USING BIOGEOCEMENTATION TO REDUCE PHYSICAL

INSTABILITY AND WIND EROSION OF MINE TAILINGS

by

Nicole Woodcock

A thesis submitted to the Department of Civil Engineering

In conformity with the requirements for

the degree of Master of Applied Science

Queen’s University

Kingston, Ontario, Canada

(January, 2021)

Copyright ©Woodcock, 2021 Abstract

The mining industry in Canada is an essential driver of economic growth as societal demands for minerals increases with advances in technology. The waste generated through these mine operations is one of the biggest environmental and safety hazards in the world, with the long- term storage of this waste in tailings dams being a geotechnical and geochemical hazard.

The biogeocementation of tailings, using calcite produced by microorganisms to create rock-like tailings deposits, is an attractive concept because a bio-cement can be formed that is durable enough to improve the mechanical behavior of the tailings. In this work, the microorganism Sporosarcina pasteurii was explored for its effectiveness at performing biogeocementation to physically stabilize gold and nickel mine tailings and prevent the wind erosion of gold mine tailings deposits.

S. pasteurii was proven to be resilient when exposed to heavy metals at low concentrations, from temperatures of 4 to 31C, and pH values from 2 to 12, indicating S. pasteurii can survive in the potentially harsh conditions of tailings dams. S. pasteurii was then utilized to strengthen undrained gold and nickel tailings. Gold tailings from the Dominican Republic showed a 6.8% increase in shear strength with bacteria inoculation whereas nickel tailings from British Columbia showed an 18% decrease in shear strength. This indicates biogeocementation was successful for the gold tailings and unsuccessful for the nickel tailings which could be due to pore clogging.

Finally, S. pasteurii was investigated for its ability to withstand wind speeds up to 15 km/h with the average percent mass lost reduced to 0.46  0.22% from 3.99  0.88%. The most effective application method for biogeocementation was found to be a single application of S. pasteurii

i followed by a continuous application of nutrient amendments, which has positive implications for field level scale-up.

The promise of biogeocementation is demonstrated in this thesis, however, future proof- of-concept work needs to be performed to further characterize the robustness of S. pasteurii and determine its effectiveness in real-world environments, such as tailings dams.

ii Acknowledgements

I would firstly like to thank my supervisor, Dr. Pascale Champagne, for giving me the opportunity to join her research group all those years ago and for her unending support along the way. She has not only provided me with guidance during my research but has been a valued mentor and friend. I am very thankful for her endless hard work and late nights staying up editing my work, writing me reference letters, chatting, and giving me great life advice.

A special thanks to Vanessa and Jason, who provided me with invaluable opportunities in my career and have been a wealth of knowledge, fun, and guidance. This research would be nowhere without you. To everyone the BGC Fredericton office, thank you for making my internship the most fun and helping me with my research.

I am grateful to Sarah and Samira, whose work on the biogeocementation project gave me inspiration. To the civil staff, Stan, Nathan, Hal, Jyoti, Sophie, and Laura, thank you for lending your hands and for great conversations I will treasure.

Thanks to David, Connor, Joaquin, Matt, Alan, Lauren, and Bailey for countless fantastic research and non-research related conversations, you are the reason I am still sane. To Claire and

Vanessa (and Fish), thank you for the support you provided me as roommates, knowing I had wonderful people to come home to made everything easier. To my friends, Abby, Alanna, Rowan,

Darcy, Holly, and Vinyas, thank you for continuous support and love.

Last but not least, to my family. To Mom and Dad, Amber and Rebecca, Grandma and

Grandpa, Nana, and Nana and Poppa, thank you for caring for me and fully supporting me (even if you didn’t always understand what I was doing). Finally, Conrad, thank you for having my back, making me laugh, and providing a much-needed escape during this time. I love you all!

iii Table of Contents

Abstract ...... ii Acknowledgements ...... iv Table of Contents ...... v List of Figures ...... viii List of Tables ...... xi List of Equations ...... xii List of Abbreviations ...... xiii Chapter 1 - Introduction ...... 1 1.1 Introduction ...... 1 1.2 Background ...... 1 1.2.1 Mining in Canada ...... 1 1.2.2 Current Issues in the Mining Industry ...... 2 1.3 Site Descriptions ...... 3 1.3.1 Gold Mine in Ontario...... 3 1.3.2 Climate and Wind Speed Data for Gold Mine in Ontario ...... 4 1.3.3 Characteristics of Mine Tailings for the Gold Mine in Ontario ...... 4 1.4 Project Description...... 5 1.4.1 Research Objectives ...... 6 1.5 Thesis Organization ...... 6 Chapter 2 - Literature Review ...... 7 2.1 Safe Management of Mine Tailings ...... 7 2.1.1 Introduction to Tailings Dams ...... 8 2.1.2 Mine Tailings Dam Design ...... 8 2.1.3 Stability Risks of Tailings Dams ...... 10 2.1.4 Management of Tailings Facilities ...... 12 2.1.5 Major Mine Disasters ...... 14 2.1.6 Gold Mine Waste ...... 15 2.2 Environmental Impact of Mine Tailings ...... 18 2.2.1 Socioeconomic Impact of Tailings Dam Failures ...... 19 2.2.2 Wind Erosion of Tailings ...... 20 2.2.3 Dust suppression reagents ...... 21

iv 2.3 Current Treatment of Mine Tailings ...... 22 2.3.1 Remediation of Tailings after Failure...... 24 2.3.2 Current Physical Treatment of Mine Tailings ...... 25 2.3.3 Current Chemical Treatment of Mine Tailings ...... 29 2.3.4 Current Biological Treatment of Mine Tailings ...... 32 2.4 Microbially Induced Calcite Precipitation (Biogeocementation) ...... 37 2.4.1 Bacteria used for Biogeocementation ...... 38 2.4.2 Use of Biogeocementation in soils ...... 40 2.4.3 Use of Biogeocementation in mine tailings ...... 46 Chapter 3 - Investigation into Sporosarcina pasteurii and the Measure of Shear Strength on Undrained Gold and Nickel Tailings...... 49 Abstract 49 3.1 Introduction ...... 51 3.2 Materials and Methods ...... 53 3.2.1 Preparation of Synthetic Mine Tailings ...... 53 3.2.2 Preparation of Mine Tailings...... 54 3.2.3 Cultivation of Sporosarcina pasteurii ...... 54 3.2.4 Preparation of Media ...... 54 3.2.5 Preparation of Sporosarcina pasteurii ...... 55 3.2.6 Kinetic Experiments for Sporosarcina pasteurii...... 56 3.2.7 Survivability Tests with Synthetic Mine Tailings ...... 56 3.2.8 Preparation of Agar Plates ...... 57 3.2.9 Plating S. pasteurii to Determine Survivability ...... 58 3.2.10 Undrained Tailings Strength Test Experimental Plan ...... 59 3.2.11 Specific Gravity and Particle Size Analysis ...... 59 3.2.12 Consolidated Undrained Triaxial Tests ...... 60 3.3 Results and Discussion ...... 62 3.3.1 Kinetic Growth Curve of Sporosarcina pasteurii ...... 62 3.3.2 Effects of Heavy Metals, Temperature, and pH on Sporosarcina pasteurii ...... 64 3.3.3 Effects of Sporosarcina pasteurii and Supplemental Media Application on Mine Tailings ...... 66 3.3.4 Effects of Sporosarcina pasteurii and Supplemental Media Application on the Penetration Strength of Gold and Nickel Mine Tailings ...... 68

v 3.3.5 Effects of Sporosarcina pasteurii and Supplemental Media Application on Particle Size of Mine Tailings ...... 72 3.3.6 Effects of Sporosarcina pasteurii and Supplemental Media on the Shear Strength of Gold and Nickel Tailings Measuring using Consolidated Undrained Triaxial Test ...... 76 3.4 Conclusions ...... 78 Chapter 4 - Biogeocementation: increased stability of mine tailings to wind erosion by microbially induced calcite precipitation...... 80 Abstract 80 4.1 Introduction ...... 81 4.2 Materials and Methods ...... 86 4.2.1 Cultivation of Sporosarcina pasteurii ...... 86 4.2.2 Preparation of Media ...... 87 4.2.3 Preparation of Mine Tailings...... 87 4.2.4 Experimental Set-up ...... 88 4.2.5 Experimental Plan ...... 89 4.2.6 Wind Erosion Tests ...... 90 4.2.7 Penetrometer Measurements ...... 90 4.2.8 Scanning Electron Microscopy Images ...... 91 4.3 Results and Discussion ...... 91 4.3.1 Effects of Sporosarcina pasteurii and Supplemental Media Application on the Prevention of Wind Erosion of Mine Tailings Beaches ...... 91 4.3.2 Effects of Sporosarcina pasteurii and Supplemental Medium Application on the Crust Strength of Gold Mine Tailings ...... 94 4.4 Conclusions ...... 96 Chapter 5 - Conclusions and Recommendations ...... 98 5.1 Conclusions ...... 98 5.2 Recommendations and Future Work ...... 100 5.3 Contributions to Science ...... 101 Chapter 6 - References ...... 102

vi List of Figures

Figure 2-1. The design options for a tailings dam (Mittal & Morgenstern, 1975) ...... 9 Figure 2-2. Combined Au–CN and Zn–CN Eh-pH diagrams which show leaching and cementation for the gold cyanidation process (H. H. Huang, 2016) ...... 16 Figure 2-3. Process flowchart for gold recovery (International Cyanide Management Institute, 2017) ...... 17 Figure 2-4. Requirements for bioremediation (Cookson, 1995) ...... 33 Figure 2-5. Aerobic biological treatment of cyanide (Akcil, 2003)...... 35 Figure 2-6. Microscopic image of S. pasteurii (2000x magnification), demonstrating the rod-shaped bacteria ...... 38 Figure 3-1. Demonstration of plating the S. pasteurii bacteria with PBS dilution factors included ...... 58

Figure 3-2. Demonstration of triaxial cell with σ3 representing the cell (confining)

pressure (kPa) and σ1 representing axial strength (kPa) of the load cell a) at the end of consolidation and b) during shearing ...... 61

Figure 3-3. Cell concentration and OD600 value over time for growth of S. pasteurii bacteria in yeast growth medium ...... 63 Figure 3-4. Classification of survivability tests with a) indicating a low to no survivability, b) indicating some growth and moderate chance of survivability, and c) indicating the bacteria have a higher chance of surviving the specific stressor ...... 64 Figure 3-5. Gold mine tailings from the Dominican Republic (GMDR) inoculated

with S. pasteurii and CaCl2 on a) Day 0, b) Day 14, noting that the water cap had evaporated, and c) Day 25 when final strength was reached ...... 66 Figure 3-6. Nickel mine tailings from British Columbia (NMBC) inoculated with S.

pasteurii and CaCl2 on a) Day 0, b) Day 14, noting that some of the water cap was still present, and c) Day 25 when final strength was reached ...... 66 Figure 3-7. Gold mine tailings from British Columbia (GMBC) inoculated with S.

pasteurii and CaCl2 on a) Day 0, b) Day 14, noting that some of the water cap had evaporated, and c) Day 25 when final strength was reached ...... 67

vii Figure 3-8. Consolidated tailings inoculated with S. pasteurii and CaCl2 (a) gold tailings from Dominican Republic, b) nickel tailings from British Columbia, and c) gold tailings from British Columbia removed from cement molds after 30 days ...... 67 Figure 3-9. Measure of strength with time for control and inoculated samples of gold mine tailings from the Dominican Republic ...... 69 Figure 3-10. Measure of strength changing over time for control and inoculated samples of nickel tailings from British Columbia at room temperature and 13°C...... 70 Figure 3-11. Measure of strength changing over time for control and inoculated samples of gold mine tailings from British Columbia ...... 71 Figure 3-12. Particle size distribution of GMDR – CIL tailings ...... 73 Figure 3-13. Particle size distribution of GMDR – HDS tailings ...... 73 Figure 3-14. Particle size distribution of GMBC – Zone 3 tailings ...... 74 Figure 3-15. Particle size distribution of NMBC tailings at room temperature ...... 75 Figure 3-16. Particle size distribution of NMBC tailings tested at 13°C ...... 76 Figure 3-17. The effective stress (p’) versus shear stress (q) for GMDR HDS tailings with shear strength values highlighted when pore pressure is maximized during phase transition (softening to hardening) ...... 77 Figure 3-18. The effective stress (p’) versus shear stress (q) for NMBC tailings with shear strength values highlighted when pore pressure is maximized during phase transition (softening to hardening) ...... 78 Figure 4-1. Example of the development of a tailings beach (Pirouz et al., 2005) ...... 82 Figure 4-2. Representative image of calcite precipitation during ureolysis (Dejong et al., 2010) ...... 86 Figure 4-3. Image of wind tunnel used to test samples ...... 90 Figure 4-4. Plot of the mass percentage of tailings displaced in 15 km/h winds vs trial number, with trial 10 occuring after 30 days (a), plot demonstrating relationship between the control samples (A) and the samples with continuous application of S. pasteurii (D) under 15 km/h wind speed (b),

viii and plot demonstrating trend of samples with continuous addition of bacteria (E & F) over time under 15 km/h wind speed (c) ...... 92 Figure 4-5. Images of control sample (a), sample D (b) and sample F (c) after 30 days of treatment ...... 94 Figure 4-6. A plot of the penetrative strength of the crust vs days of treatment for samples B, C, D, and J ...... 95 Figure 4-7. SEM images of the dried mine tailings before treatment (a) and after 30 days of treatment (b), both at 2000x magnification ...... 96

ix List of Tables

Table 1-1. Average historical climate data for each month for the Gold Mine in Ontario ...... 4 Table 1-2. Historical wind speed data from 2008 – 2019, with the maximum wind speed in 2018 highlighted (Government of Canada, 2020) ...... 4 Table 1-3. Soil characteristics for GMON tailings ...... 5 Table 1-4. Concentration of a few key dissolved metals in GMON tailings ...... 5 Table 2-1. A summary of the chemical pathways utilized by microorganisms which are capable of producing calcareous material (Kalantary & Kahani, 2015) ...... 40 Table 3-1. Concentration of metal salt used to determine the likelihood of S. pasteurii survivability ...... 57 Table 3-2. Likelihood S. pasteurii bacteria would survive in environments with different heavy metal concentrations, pH levels, or temperature ...... 65 Table 3-3. Specific gravity values at 20°C for tailing samples ...... 72 Table 4-1. Experimental setups implemented for wind erosion tests ...... 89

x List of Equations

Equation 2-1. Gold cyanidation process ...... 15 Equation 2-2. Chemical equation for the formation of acid rock drainage through the oxidation of pyrite ...... 23 Equation 3-1. Calculation of bacteria cell concentration ...... 56 Equation 3-2. Calculation of Skemton’s B-value, where Δu represents change in

pore pressure (kPa) and Δσ3 represents the change in cell pressure (kPa) ...... 61 Equation 3-3. Calculation of effective stress ...... 62 Equation 3-4. Calculation of shear stress ...... 62 Equation 4-1. Conversion of urea to ammonium and carbonic acid using the water and the enzyme urease ...... 84 Equation 4-2. Conversion of carbonic acid and ammonium to carbamic acid and ammonia ...... 84 Equation 4-3. Conversion of carbamic acid to ammonia and carbonic acid using water ...... 84 Equation 4-4. Conversion of carbonic acid to bicarbonate ...... 84 Equation 4-5. Conversion of ammonia to ammonium using water ...... 84 Equation 4-6. Conversion of bicarbonate and ammonium to carbonate and ammonium ...... 85

Equation 4-7. Calcium ions from CaCl2 attaching to a particle of mine tailings ...... 85

Equation 4-8. Calcium ions from CaCl2 combining with carbonate and attaching to a particle of mine tailings...... 85 Equation 4-9. Urea hydrolyzing to form carbonate and ammonia ...... 85

Equation 4-10. Calcium ions from CaCl2 combining with carbonate and attaching to a particle of mine tailings ...... 85

xi List of Abbreviations

APP Acid production potential ARD Acid rock drainage ALD Anoxic limestone drain σ1 Axial load BGC BGC Engineering Inc. BC British Columbia CaCO3 Calcite or calcium carbonate CaCl2 Calcium chloride CIL Carbon-in-leach σ3 Cell pressure ca. Circa CU Consolidated undrained triaxial p' Effective stress EICP Enzyme induced calcite precipitation EPS Extracellular polymeric substance FTP Flotation GMBC Gold Mine in British Columbia GMON Gold Mine in Ontario GMDR Gold Mine in the Dominican Republic GM Growth medium HDS High density sludge MICP Microbially induced calcite precipitation NMBC Nickel Mine in British Columbia PM Particulate matter PBS Phosphate-buffered saline PLA Polylactic acid REL Recommended exposure limit SEM Scanning electron microscopy q Shear stress S. pasteurii Sporosarcina pasteurii SM Supplemental medium UM Urea medium XRD X-ray diffraction

xii Chapter 1 -

Introduction

1.1 Introduction

Mining is the process of extracting a naturally occurring material from the earth to derive a profit. Typical extracted materials include metallic ores, such as iron, copper and gold, non- metallic minerals, such as sand and gravel, and fossil fuels, such as coal. Naturally occurring liquids, like petroleum and natural gas, require alternative extraction techniques than those of solid materials and can present challenges of their own. Mining has five stages: prospecting, exploration, development, exploitation, and reclamation. The first stage, prospecting, involves an inspection and measurement of the Earth’s properties to determine the location of mineral deposits. Then, through exploration, geologists determine the value of the deposit by obtaining core samples. From there, development translates planning to mine design, obtaining rights to access the land, and preliminarily preparing the site for exploitation. During exploitation, ore is continuously removed to be processed and leaves waste, known as tailings. Finally, the fifth stage, reclamation, restores the area to its natural state after all profitable material has been removed from the mine site

(Newman et al., 2010). The first four stages of mining are all well researched and developed, however it is in the fifth stage, reclamation, where the greatest potential for innovation lies.

1.2 Background

1.2.1 Mining in Canada

Canada is a dominant member in the global mining sector, particularly in mineral production, mining finance, sustainability, and safety (The Mining Association of Canada, 2017).

As of 2016, Canada was home to 1201 mines, 65 metal mines, and 1136 non-metal mines; notably potash and bitumen used for oil production in the Alberta oil sands (The Mining Association of

1

Canada, 2017). Although the mining industry in Canada provides jobs and contributes to the GDP, the harmful environmental and social implications of mining can foster a negative outlook on the overall success of the industry. Currently, Canada is considering the expansion of an existing oil pipeline from Alberta through Indigenous land to the coast of British Columbia (BC). The Albertan government is interested in this expansion and is financially invested in the pipeline, whereas the

BC government opposes the pipeline over concerns with First Nations communities and the environment (Reuters, 2019). The delays in construction of the pipeline are costly and have exacerbated the rift between the bordering provinces, halting economic growth, and pushing legal action to prevent the expansion. This debate demonstrates the issues taking place in the mining industry and the consequent financial, environmental, and social impacts.

1.2.2 Current Issues in the Mining Industry

Mining companies currently face issues with sustainability, water allocation, and waste management. In countries experiencing water insecurity, mine operations are growing to accommodate world demand, usually in areas which are becoming increasingly scarce in water resources. The advancements in technology and improved extraction techniques have allowed for the use of low-quality ores, which require more water and produce more waste than high-quality ores. The most water-intensive activities are the separation of minerals from ore, cooling drilling machinery, and dust suppression (Toledano & Roorda, 2014). The massive volumes of mine waste are difficult to manage, as they are unprofitable to mining companies and can be physically and chemically unstable.

The mining industry in Canada has an unmet need to improve tailings deposit performance during both mine operation and post-closure. Three vital concerns in the industry involve improving tailings dust suppression, the mitigation of acid mine drainage, and safe tailings dam

2 storage (Kyhn & Elberling, 2001). There are several physical, chemical, and biological strategies utilized to reach these goals. This thesis will focus on the use of a revolutionary biologic method called biogeocementation, or microbially induced calcite precipitation (MICP), to overcome issues surrounding dust suppression and safe tailings storage. This method employs the use of

Sporosarcina pasteurii (S. pasteurii) bacteria to cement the tailings material.

