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Novel two-stage biochemical process for hybrid passive/active treatment of mine-influenced water Lundquist, Lauren 2020

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NOVEL TWO-STAGE BIOCHEMICAL PROCESS FOR HYBRID PASSIVE/ACTIVE TREATMENT OF MINE-INFLUENCED WATER by  Lauren Lundquist  B.Sc., The Colorado School of Mines, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2020  © Lauren Lundquist, 2020  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  Novel two-stage biochemical process for hybrid passive/active treatment of mine-influenced water  submitted by Lauren Lundquist in partial fulfillment of the requirements for the degree of MASc in Chemical and Biological Engineering  Examining Committee: Dr. Susan Baldwin, Chemical and Biological Engineering Supervisor  Dr. James Piret, Chemical and Biological Engineering Supervisory Committee Member  Dr. Madjid Mohseni, Chemical and Biological Engineering Supervisory Committee Member  iii  Abstract The research goal was to develop and test a novel hybrid passive/active treatment system to remove metals from mine-influenced water that also contains sulfate. Current passive treatment systems for metal removal from sulfate-rich mine-influenced water include a type of biochemical reactor (BCR) called a permeable reactive barrier (PRB) which suffers from poor operational performance, such as metal sulfide retention and declining efficacy with time due to depletion of the solid organic materials.   The lab-scale work of this thesis included proof-of-concept experiments in order to validate the proposed new design that decouples the metal removal and biological sulfate reduction steps into two stages and uses a liquid carbon source. The kinetic studies for sulfate reduction with leachates were done in batch bioreactors where a laboratory standard, lactate, achieved 66% sulfate removal and leachates from hay & wood chip mix and pulp & paper mill biosolids, achieved 66% and 34%, respectively. After proving that sulfate-reducing bacteria (SRB) could successfully utilize a leachate as a carbon source, the next step was to prove that the biologically produced (biogenic) sulfide in the bioreactors was effective at reacting with metal ions in the real mine-influenced water in order to create a metal precipitate. Zinc, cadmium and lead were removed at 90% and above.   After the proof-of-concept experiments were completed in the lab, a continuous flow experiment was designed and implemented in the field at a mine site. The two-stage design decoupled the biological sulfate reduction reaction from the chemical metal precipitation reaction in a series of two packed-bed column reactors. Three different column systems were set up using a hay and iv  wood chip mix leachate, molasses and a negative control. All three systems were tested for a duration of 96 days, showing that both tested liquid carbon sources could remove sulfate to extents greater than 73% and precipitate Zn, Cd and Pb to achieve greater than 85% removal. These results will inform further pilot testing on the mine site, and implementing the design improvements will help to avoid problems associated with passive treatment systems in the past.     v  Lay Summary  Sustainable mining requires that negative impacts on the environment, such as metal leaching into water resources, be managed and mitigated. Passive treatment systems are preferred rather than tackling large sources of mine-influenced water since it can be implemented close to the sources of contamination; however, problems with current systems have occurred due to metal retention, clogging and declining efficacy with time. Thus, the purpose of this study was to develop a novel hybrid passive/active treatment system that does not suffer these shortcomings. The novel two-stage design decoupled the biological sulfate reduction reaction from the chemical metal precipitation reaction into a series of two column reactors, and used a liquid carbon source. At a mine site location, a field experiment tested the hybrid system for a duration of 96 days which proved to successfully remove sulfate and metals to below British Columbia Water Quality Guidelines. vi  Preface  This research was designed, carried out and analyzed by the graduate researcher. Site location, field equipment and financial support were made possible by collaboration with an industrial partner as part of a Mitacs Fellowship grant. vii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements ......................................................................................................................xv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................1 1.1 Mine-Influenced Water and Treatment Objectives ......................................................... 1 1.2 Metal Toxicity in Freshwater Aquatic Systems .............................................................. 2 1.3 Metal Speciation ............................................................................................................. 4 1.4 Passive Treatment Systems for Metal Removal ............................................................. 5 1.4.1 Summary of Current Passive Treatment Systems ................................................... 5 1.4.2 Biochemical Reactor ............................................................................................... 8 1.4.3 Hybrid Passive/Active System .............................................................................. 12 1.5 Sulfate Reducing Bacteria ............................................................................................. 14 1.5.1 Role in the Environment ....................................................................................... 14 1.5.2 Carbon Metabolism and Growth ........................................................................... 16 1.5.3 Taxonomy ............................................................................................................. 17 viii  1.6 Genomic Tools and Techniques for Characterization of Microbial Communities ....... 18 1.6.1 16s rRNA Gene Surveys for Phylogenetic Analysis ............................................ 18 1.6.2 Quantitative PCR for Functional Genes ............................................................... 20 1.7 Research Goals .............................................................................................................. 21 Chapter 2: Batch Proof-of-Concept Testing of Leachates, Sulfate Reduction and Metal Precipitation .................................................................................................................................23 2.1 Synopsis ........................................................................................................................ 23 2.2 Materials and Methods .................................................................................................. 24 2.3 Results and Discussion ................................................................................................. 28 2.3.1 Bulk Properties of Solid Organic Materials and Leachates .................................. 28 2.3.2 Sulfate Removal and Sulfate Reduction Rates ..................................................... 31 2.3.3 Biogenic Sulfide Production and Metal Precipitation .......................................... 34 2.3.4 Microbial Community Analysis ............................................................................ 39 Chapter 3: Continuous Flow Column Study of Hybrid System Treating Real Mine-Influenced Water .........................................................................................................................47 3.1 Synopsis ........................................................................................................................ 47 3.2 Materials and Methods .................................................................................................. 48 3.2.1 Site Description ..................................................................................................... 48 3.2.2 Experimental Set-Up ............................................................................................. 51 3.2.3 Sampling Points .................................................................................................... 54 3.2.4 Analytical Methods ............................................................................................... 55 3.2.5 Biological Inoculation ........................................................................................... 56 3.3 Results and Discussion ................................................................................................. 57 ix  3.3.1 Sulfate Removal and Sulfide Production .............................................................. 57 3.3.2 Metal Removal ...................................................................................................... 61 3.3.3 Quantitative PCR of dsrA Gene ............................................................................ 65 3.3.4 Comparisons to Hybrid Passive/Active System ................................................... 66 Chapter 4: Conclusions and Recommendations .......................................................................70 Bibliography .................................................................................................................................73 Appendices ....................................................................................................................................82 Appendix A ............................................................................................................................... 82 Appendix B ............................................................................................................................... 