18% 2.98% Cyanobacteria;c_Chloroplast;o_Stramenopiles;f_;g_ 2.05% Less than 2% 34.91% 29.84% 33.49% 20.20% 24.94% 19.92% 20.59% 21.29% 31.50% Uncategorized 8.25% 8.19% 8.47% 9.19% 5.03% 6.08% 4.10% 5.47% 4.42% sum 100% 100% 100% 100% 100% 100% 100% 100% 100%

92

Table 32: Taxonomic assignment of aerobic microcosms – Tails, Dam, and Exp

Taxon Tails7 Tails9 Dam7 Dam8 Dam9 Exp7 Exp8 Exp9 OTU3261,3800,6684 f_Hydrogenophilaceae;g_ 19.43% 18.16% 18.59% 40.54% 32.04% 45.98% 63.61% 31.72% OTU4252 Betaproteobacteria;Other;Other;Other 4.38% 2.49% 3.03% 8.09% 5.36% 4.56% 6.75% 10.02% Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ 3.22% 5.60% 2.20% 2.05% 3.93% 2.23% 3.70% Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_ 2.02% 2.82% 2.18% 3.08% 2.47% 4.07% Cyanobacteria;c_Chloroplast;o_Stramenopiles;f_;g_ 2.81% 4.91% OTU408 f_Rhodocyclaceae;g_Methyloversatilis 2.05% 2.63% OTU6467,7494 f_Gallionellaceae;g_ 3.33% 5.76% 3.66% 2.53% 2.85% 2.52% OTU3487 f_Hydrogenophilaceae;g_Thiobacillus 2.93% 6.63% 3.06% 2.42% 2.92% Nitrospirae;c_Nitrospira;o_Nitrospirales;f_Thermodesulfovibrionaceae;g_GOUTA19 2.34% 2.99% 3.16% Gammaproteobacteria;Other;Other;Other 4.87% 3.09% 4.04% 2.52% Gammaproteobacteria;o_Chromatiales;Other;Other 27.46% 3.75% 13.96% Gammaproteobacteria;o_Chromatiales;f_;g_ 7.96% OTU6263 f_Hydrogenophilaceae;Other 2.25% Alphaproteobacteria;Other;Other;Other 9.99% 3.62% Deltaproteobacteria;o_;f_;g_ 3.03% Less than 2% 44.23% 44.21% 29.31% 34.01% 30.86% 28.32% 19.49% 29.16% Uncategorized 11.46% 7.10% 3.03% 3.89% 2.92% 3.74% 2.60% 5.32% sum 100% 100% 100% 100% 100% 100% 100% 100%

93

Table 33: Taxonomic assignment of TSRA microcosms

Taxon TSRA7 TSRA8 TSRA9 TSRA10 TSRA11 TSRA12 OTU3487 f_Hydrogenophilaceae;g_Thiobacillus 41.33% 8.87% 48.33% 26.35% 19.28% 2.20% OTU7494 f_Gallionellaceae;g_ 7.88% 40.47% 30.23% 9.57% 4.63% OTU1225 Betaproteobacteria;Other;Other;Other 3.67% 15.78% 5.87% 6.73% 3.85% OTU408,2838 f_Rhodocyclaceae;g_Methyloversatilis 3.22% 3.47% 10.23% 26.76% 19.21% OTU4411 Betaproteobacteria;o_Methylophilales;f_;g_ 3.06% OTU1041,5214 Betaproteobacteria;o_Rhodocyclales;f_;g_ 2.89% 7.04% Deltaproteobacteria;o_Myxococcales;f_;g_ 2.60% Bacteroidetes;c_Sphingobacteria;o_Sphingobacteriales;f_Sphingobacteriaceae;g_ 11.83% Nitrospirae;c_Nitrospira;o_Nitrospirales;f_Nitrospiraceae;g_Nitrospira 7.38% Bacteroidetes;c_Sphingobacteria;o_Sphingobacteriales;f_;g_ 3.14% Chloroflexi;c__Anaerolineae;o_GCA004;f_;g_ 2.63% 2.37% Gammaproteobacteria;o_Chromatiales;f_;g_ 2.54% 18.96% Chloroflexi;c_Anaerolineae;o_envOPS12;f_;g_ 2.51% OTU343,7062 f_Gallionellaceae;Other 2.00% 8.64% 19.24% f_Rhodocyclaceae;Other 6.31% f_Gallionellaceae;g_Gallionella 2.74% Bacteroidetes;c_Bacteroidia;o_Bacteroidales;f_;g_ 10.80% Less than 2% 32.89% 19.49% 24.64% 17.64% 17.84% 16.68% Uncategorized 5.05% 2.28% 4.69% 2.13% 2.05% sum 100% 100% 100% 100% 100% 100%

94

Table 34: Dam second enrichment partial data - Denitrifying

Dam_2 Denitrifying Ni (ppm) Ni (ppm) Sulfate (mM) Sulfate (mM)

Days Dam Dam Dam Killed pH Mg Dam Dam Dam Nitrate Sample killed ave. N7 N8 N9 ave. ave ave. N7 N8 N9 additions Dam-NN-0 0 8.44 8.27 8.02 2.47 0.38 1.88 1.93 1.85 0.18

Dam-NN-1 16 0.00 0.00 0.00 0.00 6.62 0.00 2.19 2.22 2.22 0.39 + Dam-NN-2 26 0.67 0.73 0.53 6.39 5.05 5.11 5.14

30 +

Dam-NN-3 34 1.61 1.68 0.80 6.20 9.38 9.49 9.37

Dam-NN-4 40 2.45 2.86 1.87 6.42 10.86 11.34 11.44

44 +

58 +

Dam-NN-5 72 6.68 6.65 4.99 0.00 5.82 0.25 19.06 19.63 19.59 0.36 + 88 +

Dam-NN-6 102 30.61 31.11 31.44 +