1.3 Site Descriptions

The mine tailings studied in this thesis were provided by BGC Engineering Inc. (BGC), a consulting company which collaborates with mining operations to design remediation plans for mines in operation and post-closure. The mine tailings studied were from different gold mines; specifically, a gold mine in northern Ontario (GMON), a gold mine in the Dominican Republic

(GMDR), a gold mine in northern BC (GMBC), and a small-scale nickel mine in BC (NMBC).

Many samples types were used from the described mining environments and are described in full during the respective chapters. A thorough metal and soil characterisation were only obtained for the GMON and will be discussed in further detail in the introduction. The tailings samples obtained from the GMDR, GMBC, and NMBC were not fully analyzed due to time constraints. Due to privacy concerns, the location and client information for the mine sites was not included.

1.3.1 Gold Mine in Ontario

The gold mine in Ontario (GMON) is located in Northern Ontario. It is one of the largest gold mines both worldwide and in Canada. It spans over 42,000 hectares and has produced over

29 million ounces of gold in its lifetime (Joyce & McGibbon, 2004).

3

1.3.2 Climate and Wind Speed Data for Gold Mine in Ontario

Due to the location of the GMON, five months of the year the temperature is below freezing

(Table 1-1). The tailings ponds may freeze at these temperatures in the winter months, which can cause further challenges when considering tailings consolidation.

Table 1-1. Average historical climate data for each month for the Gold Mine in Ontario

Jan Feb Mar April May June July Aug Sept Oct Nov Dec Avg -19.6 -15.2 -7.7 1.9 10 15.2 18.1 16.7 10.3 3.7 -6.4 -16.2 Temp (C) Max -14 -9 -1.1 8.2 16.6 21.2 23.8 22.4 15.3 7.7 -2.7 -11.3 Temp (C) Min -25.1 -21.4 -14.2 -4.5 3.4 9.2 12.3 11 5.4 -0.4 -10 -21.1 Temp (C)

The average wind speed in Red Lake ranges from 11-13 km/h, however, in 2018 there was a sharp increase in the maximum wind speed experienced in the area to 84 km/h (Table 1-2). This was a concern, as wind events of this magnitude picked up the dried fines on the beach from the treatment facility and blew them to neighbouring communities.

Table 1-2. Historical wind speed data from 2008 – 2019, with the maximum wind speed in 2018 highlighted

(Government of Canada, 2020)

2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Max Wind Speed 41 46 46 44 41 40 44 41 41 55 84 45 (km/h) Avg Wind Speed 12 12 12 11 12 12 13 12 12 13 13 12 (km/h)

1.3.3 Characteristics of Mine Tailings for the Gold Mine in Ontario

The mine tailings were characterized by AGAT Laboratories in Burnaby, BC. The results of soil characterization and dissolved metal analysis for the GMON tailings are summarized in

Table 1-3 and Table 1-4.

4

Table 1-3. Soil characteristics for GMON tailings

Parameter Value Moisture Content 94.9% Soil Classification Silt Loam pH 7.9 Iron Related Bacteria (present - Y/N) Y Sulphate Reducing Bacteria (present – Y/N) Y

Table 1-4. Concentration of a few key dissolved metals in GMON tailings

Dissolved Metal Concentration (mg/L) Aluminum 0.17 Arsenic 0.06 Calcium 140 Cobalt 0.01 Copper 0.004 Lead <0.0005 Nickel 0.003 Zinc 0.002

Each of the tailings taken from gold mines has similar properties with a small particle size distribution, a pH ca. 6-8, and a moisture content ranging from 80-95%. The NMBC tailings have an initial pH of 7. A full analysis was only performed at an independent laboratory for GMON tailings due to cost; however, particle size distribution for GMDR, GMBC, and NMBC tailings was performed in Chapter 3.

1.4 Project Description

This project is in collaboration with BGC and utilizes mine tailings from a variety of mining operations to explore the application of biogeocementation as both a dust suppressant and a strengthening material.

5

1.4.1 Research Objectives

The objectives of this research are to explore the potential for biogeocementation to be used as a treatment approach for gold mine tailings. Specifically, the goals of this research are to:

1. Explore the properties of Sporosarcina pasteurii.

a. Provide kinetic data for the growth of Sporosarcina pasteurii.

b. Assess the survivability of Sporosarcina pasteurii when subjected to heavy

metals at different loadings, variations in temperature, and different pH

values.

2. Explore the ability of Sporosarcina pasteurii to increase the shear strength of

undrained mine tailings.

3. Determine the effectiveness of Sporosarcina pasteurii at suppressing dust and fine

particle formation on a simulated tailings beach.

1.5 Thesis Organization

This thesis is organized into five chapters, written in manuscript format. Chapter 1 is an introduction to the topic, including a description of the critical mine sites, along with a description of the project and research objectives. Chapter 2 provides an overview of the knowledge gaps related to the further development of this technology that will be filled by this research. Chapter 3 is an introduction to S. pasteurii and an exploration of the conditions that would affect the ability of the microorganisms to produce calcite, as well as an exploration into the effectiveness of S. pasteurii on the undrained shear strength of gold mine tailings. Chapter 4 is an assessment on the use of S. pasteurii to supress gold mine tailings dust generated on a small-scale simulated tailings beach. Finally, Chapter 5 presents conclusions, contributions to science, and proposes future work in this field.

6

Chapter 2 -

Literature Review

2.1 Safe Management of Mine Tailings

The safe disposal of waste, or tailings, produced from mining operations and the continuous management of waste from old mining operations (legacy mines) are two of the largest challenges facing the mining industry today. There are three main strategies to contain or store the waste; the

Earth’s surface, either within retaining structures (slurry or thickened tailings) or in the form of piles (paste or filtered tailings); underground in mined out voids (hydraulic fill or backfill tailings); or in a tailings dam covered in water (Cacciuttolo & Tabra, 2015).

A method of storage utilized in areas with limited space and water resources, or in areas where the geography does not support the construction of a tailings dam, is the dewatered stockpiling (dry stacking) of tailings (Gomes et al., 2016). It is argued that this is the safest method of disposal only when all the water can be removed from the tailings, effectively creating a paste

(Rico, Benito, Salgueiro, et al., 2008). If the mine tailings are dry stacked behind a dam and not enough water is removed, it can result in the liquefaction of the tailings. Liquefaction occurs when a saturated material loses strength, resulting in the solid material behaving like a liquid

(Muhammed et al., 2018). When this happens, there is an increase of pore water pressure and a decrease of effective stress, leading to disaster (Bao et al., 2019). Liquefaction generally happens after seismic activity, such as an earthquake, but can occur in tailings dams when the undrained material has been overloaded, such as saturation from heavy rainfall (Muhammed et al., 2018). To prevent the liquefaction of tailings, the shear strength of the material must be increased. This is done by considering the density of the material while building dams and using a number of different techniques like nanomaterials, bentonite clay, the addition of synthetic fibres, chemical

7 grouting, microorganisms and many more (Bao et al., 2019). For underground mines, the use of a paste for backfilling mining sites is popular. When all valuable ore has been extracted from the underground mine, the mine tailings are combined with cement and backfilled into the mine, preventing the formation of acidic rock drainage (ARD) and ensuring stability. However, the most popular and economic option for safe tailings management in open pit or above ground mines is the use of tailings dams.

2.1.1 Introduction to Tailings Dams

In mining operations where massive amounts of waste are produced due to the nature of the ore, tailings dams are employed. The waste is placed in these structures, which are constructed from readily available local sites and materials, such as dams or berms (Kossoff et al., 2014).

Tailings are pumped from the mill to the tailings dam as a thickened slurry and are stored under water to prevent dusting and the generation of ARD (Kossoff et al., 2014). The tailings can either remain in the dam or can be removed to further treat the water before discharge or reuse. On a unit cost basis, tailings dams are the least expensive when compared to other methods of storage. This is why dams are often selected for the management of waste in most open pit mines, as open pits produce the most waste compared to other methods of mining (Bascetin et al., 2016).

2.1.2 Mine Tailings Dam Design

The design of long-term tailings containment structures must protect the environment, be aesthetically pleasing, be capable of restoration after use, and stable against disruptive forces.

These include short-term events like earthquakes or floods but also take into consideration slow deterioration by wind and water erosion, freeze-thaw cycles, and disruption to the structure by biotic activity (plants or animals) (Robertson & Clifton, 1987). A unique characteristic of tailings dams is that their height increases over time, as new lifts are continuously added (Azam & Li,

8

2010). This presents a structural challenge because the dam must be designed for increased height and weight over time. As well, with the increasing number of severe weather events due to climate change, mining companies must design for worst case scenario or risk an impact to both the environment and industry (Pearce et al., 2010). Following the 2014 Mount Polley mine disaster in

British Columbia, which was the collapse of a dam that was unknowingly built on top of glacial till, the need for more rigorous regulations surrounding tailings dams was highlighted (Byrne et al., 2018). Generally, there are three types of tailings dams: the upstream, downstream, and centreline methods of construction (Figure 2-1). They are normally hydraulically filled by a spigot system, which easily deposits the tailings in the desired location.

Figure 2-1. The design options for a tailings dam (Mittal & Morgenstern, 1975)

9

These dams are created by separating the coarse fraction of tailings from the fine fraction using a hydro-cyclone. The course fraction (underflow) of the tailings is used to build the embankments with steep slopes, which saves on cost (Azam & Li, 2010). The fine fraction of the tailings (slimes or overflow) is then enclosed within the embankments and left to settle and/or treat. The upstream method of construction is when dams are build “upstream” of the starter dam and tailings material is used to build support (S. Barrera et al., 2011). They are the most economic choice since a minimum quantity of virgin sand is required, and instead, tailings material is used.

However, due to the physical characteristics of the tailings material, these types of dams are dangerous and often suffer from liquefaction when they undergo dynamic loading (i.e. earthquake) and are sometimes banned from being used in countries with known seismic activity (Bascetin et al., 2016). The downstream method of construction takes place “downstream” of the starter dam and generally contains an internal drain (Bascetin et al., 2016). This method of tailings containment is most expensive, as virgin material must be used, but are considered safer than upstream tailings dams. Finally, centreline tailings dams strike a balance between cost and stability, as they require less material than downstream dams but are more stable than upstream dams. The cyclone separation of slimes and coarse material means that there is more effective contact between grains of material, increasing the shear strength and minimizing the risk of liquefaction.

2.1.3 Stability Risks of Tailings Dams

Tailings dams are the largest engineered structures in the world and there is a lack of worldwide regulations surrounding their construction and use, therefore the risk of a catastrophic failure is high (Owen et al., 2020). An example of such disaster occurred in January 2019 in

Brumadinho, Brazil as hundreds of people lost their lives when a dam collapsed as a result of poor tailings management (Shukman, 2019). The vast majority of mine disasters result from a tailings

10 dam breach, either through the liquefaction of tailings or the collapse of a facility as a result of floods or earthquakes (Kossoff et al., 2014). The failing of tailings containment facilities is usually a result of the following four weak points; the construction of dykes from residual mining materials, the increase in production and subsequent effluent waste, the lack of regulations on design criteria, especially in developing countries, and poor maintenance as a result of high costs after mine closure (Rico, Benito, Salgueiro, et al., 2008). The two main reasons triggering tailings dams failure events are “unusual rain” and “poor management” and generally occur in dams that are “small to medium” in size (Azam & Li, 2010). The poor management of dams accounts for

10% of worldwide instances and is often the result of to poor beach management, faulty maintenance of the dam drainage structures, and inappropriate dam procedures (Rico, Benito,

Salgueiro, et al., 2008). An unusual amount of rain is a large contributor to the failure of dams as the increased volume of rain can oversaturate the tailings material and resulting in liquefaction.

The tailings must be placed according to density, with the fine tailings having a low density and more susceptibility to liquefaction (Harper et al., 1992). The upstream method of dam construction is the most susceptible to having zones of loose material, which can cause failures. The use of these structures can be exacerbated by increased societal demands for materials that can only be extracted through mining operations (Kossoff et al., 2014). This is when the poor management of tailings facilities can occur, when low quality ores are used to meet the demands for goods. In fact, the increase in metal prices has been correlated with an increased rate of tailings dam failures for two to three years following the increased demand (Kossoff et al., 2014). This correlation may refer to the mining companies striking an imbalance between production of goods and safety or legislative restrictions.

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2.1.4 Management of Tailings Facilities

The management of tailings facilities falls to several organizations. If the mine is still in operation, the onus is on the mining company to ensure the safety of the facilities. However, in mines that are old, or the company has been bankrupted, the onus falls on governments to restore the mine site. For instance, Giant Mine in the Northwest Territories has been the property of the government of Canada since 1999, when Royal Oak Mines Inc. went into receivership (Minogue,

2020). The mine left behind over 237,000 tonnes of arsenic trioxide and the government plans on spending $1 billion to restore the contaminated site (Minogue, 2020). Worldwide, it is difficult to know just how many mine sites there are, where the waste has been disposed, and the full impact on human health and the environment. For instance, there is no official global registry of tailings dams, making it nearly impossible to know just how many failures have occurred and the exact cause (Owen et al., 2020). Recent studies have suggested there are now over 12,000 dams located in China alone (Burritt & Christ, 2018). There are also a number of artisanal, or small-scale, mines around the world, often in rural communities in countries with less rigorous environmental protection plans (Heemskerk, 2005). For example, approximately 20,000 people are involved in artisanal gold mining in Mozambique, which produces 480 – 600 kg of gold annually (Dondeyne et al., 2009). The lack of regulations and education with respect artisanal mining can lead to the improper handling of waste, polluting aquatic ecosystems and creating lasting environmental impacts. The only way to manage the artisanal mining activities are to create policies that directly pertain to these mines. Policies such as restoration plans and an annual waste declaration, both of which are mandatory in small to medium size mines in Catalonia, Spain, can lead to more sustainable mining practices (Vintró et al., 2014). These include providing environmental disclosures and a commitment to sustainability.

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As larger dam disasters, like the Brumadinho dam collapse, come into the media spotlight and people understand the true impact mining can have on the environment, there can be societal and financial consequences (Owen et al., 2020). These can be costly for the mine and must be considered, as community acceptance is vital to the operation of a mine. A case study in Central

America shows that early community involvement and regular impact assessments motivate community engagement and give value to the social outcome of mining development (Sinan

Erzurumlu & Erzurumlu, 2015). In Wisconsin, USA, the mining of sand for fracking has expanded and has raised concerns among local communities with regards to environmental and health risks, the uneven distribution of benefits and costs associated with sand mining, and the removal of hills, bluffs, and picturesque farmland (Pearson, 2013). Mining operations can invoke a personal response, and as a result, several grassroots organizations were formed to resist these mining operations. This resistance can result in costly delays for the companies; therefore, the need to ensure they have considered the needs of the community to prevent backlash.

As the societal, economic, and environmental demands for safe tailings management increases, so does the need for innovative solutions. To reduce the volume of freshwater that is needed for mining operations, technologies like seawater desalination are used to supply mines

(Odhiambo, 2017). For instance, the Olympic Dam in Australia uses a new solid/liquid separation process and reuses acidic mine water via a neutralization circuit, which reduces fresh water consumption by 20% and prevents acidic water from being released to the environment (Ring et al., 2001). The management of water is essential in preventing mine tailings incidents and technological advances have focused on the removal of water from tailings, including the use of tailings paste, thickened tailings, and belt press filtration (Adiansyah et al., 2015). These new techniques can be more expensive than conventional techniques but can be more effective.

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In general, to prevent these mine disasters, regulating bodies and mining companies must adhere to policies that ensure the dams are being monitored and irregularities are being reported. The obligations of both dam owners and operators must be defined to operate safely and reduce the risk of an accident (Mara et al., 2007). The only way to ensure the safety of these facilities are to study past mine disasters to determine their cause and to work to prevent future collapses.

2.1.5 Major Mine Disasters

There have been thousands of mine disasters in every country of the world in the last two centuries. Information on the exact nature of these disasters is difficult obtain, as mining companies rarely release the details of their facilities. The most common causes of mine fatalities are the collapse of underground mines, explosions or fire from coal mines, and the breach of tailings storage dams. Tailings dams fail at a rate of 1 in 700 every year, with 2 to 5 occurring as major disasters, as minor spills generally go unreported (Burritt & Christ, 2018). There are approximately 18, 401 mines worldwide and the failure rate for the last one hundred years is 1.2%

(Azam & Li, 2010). The most impactful dam disasters result in the loss of human life. One of the deadliest tailings dam collapses in the last one hundred years occurred in 1963, as a landslide into the Vijont dam in Italy killed 1900 people (Burritt & Christ, 2018). In 1994 in South Africa, 17 people were killed in the Merriespruit tailings dam breach (Burritt & Christ, 2018). The El Cobre dam in Chile in 1965 killed over 300 people as a result of an earthquake, and in 1985, Italy saw over 250 deaths as a result of poor maintenance of a tailings facility (Kossoff et al., 2014).

Recently, two disasters in Brazil, the Samarco dam in 2015 and the Brumadinho dam in 2019, resulted in the deaths of 19 and 270 people, respectively, marking them as catastrophes with worldwide media attention (Burritt & Christ, 2018). Both mines were owned by the same

14 company, Vale, and following public outcry, Vale lost its license to operate eight dams in Brazil and its stock lost 25 percent of its value (Rolfe, 2019).

While the loss of human life defines a tragedy, the environmental impact of tailings dam collapses is also far-reaching and devastating. The activities associated with mineral and ore processing are identified as seven of the world’s top twenty pollution problems, with mercury poisoning from artisanal gold mining topping the list (Harris et al., 2011). This is due to the rigorous processes required to extract gold and the volume and toxicity of the waste left behind.

2.1.6 Gold Mine Waste

Prior to 1887, gold was mined by hand, until McArthur and the Forrest brothers filed a patent for the gold cyanidation process still used today (Logsdon et al., 1999). In this process, the gold bearing rocks are broken down to a ground ore and mixed with cyanide in the presence of activated carbon to produce a gold-cyanide complex (Mpinga et al., 2014). The overall hydrometallurgical reaction is seen in Equation 2-1 below:

Equation 2-1. Gold cyanidation process

4 퐴푢(푠) + 8 푁푎퐶푁(푎푞) + 푂2 (푔) + 2퐻2푂(푙) → 4푁푎 [퐴푢(퐶푁)2](푎푞) + 4 푁푎푂퐻(푎푞)

In an ideal scenario, the gold ions (Au+ or Au3+) would be reduced by water to form metallic gold (Nicol et al., 1987). However, the difficulty in gold recovery from this process is related to the instability of the gold cations in aqueous solutions. Therefore, cyanide is introduced as a complexing ligand and forms a more stable, Au-cyanide complex (Nicol et al., 1987).

Traditionally, gold is precipitated out of solution using zinc, although many different extraction process were explored, including ion exchange with an anionic exchange resin (Burstall et al., 1953). Now, the most commonly used method is the Merrill-Crowe process (Mpinga et al.,

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2014). This is a cementation process that utilizes an additional vacuum to remove air in the solution, and zinc dust used for greater surface area penetration (Mpinga et al., 2014). The cyanide and zinc work together to stabilize the gold in aqueous solutions so it can then be recovered and utilized in industry.

As demonstrated by the gold Pourbaix diagram (Figure 2-2), Au is not stable between the

H2O and O2 lines, indicating it cannot be extracted in an aqueous solution. The Au must therefore be leached out of solution by cyanide and then cemented using zinc, creating an Au-Zn-CN complex.