83 Appendix C ............................................................................................................................... 86 Appendix D ............................................................................................................................... 87 D.1 Zinc ........................................................................................................................... 87 D.2 Cadmium ................................................................................................................... 87 D.3 Lead ........................................................................................................................... 88 D.4 Manganese ................................................................................................................ 88 D.5 Cobalt ........................................................................................................................ 89 D.6 Iron ............................................................................................................................ 89  x  List of Tables  Table 1. Example of BC WQGs for metals including hardness equations ..................................... 3 Table 2. Examples of metal species and standard state solubility product constants ..................... 4 Table 3. Metal removal mechanisms in passive treatment systems ................................................ 6 Table 4. Examples of recently (last five years) studied passive treatment systems for mine-influenced water .............................................................................................................................. 7 Table 5. Common microbial pathways for organic matter removal based on redox potential ..... 11 Table 6. Environmental conditions that favor growth of SRB ..................................................... 15 Table 7. Commonly used carbon sources for SRB ....................................................................... 16 Table 8. µmax values for commonly used carbon sources for SRB ............................................. 17 Table 9. Batch sulfate reduction bioreactor target concentrations ................................................ 26 Table 10. Bulk properties of solid organic materials .................................................................... 29 Table 11. pH and COD results of leachates produced from solid organic materials .................... 31 Table 12. SO42-  removal percentages for liquid carbon sources in this work compared with other studies ........................................................................................................................................... 33 Table 13. Percent product yield, pH, and total dissolved sulfide in bioreactors by carbon source....................................................................................................................................................... 36 Table 14. Removal percentages for metals of interest in metal precipitation reactors by biogenic sulfide carbon source .................................................................................................................... 38 Table 15. Source water quality in metals and treatment objectives .............................................. 49 Table 16.  Source water quality in other chemical parameters and treatment objectives ............. 50 xi  Table 17. Flow descriptions of column systems and corresponding tubing color, pump name and flow rate ........................................................................................................................................ 53 Table 18. Parameters of chemical and biological columns for hybrid design .............................. 53 Table 19. Sample descriptions for column effluents in hybrid design ......................................... 54 Table 20. Sulfate removal and reduction rates for second-stage columns in hybrid system ........ 58 Table 21. Average metal removal extents for first-stage and second-stage columns ................... 63 Table 22. Biological sulfate reduction performance in hybrid passive/active system compared to both a passive and an active system .............................................................................................. 67 Table 23. Metal precipitation performance in hybrid passive/active system compared to an active system ........................................................................................................................................... 68 Table 24. Metals in soil results for solid organic materials .......................................................... 82  xii  List of Figures  Figure 1. Horizontal flow biochemical reactor design for groundwater ......................................... 9 Figure 2. Traditional passive versus hybrid passive/active treatment system .............................. 13 Figure 3. Example of microbial community analysis workflow ................................................... 19 Figure 4. Sulfate concentration over time for batch bioreactors by carbon source ...................... 32 Figure 5. Sulfide production over time for batch bioreactors by carbon source ........................... 35 Figure 6. Zinc removal over time for batch metal precipitation reactors ..................................... 37 Figure 7. Dominant phyla in the batch bioreactors summarized by carbon source ...................... 40 Figure 8. SRB genera represented in the microbial communities summarized by carbon source 42 Figure 9. Faith Phylogenetic Diversity of microbial communities by carbon source .................. 44 Figure 10. Log copy number of the 16S rRNA and the dsrA genes in the mine sediment inoculum and batch bioreactors summarized by carbon source ................................................... 45 Figure 11. Annotated photo of novel hybrid passive system experimental set-up [tubing was color coded to denote separate flow streams] ............................................................................... 52 Figure 12. Sample point schematic ............................................................................................... 55 Figure 13. Sulfate concentration over time in the biological columns’ effluents ......................... 59 Figure 14. Sulfide concentration over time in the biological columns’ effluents ......................... 60 Figure 15. Biogenic sulfide to zinc mass ratio in the hybrid systems ........................................... 61 Figure 16. Total zinc concentration over time in the first-stage (metal precipitation) columns’ effluents ......................................................................................................................................... 62 Figure 17. Total Iron concentrations over time in the first-stage (metal precipitation) columns’ effluents ......................................................................................................................................... 65 xiii  Figure 18. Log copy number of the 16S rRNA and the dsrA genes in the mine sediment inoculum and batch bioreactors summarized by carbon source ................................................... 66 Figure 19. Dissolved cadmium over time in metal precipitation batch reactors by carbon source....................................................................................................................................................... 83 Figure 20. Dissolved cobalt over time in metal precipitation batch reactors by carbon source ... 84 Figure 21. Dissolved iron over time in metal precipitation batch reactors by carbon source ....... 84 Figure 22. Dissolved lead over time in metal precipitation batch reactors by carbon source ...... 85 Figure 23. Dissolved manganese over time in metal precipitation batch reactors by carbon source....................................................................................................................................................... 85 Figure 24. Dimension for the columns used in the hybrid design. LHS is first-stage column and RHS is second-stage column. ....................................................................................................... 86 Figure 25. Total zinc over time for first and second stage columns in hybrid design .................. 87 Figure 26. Dissolved cadmium over time for the first and second stage columns in hybrid design....................................................................................................................................................... 87 Figure 27. Total lead over time for first and second stage columns in hybrid design .................. 88 Figure 28. Total manganese over time for first and second stage columns in hybrid design ....... 88 Figure 29. Total cobalt over time for both first and second stage columns in hybrid design ....... 89 Figure 30. Total iron over time for the first and second stage columns in hybrid design ............ 89 Figure 31. Dissolved iron over time for the first and second stage columns in hybrid design ..... 90  xiv  List of Abbreviations  BCR  Biochemical Reactor BC WQG British Columbia Water Quality Guidelines BOD  Biological Oxygen Demand COD  Chemical Oxygen Demand CW  Constructed Wetland MIW  Mine-Influenced Water OTU  Operational Taxonomic Unit PCR  Polymerase Chain Reaction PRB  Permeable Reactive Barrier PTS  Passive Treatment System qPCR  Quantitative Polymerase Chain Reaction SPR  Sulfide Production Rate SRB  Sulfate Reducing Bacteria SRR  Sulfate Reduction Rate TDS  Total Dissolved Sulfide TOC  Total Organic Carbon TN  Total Nitrogen  xv  Acknowledgements  I offer my gratitude to those I worked with at various mine sites along my journey who inspired me to continue my work in this field. I owe particular thanks to Dr. Susan Baldwin who sparked an interest in me for understanding microbes role in the environment. Special thanks to my parents for their endless and unwavering support of my abilities.  