Figure 2-2. Combined Au–CN and Zn–CN Eh-pH diagrams which show leaching and cementation for the gold cyanidation process (H. H. Huang, 2016)

This is a simplistic version of the many steps required to recover gold from mines, as multiple unit processes need to be used in conjunction to maximize profit and minimize waste

(Figure 2-3).

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Figure 2-3. Process flowchart for gold recovery (International Cyanide Management Institute, 2017)

This process requires many treatment steps to obtain the gold ore (dore) and results in the production of waste from the flotation tank. The slurry to be sent for underground backfill is filtered cement, fly ash, and water, which are added to form a paste that is discharged underground.

The rest of the tailings stream (flotation and oxide tailings) is mixed with other acidic fluids from the oxidation circuit, treated with lime to both precipitate some heavy metals and increase the pH before being discharged to the tailings pond (International Cyanide Management Institute, 2017).

There are many concerns about the discharge of the waste from gold mining into tailings dams. Although some of the cyanide used in this process is recycled, there is still the potential for the cyanide to leach into groundwater and end up in the water supply (Coppock, 2009). Due to its toxicity, cyanide can have detrimental health effects on both humans and animals. A lethal dose of cyanide can be as little as 1.5 mg/kg body weight (Donato et al., 2007). The long-term health effects of cyanide exposure can include heart, brain, and nerve damage. When tailings dams fail, the toxic effects of cyanide increase as the chemicals enter the waterway quickly. One such disaster occurred in January of 2000 at the Aurul gold processing plant in Romania, where the retaining wall of a tailings dam breeched and cyanide, among other dangerous materials, was released into

17 the Sasar River (Cunningham, 2005). The cyanide and heavy metals moved from the Sasar River in Romania to subsequent rivers through Hungary, the Federal Republic of Yugoslavia, and

Bulgaria, killing wildlife and poisoning the water supply (Cunningham, 2005). Hence, the safe management of mine tailings is essential to mitigate these disasters.

One of the biggest challenges in cyanide management and discharging to tailings dams is the presence of copper cyanide complexes, which are highly toxic to most forms of life even in small doses (Jeffrey et al., 2002). One method developed to overcome this issue in gold extraction is the microbial destruction of cyanide wastes, where full-scale bacterial processes have been used effectively for many years in commercial applications in North America (Akcil & Mudder, 2003).

Both GMON and GMDR are cyanide certified, a voluntary certification created in 2002 through the International Cyanide Management Code for safe management of cyanide in mine tailings and leach solutions (International Cyanide Management Institute, 2017, 2018). This is an added cost for mine companies but is important to improve the management of cyanide in gold and silver mining to protect human health and reduce the environmental impacts of heavy metal mining.

2.2 Environmental Impact of Mine Tailings

The full impact of mine collapses is measured in the short-term (hours to months) and in the long-term (years to centuries). The short-term impacts are defined as the loss of human life as a result of drowning or suffocation from dam breaks (Kossoff et al., 2014). Although the toxicity of the spills can be fatal to human life, these effects are not well documented and would normally occur years after the failure from contamination of water, soils, and sediments. Mine tailings are a source of heavy metal contamination (Cu, Pb, Zn, Cd, Cr, and As) corresponding to the type of metal being mined (Bascetin et al., 2016). The exposure to multiple heavy metals at once may produce toxic effects that can be additive, antagonistic, or synergistic (Tchounwou et al., 2012).

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ARD, heavy metal leaching, processing chemicals pollution, erosion, and sedimentation are the main causes of contamination from mining activities (Kossoff et al., 2014). The potential negative effects persist in tailings facilities that have not been breached, but the mobilization occurs slowly over time. However, if a tailings dam is breached, the negative effects associated with heavy metals in tailings can be exacerbated.

When dam failures are from small to medium facilities, the released tailings can be managed relatively easily (Azam & Li, 2010). One of the most well-known dam collapses in Canada in recent history is the Mount Polley mine. In August of 2014, a breach of the tailings dam was reported at the Mount Polley mine site in British Columbia, where approximately 25 Mm3 of tailings and water were discharged into the Hazeltine Creek (Byrne et al., 2018). The quick removal of tailings material from the primary receiving watercourse, the stabilization of the river corridor and the construction of a new river channel all minimized the environmental impact of the spill (Byrne et al., 2018). The spill lead to the release of Vanadium (V) and Copper (Cu) to the environment, where physical (V) and chemical (Cu) mobilization of these elements occurred

(Hudson-Edwards et al., 2019). Although not completely mitigated, it was found that the quick removal of tailings material could reduce the negative effects of these chemicals. However, not all sites have been as fortunate or proactive, and the reported impacts of other spills have been more pronounced. Other notable dam collapses include Omai (Guyana) in 1994; Los Frailes (Spain) in

1998; Baia Mare (Romania) in 2000; and Aitik (Sweden) in 2000 (Azam & Li, 2010).

2.2.1 Socioeconomic Impact of Tailings Dam Failures

In 2000 in Baia Mare (north-western Romania), over 100,000 m3 of cyanide-contaminated water breached dam walls, due to their poor design and excessive snow melting, spilling into the

Somes river (Mara et al., 2007). The spill extended to the Tisza River and then the Danube, which

19 killed a number of fish and wildlife in Hungary, Serbia, and Romania. The spill also affected drinking water, as the cyanide levels in these rivers were up to 100 times the safe drinking limit

(Cunningham, 2005). This spill is referred to as the worst environmental disaster since Chernobyl and has had global ramifications for the gold industry and the politics of mining (Cunningham,

2005). The mining company was given a token fine of US$150 for the ensuing damages and the compromised livelihood of millions of people, which sparked outrage (Cunningham, 2005). This

Hungarian government sued the company for US$179 million as compensation for the environmental damage (Edraki et al., 2014). The total cleanup cost was estimated to be around

€190 million (US$ 215 million) (Kossoff et al., 2014). Another example of the socioeconomic impact of dam failures is the 1998 Los Frailes event in Spain, where 4-5 million m3 of silver mine tailings spilled into the nearby River Agrio (Rico, Benito, & Díez-Herrero, 2008). The overall socioeconomic losses topped €152 million (US$ 172 million) with about €147 million (US$ 166 million) spent correcting the enviornmental impact of heavy metals on agricultural farmland (Rico,

Benito, & Díez-Herrero, 2008). As of 2014, the government had encurred the costs of clean up, with the mining company claiming the construction company was at fault (Kossoff et al., 2014).

2.2.2 Wind Erosion of Tailings

As mentioned earlier, the wind erosion of tailings can cause negative health and environmental effects. Heavy metals have the potential to become airborne when attached to dust particles, and can be inhaled by individuals causing numerous health issues (Hettiarachchi et al.,

2018). In Tanzania, the dust generated from mining activities at a small-scale gold mine resulted in the release of concentration of silica four times the recommended exposure limit (REL) in the air, and almost 337 times the REL underground (Gottesfeld et al., 2015). The decrease in particle size (< 4m) of these mine tailings increases the severity of inhalation, as smaller airborne

20 particulate matter (PM) can penetrate deeper into lung tissue and cannot be removed by the body as easily as large PM (Hettiarachchi et al., 2018). This has negative implications for individuals living or working near mine sites, as fine particles can be picked up by wind events and carried to nearby communities. The fugitive dust from the Sierrita copper mine in Arizona has forced mining companies to pay for damages in fines and clean-up costs, as the dust suppression technique used by the company did not provide fine particles containment (Smith, 2020). Similarly in Arizona, the Iron King Mine has noted lead and copper concentrations of ~ 40 ppm in topsoil 5 km from the mine, indicating the tailings have been blown great distances from the site, which has negative implications for individuals living or working near the mine (Stovern et al., 2014).

2.2.3 Dust Suppression Reagents

Dust is generated whenever large particles are broken down to smaller particles and can occur at any stage of mining including drilling, blasting, crushing, milling, screening, bagging, loading, and transportation (Cecala et al., 2012). A concern with mine dust is the potential for the dust to spontaneously combust, such as in the case of dust from underground coal mines (Ding et al., 2011). The dust generation from underground mines represents a major health hazard but is less dynamic and variable than surface mines (Colinet et al., 2010). A study reported that wind erosion occurs primarily on sloped edges and only slightly from flat surfaces, with higher recurrence of events during the summer months than in the winter (Angeles & Blight, 2008).

The application of water is the most common method employed for dust control at mine sites (Capeland & Kawatra, 2005). It is used to capture the fine particles and clumping them together, but is only effective if the particles can be completely captured (Capeland & Kawatra,

2005). This means that a separate water truck is needed to collect the water and transport it to the site, thereby, increasing labour and operational costs (Gambatese & James, 2001). This could be

21 mitigated by adding water suppression equipment to existing equipment, such as dump trucks

(Gambatese & James, 2001). Atomization using an air-and-water spray mechanism can be applied to improve the effectiveness of water (Prostański, 2013). This also reduces water use, hence, saving time and money. There are two major concerns associated with dust control or suppression; the specific properties of the tailings and moisture loss, both of which can be resolved with chemical additives. Some types of mine tailings require special attention, such as coal tailings, due to their hydrophobic nature. This is where chemical additives, such as surfactants, are necessary to reduce the surface tension of water, allowing the wettability to increase (Capeland & Kawatra,

2005). It has also been shown that non-ionic surfactants, or those with the ability to be magnetized, can reduce the contact angle of the coal dust, as well as improve the wettability (Ding et al., 2011;

Tien & Kim, 1997). One chemical additive used that is a popular choice for dust suppression is calcium chloride or magnesium chloride, as they are econ economical, hygroscopic (can absorb moisture from surroundings), deliquescent (dissolves in moisture and resists evaporation), and has a high surface tension to bind particles together (Edvardsson, 2009). Finally, binders, such as polyvinyl acetate or crude oil by-products, can be used to bind the material together after the water has evaporated (Capeland & Kawatra, 2005). While these chemical additions represent a temporary solution, a long-term solution must be implemented to fully restore the site and prevent wind or weather erosion from compromising the health and safety of workers and individuals in the community. This is where the physical, chemical, and biological treatment of tailings can be useful to stabilize tailings, prevent ARD, and reduce wind erosion.

2.3 Current Treatment of Mine Tailings

Tailings management is needed for the long-term disposal of mine waste such that the impact to the environment is minimized. The specific chemical and physical characteristics of the waste

22 must be taken into consideration, and the three areas of concern are the presence of heavy metals, the difficulty associated with separating solid and liquid material such as oil sands tailings, and the presence of sulphide heavy minerals which would be responsible for the generation of ARD, caused by the oxidation of pyrite (FeS2) or other sulphide minerals, accelerated by oxygen and water (Equation 2-2) (Johnson & Hallberg, 2005). This leads to an increase in acidity and allows heavy metals to leach into water quickly (Bois et al., 2004).

Equation 2-2. Chemical equation for the formation of acid rock drainage through the oxidation of pyrite

2− + 4 퐹푒푆2 + 15푂2 + 14퐻2푂 → 4퐹푒(푂퐻)3 + 8푆푂4 + 16퐻

The negatively charged sulphates from the oxidation of sulphides combine with positively charged metals, which increases the likelihood of the transportation of the heavy metals to waterways. The generation of ARD depends on many factors, such as pH, temperature, oxygen content in gas and water phases, chemical activity, surface area of metal sulphide, activation energy, and bacterial activity (Akcil & Koldas, 2006). ARD can still occur in tailings that have a low exposure to oxygen marking it as a significant issue for the mining industry. The sulfide- containing minerals are the main driver of acid generation and from this, the acid production potential (APP), can be directly calculated by determining the sulfide content of the tailings stream

(Michael et al., 2010).

There are a number of ways ARD can be prevented, such as diverting surface water flow from the pollution site, preventing groundwater infiltration, preventing hydrological water seepage, and by controlling placement of acid-generating waste (Akcil & Koldas, 2006). To treat ARD, an environment must be created where the pH can be increased to produce more OH- ions, pushing the reaction (Equation 2-2) to the left. This limits the production of negatively charged sulphate ions, which can no longer combine with positively charged metals for transportation to other

23 locations. The easiest way to manage tailings long-term is to introduce a wet (water cap) or dry

(vegetation or sand cap) cover over the tailings (Kossoff et al., 2014). Geotextiles can also be used as a method of containment, either over or underneath the tailings (Bascetin et al., 2016) All new tailings facilities must include a liner on the bottom and caps on top, and be designed to last for

200 to 1000 years (Abdelouas, 2006).

2.3.1 Remediation of Tailings after Failure

The clean-up of mine tailings after a disaster present an engineering challenge due to the sheer volume of material that needs to be contained and treated, as well as the slurry nature of the material. When disaster occurs and these dams release millions of tons of material into waterways, and quick action must be taken to contain the spill. The most popular option is to physically remove the spill, transferring the impacted media to a storage area, as it has been found that the quick removal of spills can reduce the environmental impact (Kossoff et al., 2014). The first step is to build barriers to keep the tailings contained, then chemicals or organic additives can be used to reduce the mobility of contaminants or to neutralize impacted waters (Kossoff et al., 2014). The tailings can then be properly treated and experimental options, such as the utilization of iron tailings for sustainable paints or the use of mine tailings for a road base, can be explored (Barros

Galv et al., 2018; Mahmood & Mulligan, 2010; Swami et al., 2007) While these methods can contain or treat the spill, emphasis must be placed on both adequate construction and ongoing management of tailings facilities to prevent disaster. These can be defined as physical, chemical, or biological techniques and can be utilized to manage the pH, salinity, heavy metal content, and presence of radionucleotides in mine tailings. These methods can also be utilized to increase the stability of the tailings or to prevent other transmission mechanisms such as wind or slope erosion.

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2.3.2 Current Physical Treatment of Mine Tailings

The first step in improving the stability of mine tailings is by exploring the geography of the locations where mine waste is dumped. By utilizing the geophysical data, obtained using remote sensing and history of the area, of the intended area for a tailings dam, mine companies can determine potential hazards or weak spots in the area (Shang et al., 2009). The elimination of instability related to the unseen geophysical properties of the mine site is a step forward to reducing the number of mine disasters. Even when the geographical properties of the site are ideal, measures need to be in place to reduce the physical and chemical instability of the dam. As ARD is generated from oxygen and water exposure, to reduce the rate of oxidation, exposure of oxygen and water must be minimized (Karaca et al., 2018). This can be done by including physical barriers like a cement foundation, grouting with cement, building gravel columns, or adding drains (Bao et al.,

2019). These are not ideal solutions due to the high capital cost and a potential for damage to the environment due to cement leaching into waterways but can drastically improve the outcome of tailings dams. The first step in remediation is finely grinding the material to create the largest surface area. As tailings material is being added to the dam, the most common methods used to increase stability are dewatering, filtration, or centrifugation to separate as much solid material from the water. The challenge with methods like these is the high amount of energy, capital, and operational costs required to increase the density of the tailings, which is sometimes not practical when considering the volume of material to be treated. The difficulty in utilizing these techniques also increases as particle size decrease and can result in filter clogging or an inefficient separation of solids and liquids. After the tailings have been placed, other methods can be employed to increase stability. In regions that experience freezing, the tailings are spread out over a large land area and undergo a freeze-thaw cycle, which increases the solids content of the material by

25 allowing water to drain from the tailings in warmer weather (Afacan et al., 2020). More experimental options include the addition of metal strips or tailings densification using the roots of plants grown after the material has been added to the dam (Bao et al., 2019). The treatment and containment of mine tailings must be cost-effective, robust, and long-lasting to be considered techno-economically viable to the mining industry due to the massive volumes of waste produced every day.

The physical treatment of tailings, such as dumping, covering, or solidifying, can be a quick and effective way to improve the soil characteristics to allow for the natural restoration of the site

(L. Wang et al., 2017). For example, the use of a hardpan layer covering the top layer of the tailings can effectively prevent water infiltration and ARD generation (Sung Ahn et al., 2011). While the mine is in operation, the water that enters mine treatment areas, such as rainwater or water from underground aquifers, is generally pumped and treated on site (Johnson, 2003). However, these methods are not always effective at increasing the strength of the material or preventing the leaching of heavy metals and can be expensive.

The simplest and most widely used method for physical or mechanical stabilization of tailings is the stabilization/solidification process where chemical or physical binders, such as cement, can be used to both increase the UCS strength and immobilize heavy metals during hydration reactions

(Minjo et al., 2007). One study found that a cement addition of 7.5wt% in mine tailings contaminated with arsenic increased the UCS strength to meet industry standards, while effectively minimizing the leaching of arsenic and lead (Choi et al., 2008). The stabilization of mine tailings and prevention of ARD using Portland cement is a common procedure and is enhanced with the addition of other materials, such as fly ash, cement kiln dust, and slag (Nehdi & Tariq, 2007).

These additions can be useful for both above ground and paste backfill applications, and it has

26 been reported that fly ash with high lime content and slag are more appropriate for paste backfill than waste products with a low lime content (Tariq & Yanful, 2013). The benefits of this are two- fold; the stability of the material is increased, along with the incorporation of otherwise useless industrial by-products. The effectiveness of this technique depends almost entirely on the physical and chemical composition of the tailings, which determines the binding and water chemistry between the two materials (Nehdi & Tariq, 2007). An example of a waste product with potential paste applications for the treatment of mine tailings is green liquor dregs, a residue from paper- pulp making, amended with fly ash. The two waste products combined increase the UCS strength and slightly decrease the hydraulic conductivity of the tailings paste (Jia et al., 2013). However, the long curing time required makes this an inefficient treatment method and the costs of inexpensive waste products versus time must be weighed before implementation. Another study utilized small quantities of potassium dihydrogen phosphate (1wt%) and ferric chloride hexahydrate (1wt%) with 5wt% Portland cement to form stable compounds that prevent the further release of metal pollutants, even in highly alkaline conditions (Desogus et al., 2013). The nano- sized magnetite coated with sodium dodecyl sulfate were found to be the more effective of the two, where a small mass of stabilizer resulted in an increased arsenic stabilization to the aluminum and iron phases of the tailings (K.-R. Kim et al., 2012). They noted that metal removal and acid neutralization occurred simultaneously and most rapidly within the first 24 h of reaction (Prakash

Mohapatra et al., 2017). This has implications for arsenic stabilization in other applications, such as groundwater and wastewater, however, it can be relatively expensive and has only been tested to successfully remove arsenic, leaving behind other heavy metals.

Using mine tailings as construction materials is also an effective way to reduce their environmental impact and generate revenue. These can include the incorporation of mine tailings

27 into cement for concrete or the formation of geopolymer bricks. The physical and chemical characteristics of mine tailings determine their effectiveness and appropriateness for incorporation into concrete, and are generally split into three categories; calcium-rich with potential activating properties, silicon-rich with potential hydraulic properties, and silicon enriched with potential pozzolanic properties (Mette et al., 2020). A thorough physicochemical analysis must be conducted on the tailings to determine their most effective application. Geopolymer bricks, on the other hand, are another building material created using mine tailings where stable amorphous polymers with interconnected Si-O-Al bonds are formed from the reaction between aluminosilicates with concentrated alkali hydroxide solutions (Ahmari & Zhang, 2013). These geopolymers are combined with mine tailings to create bricks, which theoretically could be used for construction materials as a replacement for virgin materials. Geopolymers have been shown to have high mechanical strength, can resist acid, can adhere to aggregate, and are capable of immobilizing heavy metals, which helps reduce environmental impact (Ahmari & Zhang, 2012).

These bricks have also been explored with other waste products as additives, including cement kiln dust to increase strength and durability of the bricks (Ahmari & Zhang, 2012). While this is a specific use of mine tailings, the potential for the creation of low-cost building materials, especially with the diminishing global quantity of virgin sand used for concrete, it is an attractive concept that warrants more research.

To reduce the effects of slope erosion of tailings, a vegetative cover (phyto stabilization) can be used to stabilize slopes and decrease the effects of wind and water erosion, as well as provide an esthetically pleasing landscape (Amponsah-Dacosta, 2015). This requires a stabilization of the rhizosphere, to restore the entire root zone, which is the only way the microbial community can recover and provide support to the vegetation (L. Huang et al., 2012). The success of vegetative

28 covers depend on the level of fertilization, the rate it is applied, and the type of organic amendment used (Norland et al., 1992). Plants that are native to the area generally have the most successful outcome. In a gold mine site in Quebec, the speckled alder and the green alder demonstrated the best plant cover on the site, with the speckled alder considered the most desirable, as it is native to the area (Gagnon et al., 2020). The tree species were evaluated based on their survival rates, biomass production, and their low translocation of heavy metals to leaves (Gagnon et al., 2020).

In areas where vegetation cover is not possible, such as tailings that are inhospitable to plants or where there is not enough rainfall to support their growth, non-vegetative cover, such as rock fragments, can be used (Amponsah-Dacosta, 2015). This is where a product such as biochar could be used, which works to improve the soil properties to provide a more hospitable place for plants to grow (Fellet et al., 2011). The effectiveness of this application depends on the feedstock, which could vary depending on the location of the mine site, therefore it is difficult to discern whether this is truly a promising treatment method for mine tailings. While the physical treatment of mine tailings aims to provide a long-term solution to the management of tailings, these are generally rudimentary techniques that provide some stability or containment and do not treat the root of the problem. This is where a chemical or biological additive can be included to improve the physiochemical characteristics of the mine waste, offering a solution to further restore the area.

2.3.3 Current Chemical Treatment of Mine Tailings

The chemical treatment of tailings involves the addition of organic or inorganic compounds to treat the tailings with the goal of removing heavy metals, neutralizing the pH, increasing the density of the soil material, and reducing the risk of ARD generation (L. Wang et al., 2017). In areas where densification is necessary, coagulants (used to induce a charge on the particles and microscopically bond) or flocculants (used to increase the particle size by combining the

29 microscopic particles) can be added in large doses (Afacan et al., 2020). This technology is of particular interest in Canada, where oil sands tailings, also called mature fine tailings (MFT), present a unique challenge due to the residual bitumen and material with a small particle size (C.

Wang et al., 2014). The solution is to create larger particle sizes that can settle faster to release as much water as possible. This has been recently explored with water-soluble polymer flocculants, where the addition of a solid form of the polymer was found to increase the settling rate of the tailings from nonexistent to 60 m/h (Afacan et al., 2020). The use of poly (lactic acid) (PLA) polymers, a biodegradable polymer created from corn, has also shown promise for the treatment of MFT, as the higher degradation rates of the PLA-based materials reportedly led to faster compaction of the MFT sediment (Younes et al., 2018).

To concentrate desired or undesired components of tailings, froth flotation is used, which takes advantage of the chemical properties of the tailings to the advantage of the operator. This technique focusses on the differences in hydrophobicity between different components of a slurry

(Benzaazoua & Kongolo, 2003). The ore needs to be crushed to a small grain size, then a surfactant or wetting agent is added that renders the desired compound hydrophobic. This causes the desired material to float to the top, which can then be removed and treated or harvested. This is a popular method to remove the sulphide compounds in the tailings, resulting in the desulphurization of tailings (Benzaazoua & Kongolo, 2003). This is achieved through either the non-selective or selective froth flotation. The most common non-selective reagents used for sulphide flotation are xanthate-based collectors, whereas selective reagents are tailored to remove specific compounds by conditioning the surface tension of the material (Benzaazoua & Kongolo, 2003). The sulphide material collected from froth flotation can be utilized as a component of paste backfill for underground mines. It was noted that low to medium sulphide-bearing tailings could be combined

30 with slag- and fly ash- based binders to create a mid-strength material, whereas higher fractions of sulphides required cement to create a material with similar strengths (Benzaazoua et al., 2002).

To increase the pH of the tailings, and subsequently address many of the issues related to ARD, two options are available: active or passive treatment methods. The active chemical treatment is abiotic (not containing microorganisms), and these types of treatments generally rely on the addition of lime, also known as limestone, as a viable long-term method to mitigate acid generation in tailings (A. Davis et al., 1999). Other chemicals shown to have a similar effect are marl, sandstone and calcareous crust (García-Valero et al., 2020). The use of some waste products, such as cement kiln dust, have also been explored and found to be successful, with that success positively correlated with the amount of free lime in the dust (Mackie & Walsh, 2012). The active treatment of tailings can include the direct addition of lime or other caustic ingredients to lower the pH and precipitate heavy metals or by introducing a chemical flocculent (Johnson & Hallberg,

2005). These methods may need agitation, which can be achieved by spigot. An active treatment is generally chosen if the metal concentration of the waste is high, as it requires a high cost, external energy, and chemical inputs (Bone, 2010). If the continuous addition of caustic chemicals is cost- prohibitive or not feasible due to location or supply, a passive treatment system can be implemented, which generally involves the use of a drain or channel. This drain are usually lined with limestone, referred to as anoxic limestone drains (ALD), and as the water flows over the drain, the limestone becomes eroded, taking negatively charged ions with it, neutralizing sulphides, and subsequently capturing heavy metals (Hedin & Watzlaf, 1994). They are used most before a wetland to control the pH of the water. These channels offer a shorter retention time for the drainage, however the gas transfer, heat transfer, and suspension of ferric oxides have also been reported to be beneficial in increasing pH over the simple addition of limestone (Dempsey et

31 al., 2001). They are not truly a solution designed to last forever because the channel can become clogged and will need maintenance every few years. While the precipitation of heavy metals using lime is commonly used and very effective, it can also have disadvantages, including the need for large doses of lime and costly sludge disposal fees (Matlock et al., 2002).

Sometimes the combination of two waste products can produce the same effect without the use of pure chemicals. For example, the addition of manganese tailings to the ARD produced from gold mining in South Africa was found to increase the pH and achieve an almost 99% reduction in inorganic species (sulphide was ~ 80%) through chemisorption (Masindi, 2016). In order to make the addition of a calcium carbonate more effective, another study explored the wet ball milling of CaCO3 to mechanochemically activate the carbonate (Zeng et al., 2020). The increase in surface area and reactivity allowed for a more effective capturing of Pb2+ to separate it from other heavy metals, most notably zinc. While the addition of chemicals can be effective for the treatment of mine tailings, this requires the purchase and transportation of chemicals or materials, which can be expensive and difficult to ensure a continuous supply depending on the location of the mine. As well, this creates a high volume of sludge that requires disposal. A solution that has been heavily explored for several years for the treatment of mine tailings is microorganisms.

2.3.4 Current Biological Treatment of Mine Tailings

While physical and chemical treatments are useful to the mining industry to treat tailings, these methods are finite, require continuous monitoring and additives, and can be expensive. They also produce sludges which can contain high concentrations of heavy metals or neutralization chemicals that must be specially disposed and treated (Li et al., 2019). Bioremediation, the use of microorganisms, plants, fungi, or enzymes to reclaim contaminants in soil and other environments, has been increasingly revered as an effective and economical alternative to physical or chemical

32 treatments (Gouma et al., 2014). For bioremediation to be an effective mitigation approach, a number of factors must be taken into consideration (Figure 2-4).

Figure 2-4. Requirements for bioremediation (Cookson, 1995)

Bioremediation can be performed either ex situ or in situ, where ex situ requires for the contaminated material to be removed and transported for treatment to bioreactors, land farming, or composting facilities; while in situ occurs in place, like bioventing or bio stimulating (Zouboulis

& Moussas, 2011). Typically, ex situ treatment options are more expensive, as they require shipping and handling. A bio stimulation approach is especially attractive because it utilizes the properties of indigenous microorganisms without the need to adapt new strains to the environment.

The effectiveness of gram-positive (thicker peptidoglycan layer in the cell wall) versus gram- negative bacteria is still debated in the literature, but studies have found that, in general, gram- negative bacteria have a higher tolerance to high metal concentrations (Srivastava et al., 2014). As well, one study showed gram-negative bacteria were more tolerant to mixtures of hydrocarbons than gram-positive bacteria (Lazaroaie, 2010). Although this can be the trend, the most effective bacteria are generally those that have been adapted to the specific environment and contaminant requiring degradation. The most effective bioremediation pathways are typically aerobic (with oxygen), however, a few anaerobic (without oxygen) pathways have also been reported (Zouboulis

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& Moussas, 2011). Under anaerobic conditions, an alternative electron acceptor, such as nitrate or sulfate, must be available in order to satisfy the energy requirements of the microbes through oxidation/reduction reactions (Zouboulis & Moussas, 2011). Methanogenic bacteria, for example, utilize carbon dioxide and hydrogen gas under anaerobic conditions to produce methane

(Holowenko et al., 2000). Hence, different types of microorganisms could be present at varying depths of material depending on the level of oxygen present. The reclamation of mine tailings is a unique problem, as it also requires microorganisms to thrive in extreme environments. This can include a high or low temperature, high or low pH, and high heavy metal concentrations. However, even under extreme conditions, many microbial species thrive and create specialized pathways to adapt to their environment. As discussed, ARD is a major concern in the mining industry, where minerals containing sulfide are oxidized by air and water to generate sulphates. Sulphate reducing bacteria (SRB) utilize sulphate as an oxidizing agent in their anaerobic metabolic pathway (Dvorak et al., 1992), where the sulphate is reduced in an anaerobic environment generated sulfide, which in the presence of heavy metals and an organic substrate as a carbon source, produces highly insoluble metal sulfides (Praharaj & Fortin, 2004). A wide range of SRB species have been found in tailings, however strains that belong to the genus Desulfovibrio are most common (H. Wang et al., 2016). These bacteria work in conjunction with other bacteria species to treat tailings.

This thesis focuses on gold mining tailings, which can be high in cyanide from the mineral processing techniques used to extract gold. The chemical degradation of cyanide is expensive and complex, and can include the application of sulphur dioxide/air (INCO process), peroxides, or chlorination (Akcil, 2003). These processes require chemical additives and can produce harmful by-products. The microbial degradation of cyanide is a more attractive option, as microbes can break down the cyanide to acceptable environmental levels without the generation of harmful by-

34 products (Akcil & Mudder, 2003). Although there can be a high capital costs compared to chemical approaches, the operating costs are typically lower (Dash et al., 2009). Different strains of microorganisms can breakdown cyanide in both aerobic and anaerobic environments, which diversifies the effectiveness of biological species (Akcil & Mudder, 2003). The naturally occurring Pseudomonas sp. strains of bacteria are most used; however, many species of bacteria can be utilized including Fusarium solani and mixed cultures of fungi, including F. solani,

Fusarium oxysporum, Trichoderma polysporum, Scytalidium thermophilum and Penicillin miczynski (Dash et al., 2009). These bacteria are competitive with chemical treatment processes by degrading cyanide via hydrolytic, oxidative, reductive, and substitution/transfer pathways utilizing cyanide as nitrogen and carbon sources, converting to them ammonia and carbonate

(Figure 2-5) (Akcil et al., 2003).

Figure 2-5. Aerobic biological treatment of cyanide (Akcil, 2003)

When cyanide concentrations are above the maximum survival limit for the bacteria, biological treatment may not be a viable option and alternative methods of cyanide removal may

35 need to be employed upstream of a biological treatment (Dash et al., 2009). However, the robustness of bacteria can be improved by implementing a co-culture, such as varying strains of Halomonas, which were capable of surviving a cyanide concentration of 350 ppm (Khamar et al., 2015). This adaptation to withstand high levels of cyanide has also been shown to occur over time, as aged gold mine tailings showed higher concentrations of cyanide-resistant bacteria and a lower concentration of cyanide (Zagury et al., 2004). This would suggest that bacteria are capable of adapting to their environment and become more effective over time.

Phytoremediation is an extension of bioremediation that harnesses the biologic functions of plants and energy from the sun to remove or degrade contaminants from soils, wastewaters and mine tailings (L. Wang et al., 2017). Contaminants are translocated from the tailings through the roots of the plants (phytoextraction) and can accumulate in the leaves, at which point they can then be disposed as hazardous waste or utilized for metal recovery (L. Wang et al., 2017). Some contaminants can be transported with water and volatilized to the atmosphere (phytovolatilization).

Finally, phytostabilization is one of the most important plant function in mine tailings disposal, where plants can be used as a vegetation cap to reduce mobility of contaminants and roots can stabilize the tailings (L. Wang et al., 2017). The effectiveness of each type of phytoremediation process is dependent on the environmental conditions. In temperate climates, for example, phytoextraction is more effective as tailings are a source of heavy metals and ARD generation, whereas in arid environments, phytostabilization is more vital as tailings are subject to wind and water erosion (Mendez Ae & Maier, 2007). The ideal strategy for mine tailings mitigation would involve an approach that can address a wide range of issues, from providing stability against liquefaction and wind erosion, to the capture of heavy metals, to the decrease of pH. New strains

36 of bacteria are continuously being discovered and proposed to overcome several issues associated with the mining industry.

2.4 Microbially Induced Calcite Precipitation (Biogeocementation)

Microbially induced calcite precipitation (MICP or biogeocementation) is a recently developed branch of geotechnical engineering that utilizes naturally-occurring microorganisms to improve the properties of soil, wastewater, and mine tailings, as well as remove heavy metals

(Mujah et al., 2017; Torres-Aravena et al., 2018; Zamani et al., 2018). MICP has been used for the restoration of calcareous stone materials (Rodriguez-Navarro et al., 2003), wastewater treatment

(Hammes et al., 2003), strengthening of concrete (Bang et al., 2010) plugging (Frederick G. Ferris

& Lester G. Stehmeier, 1992), and oil recovery (Nemati et al., 2005). Generally, the bacteria produce a carbonate structure, which then bonds with alkali earth metals, calcium (Ca) and/or magnesium (Mg), to create carbonate crystals with lattice structures. Calcite (CaCO3) and dolomite

(CaMg(CO3)2) are the two most common structures formed from MICP (Al Disi et al., 2017), both

2- of which form a trigonal lattice structure with alternating Ca/Mg and CO3 groups. They are can be further characterized as polymorphs by identifying different space and point groups. Calcite crystals can form many distinct structures; including prismatic, rhombohedral, and scalenohedral

(Cordier, 2004). The two most common calcite structures reported in one study (confirmed by

SEM) were rhombohedral crystals and spheroids (Al-Thawadi, 2008). The spheroids were fragile, and the strength of the material increased with higher rhombohedral crystal ratio (Al-Thawadi,

2008). These crystals create a bridge between particles to strengthen materials and can also be used to incorporate heavy metals into the lattice structure of the crystal for effective removal.

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2.4.1 Bacteria used for Biogeocementation

In 1973, Boquet et al. determined that crystal formation “is a function of the medium used and that under suitable conditions most bacteria can form calcite crystals”. Many types of bacteria have been observed to produce calcite since, but a few main species have proven successful in literature. The six pathways microorganisms can undergo to induce carbonate precipitation are sulphate reduction, photosynthesis, ammonification, denitrification, metabolization conversion of organic acids, and ureolysis (Seifan and Berenjian 2019). There are a variety of microorganisms that can perform biogeocementation. Some notable species include ureolytic bacteria Bacillus sphaericus and Bacillus megaterium, the ammonification bacterium Myxococcus xanthus,, and a photosynthetic cyanobacterium Synechococcus sp. PCC 7002 (Heveran et al., 2019; Seifan &

Berenjian, 2019). A variety of the Bacillus species, Sporosarcina pasteurii (S. pasteurii), has been used extensively for the production of calcite (Mutitu et al., 2019) and as shown in Figure 2-6. The genus Bacillus are Gram positive, rod-shaped aerobic or facultatively anaerobic, endospore- forming bacteria (Lu et al., 2005).

Figure 2-6. Microscopic image of S. pasteurii (2000x magnification), demonstrating the rod-shaped bacteria

Bacillus species are so resilient, they have been found on the Mars Space Odyssey and are resistant to desiccation (100% survival), H2O2 (26% survival), UV (10% survival) and gamma radiation (0.4% survival) (La Duc et al., 2004). The bacteria spores are heat resistant (Palop et al.,

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1999), for instance the strain Bacillus amyloliquefaciens is resistant to high temperatures, with a maximum dry heat tolerance of 420°C (Beladjal et al., 2018). The ability of the Bacillus species to withstand extreme conditions is important when considering their application to the mitigation of mine tailings.

S. pasteurii (formerly Bacillus pasteuri (Gibson, 1934)) is an attractive option for MICP due to its optimal growth pH of 9.0 and robust nature that can tolerate extreme conditions (Anbu et al., 2016; De Muynck et al., 2010; Jonkers et al., 2010). It has been shown to outperform other types of bacteria when it comes to MICP (Saricicek et al., 2018). S. pasteurii produces calcite through a ureolytic pathway, using the hydrolyzation of urea as the nitrogen source. The pathway begins with the bacterial production of the enzyme urease. The hydrolysis of urea is slow but in the presence of the enzyme urease the reaction is about 1014 times faster (Kalantary & Kahani,

2015). The bacteria use urease to hydrolyze urea to carbonates, which then combine with calcium ions to create calcium carbonate. During this reaction the pH of the solution decreases as the double layer thickness of the particles also decreases, contributing to the ability for the particles to form calcite (Moravej et al., 2017).

Another advantage of the decreased double layer thickness when using ureolytic organisms is the ability to remove heavy metals from aqueous solutions and soils immobilizing these toxic metals, independent of metal valence state, toxicity, or redox potential (Kumari et al., 2016). This is accomplished by incorporating heavy metals to the lattice structure of calcite (Di Benedetto et al., 2006; Lee et al., 2007) or through the formation of insoluble metal carbonates (Phillips et al.,

2013). To bypass the use of bacteria, these experiments have also been performed using only the enzyme urease, generally obtained from the jack bean plant (Canavalia ensiformis) (Yasuhara et al., 2012). It has been used for dust control, where the topically applied enzyme solution increased

39 the resistance of soils to erosion (Hamdan & Kavazanjian, 2016). Although this solution simplifies

MICP (now EICP, enzyme induced calcite precipitation), it can limit the applications of EICP, as the enzyme is not as effective when incorporated into the soil and can only be sprayed or injected into samples. The enzyme cannot travel as well as bacteria and this leads to non-uniform treatment of bio-cemented material and results in weaker treated materials than those that utilize MICP

(Neupane et al., 2015). As well, the commercially available urease utilized can be expensive and limited in quantity, both drawbacks when considering their application on a large scale (Hoang,

2018). It was also noted that MICP performed by bacteria had a higher efficiency than when EICP was utilized (Zhao et al., 2014). MICP has had success in both the treatment of soils and mine tailings, but more research needs to be performed to be able to utilize the solution at a large scale.

2.4.2 Use of Biogeocementation in Soils

Biogeocementation has been explored on many types of soils with many types of bacteria.

The chemical pathways used to create cement-like material depend on the type of bacteria and the environmental conditions (Table 2-1). The urea-driven reaction is the most popular choice in literature for use in MICP applications as it is the thermodynamically favourable option and can occur aerobically, especially when compared to the denitrification pathway (Pham et al., 2016).

Table 2-1. A summary of the chemical pathways utilized by microorganisms which are capable of producing calcareous material (Kalantary & Kahani, 2015)

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The use of indigenous bacteria or a consortium of bacteria species is preferred for the treatment of soils as this can increase the effectiveness of the technology by reducing the need for adapting a pure strain to the specific treatment environment. For instance, the soil-isolated bacteria Bacillus sp. UTMC 2623 performed similarly to S. pasteurii when subjected to strength, permeability, and wind erosion tests (Bahmani et al., 2019), suggesting that the bio-stimulation of native bacteria could be an alternative to introducing non-native bacteria (pure strain of S. pasteurii), but more research is needed to control the calcium and carbon sources for the bacteria.