xvi  Dedication  I  dedicate this to my father.1  Chapter 1: Introduction  1.1 Mine-Influenced Water and Treatment Objectives In British Columbia, the Ministry of Forests, Lands and Natural Resource Operations (FLNR) has investigated a total of 84 industrial sites, which includes 18 sites where remediation is complete and 16 sites where investigation and remediation is ongoing – approximately 90% of these sites are mines [Crown Contaminated Sites Program, 2016]. Once water contamination is initiated at these legacy sites, it is challenging to reduce because metal leaching is a process that will continue and possibly accelerate until one of the reactants (such as the metal ore, oxygen or water) is exhausted. Consequently, mine-influenced water can be discharging into receiving environments for decades or centuries after the mining operation has ceased. Released metals can either originate in surface water run-off over mining waste or from underground as water seeps through the mine workings. Treatment solutions to remove metals are most effective to reduce contamination when carried out as close to the point of deterioration in water quality, also known as source reduction.   The types of metals released will depend on the minerology of the mined area and each site will undergo its own investigation for remediation strategies. Most remediation strategies require that the site be restored to the levels acceptable to the surrounding environment which is most often a freshwater basin in British Columbia. Background information on metal toxicity in freshwater environments, metal removal mechanisms and point-source treatment systems will be further outlined in the following sections.   2  1.2 Metal Toxicity in Freshwater Aquatic Systems In British Columbia, the Ministry of Environment and Climate Change Strategy is responsible for the effective protection, management and conservation of B.C.’s environment, which includes determining approved water quality guidelines (WQGs). Specifically, for freshwater ecosystems, the water quality guidelines outline concentrations that protect the most sensitive species at its most sensitive life-stage during both short-term (acute) and long-term (chronic) exposures [Water Protection & Sustainability Branch, 2019]. The guidelines by the government will be used as treatment objectives for this thesis in order to be mindful of the toxic effect of metals on aquatic life.  The guidance documents account for metals and metalloids , e.g., As, Al, Cd, Co, Fe, Pb, Hg, Mn, Ni, Se, Zn, etc., since they have a tendency to be reactive, and consequently, biochemically active. The guidelines for Cd, Pb, Mn and Zn are unique since they are given as an equation that includes a parameter for the background level of hardness in the water. Hardness, or the concentration of calcium and magnesium ions, is known to reduce the negative effects of the specific metals on aquatic organisms [Water Protection & Sustainability Branch, 2019]. Table 1 summarizes an example list (not comprehensive) of metal contaminants and corresponding guidelines, including the hardness based equations where applicable.        3  Table 1. Example of BC WQGs for metals including hardness equations Metal BC Water Quality Guidelines for Freshwater Aquatic Life Arsenic (As) Maximum (µg/L total As) = 5 Cadmium (Cd) Long-term Chronic (µg/L dissolved Cd): WQG = e[0.736 x ln(hardness*) – 4.943] Short-term Acute (µg/L dissolved Cd): WQG = e[1.03 x ln(hardness*) – 5.274] Copper (Cu) (Water hardness = 150 mg/L CaCO3, pH=6.5) Long-term Chronic (µg/L dissolved Cu) = 0.2 Short-term Acute (µg/L dissolved Cu) = 1.6 Cobalt (Co) Long-term Chronic (µg/L total Co) = 4 Short-term Acute (µg/L total Co) = 110 Iron (Fe) Short-term Acute (mg/L Fe): Total Fe = 1, Dissolved Fe = 0.35 Lead (Pb) (Water hardness > 8 mg/L CaCO3) Long-term Chronic (µg/L total Pb): WQG £ 3.31 + e[1.273 x ln(hardness*) – 4.704] Short-term Acute (µg/L total Pb): WQG = e[1.273 x ln(hardness*) – 1.460] Mercury (Hg) Long-term Chronic (µg/L total Hg): WQG = 0.0001/(MeHg/total Hg) where MeHg is methyl mercury Manganese (Mn) Long-term Chronic (mg/L total Mn): WQG £ 0.0044 x hardness* + 0.605 Short-term Acute (mg/L total Mn): WQG £ 0.01102 x hardness* + 0.54 Zinc (Zn) (Water hardness > 90 mg/L CaCO3) Long-term Chronic (µg/L total Zn): WQG = 7.5 x 0.75 (hardness* – 90) Short-term Acute (µg/L total Zn): WQG = 33 x 0.75 (hardness* – 90) Source: British Columbia Approved Water Quality Guidelines: Aquatic Life, Wildlife & Agriculture, Summary Report, August 2019. *The value of hardness is relevant for applicability of chronic and acute calculations.  It can be seen from the guidelines that each metal can have different species (methylated, particulate, soluble, etc.) that affect toxicity levels, as well as the environmental conditions such as pH and hardness. Metals can have different toxic mechanisms. For example, zinc interferes with calcium uptake by organisms resulting in cellular damage, decreased metabolic activity and adverse effects on osmoregulation [Water Protection & Sustainability Branch, 1999]. It can be hypothesized that increased hardness would therefore ameliorate the competitive nature of zinc to bind preferentially in organisms as opposed to the desired element of calcium.  4  1.3 Metal Speciation Toxicity of a metal is related not only to its concentration, which can be measured by sensitive analytical techniques, but also to its chemical speciation. The speciation of metal ions helps to  distinguish their distribution as soluble labile complexes versus inorganic solid phases, and is generally determined through methods that combine a measurement of the free metal ion concentration with thermodynamically based models. Equation 1 shows how metals can be in equilibrium between the solid and aqueous phases with the degree of solubilization represented by the solubility product constant (Ksp).  Equation 1. 𝑴𝒎𝑨𝒏	(𝒔) ↔ 𝒎𝑴(𝒂𝒒)𝒏, + 𝒏𝑨(𝒂𝒒)𝒎. 	𝒘𝒉𝒆𝒓𝒆	𝑲𝒔𝒑 = [𝑴𝒏,]𝒎 ∗ [𝑨𝒎.]𝒏 Table 2 shows solubility product constants that rely on standard state thermodynamic data, and any difference from standard conditions will inject some amount of uncertainty in the predicted speciation.  Table 2. Examples of metal species and standard state solubility product constants Chemical Name Formula Ksp Cadmium hydroxide1 Cd(OH)2 7.2*10-15 Cadmium sulfide2 CdS 8*10-27 Copper(II) hydroxide1 Cu(OH)2 1*10-20 Copper sulfide2 CuS 6.3*10-36 Iron(II) hydroxide1 Fe(OH)2 4.87*10-17 Iron(III) hydroxide1 Fe(OH)3 2.79*10-39 Iron(II) sulfide2 FeS 6.3*10-18 Zinc carbonate1 ZnCO3 1.46*10-10 Zinc hydroxide1 Zn(OH)2 3*10-17 Zinc sulfide 2 ZnS 2.93*10-25 Source 1: Engineering Toolbox: Calculated Ksp values for common ionic substances at 25 ºC using standard state thermodynamic data and the equations. Source 2: Zhang, M et al., 2016 5  The solubility product constants are the value of the reaction quotient at equilibrium, therefore if it is large then it is product favored or more soluble. Based on equilibrium values it can be seen that metal sulfides have a lower Ksp compared to their other complexes (hydroxides), indicating that sulfide solids can be formed and are more stable. Under natural environmental conditions in rivers it was shown that the majority of Cu, Hg, Cr and Pb were associated with suspended particulates (60-80%), while Zn and Cd are likely to remain predominantly in solution [Forstner, 1977]. There is general consensus that the free (hydrated) metal ion is the most toxic and bioavailable form, which has been related to its affinity for various biological functional groups, such as amino acids [Bubb, 1991]. This makes it very important that these free metal ions are transformed into a stable solid that will not re-oxidize into the environment.   1.4 Passive Treatment Systems for Metal Removal 1.4.1 Summary of Current Passive Treatment Systems Passive treatment systems (PTSs) were developed based upon natural ecosystems such as marshes and use both chemical and biological processes; thereby, reducing the need for sophisticated process technology [Barton, 2007]. Passive treatment is preferred over active treatment since it can be implemented at many places on the mine site and close to the sources of contaminated water, requiring less maintenance. The significant mechanisms for metal removal stem from various biogeochemical processes, including, but not limited to those in Table 3.       6  Table 3. Metal removal mechanisms in passive treatment systems Metal Removal Mechanisms References Adsorption onto organic matter Burakov, A., 2018 Interception/filtration and flocculation/sedimentation of suspended solids Dotro, G., 2017 Formation of carbonates (biological and chemical) Braissant, O., 2007 Association with iron and manganese oxides Liu, J., 2016 Reduction to nonmobile forms such as hydroxides  Blowes, D., 2000 Formation of insoluble metal sulfides Logan, M.V., 2005; Zhang, M., 2016 Biological methylation Baldwin, S.A., 2015  Identifying and quantifying these processes provides a basis for the engineered design of PTSs and informs on the stability and biological availability of the contaminants they retain [Sobolewski, 1991]. Engineered passive systems involve all aspects of these mechanisms; however, a few selected ones such as the formation of insoluble metal sulfides will be the focus of this research. It has been determined that the desired type of bacteria in a biochemical reactor for metal removal by sulfide precipitation are sulfate reducing bacteria (SRB). Equation 2 represents the metabolic activity of SRB to perform anaerobic respiration utilizing sulfate as the terminal electron acceptor and reducing it to hydrogen sulfide.  