Another study performed in Iran found a native bacteria (Acinetobacter calcoaceticus strain Nima) isolated from a sandy soil outperformed S. pasteurii in reducing the internal friction angle, increasing shear strength, and increasing adhesion between particles (Mohammadizadeh et al.,

2019). Another soil-isolated strain of bacteria from Iran, Staphylococcus pasteuri HF 2011 (a facultative, non-spore forming anaerobe) was found to produce similar geotechnical results when compared with S. pasteurii, however, the applicability of this bacteria was reduced, as it could not exist as a spore and wouldn’t be able to survive under unfavorable conditions (Badiee et al., 2019).

A local ureolytic bacteria from Japan, Pararhodobacter sp., was tested for its ability to undergo

MICP and it was noted that the conditions yielding the strongest MICP formation were a curing

2+ temperature of 30°C and daily injections of CaCl2 at a concentration of 0.5 M Ca (Amarakoon

& Kawasaki, 2018). In Qatar, indigenous bacteria B. cereus strains, QBB4 and QBB5, were shown to be able to adapt to the harsh conditions and induce MICP, leading to a 7% increase in soil strength (Oualha et al., 2020). Another study comparing the effectiveness of single cultures versus mixed cultures, B. pasteurii and B. pasteurii + B. subtilis was undertaken to stabilize dune sand

(Khaleghi & Rowshanzamir, 2018). The treated sand was found to be stronger than untreated sand, and the mixed culture was more effective compared to the single culture. This concept has also

41 been investigated with algal species and it was found that a Chlorella sp. and S. pasteurii co- culture enhanced the algal biomass production and improved the efficiency of the MICP when compared to a monoculture (Xu et al., 2020). In soil environments where a pure culture of S. pasteurii is required, it must be adapted to the specific conditions in which it will be used. S. pasteurii is generally cultivated and utilized with lab-grade medium components, however, a few studies have shown the effective use of low-cost growth material to reduce costs and increase the potential for MICP application at a field scale (A. I. Omoregie et al., 2019). The S. pasteurii grown in technical-grade reagents performed just as well as the culture grown in lab-grade materials and was able to reduce cost 47-51 fold (A. Omoregie et al., 2019). Along with growth media adaptations, a variety of calcium sources required by the bacteria have been explored. These

MICP-producing bacteria can also be isolated from marine sediments, and it is believed that the environment they live in dictate their ability to produce calcite (Wei et al., 2015). The bacteria

Bacillus sp., found in marine sediments, was utilized for MICP with urea and synthetic seawater provided as a calcium source (Cheng à et al., 2014). Although the amount of urea supplied to the bacteria was low to stoichiometrically correspond to the low concentrations of calcium in seawater, it was still found that the soil could reach a strength of about 300 kPa, after 200 flushes with amendments. This is a method that could potentially improve the economics of the treatment by eliminating the need to provide the bacteria with a calcium source, however, the process was slow in comparison to other MICP methods (Cheng à et al., 2014).

There are several different types of soils and sands where MICP has been successfully implemented. MICP can be performed with organic soils, lower shear strengths have been reported when compared to sandy soils of similar composition (Canakci et al., 2015). Research regarding the application of MICP to organic soils is lacking as the composition requires a specific approach

42 to ensure the calcite-producing bacteria can out compete the robust microorganisms already present.

Generally, treated fine-grained sand has a lower unconfined compressive strength and stiffness compared to a porous, coarser-grained material (Terzis & Laloui, 2018). In a study by

Terzis and Laloui, with a calcite content of 5 – 10% for both types of soil, the bio-treated medium- grained soil reached a USC strength of 3 – 12 MPa, whereas the fine-grained material only reached a USC strength of 2.5 MPa compared to the control. They proposed that larger particle sizes indicate larger bond particle diameters and a lower value of inter-particle stress. When larger plane surfaces are present, the forces that resist shearing and promote particle interlocking are developed and are useful for liquefaction resistance. In sand from the Fraser River, MICP was found to decrease susceptibility to cyclic liquefaction, as a larger number of water cycles were required to induce liquefaction in treated sand than untreated sand (Riveros & Sadrekarimi, 2020).

This leads to arguably the biggest setback in MICP technology; the method used to incorporate bacteria and amendment solutions. There are three techniques currently employed for the introduction and retention of the bacteria inside the soil matrix to allow for evenly dispersed

MICP; injection, surface percolation, and premixing methods (Mujah et al., 2017). The injection method is the most common MICP treatment method, as the conditions (i.e., flow, pressure, and hydraulic gradient) can easily be controlled; however, this leads to uneven distribution of bacteria resulting in non-uniform treatment of bio-cemented material. The percolation method does not require heavy machinery, however low infiltration rate and permeability affect the treatment for fine-grained materials and is better applied for coarse material or an application that does not require a deep penetration. Finally, premixing solves the issues with homogeneity, however, it requires the existing soil to be moved and treated, which increases costs (Mujah et al., 2017). The

43 application of MICP varies in accordance with where the calcium source is incorporated to the material. For instance, CaCl2 incorporated at the surface of sand resulted in a strong, thin crust, while calcium incorporated within the bulk of the sand matrix resulted in a more uniform distribution of calcium carbonate within the sand structure (Chu et al., 2012). These methods have benefits for specific applications; a crust will be better suited for an application where wind erosion is a concern, whereas strength in the bulk of the material would be more useful for liquefaction resistance.

The stabilization of sand against wind and slope erosion is an emerging application of

MICP with promising results. When a silty loam and silty sand were treated with a variety of

MICP-producing bacteria it was found that treated material could withstand windspeeds up to 97 km/h compared to untreated soil that could only withstand 8 – 10 km/h (Rajabi Agereh et al.,

2019). It was reported that optimal dust suppression occurs in a low humidity, high temperature environment, like that of a desert (Meyer et al., 2011). As well, to investigate the ability of sand to withstand slope erosion, samples of sandy soil were arranged to represent slopes at 48°, 51° and

53° and underwent thirty tidal cycles (Salifu et al., 2016). It was found that soils treated with S. pasteurii (MICP) were significantly more stable and less prone to erosion than the untreated soil, indicating this technology could be utilized to stabilize sandy slopes in the field.

For an application of MICP to the bulk of the sand, low calcium concentrations in the treatment solutions were found to be more effective than higher concentrations (Gomez et al.,

2015). This was most likely due to the plugging of pore spaces by calcite, which formed too quickly at the surface instead of allowing deeper penetration. One study found the ideal concentration of calcium was 0.25 M, as the MICP process could be repeated four times, when the calcium concentration increased two or three fold, the material could only be treated twice by

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MICP due to clogging (Wen, Li, Liu, et al., 2019). To overcome clogging, one study sequentially flushed the treated soil after injecting with bacteria in solution (Al-Thawadi, 2008). This resulted in a uniform cementation along a 1 m packed column, but this method was less successful when a finer sand was utilized. The infiltration rate determined the effectiveness of the treatment; as the ureolytic bacteria Bacillus sphaericus was percolated through unsaturated soil and it was found the cementing depth was dependent on the infiltration rate of the bacteria and amendment solutions

(Cheng & Cord-Ruwisch, 2014).

The use of cyclic treatment methods, applying the bacteria and amendment solutions concurrently, has been explored extensively to reduce the instances of plugging. For example, multiple treatment cycles doubled the unconfined compressive strength of treated soil compared to one treatment cycle (Wen, Li, Zhang, et al., 2019). The CaCO3 was precipitated after each additional cycle as a layer-by-layer deposition, which encased the inactive bacteria and wrapped around the sand particles for stabilization (Wen, Li, Zhang, et al., 2019).

Expansive soils like bentonite clay are often used for backfilling due to their low permeability and high potential for swelling. However, when these soils are dried, the moisture content decreases and leads to desiccation cracks (Vail et al., 2019). These cracks negatively impact the mechanical and hydraulic behavior of clayey soils, rendering them less useful for these applications. It was found that MICP could delay crack initiation and mitigate desiccation cracking

(Liu et al., 2020; Vail et al., 2019). As well, multiple MICP treatment cycles have also been shown to remediate cracks in soil due to desiccation (Liu et al., 2020). This has positive implications for colder climates which undergo freeze-thaw cycles. The continuous melting and freezing can result in an increase in soil fractures compared to soil in warm climates. The fractures that occur in fine- grained soils, such as clays which also have expansive properties, can greatly reduce the hydraulic

45 conductivity of the material (ability of a fluid to move through pore spaces) (Harbottle et al., 2014).

The self-healing of cemented soils via MICP demonstrates the ability of the material to restore itself after undergoing physical or chemical stresses by producing more calcite to fill in pore spaces

(S. Botusharova et al., 2019). One of the ways this is accomplished is through the natural preservation of the bacteria as spores. The ability of S. pasteurii to form spores has not been reported definitively in literature, but the Bacillus genus are spore-forming bacteria (S. P.

Botusharova, 2017). To overcome this, both a sporulating and carbonate-precipitating microorganisms could be utilized in conjunction, in this case Sporosarcina ureae, a spore-forming, aerobic, ureolytic bacterium was used alongside S. pasteurii and was able to survive harsh conditions, starvation for periods of 6 months, and able to produce calcite after a starvation period once media was reapplied (S. Botusharova et al., 2019).

MICP has been found to increase the strength of sands and soils, both in the bulk of the material and at the surface. MICP is still an emerging technology and there are four main factors that could limit the success of the technology: calcium concentration, pH, availability of nucleation sites, and urease activity.

2.4.3 Use of Biogeocementation in Mine Tailings

The applications of MICP in sands and soils have become the subject of many publications and span a range of industries. The exploration into the use of S. pasteurii to treat mine tailings, however, has been less expansive and is similarly promising. The results found from studies with sands has a direct implication for its effectiveness in mine tailings. MICP has the potential to improve the deficiencies often associated with tailings and, more specifically, tailings dams such as heavy metal contamination, surface erosion, static liquefaction, and dynamic liquefaction

(Zamani et al., 2018). The difficulty with determining the applicability of MICP to mine tailings

46 is the range of chemical and physical properties between different types of mine tailings. The grain size, pH, temperature, and heavy metal content can vary wildly between tailings depending on the type of mine they come from. To encourage MICP, native bacteria can be adapted to specific conditions to improve survival, such as introducing a particular heavy metal in small concentrations to the growth media (Achal et al., 2009). Alternatively, indigenous bacteria can be isolated from these harsh environments and utilized to perform MICP (J.-H. Kim & Lee, 2019).

This has been done at a copper mine, where indigenous bacteria were isolated from the acidic tailings and utilized for the bio-immobilization of Cu, Pb and Cd (Yang et al., 2016). The treatment with bacteria reduced the exchangeable fractions of the heavy metals and increased the carbonate- bound fraction, indicating the feasibility for utilizing MICP to immobilize heavy metals in mine tailings. This was further demonstrated using the strain Sporosarcina luteola, which was isolated from highly acidic mill tailings and utilized to immobilize heavy metals such as lead and cadmium

(Cuaxinque-Flores et al., 2020). Other strains of indigenous bacteria used for MICP are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, acid-loving bacteria found in sulfide mine tailings (Piervandi et al., 2020). The bio-immobilization of heavy metals by these bacteria was successful, as Mn solubility started at 99% and after 22 days was reduced to 30%, with similar results for Cr, Fe, and Cu (Piervandi et al., 2020). This has positive implications for the use of these bacteria to prevent and remediate sulfide-bearing tailings at an affordable cost

(Cuaxinque-Flores et al., 2020).

In Canada, Alberta holds the largest oil sands reserve containing over 2.5 trillion barrels of oil (J. Liang et al., 2014). This oil is extracted as bitumen from the oil sands and results in large quantities of tailings containing residual bitumen, quartz sands, clays, and silts (J. Liang et al.,

2014). Due to the slurry nature of these tailings, they take many years to settle and are difficult to

47 work with. MICP has been shown to reduce the time it takes for diluted samples to settle and can slightly increase the shear strength of the consolidated sludge (Jiaming Liang et al., 2015). While not a perfect solution, it is a cheaper alternative to chemical flocculants with a similar effectiveness. For fine-grained uranium tailings, S. pasteurii has been show to increase the shear strength of tailings by up to 84% (Gui et al., 2018). An indigenous bacterium, Pararhodobacter sp., was isolated from mine waste from Zambia and utilized to increase the strength of mine tailings to 8 MPa for a coarse-grained sample and 4 MPa for a fine-grained sample (Mwandira et al., 2019).

To improve slope stability and the ability to withstand wind erosion, MICP has been employed to reduce the erodibility of the tailings (Zamani et al., 2018). From preliminary forecasting experiments on mine tailings, it is found the fugitive dust from emissions from mine sites can be far reaching (Kon et al., 2007b). The dust suppression of tailings is a relatively unexplored branch of MICP which has promising benefits based on the successful results of similar experiments performed on soils and sand. The prospect of utilizing MICP on tailings has only recently been explored in literature, however, the potential applications are vast and could help resolve some of the major issues in the mining industry.

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Chapter 3 -

Investigation into Sporosarcina pasteurii and the Measure of Shear Strength

on Undrained Gold and Nickel Tailings

Abstract

Technological advancements in the mining industry have resulted an increased production of mine waste. This waste is managed using tailing dams, which contain the unusable material until it can be treated. Tailing dams are a unique engineering challenge because the height and weight of the dam increases over time. The continuous addition of lifts of material can result in a hazardous situation when the tailings remain partially or fully saturated. The partial saturation of tailings material can result in liquefaction, where loading causes an increase of pore water pressure between grains of material. This causes a decrease in the effective stress between particles and a critical loss of strength in the tailings stack, leading to a deadly flow slide of mine waste with catastrophic environmental impacts and loss of human life.

Sporosarcina pasteurii, a bacterial species that has the potential to undergo microbially- induced calcite precipitation, was explored for its ability to increase the shear strength of gold and nickel mine tailings, which could decrease the risk of a tailings dam failure. The bacteria were first tested for their ability to survive harsh environmental stressors such as elevated heavy metal concentrations, high or low pH, and high or low temperatures. The bacteria were found to survive under low concentrations of several heavy metals found in tailing dams, at a pH of 2-12 and temperatures of 4 - 37°C. This has positive implications for the use of this bacteria at mine sites, as the environmental conditions are not always favourable for bacteria growth and function.

49

S. pasteurii bacteria was then added to gold and nickel mine tailings to determine their effectiveness at increasing the shear strength of the tailings. It was found that the particle size distribution was similar between control (no bacteria) and inoculated samples for each type of tailings, except for nickel tailings inoculated at room temperature, where the particle size distribution decreased with inoculation. However, nickel tailings inoculated at 13°C showed no increase in particle size, indicating the bacteria may be less effective at lower temperatures. For nickel tailings at room temperature, the penetration strength of both the control and the inoculated

2 samples reached a similar value of 4.5 kg/cm (ca. 440 kPa) when tested with a pocket penetrometer over 25 days. Consolidated undrained triaxial testing was also performed on the control and inoculated gold and nickel tailings. The gold tailings of smaller particle size distribution showed a 6.8% increase in shear strength from 263 kPa to 281 kPa when inoculated with bacteria, suggesting successful biogeocementation. The nickel tailings of larger particle size distribution showed an 18.2% decrease in shear strength from 181 kPa to 148 kPa, indicating that biogeocementation was not effective. This was likely a result of pore clogging by the bacteria during calcite precipitation due to the small grain size of the tailings. Future testing should be performed on samples of different particle size distribution to determine the ideal grain size for biogeocementation.

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3.1 Introduction

The safe disposal of waste produced from mine operations and the continuous management of waste present from legacy mines are two of the largest challenges facing the mining industry today. This mine waste is either stored within a retaining structure as a slurry or thickened tailings, underground in mined out voids, or in a tailing dam covered with a water cap (Cacciuttolo & Tabra,

2015). When all the water can be removed from the tailings, the dewatered stockpiling (dry stacking) of tailings is the safest method of disposal (Gomes et al., 2016). This can be difficult, as the ore is ground finely and then chemically inoculated to allow for maximum mineral extraction; and fine-grained material is more difficult to dewater. However, advancements in hydrometallurgy allow for improved processing techniques for low-quality ores, which means more water is used in the extraction process and more waste is produced than with high-quality ores (Dudeney et al.,

2013). This material requires more time, energy, and costs to be dewatered before placing in waste management facilities. This fine-grained material has a low hydraulic conductivity, where water cannot drain easily through the pore spaces and becomes trapped, increasing saturation (C. Wang et al., 2014), which can cause the static liquefaction of tailing stacks, where the saturated material suddenly loses strength, resulting in the solid material behaving like a liquid (Muhammed et al.,

2018). Liquefaction can occur statically, from loosely packed tailings saturated from events like heavy rainfall, or cyclically, from events like earthquakes (Muhammed et al., 2018). In static liquefaction, the increase of pore water pressure leads to a decrease in effective stress between loose particles, resulting in a loss of critical strength and leading to flow slide and potential disaster

(Bao et al., 2019). Static liquefaction can be dangerous, as was seen in Brumadinho, Brazil on

January 25, 2019 where hundreds of people lost their lives when a dam collapsed as a result of poor tailings management (Shukman, 2019). As dam failures like Brumadinho receive media

51 attention and worldwide scrutiny, the mining industry must focus its attention on the regulation of tailings and the prevention of tailing-related disasters to ensure the longevity of the industry.

In response to mine disasters, the mining industry is turning toward new technologies to prevent the liquefaction of tailings. The key to prevention is to increase shear strength between particles, thus increasing the shear strength of the overall material. This is achieved using several different techniques like incorporating nanomaterials, bentonite clay, the addition of synthetic fibres, chemical grouting, cement grouting, and the use of microorganisms (Bao et al., 2019).

One microorganism in particular, Sporosarcina pasteurii (S. pasteurii), has been used extensively in literature for its ability to perform biogeocementation, or microbially-induced calcite precipitation (MICP), to producing calcite, or bio-cement (Mutitu et al., 2019). This bacterial species is robust and can survive harsh conditions (Anbu et al., 2016). S. pasteurii produces the enzyme urease, which hydrolyses urea to form carbonates. These carbonates bond with alkali earth metal ions calcium (Ca2+) and/or magnesium (Mg2+) ions introduced to the system to form calcium or magnesium carbonate (CaCO3 or (MgCaCO3)2) crystals with lattice structures

(Al Disi et al., 2017). This urea-driven reaction is the most popular choice in literature for use in

MICP as it is the fastest option and can occur aerobically (Pham et al., 2016). MICP has been found to increase the strength of sands and soils, both at the surface of the material, and within the bulk of the material, where an increase in strength is valuable to increase resistance to liquefaction

(Gomez et al., 2015; Salifu et al., 2016).

There are three techniques currently employed for the introduction and retention of the bacteria inside a material matrix to allow for evenly dispersed MICP; injection, surface percolation, and premixing methods (Mujah et al., 2017). The injection method is the most common MICP treatment method as the conditions (i.e., flow, pressure, and hydraulic gradient)

52 can easily be controlled; however, this leads to an uneven distribution of bacteria resulting in non- uniform treatment of bio-cemented material. The percolation method does not require heavy machinery, however low infiltration rate and permeability affect treatment for fine-grained materials and is, therefore, better applied for coarse material or an application that does not require a deep penetration. Finally, premixing solves the issues of homogeneity, however, it requires further handling of the local material, which increases costs (Mujah et al., 2017). The performance of MICP also varies depending on where the calcium source is incorporated in the material. For instance, the incorporation of calcium ions at the surface of sand results in a strong, thin crust and calcium incorporated within the bulk of the matrix results in a more uniform distribution of calcium carbonate within the tailing structure (Chu et al., 2012).

This paper explores the properties of S. pasteurii and its ability to survive under different conditions that may be present in tailing dams, such as elevated heavy metal concentrations, high or low pH, and high or low temperatures. Then, S. pasteurii will be incorporated into gold and nickel tailings using the surface percolation method to investigate the potential for MICP to strengthen the tailings. In the mining industry where billions of tons of waste are produced worldwide every year, even a small increase in the shear strength of tailings could be significantly beneficial to the industry as a whole (Edraki et al., 2014).