Equation 2. 𝑺𝑶𝟒	(𝒂𝒒)𝟐. + 𝟐𝑪(𝒐𝒓𝒈𝒂𝒏𝒊𝒄) + 𝟐𝑯𝟐𝑶(𝒂𝒒) 𝑺𝑹𝑩E⎯G𝑯𝟐𝑺(𝒂𝒒) + 𝟐𝑯𝑪𝑶𝟑	(𝒂𝒒).  In capped or covered designs, such as an underground biochemical reactor, the hydrogen sulfide will remain in aqueous form and be very reactive with metal ions in the water as shown in Equation 3. Equation 3.    𝑴(𝒂𝒒)𝟐, + 𝑯𝟐𝑺(𝒂𝒒) ↔ 𝑴𝑺(𝒔) + 𝟐𝑯(𝒂𝒒),  7  Equation 3 is a simplified form of metal sulfide formation which is arguably a multi-stage process and variations of the solubility product constant Ksp can exist between sulfide mineral forms of the same metal ion [Barton, 2007].  According to the Yukon College Review in 2014, the technology behind passive treatment systems has evolved considerably in the last 20 years and many adaptations have been implemented to avoid hydraulic failure, establish microbial communities and reduce organic matter decomposition [Ness, 2014]. Table 4 presents some examples of passive treatment systems studied in the 5 years subsequent to that review.  Table 4. Examples of recently (last five years) studied passive treatment systems for mine-influenced water Passive Treatment Type Location Carbon Source Scale Metal Removal Rate Reference Cascade aeration, oxidation pond Sabuk, South Korea n/a Field Demo 95% Fe Oh, C., 2016 Hybrid: aeration rock channel, peat biofilter, bioreactor California, USA Peat, mulch, compost Pilot 34% Cu, 79% Fe, 80% Mn, 27% Ni, 12% Zn Clyde, E.J, 2016 Bioreactor Yukon Molasses Insitu 85% Cd, 89% Zn Nielsen, G., 2018 Settling pond and vegetated bioreactor Glasgow, UK Mushroom compost Insitu 30% Al, Fe, Mg, Mn Sandlin, W., 2020  The recent research includes studies on iron removal strategies that prevent its re-release, hybrid passive/active designs, and site specific designs. For example, a study on iron showed that suspended Fe(OH)3 is unstable, and it can be re-dissolved into ionic form at low pH and oxygen 8  levels; therefore, the authors recommended to install a constructed wetland after the oxidation pond in order to eliminate the colloidal particles by settling [Oh, 2016]. They showed that an active component in the oxidation pond, a cascade aerator, could more efficiently facilitate the oxidation of ferrous iron to ferric but required further settling of the suspended iron oxyhydroxide precipitates in the subsequent passive component. A site specific pilot-scale biochemical reactor study demonstrated that stimulation, through carbon addition, of native microorganisms in the cold climate of the Yukon was possible and achieved Zn removal efficiency varying between 20.9% in the winter and 89.3% in the summer [Nielsen, 2018].   Other studies highlight the pitfalls that can plague passive treatment systems. For example, a bioreactor that was removing metals at 30% declined in efficacy over a 6 month period as the adsorbed metals and mineral precipitates were hypothesized to be re-oxidized, resulting in no reduction in sulfate or metal concentrations [Sandlin, 2020]. Despite the currently available options for treating metal laden wastewater, existing challenges with operation and reliability mean that new or modified treatment systems are being sought, specifically those that can address these two pitfalls: 1) Generation of deleterious byproducts such as dissolved Fe 2) Plugging due to metal precipitation  1.4.2 Biochemical Reactor Biochemical reactors are passive treatment systems designed for sub-surface flow and can be contained in an excavated pit, tank or even abandoned mine shaft. Current designs promote and retain biological growth by including a solid reactive mixture containing [Ness, 2014]:  9  1) sand or gravel as a porous medium  2) solid organic carbon source  3) bacterial inoculum  4) an essential nutrient source of nitrogen, phosphorous and pH adjustment  Most often used carbon sources are solids such as mushroom compost, cow manure, sawdust, wood chips or other organic residues from nearby industries [Vasquez, 2015]. Influent water containing metals and sulfate will percolate through the reactive mixture along the normal gradient of groundwater flow, creating the proper reducing conditions required for the desired microorganisms to thrive. Figure 1 shows a design for a PRB promoting anaerobic conditions, which includes a soil cover.  Figure 1. Horizontal flow biochemical reactor design for groundwater  The solid organic material in the PRB decomposes over time due to the activity of cellulose degrading and fermentative bacteria. The degradation products of decomposition are used by sulfate reducing bacteria for their growth and activity, and therefore the type and rate of their production affects reactor performance. For example, in a study comparing two different wood-10  hay mixtures, the hay-rich mixture provided higher concentrations of dissolved organic carbon which fueled higher sulfate reduction rates. The same study showed that, over the long-term, hay-rich bioreactors appeared to degrade 2-3 times faster than wood-rich and only had observable biodegradation for the first 175-230 days [Mirjafari, 2016]. Another study showed decline in performance after 524 days due to decline in pH conditions and microbial activity [Jeen, 2014]. Depending on the solid organic material used it will require replacement, even as early as one year later, for the treatment system to continue to support sulfate reduction and metal removal. Organic matter replacement is a major operational undertaking and also disturbs the ideal anaerobic microbial community, which establishes itself over a long time (weeks or months) in order to grow and adapt to the bioreactor environment and mine-influenced water.   In microbial metabolism, electrons are transferred from the organic compound (also referred to as the electron donor) to another specific compound (called the electron acceptor), in a process that releases energy for the cell. The types of metabolic pathways taking place in a biochemical reactor vary depending on whether conditions are aerobic or anaerobic [Dotro, 2017]. Each energetic metabolic process has an optimal redox potential, and therefore, may be active in different locations of an ecosystem, also called redox zones. Table 5 lists the electron transfer or respiration pathways of microorganisms in order of decreasing energy yield.       11  Table 5. Common microbial pathways for organic matter removal based on redox potential Electron Transfer or Respiration Pathway Reaction (Electron Acceptor/Reduced Species) Redox Potential at pH = 7 (mV) Aerobic respiration 𝑂J/𝐻J𝑂 810 Manganese Reduction 𝑀𝑛𝑂J/𝑀𝑛,J 580 Denitrification 𝑁𝑂P/𝑁J 750 Iron Reduction 𝐹𝑒(𝑂𝐻)P/𝐹𝑒,J -80 Alcohol Fermentation 𝐶𝐻J𝑂/𝐶𝐻P𝑂𝐻 -180 Sulfate Reduction 𝑆𝑂U./𝐻J𝑆 -210 Methanogenesis 𝐶𝑂J/𝐶𝐻U -240 Source: ITRC-Overview of In Situ Bioremediation of Chlorinated Ethene DNAPL Source Zones, 2005  Alcohol fermentation occurs at a slightly less negative redox value than sulfate reduction. Therefore, to allow optimization of sulfate reduction separate from the biodegradation of the solid organic materials, they need to occur in separate redox zones. Additionally, having fermentation and sulfate reduction taking place in the same physical location depletes the fermentation byproducts over time and results in loss of control over biological sulfide production and consequent metal precipitation.  There are several major impediments to effective application of PTSs and BCRs. Biological activity is not reliably supported by solid waste organics because as they degrade over time, the supply of smaller dissolved organic carbon molecules that sulfate reducing bacteria can use diminishes and treatment can fail. Additionally, the metal sulfide solids accumulate within the PTS or BCR over time and cause metal toxicity to the microorganisms along with plugging issues. Typically, traditional PTS and BCR systems operate effectively over only short periods of time and when they cease to remove metals, any contaminants that have been captured in the 12  BCR must be dug up and disposed of elsewhere. Thus, there is incentive to develop new innovative systems that enable more sustainable and reliable treatment.  1.4.3 Hybrid Passive/Active System Hybrid systems incorporate mechanical, chemical or physical devices that will enhance an existing passive treatment technology. Potential devices that are explored in this thesis for enhancing a biochemical reactor for metal removal are: 1) separate compartment for metal solids capture  2) external liquid carbon source for biological reaction 3) feedback loop for biologically produced (biogenic) sulfide Combining these components together in assimilation with a classic PRB – or BCR – type design would still allow for an inground, naturally driven biogeochemical process to be installed, but with enhancements that address the main shortcomings of PRBs and BCRs as described in Section 1.4.1 and 1.4.2. The traditional system versus a hybrid system incorporating both passive and active components is shown in Figure 2.  13   Figure 2. Traditional passive versus hybrid passive/active treatment system For metal removal and recovery, the required amount of sulfide to be produced by SRB in the biochemical reactor depends on the composition of the wastewater to be treated, i.e. its metal concentration. Steering the sulfide production toward this required stoichiometric amount in bioreactors is highly relevant to avoid overproduction of H2S which increases operational costs and may require a sulfide removal post-treatment step [Cassidy, 2015]. The development of a control strategy based on the sulfide concentration as the controlled variable is a more direct approach for the control of the sulfate reducing bioreactors rather than pH or hardness concentration. A study found that an increase in the organic loading rate or Chemical Oxygen Demand (CODin) showed a rapid response in sulfide concentration change, proving that control over amount of liquid carbon source added in a hybrid system is an important control strategy [Villa Gomez, 2014].   The sulfide produced in the biological sulfate reducing reactor can then be used to precipitate metals in the sand filter by means of a feedback loop. A study found that a sulfide ion (S2-) 14  concentration of 10-15 M was optimal for zinc sulfide precipitation, resulting in a 99% removal for both chemical Na2S and biogenic sulfide solutions. The reaction rates of this study showed the ZnS precipitate achieving a steady-state in 1 hour [Esposito, 2006]. This shows promise for a smaller sand filter cartridge to be used for metal solids capture which can be more easily removed and replaced. Potential metal recovery from the sand is desirable for both sustainability and economic reasons, and can be achieved since many metal-refining operations are able to process sulfide ores.  1.5 Sulfate Reducing Bacteria  1.5.1 Role in the Environment A biological treatment system, especially a passive type which simulates natural environments containing indigenous microorganisms, will contain a microbial community consisting of thousands of different bacterial species with SRB only contributing to a small percentage of the total population. For example, one study of 4 different biochemical reactors on mine sites found that SRB comprised between 0.3% and 8.3% of all bacteria in the total population [Rezadehbashi, 2008]. Another study showed that low-abundance SRB genera stayed below or around 0.1% in the native soil and could be increased in sulfate-amended incubations to between 0.26% and 0.46% [Hausmann, 2016].   A bioreactor utilizing biological sulfate reduction should be designed with certain process conditions to promote the growth and activity of these low-abundance groups. Table 6 outlines environmental conditions that favor the growth of SRB.  15  Table 6. Environmental conditions that favor growth of SRB Parameter Literature Values Nitrogen Content (mass %)1 0.1 – 1.6 C:N Ratio1 (g/g) 6.4 - 179 COD:SO4 Ratio2 (g/g) 0.8 – 1.2 Redox Potential at pH=73 (mV) -210 pH2 6 - 8 Source 1: Bhattacharya, 2018 Source 2: Kosinska, 1999 Source 3: The Interstate Technology & Regulatory Council Bioremediation of DNAPLs Team Once SRB are established they play an important role in the sulfur, carbon and nitrogen cycles. In the biological sulfur cycle, 𝑆𝑂U	J. is reduced to 𝑆J. by SRB through dissimilatory sulfate reduction which then allows sulfide-oxidizing bacteria to transform it to elemental sulfur (𝑆V) and eventually back to sulfate. In assimilatory sulfate reduction, the sulfur of the 𝑆𝑂U	J. passes through the sulfide level of oxidation and becomes incorporated into amino acids before being built into microbial protein. This can be eaten by animals and plants and returned to the cycle as sulfide formed during the breakdown of dead organisms by bacteria. Carbon is utilized by SRB as electron donors for sulfate reduction, and often simultaneously as carbon sources for cell synthesis. One study reported that an up-flow anaerobic sludge-bed bioreactor used an estimated 36% of the silage leachate carbon directly for sulfate reduction [Li, 2011]. SRB were shown to utilize nitrogen compounds such as 𝑁𝐻U,, 	𝑁𝑂P. and 𝑁𝑂J.. The maximum growth of SRB is shown to be supported by 𝑁𝐻U,, while 𝑁𝑂P. and 𝑁𝑂J. contribute less and are also able to be used as electron acceptors in the absence of sulfate [Bhattacharya, 2018].  16  1.5.2 Carbon Metabolism and Growth SRB lack the ability to metabolize complex organic compounds, and in a mixed culture the fermentative bacteria will help to completely metabolize larger carbon compounds [Bhattacharya, 2018]. The most commonly used carbon sources that SRB can easily oxidize are shown in Table 7.  Table 7. Commonly used carbon sources for SRB Carbon Source Chemical Names Monocarboxylic Acids Lactic, Formic, Propionic, Butyric, Acetic, Pyruvic Dicarboxylic Acids Succinic Fumaric, Malic, Oxalic Monovalent Alcohols and Polyols Ethanol, Methanol, Propanol, Glycerol Amino Acids Alanine, Glycine, Serine, Cysteine Source: Bhattacharya, 2018 A common competitor with SRB are other anaerobes, including methanogens, which use an even more limited number of electron donors of which hydrogen, carbon dioxide and acetate are the best-known [Muyzer, 2008]. Lactate, in terms of energy and biomass yield, is reported as the best-suited carbon source, as many species of sulfate reducers can use it [Postgate, 1983]. The organic matter or electron donor selected for a sulfate removal system should consider that SRB require simple compounds for growth.  Mathematical models can help to describe kinetics of bacterial metabolism and the Monod model has been widely accepted, and often used as a baseline for its mathematical simplicity. In this model the growth rate of the specific bacteria (µspecies) is related to the concentration of the limiting substrate (Si), as seen in Equation 4. Equation 4. 𝝁𝒔𝒑𝒆𝒄𝒊𝒆𝒔 = 𝝁𝒎𝒂𝒙 𝑺𝒊𝑲𝒔,𝒊,𝑺𝒊 17  Where µmax (d-1) is the maximum specific growth rate for the bacteria and Ks,i (g/L) is the affinity constant for the bacteria with respect to the substrate. In the case of sulfate reducing bacteria the limiting substrate in their respiration can either be sulfate or the carbon source. A variety of studies have reported on the kinetic effect of the carbon source on growth and µmax can vary depending on the type as seen in Table 8. Table 8. µmax values for commonly used carbon sources for SRB Carbon Source µmax Range (d-1) Lactate 3.12 – 8.064 Acetate 0.025 – 1.416 Hydrogen 0.3 - 5  Source: Cassidy, 2015 These studies show that lactate has greater potential for promoting SRB growth than either acetate or hydrogen.   1.5.3 Taxonomy As of 1983, there were two established genera of SRB that were largely studied and those were Desulfovibrio and Desulfotomaculum, with several new genera being identified [Postgate, 1983]. However, taxonomy in 1983 was largely imperfect because it is not possible to obtain most bacteria in pure lab culture and relatively few phenotypic or morphological qualities can be assigned to SRB. Now, most information on natural and engineered environments containing SRB has been obtained with genomic techniques using marker genes, the most common one being 16S ribosomal RNA (rRNA) [Muyzer, 2008].  18  Recently, there has been great progress in defining a complete taxonomy of bacteria and archaea, which has been enabled by improvements in DNA sequencing technology and new bioinformatic techniques. An alternative taxonomy database, the Genome Taxonomy Database (GTDB), was proposed in 2018 and incorporates only sequence homology of conserved genes (concatenated protein phylogeny rather than 16S) and normalizes taxonomic rank based on evolutionary divergence [Prodan, 2020]. Taxonomy inferred from the concatenation of single-copy vertically inherited proteins provides higher resolution than that obtained from a single phylogenetic-marker gene and is more representative of microbial diversity [Parks, 2018]. The GTDB taxonomy has been adopted by SILVA database and as a consequence the following groups were prone to significant adaptations: Archaea, Enterobacterales, Deltaproteobacteria, Firmicutes, Clostridia [SILVA, 2019]. The Deltaproteobacteria where many SRB were located has been reclassified, and there is a new phylum called Desulfobacterota, additionally, the phylum Firmicutes has two orders: Desulfitobacteriia and Desulfotomaculia that contain the spore forming SRB. The primary taxonomic character of SRB is the ability to perform dissimilatory sulfate reduction. This process can also be called sulfate respiration and this type of respiration is not seen in any other organism other than SRB.   1.6 Genomic Tools and Techniques for Characterization of Microbial Communities 1.6.1 16s rRNA Gene Surveys for Phylogenetic Analysis Microbial DNA amplicon sequencing studies are an important tool in biological research. Using a single gene, in this case, a specific variable region of the 16S rRNA, a tree of the prokaryotic organisms can be reconstructed giving a statistical probability of relatedness. Widespread 16S rRNA gene microbial surveys have shed light on the microbial community structure of many 19  ecosystems inhabited by bacteria, including bioreactors. Better understanding of the microbial community structure and function can elucidate more information about the dynamics of the bioprocesses such as kinetics and mechanisms. An example of the workflow required for microbial community analysis is shown in Figure 3.   Figure 3. Example of microbial community analysis workflow The bioinformatic pipeline software, Qiime 2, converts raw sequencing data into biologically meaningful information such as tables of relative abundance of bacterial types. There are various bioinformatic pipelines available that are free and open-source, and they all have their own potential limitations and biases. The pipelines used in this thesis were Qiime2-Deblur and DADA2 which both attempt to reconstruct the exact biological sequences present in the sample, known as Operational Taxonomical Units (OTUs) [Prodan, 2020].   