3.2 Materials and Methods

3.2.1 Preparation of Synthetic Mine Tailings

Synthetic mine tailings were prepared by combining kaolin clay and silica flour in a 60/40 wt.% ratio and oven drying at 100C for 24 hours, then adding distilled water to a 60% w/w moisture content, and placing on a shaker table for 24 hours (Hamade & Bareither, 2019).

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3.2.2 Preparation of Mine Tailings

The mine tailings utilized in this study were obtained from different locations; gold mine tailings from mines in the Dominican Republic (GMDR) and British Columbia (GMBC), and nickel tailings from a mine in British Columbia (NMBC). The GMDR tailings consisted of HDS

(high density sludge), CIL (carbon-in-leach), and FTP (flotation) tailings, representing outputs of different stages of the processes undergone in the tailing facility. The GMBC tailings were designated as Zone 3 and Primary Underflow, indicating the locations from which the tailings were collected. The gold mine tailings arrived in buckets with the water cap from the tailings pond. The tailings were scooped out and well mixed on a shaker table to make them homogenous. The nickel mine tailings arrived pre-dried and were reconstituted by adding the required distilled water to achieve a water content of 70% w/w, which is modelled after real world conditions. The tailings were placed on a shaker table for 24 hours to become saturated them before use.

3.2.3 Cultivation of Sporosarcina pasteurii

Sporosarcina pasteurii (ATCC 11859) was cultivated from freeze-dried S. pasteurii populations in ATCC recommended yeast growth medium (GM – see below) (Bhaduri et al.,

2016). The cells were incubated in an autoclaved 150 mL flask on a shaker table at 31°C until turbid (2-3 days) and then a 50 mL aliquot was transferred to a 250 mL autoclaved flask with an additional fresh 100 mL GM. The cells were left on a shaker table at 31°C until turbid (2-3 days).

3.2.4 Preparation of Media

The GM was prepared by combining 15.75 g tris-base with 1 L of deionized water to make

1 L of 0.13 M of tris-buffer (Bhaduri et al., 2016). Then, 0.5 M HCl was added dropwise to obtain a pH of 9. The 1 L buffer solution was divided into two equal parts – 10 g of ammonium sulfate

((NH4)2SO4) added to 500 mL of buffer solution and 20 g of yeast extract added to the other 500

54 mL of buffer solution. The solutions were autoclaved separately at 121C for 1 hour, cooled, combined and stored in autoclaved medium jars in the refrigerator (at 4°C) until use.

The urea medium UM was prepared by dissolving 3 g of nutrient broth, 10 g of (NH4)2SO4 and 2.12 g of sodium bicarbonate (NaHCO3) in 1 L of deionized water and autoclaving at 121°C for 1 hour. Then, in a separate container, 20 g of urea was dissolved in 25 mL of autoclaved deionized water and filter sterilized (0.22 m) into the autoclaved UM after cooling to prevent degradation of the urea at elevated temperatures. The solution was stored in autoclaved medium jars in the fridge until use.

The supplemental medium (SM) was prepared by dissolving 14 g of calcium chloride

(CaCl2) in 100 mL deionized water and autoclaving at 121C for 1 hour. This was used to provide a source of calcium ions to the carbonates produced by the hydrolyzation of urea. The solution was stored in autoclaved medium jars in the refrigerator (at 4°C) until use.

The phosphate-buffered saline (PBS) solution, used for dilutions, was prepared by combining 1 L of distilled water with 8.0 g of sodium chloride (NaCl), 0.2 g of potassium chloride

(KCl), 1.44 g of disodium phosphate (Na2PO4), and 0.24 g of monopotassium phosphate (KH2PO4) in a 1 L Erlenmeyer flask. The pH was then adjusted to 7 and autoclaved at 121C for 1 hour before sealing in the flask and placed in the refrigerator (at 4°C) until use.

3.2.5 Preparation of Sporosarcina pasteurii

To prepare the S. pasteurii for use in MICP, the GM must be replaced with the UM. Hence,

9 mL of S. pasteurii (OD600 of 0.6) in GM solution was placed into a 10 mL centrifuge tube and centrifuged for 15 mins at 1500 rpm and room temperature. The supernatant was removed and

55 replaced with 9 mL of UM and the bacteria were resuspended using a vortex. It was then used immediately.

3.2.6 Kinetic Experiments for Sporosarcina pasteurii

The kinetic growth of S. pasteurii was determined using a UV/Visible spectrophotometer with a 10 mm path length (Thermo Scientific™ GENESYS™ 10S UV-Vis Spectrophotometer) at a wavelength of 600 nm. The S. pasteurii was prepared according to above and a 10 mL aliquot was placed in 100 mL of sterilized GM in a 250 mL Erlenmeyer flask with a foam plug to begin with an OD600 of approximately 0.08. The S. pasteurii was cultivated and 1 mL samples were taken at 0.5, 1, 3.5, 5, 6.5, 8, 9, 10, 11, 12.5, 13.5, 24, 26, 28.5, 29.5, 31, and 32.5 h for UV-Vis measurements. The UV-Vis results were plotted against time to determine the kinetic growth curve. The cell density was calculated using Equation 3-1.

Equation 3-1. Calculation of bacteria cell concentration

푐푒푙푙푠 퐶푒푙푙 푐표푛푐푒푛푡푟푎푡푖표푛 ( ) = (9푥107) ∗ 푂퐷1.18 푚퐿 600

3.2.7 Survivability Tests with Synthetic Mine Tailings

Approximately 20 mL of synthetic tailings were prepared as above and added into separate

9 mL test tubes. Then, 1 mL of S. pasteurii measuring an OD600 of 0.6 in GM were added to each of the test tubes and well mixed. They were loosely sealed with foam stoppers and left in the incubator at 31°C for 24 hours, a control sample was included using DI water in place of S. pasteurii. Stock solutions were made of the metal salts to achieve the metal concentrations required

(Table 3-1). Then, the test tubes were labelled with a metal (aluminum, arsenic, calcium, cobalt, copper, pot. ferricyanide, pot. thiocyanide, nickel, lead or zinc) in high, mid, or low concentrations, a temperature of -18, 4, 25, or 31C, or a pH of 2, 7, or 12. For test tubes where pH was the

56 measured variable, either 0.5 M hydrochloric acid or 0.5 M sodium hydroxide was utilized to adjust the pH, measured using a pH meter. Test tubes where temperature was the controlled variable were placed in the freezer (-18C), fridge (4C), on the benchtop (25C), or in the incubator (31C), measured using a thermometer. Experiment were conducted in triplicates for each stressor.

Table 3-1. Concentration of metal salt used to determine the likelihood of S. pasteurii survivability

Metal Salt Low Concentration Mid Concentration High Concentration (mg/L) (mg/L) (mg/L)

a Aluminum sulfate (Al2(SO4)3) 0.017 0.17 1.7

a Arsenic trioxide (As2O3) 0.006 0.06 0.60

a Calcium carbonate (CaCO3) 14 140 1400

a Cobalt chloride (CoCl2) 0.001 0.01 0.10

a,b Cupric sulphate (CuSO4) 0.004 0.04 0.4

Potassium ferricyanide 2.0 20 c 200 (K₃[Fe(CN)₆])

Potassium thiocyanide 2.0 20 c 200 (KSCN)

a Nickel sulphate (NiSO4) 0.01 0.003 10

a,b Lead sulphate (PbSO4) 0.005 0.05 0.05

a Zinc sulphate (ZnSO4) 0.0002 0.002 0.02 a These values were adapted from the metal analysis shown in the introduction b Concentration of lead and copper too low to make an accurate dilution so values were increased 10x for copper and 100x for lead c These values were obtained from the average cyanide concentration found in gold mine tailings (Zagury et al., 2004)

3.2.8 Preparation of Agar Plates

Agar plates were prepared using the same methodology as that for the GM preparation, by creating a 1 L 0.13 M tris buffer solution then dividing into two equal parts – 10 g of ammonium sulfate ((NH4)2SO4) was added to 500 mL of buffer solution and 20 g of yeast extract and 20 g of

57 agar into the other 500 mL of buffer solution. The solutions were autoclaved separately at 121C for 1 hour, cooled, combined, and poured into 100 mm x 15 mm Petri dishes, sealed, and placed in the refrigerator before use. On the bottom of the Petri dish, using a permanent marker, 7 dark lines were drawn approximately 14 mm apart to separate the Petri dish into sections, each representing a dilution factor (Figure 3-1). The bacteria were incorporated in the synthetic tailings for 24 hours and then subjected to the desired stressor (metal, pH, or temperature). They were then left in the incubator at 31°C for 48 hours, after which they were placed on the benchtop for 1 hour to return to room temperature while pipette tips and test tubes were autoclaved at 121C for 1 hour.

Then, the supernatant of the synthetic tailings with specific stressors containing S. pasteurii were diluted using PBS solution and plated according to the instructions below.

Figure 3-1. Demonstration of plating the S. pasteurii bacteria with PBS dilution factors included

3.2.9 Plating S. pasteurii to Determine Survivability

The first row of the Petri dish was designated as the “undiluted culture” space, and three

1-2 µL drops of undiluted supernatant were added (Figure 3-1). Then, 1 mL of undiluted culture was transferred to the first test tube and 9 mL of PBS buffer solution was added and mixed, then three 1-2 µL drops were placed onto the second row of the plate for a 10-1 dilution. From there, another 1 mL of 10-1 diluted culture and 9 mL of PBS buffer solution was combined, and three 1-

58

2 µL drops were placed on the third row of the plate. This procedure was continued until the plate was full and had a final dilution of 10-7. The dishes were then left in the incubator at 31°C for 5 days. The control sample was also plated in the same way to account for the occurrence of bacterial contamination. Another control plate was added in the incubator with no bacteria or PBS to account for spore contamination of the samples. After 5 days, the Petri dishes were removed from the incubator and a visual inspection was performed to determine the degree of survivability.

3.2.10 Undrained Tailings Strength Test Experimental Plan

To measure the undrained shear strength of the tailing samples, four Hoskin Scientific

Concrete Test Bio-Cylinder™ Molds were used per tailings type. One was used as a control, where no bacteria or CaCl2 was added, the other three were inoculated with bacteria and CaCl2. The tailings were equally measured by weight into each of the four molds and placed on a shaker table for one hour to ensure homogeneity. Then, 50 mL of S. pasteurii bacteria at an OD600 of 0.6 in UM and 15 mL of SM were added to the three inoculated molds. The molds were then placed on a shaker table for 1 hour before being placed in the fume hood for four weeks. This experiment was also performed for NMBC tailings in a temperature-controlled room at 13°C to simulate cooler ambient temperatures which may be experienced in real-world scenarios. The NMBC samples placed in the cold room were left for longer than samples at room temperature. The strength of the material was measured once per week using an AMS 59032 pocket penetrometer for a rough estimate of strength, taken when the surface could no longer withstand the force and cracked.

3.2.11 Specific Gravity and Particle Size Analysis

To measure the particle size of the undrained tailings before and after treatment with bacteria, the trimmings from preparing the consolidated undrained (CU) triaxial tests were taken and dried in a 60°C oven overnight. Then, they were prepared for a hydrometer test according to

59

ASTM D7928 (ASTM International, 2017) by crushing and sieving the sample through a No. 10

(2.0 mm) sieve. To determine the hygroscopic moisture, 20 g of material passing a No. 10 sieve was placed in a 100 ± 5°C oven for 24 hours with the weight recorded before and after drying. To determine the specific gravity of the samples, 100 g of material was weighed and placed in a 100

± 5°C oven for 24 hours then measured using a pycnometer, following ASTM D854 (ASTM

International, 2000). The specific gravity measurement was adjusted from room temperature to

20°C for consistency.

For the hydrometer test, 50 g of material passing the No. 10 sieve was weighed into a glass jar and 125 mL of 40 g/L sodium hexametaphosphate was added and well-mixed, then left overnight to form a slurry. The hydrometer test was performed using a 152H hydrometer with readings taken at 1, 2, 5, 8, 15, 30, 60, 240, and 1440 minutes. Then, the sample was washed on a

No. 200 (0.074 mm) sieve and mass retained was weighed and dried in a 100 ± 5°C oven overnight.

Finally, the sample was sieved in a stack containing No. 10, 20, 40, 60, 100, 140, and 200 (2.00,

0.850, 0.425, 0.250, 0.150, 0.106, 0.075 mm) sieves, with the mass retained on each recorded.

3.2.12 Consolidated Undrained Triaxial Tests

To quantitatively determine the differences in shear strength and stiffness between the control and inoculated gold and nickel tailings, a consolidated undrained (CU) triaxial test was conducted on the control and inoculated GMDR - HDS tailings, and on the control and inoculated

NMBC tailings. To prepare the samples, the tailings were carefully removed from the cement molds and trimmed using a knife to the desired size. The triaxial tests were performed in accordance with ASTM D4767 (ASTM International, 2003), however, the cylindrical size of the samples did not reach a 2:1 ratio as indicated in ASTM standard, which could result in inconsistencies with the data. The samples were contained within a flexible rubber membrane and

60 were back saturated to a pressure of 1000 kPa until Skempton’s B-value was ≥ 0.95. Skempton’s

B-value (or B-check) determines when the sample has been adequately saturated and was calculated by raising the cell pressure by 100 kPa and determining the ratio between pore pressure and cell pressure (Equation 3-2). When the ratio between pore pressure and cell pressure was close to 1, the sample was noted as fully saturated.

Equation 3-2. Calculation of Skemton’s B-value, where Δu represents change in pore pressure (kPa) and Δσ3 represents the change in cell pressure (kPa)

∆푢 퐵 − 푣푎푙푢푒 = ∆𝜎3

Once the Skempton’s B-value was ≥ 0.95, the sample was consolidated to 1000 kPa to reach the effective stress state required for the next step, shearing. At the end of consolidation, the confining pressure of the cell (σ3) is equal to the axial load (σ1) (Figure 3-2a). The cell pressure was increased while maintaining back pressure until the volume change (ΔV) of the external burette was insignificant.

a) b) σ1 σ3

σ3 σ3 σ3 σ3

σ3 σ1

Figure 3-2. Demonstration of triaxial cell with σ3 representing the cell (confining) pressure (kPa) and σ1 representing axial strength (kPa) of the load cell a) at the end of consolidation and b) during shearing

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Next, the sample was sheared by applying an axial load with a constant confining pressure of 1500 kPa at a rate of 2.5%/hour for 10 hours (Figure 3-2b). The effective stress was then calculated using Equation 3-3 and the shear stress using Equation 3-4.

Equation 3-3. Calculation of effective stress

𝜎′ + 𝜎′ 𝜌′ = 1 3 2

Equation 3-4. Calculation of shear stress

𝜎 − 𝜎 푞 = 1 3 2

These parameters were then plotted with effective stress on the x-axis and shear stress on the y-axis to obtain. A value for shear strength was determined when the pore pressure value is maximized during phase transition.

3.3 Results and Discussion

3.3.1 Kinetic Growth Curve of Sporosarcina pasteurii

A key parameter that is necessary to ensure reproducibility and consistency of results is the concentration of S. pasteurii present in solution before use. During CaCO3 precipitation, the bacteria themselves provide nucleation sites for crystal formation and growth so it is important to regulate the concentration of bacteria, and therefore the number of nucleation sites (Al-Thawadi,

2008). The kinetic growth curve of the S. pasteurii bacteria (Figure 3-3) can help determine the concentration of cells as a direct correlation of the OD600 value, as determined using Equation 3-1.

.

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1.8 1.60E+08

1.6 1.40E+08 1.4 1.20E+08 1.2 1.00E+08 1 (A) 8.00E+07

600 0.8

OD 6.00E+07 0.6 4.00E+07

0.4 Cell Concentration (cells/mL) Cell Concentration 0.2 2.00E+07

0 0.00E+00 0 5 10 15 20 25 30 35 Time (h)

Figure 3-3. Cell concentration and OD600 value over time for growth of S. pasteurii bacteria in yeast growth medium

This growth curve follows the traditional phases of bacterial growth, with a lag phase occurring until approximately 8 hours, where the cell concentration was 2.56 x 107 cell/mL. The exponential phase was maintained until reaching stationary phase within 33–35 h of incubation.

One study indicated that when the OD600 value of S. pasteurii increases, the urease activity also gradually increases (Zhao et al., 2014). However, the specific urease activity only rapidly increases when the OD600 increases from 0.3 to 0.6, indicating that a higher OD600 value does not necessarily mean imply that more urease enzyme will be produced (Zhao et al., 2014). Since enzyme activity is critical to the success of MICP the bacteria do not need to be incubated for 24 hours to reach the desired cell concentration, but instead should be used when the OD600 is 0.6. To ensure a similar cell concentration, all subsequent experiments were performed at an OD600 value of ca. 0.6, corresponding to a cell concentration of ca. 6 x 106 cell/mL, reached after ca. 10 hours.

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3.3.2 Effects of Heavy Metals, Temperature, and pH on Sporosarcina pasteurii

Next, the robustness of the S. pasteurii to different environmental stressors was evaluated.

If the bacteria were sensitive to a heavy metal or could not survive at a given pH or temperature, their effectiveness could be limited in a real-world tailings dam condition. This robustness was tested by subjecting the bacteria to an environmental stressor and determining the cell concentration left on the agar plate through visual observation. As this evaluation was qualitative rather than quantitative, the plates were categorized in three ways: as having little to no chance of survival if subjected to this stressor in the environment, having a moderate chance of surviving, or having a strong chance of surviving (Figure 3-4). The experiments were performed in triplicates and this provided a range of survivability.

a) b) c)

Figure 3-4. Classification of survivability tests with a) indicating a low to no survivability, b) indicating some growth and moderate chance of survivability, and c) indicating the bacteria have a higher chance of surviving the specific stressor

The control plates included with each set of experiments showed no bacteria growth, indicating there was no contamination from other species. Therefore, if bacteria growth was observed on the agar plates, it was assumed to be S. pasteurii. The darkest colours in Table 3-2 indicate that when S. pasteurii were subjected to the specific environmental stressor, there would

64 be little chance they would be able to survive. Lighter colours indicate S. pasteurii would be more likely to survive exposure to that environmental stressor.

Table 3-2. Likelihood S. pasteurii bacteria would survive in environments with different heavy metal concentrations, pH levels, or temperature

High Concentration Mid Concentration Low Concentration Metal Survivability Survivability Survivability Aluminum

Arsenic

Calcium

Cobalt

Copper

3- Cyanide ([Fe(CN)6) ])

Cyanide ([SCN-])

Nickel

Lead

Zinc

pH Survivability Temperature (℃) Survivability

2 -18 (freezer)

7 4 (refrigerator)

12 25 (room temp)

/ 31 (incubator)

Low chance of survivability High chance of survivability

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3.3.3 Effects of Sporosarcina pasteurii and Supplemental Media Application on Mine Tailings

The gold mine tailings from British Columbia and the Dominican Republic, and the nickel tailings from British Columbia were inoculated with S. pasteurii and SM using the well-mixed method to induce calcite precipitation and promote cementation. The tailing samples were left in the fume hood at room temperature for 25 days, each sample required about 14 days for the water cap to evaporate and 25 days for the samples to become consolidated. All samples were tested in this manner with the GMDR HDS, NMBC, and GMBC showing the most promising results as shown in Figure 3-5, Figure 3-6, and Figure 3-7 respectively.

a) b) c)

Day 0 Day 14 Day 25

Figure 3-5. Gold mine tailings from the Dominican Republic (GMDR) inoculated with S. pasteurii and CaCl2 on a)

Day 0, b) Day 14, noting that the water cap had evaporated, and c) Day 25 when final strength was reached

a) b) c)

Day 0 Day 14 Day 25

Figure 3-6. Nickel mine tailings from British Columbia (NMBC) inoculated with S. pasteurii and CaCl2 on a) Day 0, b) Day 14, noting that some of the water cap was still present, and c) Day 25 when final strength was reached

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a) b) c)

Day 0 Day 14 Day 25

Figure 3-7. Gold mine tailings from British Columbia (GMBC) inoculated with S. pasteurii and CaCl2 on a) Day 0, b) Day 14, noting that some of the water cap had evaporated, and c) Day 25 when final strength was reached

After 30 days the inoculated samples were removed from the molds (Figure 3-8) and visually inspected. The GMDR – HDS and NMBC were selected because both the control and inoculated samples were intact sample and could be compared, they were then trimmed to size for

CU triaxial tests and the remaining material was crushed for particle size analysis.

a) b) c)

Figure 3-8. Consolidated tailings inoculated with S. pasteurii and CaCl2 (a) gold tailings from Dominican Republic, b) nickel tailings from British Columbia, and c) gold tailings from British Columbia removed from cement molds after

30 days

The surface well-mixed method of bacteria and SM application can lead to pore clogging by the deposited calcite and therefore to non-uniform strength. The bacteria are aerobic and when

67 pore spaces become clogged due to calcite deposition it prevents oxygen, water, and nutrients availability for the bacteria becomes limited. As well, the coarser particles can settle during the experiment, which could lead to an uneven distribution of calcite. From Figure 3-8, it would appear that the top 3 - 4 cm of each sample has less pore space between particles when compared to the lower 8 - 10 cm of the samples.