The metabolic preference of microorganisms can be inferred from their taxonomy and they can then be identified as key functional players. A study compared the clusters of orthologous groups 20  (COGs) of proteins within the lineages of 13,735 microorganisms and reported that 41.4% of the variation in COGs relative abundance is explained by taxonomic rank, with domain, phylum, class, order, family, and genus explaining, on average, 3.2%, 14.6%, 4.1%, 9.2%, 4.8%, and 5.5% of the variance, respectively [Royalty, 2019]. This quantifies the variance in metabolic potential contributed by individual taxonomic ranks, and ranks phylum highest in terms of informing function. Once the dominant phyla have been identified then the specific genera of those types of bacteria can be further researched for their niche adaptations such as metal tolerance. For example, one study on alfalfa and woodchip PRB showed that there was an ecological transition of an increase in the phylum Firmicutes (26% to 39%) and a decrease in Bacteroidetes (35% to 28%) which correlated to the amount of sulfide-bound zinc [Drennan, 2017]. This shows that maximal zinc precipitation is influenced by microbial interactions between cellulolytic bacteria (Phylum - Bacteroides) and fermenters (Phylum - Firmicutes) and potential monitoring tools for genes of interest could help monitor performance. Another study tracked the microorganisms in a pulp mill biosolids fed biochemical reactor over the period of one year and observed known sulfate-reducing OTUs (Desulfovibrio, Desulfobulbus) utilizing the fermentation byproducts from fermenters (Phylum – Firmicutes, Genera – Acidaminococcus, Saccharofermetans, Anaerfilum) establishing the relationship between bacterial groups [Baldwin, 2016].   1.6.2 Quantitative PCR for Functional Genes Using forward and reverse primers for a specific functional gene of interest, real time polymerase chain reaction (PCR) can be used as a quantitative method for detecting and enumerating organisms capable of that particular function in natural environments or bioreactors. 21  In order to identify sulfate-reducing bacteria, the dissimilatory sulfite reductase gene (dsr) can be used. Dissimilatory sulfite reductase is a key enzyme in dissimilatory sulfate reduction, as it catalyzes the reduction of sulfite to sulfide in the final step of sulfate respiration. One study used  real time PCR to prove that high densities of SRB ranging from 0.2 – 5.7 x 108 cells/mL were established in sediment and able to achieve sulfate reduction rates within the range of pure cultures of SRB [Kondo, 2004].   1.7 Research Goals  The research goal was to develop and test a novel hybrid passive/active treatment system to remove metals from mine-influenced water that also contains elevated above background concentrations of sulfate. Current passive treatment processes for metal removal from sulfate-rich mine-influenced water include a type of biochemical reactor (BCR) called a permeable reactive barrier (PRB) which suffers from poor operational performance. A PRB is installed in the flow path of contaminated water and contains a solid organic material that acts as a carbon source to promote the activity of sulfate reducing bacteria (SRB) for sulfate reduction to sulfide and subsequent metal sulfide precipitation. The PRB requires a one-time application of a solid organic material, which after it has degraded, must be removed and disposed of appropriately. Operational limitations can occur with a one-time application of solid organic materials as they will become resistant to decomposition over time resulting in unreliable treatment. In addition to depletion of carbon sources for sustaining SRB, these current systems are also prone to plugging due to the accumulation of metal precipitates in them. To overcome these shortcomings a new treatment process was developed and tested in this study that incorporates added hybrid components, such as liquid carbon addition, biogenic sulfide feedback loop and sand filter for 22  metal capture and removal. First, lab-scale batch experiments would provide a proof-of-concept for using a liquid carbon source from fermented forestry residues to promote sulfate reduction and for metal precipitation with the biogenic sulfide. The key questions relating to the batch experiments were:  1) Will a soluble liquid fermentation product (leachate) from pulp mill biosolids or forestry/agricultural wood byproducts support sulfate removal extents similar to laboratory standard such as lactate?  2) Does the type of carbon source affect the microbial community and/or the percentage of sulfate reducing bacteria that are a part of that community? 3) Will biogenic sulfide effectively remove metals in real mine-influenced water? After completion of the proof-of-concept experiments, a continuous flow experiment of the proposed two-stage design was implemented in the field at a mine site. Key questions relating to column experiment were:  1) Will biogenic sulfide produced in the second-stage biological column provide reliable metal removal when fed back into the first-stage metal precipitation column?  2) Does the type of liquid carbon source influence the sulfate reduction rates and the metal removal extent?  3) How will a hybrid passive/active system compare to a passive system and an active system in regards to sulfate reduction rates and metal removal extents? By successfully testing and implementing the design improvements, the goal is to avoid problems associated with passive treatment systems in the past and improve upon current technologies.  23  Chapter 2: Batch Proof-of-Concept Testing of Leachates, Sulfate Reduction and Metal Precipitation 2.1 Synopsis The first objective of this study was to perform proof-of-concept experiments in order to validate the novel aspects of the new proposed hybrid passive/active design, including genomic analyses to confirm the presence of the desired microbial groups. First, solid organic residues were sourced locally from neighboring industries to mine sites and characterized for their suitability to promote biological activity of sulfidogenic bacteria. Based on their carbon and nitrogen content, two of the considered solid organic materials were chosen for fermentation to produce a soluble liquid product or leachate that was then used in batch bioreactors to prove its practicability as a carbon source. The kinetic studies for sulfate reduction with leachates were done in batch bioreactors which included:  1) an inoculum of microorganisms from mine sediment  2) the chosen leachates or lactate standard as carbon sources  3) a synthetic growth medium containing metal sulfate salts  Each bioreactor targeted a COD:SO4 mass ratio of 2 in order to ensure that the amount of carbon was not rate limiting. This experiment would elucidate information on the percentage sulfate removal and sulfate reduction rates (SRRs) with each specific carbon source. Biological samples were taken from each bioreactor at the end of the 16 day experiment and the DNA was extracted from these samples for both Illumina Miseq sequencing of the 16s rRNA gene and qPCR of the dsrA gene.  24  These genomic analyses were done in order to determine how the carbon source changed the microbial community from that intrinsic to the mine sediment inoculum. After proving that SRB could utilize leachates as a carbon source, the next step was to prove that the sulfide produced in these bioreactors could promote the formation of metal precipitates in the real mine-influenced water. Environmental waters are known to contain naturally occurring inhibitory compounds and therefore using the actual source water from the mine site was important to proving that the hybrid design could work in the field. This batch experiment involved adding a liquid sample of the biological reactors to the real mine-influenced water and monitoring the dissolved metal concentrations over time in order to determine metal removal extents.   2.2 Materials and Methods For characterization, the solid organic materials were analyzed by ALS in Burnaby, BC. Total Organic Carbon (TOC) was calculated by the difference between total carbon and total inorganic carbon, method CSSS (2008) 21.2. The metals in the organic materials were measured by Collision/Reaction Cell Inductively Coupled Plasma Mass Spectrometry (CRC ICPMS), method EPA 200.2/6020A. Total Nitrogen (TN) was measured by combustion, method SSSA (1996) P. 973-974. The pH of the materials was measured by an extraction procedure which involved mixing the dried (at <60 C) and sieved (No. 10/2mm) sample with deionized water at 1:2 ratio of sediment to water, and subsequently measured using a standard pH probe.   For the fermentation to produce leachates, the solid organic materials were mixed with river water from Spanish Banks in Vancouver, BC with a pre-determined solid to liquid ratio in a 1L glass bottle. Once sealed, the bottle was placed in a box at 25 ºC for 5 days. The box was used to 25  ensure no light reached the fermentation and to prevent photosynthetic reactions. The river water collected from a natural environment contained micronutrients and aquatic microorganisms required for promoting biological activity. After 5 days, the solid materials were removed by sieving and then filtering through 0.45 µm. The COD was measured by USEPA Reactor Digester Method (Method 8000).   For the sulfate reduction kinetic studies, bioreactors were set up in 500 mL glass bottles with butyl rubber stoppers to prevent gas release during sampling. The synthetic mine-influenced water medium or metal sulfate salt solution was modeled from the Postgate G medium [Postgate, 1983]. The bottles were filled with the Postgate G salt solution, mine sediment inoculum and liquid carbon source. The salt solution, bottles and carbon source were autoclaved to prevent contamination from any organisms except for the inoculum. This sediment inoculum was collected from a marsh on the mine site being studied which was located in the flow path of mine influenced water targeted for treatment using the new hybrid treatment process. It was hypothesized that a sediment taken from this area would contain microorganisms adapted to higher concentrations of metals. Water saturated soil that is black in color and emits a rotten egg smell when disturbed indicates the presence of sulfate reduction and a source of sulfate reducing bacteria. Approximately 5 cm of top soil was removed and the black smelly soil directly underneath was collected into a plastic (sterile) bottle filled to the brim to exclude any air and stored at room temperature before being used for inoculation. Table 9 summarizes the target concentrations of bioreactor parameters.   26   Table 9. Batch sulfate reduction bioreactor target concentrations  Parameter Target Concentration Chemical SO4 2,000 mg/L CaSO4, MgSO4 COD 4,000 mg/L Liquid Carbon Source Fe 100 mg/L FeSO4 PO4 500 mg/L KH2PO4 NH4 1,000 mg/L NH4Cl  Once filled, autoclaved and inoculated, the bioreactor bottles were kept at 28 ºC for 16 days. The bottles were kept in secondary containment of a plastic bag for gas leaks and a foil cover to prevent light penetration. In order to ensure an anaerobic environment in the bottles, the liquid medium was purged with nitrogen gas for at least 15 min, and the reducing agents thioglycolic acid and ascorbic acid were added to scavenge any remaining oxygen as specified in the Postgate G medium recipe [Postgate, 1983]. Liquid samples were taken approximately every 3 days and filtered through 0.45 µm to be analyzed immediately. Dissolved sulfate was measured by Turbidimetric Method 4500 [American Public Health Association, 1999], pH by a test strip, dissolved sulfide by the methylene blue method (Hach method 8131), and dissolved COD by mercury digestion (Hach method 8000). Sulfate, sulfide and COD analysis were performed with lab triplicates in order to determine measurement error for each lab technique. Each type of organic material leachate was tested in triplicate bottles. This was done to validate the empirical data and to ensure the mine sediment inoculum would not vary the results from the type of carbon source. This allowed the relative differences between bottles to be compared as an 27  average of the bottle triplicates. Upon completion of 16 days the bioreactor contents were filtered through 0.45 µm in order to capture biological solids on the surface of the filter paper. These filter paper samples were saved and frozen for subsequent DNA extraction. DNA extraction was performed by FASTDNA extraction kit (as per manufacturer’s instructions). DNA sequencing was done using the 515f/926r primer pair and V3-600 chemistry on the Illumina Miseq. Microbial community analysis was performed using Qiime 2 for quality filtering, Deblur package for clean-up and SILVA database for building taxonomy. R ggplot2 package was used for generating phylum and genus plots and Qiime 2 Viewer was used for faith diversity plots. Quantitative PCR with 1 ng DNA was performed on Bio-Rad CFX instrument using SsoFast EvaGreen enzyme with both dissimilatory sulfite reductase sub-unit A (dsrA) primer pair (F-ACSCACTGGAAGCACGGCGG/R-GTGGMRCCGTGCAKRTTGG) and a total bacteria (16S) primer pair (F-CGGTGAATACGTTCYCGG/R-GGWTACCTTGTTACGACTT).   For the chemical metal sulfide precipitation reactors, 100 mL of real mine-influenced water was aliquoted into 500 mL glass bottles. Using the real mine-influenced water from the mine site was important in order to determine if any natural inhibitors exist in the source water. Once these bottles were filled, liquid samples from the bioreactors were collected and immediately filtered with a 0.45 µm syringe directly into the real mine-influence water. The sample volumes from the bioreactors were measured and allocated in order to target a 1:1 mass ratio of the amount of total dissolved sulfide (TDS) to the amount of zinc in the source water. Bottle triplicates were done to account for any variations in the source of the biogenic sulfides. Dissolved metal samples were taken at time points of 30 minutes, 1 hour, 5 hours and 24 hours. The soluble fractions of the metals Cd, Co, Fe, Mn, Pb and Zn were measured in the metal precipitation reactors over a 28  period of 24 hours so as to confirm the stability of the precipitates formed. Metal concentrations were analyzed at Earth and Ocean Sciences at University of British Columbia using Inductively Coupled Plasma – Optical Emission Spectrometry (ICP OES).  2.3 Results and Discussion 2.3.1 Bulk Properties of Solid Organic Materials and Leachates The solid organic materials tested included: spruce-pine-fir (SPF) wood chips, pulp and paper mill wastewater treatment solids, municipal landfill yard compost, spent brewery grain, and a 50/50 hay and grass mix. After collection, they were analyzed for bulk properties such as total organic carbon (TOC), total nitrogen (TN), pH and trace metals. These were all compared to a sample of the mine sediment, as a baseline, in order to determine the deficiencies intrinsic to the environment on the mine site. Trace metals were also analyzed in order to determine whether the organic materials met Organic Matter Recycling Regulation (OMRR) and Soil Amendment Code of Practice (SACoP) land application guidelines [SYLVIS, 2008]. This would ensure that any material put in the ground at the mine site would still meet guidelines and not in turn be detrimental to the environment.         29   Table 10. Bulk properties of solid organic materials Solid Organic Material C:N TOC TN pH Trace Metals* Ratio % % pH units  Pulp & paper mill -wastewater treatment primary solids 700 36 Below detection limit 10 ✓ SPF wood chips 250 49 0.2 4.3 ✓ Mine sediment 36 36 1 7 ✕ Municipal landfill compost 20 16 0.9 7.6 ✓ Pulp & paper mill -wastewater treatment secondary biosolids 15 43 3 7.5 ✓ Hay & grass 50/50 mix 15 42 3 5.6 ✓ Spent brewery grain 12 48 4 5 ✓ *Check mark indicates meeting guidelines [Detailed results in Appendix A] The recommended C:N ratio for a biological anaerobic process is 20 to 35 [Ciron-Vervas, 2019]. It can be seen in Table 10 that only the municipal landfill compost and the mine sediment are within the range, as expected since they were collected from well-established anaerobic soil environments [SYLVIS, 2008]. Another metric is TOC and TN, which contribute to ensuring adequate levels of dissolved carbon and nitrogen molecules produced during biodegradation or fermentation. The SPF wood chips had one of the highest TOC and the hay & grass 50/50 mix had one of the highest TN, as well as slightly lower pH values than the other materials. Therefore, the characteristics of a mixture of SPF wood chips and hay & grass 50/50 mix 30  together would have both high available carbon and nitrogen contents, and minimal impact on pH neutrality. Consequently, this mixture was chosen as the first solid organic material for fermentation into a soluble leachate product. The pulp & paper mill residues also had desirable properties and one study showed that mine tailings amended with pulp & paper mill wastewater treatment primary solids produced a greater amount of bioavailable Ca and Mg while also generating an alkaline pH thus favoring plant growth for remediation [Asemaninejad, 2018]. However, the C:N ratio and pH of the primary solids were well above the recommendations for anaerobic biological processes promoting SRB; therefore, the pulp & paper mill wastewater treatment secondary biosolids were chosen as the other solid organic material for use in subsequent experiments. All the solid organic materials met guidelines for metal concentration limits in British Columbia except for the mine sediment, and the results of metal concentrations are detailed in Appendix A.   The study previously mentioned on pulp & paper mill residue amendments showed that the direct application of these in mine tailings can form organo-metallic complexes, which can increase the bioavailability of metals unless the rate of organic matter decomposition is stabilized, thus the fermentation of organic materials prior to in-ground application is recommended [Asemaninejad, 2018]. The author’s recommendation was addressed in this thesis by performing a 5-day fermentation to decompose longer chain, high molecular weight carbon compounds into lower molecular weight, soluble molecules that SRB can use (see Section 1.5.2). The solid organic materials ratios used to obtain a C:N ratio of 20 to 35, the final pH, and the final COD after a 5-day fermentation for the chosen solids are listed in Table 11.  31    Table 11. pH and COD results of leachates produced from solid organic materials Leachate Solid:Liquid Ratio (wt %) pH COD (mg/L) Hay and Wood Chip 50/50 Mix 1:6 6 7,000 Pulp & Paper Mill Wastewater Treatment Secondary Biosolids 1:1 8 4,000  These two products, henceforth called hay leachate and biosolid leachate, produced 7,000 mg/L and 4,000 mg/L COD, respectively. These concentrations are more than enough for complete reduction of the approximately 1,700 mg/L sulfate used in the batch bioreactors, based on the recommended requirement of 0.8 to 1.2 gCOD gSO4-1. Furthermore, the pH remained stable at 6-8 during the 5-day fermentation which is within the range for optimum SRB growth. The leachates now contain a portion of fermentation products such as acetate, propionate, lactate, butyrate, alcohols, hydrogen and amines that can be used to culture the microorganisms important for sulfate reduction [Mirjafari, 2016].   2.3.2 Sulfate Removal and Sulfate Reduction Rates The leachates produced from the 5-day fermentation were tested in comparison to a lactate standard for sulfate reduction. Sulfate was measured approximately every 3 days in bioreactors for an entire duration of 16 days in order to determine the kinetic rate of sulfate reduction for the leachate carbon sources. Kinetic information is needed in order to determine the hydraulic retention time required for a continuous flow bioreactor in further column studies. Figure 4 presents sulfate concentration plotted over time for each liquid carbon source, including the laboratory standard.  