3.3.4 Effects of Sporosarcina pasteurii and Supplemental Media Application on the Penetration

Strength of Gold and Nickel Mine Tailings

The penetration strength of the tailings was measured over 25 days. The limit of the pocket penetrometer was 4.5 kg/cm2 and was reached by both the control and inoculated samples for the

NMBC and GMDR (Figure 3-9) tailings. The GMBC tailings achieve this strength, only reaching a strength of approximately 3.5 kg/cm2 (ca. 340 kPa). For all tailings samples, the additional volume of liquid from S. pasteurii in UM and SM resulted in the control samples consolidating earlier than the inoculated samples.

68

5

4.5

4

3.5

) 2

3 Control CIL 2.5 HDS

2 FTP Limit of Pocket Penetrometer

1.5 Penetration (kg/cm Strength Penetration

1

0.5

0 0 5 10 15 20 25 Time (days)

Figure 3-9. Measure of strength with time for control and inoculated samples of gold mine tailings from the

Dominican Republic

The HDS and CIL samples gradually increased in strength over time, with the FTP samples taking longer to reach similar strengths. This could be attributed to the volume of water in the cement molds, the moisture content of the FTP tailings compared to the CIL and HDS tailings, or the particle size of the material, as coarser material tends to be more effective.

69

5

4.5

4

3.5

3

2.5

2 Control at Room Temperature

1.5 Treated at Room Temperature Penetration Strength Penetration (kg/cm2) 1 Control at 13C Treated at 13C 0.5 Limit of Pocket Penetrometer

0 0 20 40 60 80 100 120 Time (days)

Figure 3-10. Measure of strength changing over time for control and inoculated samples of nickel tailings from British

Columbia at room temperature and 13°C

Next, tailings from the NMBC were tested under the same conditions to determine the penetration strength of the material over 25 days (Figure 3-10). The water cap present in the

NMBC tailings took longer to evaporate than for that of the gold tailings. In addition, it should be noted that the NMBC tailings needed to be reconstituted with distilled water and did not contain pore water from the mine, which may change the metal content. The fine particles of the material were difficult to rehydrate, and this may have resulted in the clumping of tailings, which prevents bacteria penetration and uneven distribution of calcite. The NMBC tailings placed in the cold room at 13°C could only be examined after 124 days due to restrictions (COVID-19) which prevented further laboratory work. However, there were similar results to the NMBC tailings at room

70 temperature, as the control and inoculated samples both reached a penetration strength of 4.5 kg/cm2 (ca. 440 kPa).

5

4.5

4 Control - Primary Underflow Treated - Primary Underflow

3.5 Control - Zone 3 ) 2 Treated - Zone 3 3 Limit of Pocket Penetrometer

2.5

2

1.5

Penetration Strength Penetration (kg/cm 1

0.5

0 0 5 10 15 20 25 Time (days)

Figure 3-11. Measure of strength changing over time for control and inoculated samples of gold mine tailings from

British Columbia

Then, the inoculated and control tailings from the GMBC were tested to determine their penetrative strength after 25 days (Figure 3-11). The results for the GMBC show that the inoculated

Primary Underflow tailings reach similar strengths to the inoculated Zone 3 tailings. In both instances, the control samples reach a higher penetrative strength value than the inoculated samples. Overall, the strength results are inconclusive, as the control samples generally reach the same or a higher penetrative strength as the inoculated samples.

71

3.3.5 Effects of Sporosarcina pasteurii and Supplemental Media Application on Particle Size of

Mine Tailings

Further geotechnical testing is required to determine if the samples have been consolidated

(the partial expulsion of pore water from the tailings) or cemented (water in pore spaces replaced with CaCO3). The particle size testing and triaxial testing on the GMDR – HDS and NMBC tailings, chosen as they were intact, will provide further insight. The specific gravity of the control and inoculated tailings samples were first measured using the pycnometer method and are displayed in Table 3-3. An increase in specific gravity may be indicative of successful biogeocementation, as the deposited calcite around the particles may increase the overall mass, leading to a larger relative density and thus larger specific gravity.

Table 3-3. Specific gravity values at 20°C for tailing samples

Tailing Sample Specific Gravity at 20°C Tailing Sample Specific Gravity at 20°C

GMDR - CIL Control 2.90 GMDR - CIL Inoculated 2.70

GMDR - HDS Control 2.95 GMDR - HDS Inoculated 2.82

NMBC - Nickel Control 2.60 NMBC - Nickel Inoculated 2.60

GMBC - Zone 3 Control 2.79 GMBC - Zone 3 Inoculated 2.76

The specific gravity was generally higher for control samples than the inoculated samples, which contradicted the earlier hypothesis that specific gravity would increase with bacteria inoculation. To examine this further a particle size analysis of the tailings was performed using the hydrometer method for NMBC and GMDR –HDS tailings (Figure 3-12, Figure 3-13).

72

Figure 3-12. Particle size distribution of GMDR – CIL tailings

Figure 3-13. Particle size distribution of GMDR – HDS tailings

73

The particle size distribution of GMDR-CIL and -HDS samples was not appreciatively different between the control and inoculated samples. This would indicate the carbonate production by the bacteria was not enough to alter the particle size of the tailings. The tailings are fine-grained, as indicated by the fact that 99% of the material is smaller than 0.25 mm, which could minimize the effectiveness of the bacteria due to pore clogging and limited oxygen, water and nutrient availability at lower depths. As the bacteria precipitate calcium carbonate in the pore spaces of the tailings in the top layer, this layer becomes clogged and the bacteria cannot penetrate further into the material matrix (Bhaduri & Montemagno, 2018). This can create uneven distribution of bacteria and strength within the material matrix. The GMBC tailings were also tested for particle size distribution (Figure 3-14).

Figure 3-14. Particle size distribution of GMBC – Zone 3 tailings

The GMBC Zone 3 tailings consisted of small particles, with 99% of both the control and inoculated samples having particle sizes smaller than 0.075 mm. The penetrative strength showed

74 that these tailings were unable to reach the same strength as other tailings samples inoculated with

S. pasteurii (Figure 3-11). The small particles may have yielded a less effective bacterial treatment due to the reduced permeability within the tailings matrix, which reduces the mobility of the bacteria (Mortensen et al., 2011). Finally, the NMBC were inoculated at room temperature and

13C with S. pasteurii and CaCl2, then particle size analysis was performed on the tailings (Figure

3-15, Figure 3-16).

Figure 3-15. Particle size distribution of NMBC tailings at room temperature

75

Figure 3-16. Particle size distribution of NMBC tailings tested at 13°C

The nickel tailings tested at room temperature showed a decrease in particle size distribution with the inoculated tailings sample compared to the control. The nickel tailings tested at 13C showed no such shift between the inoculated and control samples, which could suggest that S. pasteurii is have been less effective at a lower temperature. Although the survivability test did indicate that they could survive at 13C (Table 1-1), the efficiency of the bacteria may be impacted at low temperatures. It would have been beneficial to extend this testing, however,

COVID-19 restrictions and limited lab access preventing the exploration.

3.3.6 Effects of Sporosarcina pasteurii and Supplemental Media on the Shear Strength of Gold

and Nickel Tailings Measuring using Consolidated Undrained Triaxial Test

A consolidated undrained (CU) triaxial test was performed on the GMDR HDS tailings, then the effective stress (Equation 3-3) and shear stress (Equation 3-4) were calculated, using the

76 axial load and pore pressure values. The shear strength was determined from Figure 3-17 where the pore pressure is maximized, and the effective stress is minimized, providing values of 263 kPa for the control sample of the GMDR HDS tailings and 281 kPa for the inoculated sample. This would suggest that the shear strength increased 6.8% with bacteria inoculation for the GMDR HDS tailings.

400

350

Control 300 Treated

250

]

2

/

)

3 σ

- 200

1

σ

(

[

q 150

100

50

0 0 100 200 300 400 500 600 700 p' [(σ1'+σ3')/2]

Figure 3-17. The effective stress (p’) versus shear stress (q) for GMDR HDS tailings with shear strength values highlighted when pore pressure is maximized during phase transition (softening to hardening)

This experiment was repeated for the NMBC control and inoculated tailings then the effective and shear stresses were calculated and plotted (Figure 3-18).

77

400

350 Control

Treated 300

250

]

2

/ )

3 200

σ

-

1

σ

(

[

q 150

100

50

0 0 100 200 300 400 500 600 700

p' [(σ1'+σ3')/2]

Figure 3-18. The effective stress (p’) versus shear stress (q) for NMBC tailings with shear strength values highlighted when pore pressure is maximized during phase transition (softening to hardening)

The shear strengths for the control and inoculated samples of the NMBC tailings were 181 kPa and 148 kPa, respectively, which would represent an 18% decrease in shear strength with bacteria inoculation. This further supports the theory that biogeocementation was unsuccessful for the NMBC tailings. This could be due to the bacteria clogging which may have occurred during treatment.

3.4 Conclusions

This study investigated some properties of Sporosarcina pasteurii bacteria, including its kinetic growth curve and its ability to survive environmental stressors. An OD600 of 0.6 was determined to be the ideal value for urease enzyme activity and could be reached after 10 hours of

78 incubation. This information was used for subsequent experiments to ensure a consistent bacteria cell concentration was applied to each sample.

The ability of S. pasteurii to survive several harsh environmental stressors was examined using synthetic mine tailings and it was found S. pasteurii were still able to grow at low concentrations of several heavy metals and high concentrations of heavy metals including arsenic, calcium, cyanide, and nickel, at a pH of 2 – 12, and at a temperature of 4 - 31°C. This has positive implications for the use of this bacteria on mine sites, as the environmental conditions are not always favourable for bacterial growth and function.

The S. pasteurii bacteria was then tested using the well-mixed method on gold and nickel tailings to determine the increase of shear strength of the material. It was found that the particle size distribution was similar between the control and inoculated samples for all tailing types except for nickel tailings. The particle size decreased after being inoculated with bacteria, with the change indicating biogeocementation may have been successive. This investigation was inconclusive, as the penetration strength of both the control and the inoculated samples for nickel tailings reached a similar value of 4.5 kg/cm2. Further advanced geotechnical testing was performed on the control and inoculated materials for GMDR and NMBC to quantify the degree of consolidation and the changes in shear strength. It was found that GMDR HDS tailings showed a 6.8% increase in shear strength with bacteria inoculation whereas the NMBC tailings showed an 18% decrease in shear strength. This could be due to differences in particle size distribution and in future studies, the effectiveness of MICP should be performed on different grain sizes to establish an ideal range for maximum strength values.

79

Chapter 4 -

Biogeocementation: increased stability of mine tailings to wind erosion by

microbially induced calcite precipitation

Abstract

The wind erosion of tailings from mine sites can be an environmental, health, and geotechnical hazard. The fine material may contain heavy metals and can be spread to neighboring communities, resulting in contamination of waterways and adverse health effects. Current industrial practices employed to contain the fine particles include the application of water or chemical dust suppressants, which can be expensive and ineffective. The bacterial species

Sporosarcina pasteurii, used to induce calcite precipitation, was evaluated to minimize the wind erosion of gold mine tailings. S. pasteurii and calcium chloride solutions were applied to the surface of the tailings in six different combinations, then subjected to a wind speed of 15 km/h.

The resistance to wind erosion was quantified by comparing the mass lost by the control samples to the treated samples. It was found that a single application of S. pasteurii followed by the continuous application of calcium chloride resulted in the strongest crust (ca. 250 kPa) when compared to other application combinations. The continuous application of S. pasteurii over time was the least ideal application approach as it resulted in the formation of a biofilm and a weak crust (<1 kPa). The average mass lost for the control sample was 3.99  0.88% and was reduced to 0.46  0.22% by the continuous application of calcium chloride. In addition, SEM imaging confirmed the presence of CaCO3 on the surface of the sample. This investigation indicates that mine tailings treated with S. pasteurii were able to withstand wind erosion better than untreated samples and that the single addition of S. pasteurii with continuous additions of calcium chloride produced the strongest crust.

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4.1 Introduction

The airborne dust production that occurs from wind events at mine sites has become an increasing challenge for the mining industry. For years, underground coal miners have been exposed to coal dust, resulting in chronic lung disease for those working on site (Petsonk et al.,

2013). Additionally, underground gold mining and the subsequent inhalation of silica can cause deadly silicosis and can lead to lung cancer (Verma et al., 2014). Although less drastic, this same effect can be seen above ground for individuals working at mine sites. In Tanzania, the dust generated from mining activities at a small-scale gold mine has resulted in an air concentration of silica 4 times the recommended exposure limit (REL), while underground it is almost 337 times the REL (Gottesfeld et al., 2015). Heavy metals also have the potential to become airborne on dust particles and can be inhaled by individuals causing numerous health issues (Hettiarachchi et al.,

2018). Smaller particle sizes (< 4m) of these mine tailings increases the severity of inhalation, as smaller airborne particulate matter (PM) can penetrate deeper into lung tissue and cannot be removed by the body as easily as large PM (Hettiarachchi et al., 2018). This has negative implications for individuals living or working near mine sites, as fine particles can be picked up by wind events and carried to nearby communities. The fugitive dust from the Sierrta copper mine in Arizona has forced mine companies to pay almost half a million dollars in damages, fines and clean-up costs, as the dust suppression technique used by the company did not contain the fine particles (T. Davis, 2019). Similarly in Arizona, the Iron King Mine Tailings resulted in lead and copper concentrations of ~ 40 ppm in topsoil 5 km from the site, indicating the tailings had been blown from the mine site (Stovern et al., 2014). The need for innovative dust suppression techniques is essential to contain this environmental, geotechnical, health, and economic hazard.

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In mining, after the ore is extracted from either an open pit or an underground mine, it is processed to recover the valuable material from the waste called the gangue (Kossoff et al., 2014).

This waste, known as tailings, is a mixture of crushed rock and processing fluids from mills, washeries or concentrators that remain after the extraction of economic materials (Kossoff et al.,

2014). Tailings are difficult to treat, due to their inherent chemical and physical instability. The most common treatment method for tailings is to place them in dams, which are constructed on site and built to contain the waste until treatment can occur (Kossoff et al., 2014). Tailings are pumped from the mill to the dams as a slurry and stored underwater to prevent dusting and minimize the formation of acid mine drainage (Kossoff et al., 2014). Normally the slurry is continuously added from a single elevated point of discharge and this causes a tailings stack, or beach, to develop (Figure 4-1).

Figure 4-1. Example of the development of a tailings beach (Pirouz et al., 2005)

The slurry often contains heavy metals and other potentially toxic materials from mineral processing that can have detrimental effects on human health and the environment. The dust produced from the slurry and dried on tailings beaches can be hazardous, as fine tailings particles have the potential to be spread during wind events. To minimize erosion, dust suppressants are used to bind the top layer of material together. Many dust suppression products are commercially available, some simply form a crust on the surface, while others penetrate to the bulk of the

82 material (Piechota et al., 2004). The effectiveness of a dust suppressant is defined by the material’s ability to keep particles on the surface when subjected to an erosive force (Piechota et al., 2004).

Some methods of dust suppression include the application of waste products, such as crude oil, modified waxes, petroleum resins, enzymes, polysaccharides, microbes, surfactants, polymers, biopolymers, and, most commonly, salt solutions (Edvardsson, 2009). They are either applied topically or mixed in with a top layer of soil, however, these techniques can be expensive, time consuming, and ineffective.

The biogeocementation, or microbially induced calcite precipitation (MICP), of tailings is an attractive concept for dust control because a crust can be formed on the surface that is durable enough to improve the mechanical behavior of the tailings. It is long-lasting and has the potential to heal itself if cracks develop (Seifan et al., 2016). This technology has been used with a variety of microbial species for the dust suppression of soils (Arab, 2019; Castro-Alonso et al., 2019;

Hoang, 2018; Rajabi Agereh et al., 2019; Rajasekar et al., 2017) and mine tailings (Farashahi et al., 2019; Kon et al., 2007a; Stovern et al., 2014).

The microorganism Sporosarcina pasteurii (formerly Bacillus pasteurii) has been utilized extensively in the literature to improve the properties of soil (Mujah et al., 2017), and has only recently been used with mine tailings as a strengthening material and for dust suppression. S. pasteurii is an attractive option for MICP due to its optimum growth pH at 9.0 and ability to tolerate extreme conditions (Anbu et al., 2016; De Muynck et al., 2010; Jonkers et al., 2010). S. pasteurii produces calcite through a ureolytic pathway, using urea as the main nitrogen source.

The pathway begins with the production of the enzyme urease. Urease catalyses the hydrolysis of urea to ammonium and carbamate, which are an unstable compounds (Equation 4-1), and rearrange to form ammonia and carbonic acid (Equation 4-2) (Kumari et al., 2016). Then,

83 ammonia and carbonic acid are further hydrolyzed to form bicarbonate, a second mole of ammonium, and hydroxyl ions (Equation 4-3, Equation 4-4, Equation 4-5). This creates a micro- gradient of carbonate concentration and results in a pH increase as more hydroxyl and carbonate ions are formed (Mutitu et al., 2019). The level of dissolved inorganic carbon is also increased due to the CO2 produced from cell respiration (Hammes & Verstraete, 2002), which enhances CaCO3 precipitation both around the cells and in the media. Finally, the carbonate ions combine with calcium ions to form calcite and ammonium is released as ammonia gas (Equation 4-6, Equation

4-7). Generally, the calcium source utilized comes from calcium chloride (CaCl2), as this is more effective at producing MICP when compared to either calcium acetate or calcium nitrate additions

(Abo-El-Enein et al., 2012). The ammonia gas removed pushes the reaction further to the right, producing CaCO3 until the system runs out of either calcium or carbonate ions (Equation 4-8).

Equation 4-1. Conversion of urea to ammonium and carbonic acid using the water and the enzyme urease

푢푟푒푎푠푒 − + 퐶퐻4푁2푂 + 퐻2푂 → 푁퐻2퐶푂푂 + 푁퐻4

Equation 4-2. Conversion of carbonic acid and ammonium to carbamic acid and ammonia

− + 푁퐻2퐶푂푂 + 푁퐻4 → 푁퐻2퐶푂푂퐻 + 푁퐻3

Equation 4-3. Conversion of carbamic acid to ammonia and carbonic acid using water

푁퐻2퐶푂푂퐻 + 퐻2푂 → 푁퐻3 + 퐻2퐶푂3

Equation 4-4. Conversion of carbonic acid to bicarbonate

− + 퐻2퐶푂3 ↔ 퐻퐶푂3 + 퐻

Equation 4-5. Conversion of ammonia to ammonium using water

+ − 2푁퐻3 + 2퐻2푂 ↔ 2푁퐻4 + 2푂퐻

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Equation 4-6. Conversion of bicarbonate and ammonium to carbonate and ammonium

− + + − 2− + 퐻퐶푂3 + 퐻 + 2푁퐻4 + 2푂퐻 ↔ 퐶푂3 + 2푁퐻4 ↑ +2퐻2푂

Equation 4-7. Calcium ions from CaCl2 attaching to a particle of mine tailings

퐶푎2+ + 푃푎푟푡푖푐푙푒 → 푃푎푟푡푖푐푙푒 − 퐶푎2+

Equation 4-8. Calcium ions from CaCl2 combining with carbonate and attaching to a particle of mine tailings

2+ 2− 푃푎푟푡푖푐푙푒 − 퐶푎 + 퐶푂3 → 푃푎푟푡푖푐푙푒 − 퐶푎퐶푂3

The overall reactions (Equation 4-9, Equation 4-10) show urea hydrolyzing to form carbonate and ammonia, which will lose a hydrogen atom to become ammonia and will leave the system as a gas. Two moles of ammonia gas are produced per mole of urea, which can have implications for scale up because ammonia can be toxic to humans and aquatic life in large quantities (Afrox, 2015).

Equation 4-9. Urea hydrolyzing to form carbonate and ammonia

푢푟푒푎푠푒 퐶퐻4푁2푂 + 2퐻2푂 → 퐻2퐶푂3 + 2푁퐻3 ↑

Equation 4-10. Calcium ions from CaCl2 combining with carbonate and attaching to a particle of mine tailings

2+ 2− 푃푎푟푡푖푐푙푒 − 퐶푎 + 퐶푂3 → 푃푎푟푡푖푐푙푒 − 퐶푎퐶푂3

The calcite bridge is formed between particles by nucleation sites; attaching to the material and adding precipitates until the voids are filled. The precipitation occurs at the particle contact points as well as the particle surfaces (Do et al., 2019). A representative example of this process can be seen in Figure 4-2 with microbial calcite precipitate attaching to the material of interest.

85

When the pH is decreased during the MICP reactions, the double layer thickness of the particles is also decreased, contributing to the ability for the particles to form calcite (Moravej et al., 2017).

Figure 4-2. Representative image of calcite precipitation during ureolysis (Dejong et al., 2010)

When the tailings beaches are formed, a solution of microorganisms and a calcium source

(CaCl2) can be sprayed overtop, binding the material together and creating a crust which will prevent the particles from blowing away. The penetrative strength of the surface of the mine tailings can be increased, thus generating an effective dust control technique.

4.2 Materials and Methods

4.2.1 Cultivation of Sporosarcina pasteurii

S. pasteurii (ATCC 11859) was cultivated by propagating freeze-dried S. pasteurii in

ATCC-recommended yeast growth medium (GM) (Bhaduri et al., 2016). The cells were incubated in an autoclaved 150 mL flask on a shaker table at 31°C until turbid (2-3 days) and then a 50 mL aliquot was transferred to a 250 mL autoclaved flask with an additional fresh 100 mL of GM. The

86 cells were again placed on a shaker table at 31°C until turbid (2-3 days). The cell count was maintained at an OD600 of approximately 0.6.

4.2.2 Preparation of Media

The GM was prepared by combining 15.75 g tris-base with 1 L of deionized water to make

1 L of 0.13 M of tris-buffer. Then, 0.5 M HCl was added dropwise to obtain a pH of 9. The 1 L buffer solution was divided into two equal parts – 10 g of ammonium sulfate ((NH4)2SO4) was added to 500 mL of the buffer solution and 20 g of yeast extract to the other 500 mL of buffer solution. The solutions were autoclaved at 120°C for one hour separately, cooled, combined and stored in autoclaved medium jars in the refrigerator (at 4°C) until use.

The urea medium (UM) was prepared by dissolving 3 g of nutrient broth, 10 g of

(NH4)2SO4 and 2.12 g of sodium bicarbonate (NaHCO3) in 1 L of deionized water and autoclaving at 121°C for one hour before cooling. Then, in a separate beaker, 20 g of urea was dissolved in 25 mL of autoclaved deionized water and filtered (0.22 m) into the autoclaved UM. The solution was stored in autoclaved medium jars in the refrigerator (at 4°C) until use.

The supplemental medium (SM) was prepared by dissolving 14 g of calcium chloride

(CaCl2) in 100 mL deionized water and autoclaving at 120°C for one hour. The solution was stored in autoclaved medium jars in the refrigerator (at 4°C) until use.

4.2.3 Preparation of Mine Tailings

Mine tailings were sampled from a gold mine in Northern Ontario. Soils analysis by AGAT

Laboratories indicated the tailings are a silty loam with a moisture content of 94.9%, and 45% of the material is finer than a No. 200 (75m) sieve. AGAT also provided X-ray diffraction (XRD) analyses of the tailings, indicating the tailings contained mainly gypsum (20%), quartz (18%),

87 muscovite (18%), chlorite (17%), dolomite (11%), and a variety of clays in minor quantities. The tailings arrived under a water cap, which was removed before being placed in a 120°C oven for 24 hours to dry. The tailings were then broken up and placed back in the oven to dry for another 48 hours. The drying process was finished when the weight of the tailings was < 1% over 24 hours.

Then, the tailings were put through a No. 200 (0.074 mm) sieve.

4.2.4 Experimental Set-up

To prepare the S. pasteurii for use, the GM must be replaced with the UM. To prepare the bacteria, 9 mL of S. pasteurii (OD600 of 0.6) in GM solution was placed into a 10 mL centrifuge tube and centrifuged for 15 mins at 1500 rpm and room temperature. The supernatant was removed and replaced with 9 mL of UM where the bacteria were resuspended using a vortex. It was then immediately used as dictated below.

To prepare the samples, approximately 15 g of dried tailings was weighed out individually into 60 small tin containers. Then, S. pasteurii in UM, and SM were added in different combinations as outlined in Table 4-1 by dripping the solutions using a pipette evenly over the surface of the tailings.

88

Table 4-1. Experimental setups implemented for wind erosion tests

Original Subsequent # Description of Tray Components Additions Additions

Tailings (g) 15 A Control (no SP or SM) SPa (mL) 0 SM (uL) 0 Tailings (g) 15 B Only SP added once SP (mL) 9a SM (uL) 0 Tailings (g) 15 C SP and SM added once SP (mL) 9 SM (uL) 560 Tailings (g) 15 SP and SM added once + D SP (mL) 9 every third day add more SM SM (uL) 560 280 Tailings (g) 15 SP and SM added once + E SP (mL) 9 5a every third day add more SP SM (uL) 560 Tailings (g) 15 SP and SM added once + F SP (mL) 9 5 every third day add more SP and SM SM (uL) 560 280 Tailings (g) 15 J Water added once DI (mL) 9 a 6 This refers to bacteria at an OD600 of 0.6, approximately 10 CFU/mL of bacteria

4.2.5 Experimental Plan

Each sample was prepared according to Table 4-1. The samples were left undisturbed in an incubator at 25C for the duration of the experiment. One sacrificial sample from every category

(A-F) was taken every three days, for a total of thirty days. These samples were tested, and additions were made according to Table 4-1. (For example, A6 is a control sample that was tested

89 on Day 18. E9 is a sample that had an initial addition of S. pasteurii and SM on Day 0 and S. pasteurii was every three days and was tested on Day 27.) The samples labeled “J” were tested to simulate a current industrial dust suppression technique of watering, where water is sprayed over the tailings beaches to minimize dusting.

4.2.6 Wind Erosion Tests

On the day of testing, the sample was removed from the incubator and weighed, then placed in a wind tunnel (Figure 4-3), which was comprised of a flow straightener and a box fan, to ensure the turbulent air was directed to a uniform, laminar flow. The fan was turned onto 15 km/h

(measured using a HOLDPEAK 866B Digital Anemometer) and the sample was exposed for 30 seconds. A minimal wind speed of 15 km/h was selected for testing as proof-of-concept. The sample was weighed again after the fan was turned off and the change in mass was recorded. The sample was then dried in the oven at 120C for 24 hours and placed in a scintillation vial.

Figure 4-3. Image of wind tunnel used to test samples

4.2.7 Penetrometer Measurements

The strength of the crust was measured using an AMS 59032 pocket penetrometer.

Penetrometer measurements were taken when the crust could no longer withstand the force and cracked.

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4.2.8 Scanning Electron Microscopy Images

Scanning electron microscopy (SEM) imaging was performed for the investigation of the calcite formation on the surface of the tailings. The imaging was performed on a Hitachi S-2300

(Hitachi High-Tech, Tokyo, Japan) SEM and samples were gold sputtered before imaging.

4.3 Results and Discussion

4.3.1 Effects of Sporosarcina pasteurii and Supplemental Media Application on the Prevention

of Wind Erosion of Mine Tailings Beaches

To determine the effect of S. pasteurii on the ability of mine tailings to withstand wind erosion, tailings samples were treated with various combinations of bacteria and supplemental media. They were then subjected to wind speeds of 15 km/h at intervals of 3, 6, 9, 12, 15, 18, 21,

24, 27, and 30 (Trial 1 through 10, respectively) days. The control samples exhibited a mass loss of 3.99  0.88%, whereas the treated samples with the highest mass loss were E with 1.15  0.61% and the lowest were D with 0.46  0.22% over 30 days (Figure 4-4).

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6

5 A B 4 C D

3 E

Mass Lost Lost Mass(%) F J 2

1

0 1 2 3 4 5 6 7 8 9 10 (a) Trial Number

6 6 A E 5 D 5 F 4 4

3 3

2 2

Mass Lost Lost Mass(%) Mass Lost Lost Mass(%) 1 1

0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 (b) (c) Trial Number Trial Number

Figure 4-4. Plot of the mass percentage of tailings displaced in 15 km/h winds vs trial number, with trial 10 occurring after 30 days (a), plot demonstrating relationship between the control samples (A) and the samples with continuous application of S. pasteurii (D) under 15 km/h wind speed (b), and plot demonstrating trend of samples with continuous addition of bacteria (E & F) over time under 15 km/h wind speed (c)

92

The percent mass loss of mine tailings decreased when samples were treated with bacteria.

Samples treated with only water (J) showed a smaller percent mass loss than the control, but a larger percent mass lost when compared to the samples treated with bacteria. The average mass lost observed for the control sample (A) was 3.99  0.88% and for the continuous application of

CaCl2 (D) was 0.46  0.22%. This was the most successful treatment method, as the percent mass lost was the lowest compared to other treatment methods. The control sample (Figure 4-5) is a fine-grained, material and has no penetrative strength, similar to a soft soil. After an initial application of S. pasteurii and SM, followed by SM every three days for 30 days (Sample D10), a crust of approximately 0.5 cm developed, encasing most of the fine grain material (Figure 4-5).

However, the same phenomenon was not observed when S. pasteurii was continuously added over thirty days. Samples E and F both had S. pasteurii added every third day which resulted in a percent mass loss that increased over time. A weak crust was formed on the surface, leaving most of the fine-grained material underneath (Figure 4-5). This was most likely due to a system that was overloaded with S. pasteurii and the biofilm produced by the secreted extracellular polymeric substance (EPS) of the bacteria (Dufrêne & Persat, 2020). The S. pasteurii were stressed out and could not produce calcite and instead formed a biofilm which looked like a crust but was much weaker (<< 5 kPa) than that of sample D (> 250 kPa) (Figure 4-6).

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(a) (b) (c)

Figure 4-5. Images of control sample (a), sample D (b) and sample F (c) after 30 days of treatment

4.3.2 Effects of Sporosarcina pasteurii and Supplemental Medium Application on the Crust

Strength of Gold Mine Tailings

After the first round of treatment was completed, the continuous addition of S. pasteurii (E and F) was deemed an ineffective MICP approach for dust suppression of tailings, as the bacteria created a biofilm instead of producing calcite, which was characterized by a weaker crust that could not be tested with the penetrometer. As such, tests B, C, and J were repeated and examined at 10, 20, and 30 days to investigate the crust strength over time (Figure 4-6). Since sample D had the least percent mass loss compared to the other treated samples, these strength measurements were performed at 6, 12, 18, 24, and 30 days.

94

300

250

200

B 150 C D

100 J Penetrometer Strength Penetrometer (kPa)

50

0 0 5 10 15 20 25 30 35 Days

Figure 4-6. A plot of the penetrative strength of the crust vs days of treatment for samples B, C, D, and J

The penetrative strength of the crust increased over time for sample D from 0 kPa at 0 days to < 250 kPa after 30 days. Samples B and C increased from 24 kPa after 10 days to approximately

50 kPa after 20 days and maintained this strength for the remaining of the testing period. Sample

J, which had only water applied on Day 0, maintained a crust strength of approximately 50 kPa over 30 days. The penetrative strength of the crust increases as the percent mass of tailings lost decreases, i.e., the less mass lost from wind erosion indicates a stronger crust. From this, it can be interpreted that the method of continuously applying a calcium source (SM) to the tailings yielded in the strongest crust and could, therefore, be the most effective approach for dust suppression.

The SEM images of the tailings before and after treatment (Figure 4-7) show calcite crystal formation 30 days after treatment.

95

Calcite Crystal Formation

(a) (b)

Figure 4-7. SEM images of the dried mine tailings before treatment (a) and after 30 days of treatment (b), both at

2000x magnification

As seen, the sample treated for 30 days showed the presence of calcite crystal formation when compared to the control sample, confirming the microorganisms induced calcite precipitation.

4.4 Conclusions

This paper studied the effect Sporosarcina pasteurii bacteria on the stabilization of gold mine tailings for the prevention of wind erosion. It can be inferred that gold mine tailings treated with

S. pasteurii could withstand wind speeds up to 15 km/h better than tailings that had not been treated. The most effective treatment method was to perform a single application of bacteria and then to continuously add CaCl2 (a calcium source), decreasing the percent mass lost from 3.99 

0.88% to 0.46  0.22% over 30 days of treatment. The application of water to the tailings resulted in a decreased dust production compared to the control but is not a desirable method of dust suppression as it cannot bind the top layer of tailings as effectively as S. pasteurii. This has positive implications for scale-up treatment because the bacteria can be easily supplemented and does not

96 need to be continuously applied, which can be costly, time consuming, and was deemed ineffective.

The relationship between the decreasing percentage of tailings lost and an increase of the penetrative strength of the crust indicates that the penetrometer test is a good indication of the effectiveness of the treatment method. This research could lead to the possible commercialization of this dust suppression technique and future studies should be explored at a faster wind speed and at a larger scale to continue the proof-of-concept work.

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Chapter 5 -

Conclusions and Recommendations

5.1 Conclusions

This thesis explored the use of Sporosarcina pasteurii bacteria, which undergoes microbially induced calcite precipitation or biogeocementation, to cement gold and nickel mine tailings. The tailings sampled were from gold mines in Ontario (GMON), British Columbia (GMBC), and the

Dominican Republic (GMDR), and a nickel mine in British Columbia (NMBC).

In Chapter 3 the kinetic growth curve of S. pasteurii was investigated, with an OD600 value of

0.6 determined to be the target bacteria concentration, which was used in subsequent experimentation. S. pasteurii was then tested for its ability to survive environmental stressors, such as heavy metals, high or low temperature, and high or low pH. The bacteria were still able to grow at low concentrations of all heavy metals tested, between a pH of 2-12, and between 4 and 31°C.

While the bacteria exhibit reduced function at these conditions, it has positive implications for their use on mine sites, which generally have unfavourable conditions.

The bacteria were then utilized to inoculate gold and nickel tailings using a well-mixed method.

There were two samples tested per tailings sample; control with no bacteria, and inoculated, where bacteria and calcium chloride were incorporated using the well-mixed method. The control and inoculated tailings samples were left to dry over 25 days and penetrative strength measurements were taken periodically after the water cap on the samples had evaporated. A pocket penetrometer was used to test the material’s ability to resist penetration. Both the control and inoculated GMDR

HDS and NMBC tailings reached an estimated penetrative strength of 4.5 kg/cm2 (ca. 440 kPa).

The GMBC control and inoculated tailings only reached strengths of ca. 3.5 kg/cm2 (ca. 340 kPa),

98 so the particle size distribution was determined using the hydrometer method. The particle size distribution was found to change minimally between the control and inoculated samples for all gold mine samples. Nickel tailings, however, experienced a decrease in particle size distribution after bacterial treatment, which may be indicative that biogeocementation was unsuccessful. The

GMBC tailings had the smallest particle size distribution when compared to the other gold and nickel tailings, and for this reason most likely did not reach similar strengths.

To get more information on the change in shear strength between the control and inoculated samples, further advanced geotechnical testing was performed on GMDR HDS and NMBC tailings. A consolidated undrained (CU) triaxial test was performed on the control and inoculated samples from each type of tailings. It was found the shear strength of the GMDR HDS control was

263 kPa and 281 kPa for the inoculated tailings. This is a 6.8% increase in shear strength with bacteria application, indicating biogeocementation was successful. For the NMBC tailings there was an 18.2% decrease in shear strength from 181 kPa to 148 kPa. This indicates biogeocementation was not successful for NMBC tailings.

In Chapter 4 S. pasteurii bacteria was used for the stabilization of gold mine tailings to prevent wind erosion. GMON tailings treated with S. pasteurii and calcium chloride were able to withstand wind speeds up to 15 km/h better than tailings that had not been treated. Five different treatment application methods were tested, and it was found the most effective treatment method was a single application of bacteria and then continuous applications of calcium chloride over 30 days. The percent mass of tailings lost decreased from 3.99  0.88% to 0.46  0.22% over 30 days of treatment. The application of water to the tailings was also tested and resulted in a decreased dust production compared to the control. However, water is not a desirable method of dust suppression as it cannot bind the top layer of tailings as effectively as S. pasteurii. This has positive

99 implications for scale-up treatment because the bacteria can be easily supplemented and does not need continuous application, which can be costly, time consuming, and was deemed ineffective.

5.2 Recommendations and Future Work

The use of Sporosarcina pasteurii to increase the strength of mine tailings is a promising field of research. The work performed in Chapter 3 of this thesis has shown the calcite produced by S. pasteurii bacteria can increase the shear strength of gold mine tailings when compared to samples with no microbially produced calcite. However, this result was not replicated with nickel tailings and not enough data could be collected to fully demonstrate the success of biogeocementation. Therefore, it would be of value to perform future studies where the tailings samples would be inoculated with different concentrations of bacteria and calcium chloride.

Finding the ideal ratio between bacteria and calcium chloride and performing these tests in in triplicates should give a bigger picture as to the strength increase possible by the bacteria. The sample preparation in the future should be altered to ensure the correct 2:1 height to diameter ratio can be reached for triaxial tests, as well as ensuring the tests are performed in triplicate.

The dust suppression trials that occurred in Chapter 4 of this work were successful, as the issues of pore clogging that affected the undrained strength tests are beneficial to the development of a crust to decrease dust production. In future trials, this experiment should be repeated at a higher wind velocity to simulate real-world conditions where winds can be upwards of 80 km/h.

As well, it would be beneficial to perform a trial using only calcium chloride, as salt solutions are used in real world applications to prevent dust generation. \

Finally, the exploration of S. pasteurii to produce bio-cement should be utilized for the treatment of the chemical instability of mine tailings. Biogeocementation has been used to

100 remediate heavy metals in other industries and future studies should research its application for the chemical treatment of mine tailings.

5.3 Contributions to Science

The biogeocementation of sands and soils is a well-explored branch of geotechnical engineering, however, the biogeocementation of tailings is an emerging application with promising results. The dust suppression of tailings is of interest, as it is a quick and easy application and could prevent considerable environmental and health issues related to dust generation from tailings beaches. The dust suppression work will be submitted to the Journal of Industrial Microbiology and Biotechnology for consideration.

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Chapter 6 -

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