32   Figure 4. Sulfate concentration over time for batch bioreactors by carbon source Error bars represent the standard deviation from the mean for triplicate culture bottles for each organic material.  Sulfate removal was observed in all of the batch bioreactors. In the bioreactors containing hay leachate as a carbon source, the SO42- concentration decreased from 1,971 mg/L to 677 mg/L (66% removal) where it then plateaued after 14 days. In the bioreactors containing biosolid leachate as a carbon source, the SO42-  concentration decreased from 1,460 mg/L to 966 mg/L (34% removal) where it plateaued after only 11 days. The lactate containing bioreactors achieved 66% SO42-  removal, similar to the hay leachate; however, the lactate bioreactors differed because they continued to show a downward trend in percentage removal until day 16. Despite this, the batch bioreactors were all stopped after 16 days since the leachates were no longer achieving observable changes in SO42-  concentrations and their reaction kinetics were of interest. The percentage SO42-  removal for the tested liquid carbon sources in this work are compared to other commonly studied carbon sources in Table 12.   33    Table 12. SO42-  removal percentages for liquid carbon sources in this work compared with other studies Liquid Carbon Source SO42- Removal % Hydraulic Retention Time (HRT) or Batch Duration Temperature (ºC) Reference Lactate - 99% - 69% - 66% - 65% - 10 hour HRT - 6.6 hour HRT - 16 days - 15 days - 35 ºC - 10 ºC - 28 ºC - 25 ºC - Zhao, 2008 - Tsukamoto, 1999 - This work - Paganelli, 2012 Methanol 90% 20 days 30 ºC Glombitza, 2001 Ethanol 65% 35 days 25 ºC Pagnanelli, 2012 Hay Leachate 66% 14 days 28 ºC This work Biosolid Leachate 34% 11 days 28 ºC This work  It can be seen in Table 12 that direct, simple organic carbon sources (lactate, methanol, ethanol) support high percentage SO42- removal, and the hay leachate can perform at a similar level, notwithstanding slight variations in temperature, time or inoculum used. The percentage SO42-  removal does not take into account the time of reaction and amount of carbon utilized; consequently, closed systems have rates for biological sulfate reduction that can be approximated by sulfate-removal rates or SRRs (g L-1 day-1 gCOD-1). The rates are considered approximate because other processes such as adsorption of SO42-  onto ferric oxyhydroxides, acclimation periods and levels of SO42- and H2S also affect the calculated SRRs [Waybrant, 1998]. Initially, between days 0 and 3, the SRR of both the hay leachate and lactate standard were 0.30 and 0.52 g L-1 day-1 gCOD-1, respectively, while the biosolid leachate had no detectable rate of change. After this initial activity, there was a lag period of little activity (less than 0.1 g L-1 day-1 gCOD-1) for all the carbon sources until day 9. At this time point, the hay leachate SRR peaked at 1.13 g L-1 day-1 gCOD-1 and the biosolid leachate SRR peaked at 1.20 g L-1 day-1 gCOD-1; however, the 34  lactate standard did not surpass its initial rate and remained constant. The peak SRRs for the leachates are a factor of 1000X faster than those reported by the authors Waybrant and Schmidtova on similar batch bioreactors using solid (rather than liquid) organic materials as carbon sources [Waybrant, 1998][Schmidtova, 2011]. The lag time, also called acclimation period, of 9 days for all liquid carbon sources could be explained by the bottle bioreactor conditions differing from the environment the bacteria are adapted to in the mine sediment used as the inoculum. The mine sediment as a seed bacteria required approximately one week to adapt to environmental conditions inside the bottle bioreactors in order to reach peak SRR rates. The type of carbon source does not appear to affect the duration required for acclimation period; however, it does affect the percentage SO42-  removed and peak SRR. Overall, the hay leachate out-performed the other tested liquid carbon sources in both percentage SO42-  removed and SRR, because it is assumed to contain dissolved carbon molecules that can be readily utilized by the SRB in the mine sediment inoculum, as evidenced by an initially high SRR.  2.3.3 Biogenic Sulfide Production and Metal Precipitation Since sulfide is the reduced product of biological sulfate reduction, the dissolved sulfide was measured over time to determine percentage product yield for the reaction. Initially, the total dissolved sulfide (TDS) concentrations were barely detectable and then increased to 160 mg/L, 433 mg/L and 468 mg/L for biosolid leachate, hay leachate and lactate, respectively. The measurements for sulfide over time are shown in Figure 5.  35   Figure 5. Sulfide production over time for batch bioreactors by carbon source Error bars represent the standard deviation from the mean for triplicate culture bottles for each organic material.  All bioreactors showed hydrogen sulfide production increasing over time as sulfate was being removed. In theory, approximately 1/3 of the sulfate removed should be transformed into sulfide as evidenced by the ratio of the molecular weights of the product to reactant (Equation 2) which is further detailed in Equation 5. All bioreactors were able to achieve approximately 100% yield for sulfide production which takes into account the theoretical yield as shown in Equation 6. Equation 5.   𝑴𝒐𝒍𝒆𝒄𝒖𝒍𝒂𝒓	𝑾𝒆𝒊𝒈𝒉𝒕	𝑺𝑴𝒐𝒍𝒆𝒄𝒖𝒍𝒂𝒓	𝑾𝒆𝒊𝒈𝒉𝒕	𝑺𝑶𝟒 = 𝟑𝟐.𝟎𝟏	𝒈/𝒎𝒐𝒍𝟗𝟔.𝟎𝟔	𝒈/𝒎𝒐𝒍 = 𝟎. 𝟑𝟑 Equation 6.   %	𝒀𝒊𝒆𝒍𝒅 = 𝑨𝒄𝒕𝒖𝒂𝒍	𝒀𝒊𝒆𝒍𝒅𝑻𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍	𝒀𝒊𝒆𝒍𝒅 = [𝑯𝟐𝑺]𝒇𝒊𝒏𝒂𝒍𝟎.𝟑𝟑∗([𝑺𝑶𝟒]𝒇𝒊𝒏𝒂𝒍−[𝑺𝑶𝟒]𝒊𝒏𝒊𝒕𝒊𝒂𝒍) High percentage yields for sulfide production suggests that a majority of the sulfide produced stayed in solution in the bottles and was not released as volatilized H2S or transformed into intermediate S compounds such as sulfite or thiosulfate [Barton, 2007]. The sulfide produced may be in the form of  H2S, HS- and S2-, which when referred to as a whole are termed total 36  dissolved sulfide (TDS). Aqueous H2S is volatile and will always equilibrate between gas and aqueous phases with its ultimate distribution depending on Henry’s Law. Equation 7 shows H2S equilibrium between aqueous, gaseous and its anions HS- and S2-.  Equation 7.   𝑯𝟐𝑺(𝒈) ↔ 𝑯𝟐𝑺(𝒂𝒒) 𝑲𝒂𝟏iG 𝑯𝑺− + 𝑯+ 𝑲𝒂𝟐iG 𝑺𝟐− + 𝟐𝑯+ Literature reports pKa1 values varying from 6.97 to 7.06 at 25 °C and pKa2 from 12.5 to 15 [Li, 2013]. Therefore, assuming a solution pH of 7, it can be calculated that approximately 50% of the total hydrogen sulfide exists as H2S and 50% is in the form of HS-, and the high pKa2 value indicates that S2- is negligible. Table 13 summarizes the percentage product yield, pH and the final TDS concentrations for all three carbon sources. Table 13. Percent product yield, pH, and total dissolved sulfide in bioreactors by carbon source Parameter Lactate Hay Leachate Biosolid Leachate % Product Yield 130 ± 22.7% 100 ± 3.8% 97 ± 6.8% pH 7 6 8 Final TDS Concentration (mg/L) [TDS]= 468 mg/L [TDS]= 433 mg/L [TDS]= 160 mg/L  Based on the metal concentrations in the synthetic growth medium approximately 2% of sulfur will end up as metal sulfides by weight, since the amount of metal as iron in the bioreactors is approximately 2% by weight. Any sulfide not being measured by a dissolved sulfide assay is assumed to be deposited as a metal sulfide precipitate and based on percent yield results, it was negligible.  After producing biogenic sulfide in the synthetic media, a sample from each bioreactor was added to a bottle containing real mine-influenced water with environmentally relevant levels of 37  metal contaminants. The ratio of total dissolved sulfide (TDS) does appear to affect the metal precipitation reaction; although few studies report on the ratio required. Generally, dissolved metals in contaminated groundwater are at µg/L levels and sulfide is produced at mg/L levels therefore in these treatment systems, the sulfide is an order of 1000X greater [Barton, 2007]. However, the actual mine-influenced water in this experiment has mg/L levels of Zn and contributes more significantly to the ratio. The biogenic sulfide was added to the metal precipitation bottles at a mass ratio of 1:1 and at a pH of 7. Figure 6 shows the results for Zn and the other metals Cd, Co, Fe, Mn and Pb can be seen in Appendix B.  Figure 6. Zinc removal over time for batch metal precipitation reactors Error bars were negligible in the case of zinc for triplicate mine water bottles of each biogenic sulfide source.  Zinc was shown to be removed from 24 mg/L to less than 1 mg/L for lactate containing bioreactors and less than 0.1 mg/L for leachate containing bioreactors. No changes in removal percentage were observed between the first sampling point of 30 minutes and the final time point of 24 hours which was a similar trend seen in all metals except for iron. All biogenic sulfide sources were effective in removing zinc (greater than 95% removal); however, stringent water 38  quality guidelines for the chronic toxicity levels may require the lower levels of less than 0.1 mg/L that only the biogenic sulfide from leachates were able to achieve. Table 14 summarizes the removal percentages in the metal precipitation reactors.  Table 14. Removal percentages for metals of interest in metal precipitation reactors by biogenic sulfide carbon source Metal Biogenic Sulfide Carbon Source Lactate Hay Leachate Biosolid Leachate Cd 94 ± 4% 90 ± 2% 90 ± 0.3% Pb 98 ± 4% 100 ± 4% 100 ± 2% Zn 95 ± 2% 99 ± 0.1% 99 ± 0.4% Fe 78 ± 48% -10 ± 28% -5 ± 48% Mn 5 ± 1% 12 ± 1% -5 ± 2% Co -521 ± 108% -71 ± 50% 50 ± 73% Error bars represent the standard deviation from the mean for triplicate mine water bottles for each biogenic sulfide source.  All biogenic sulfide sources were able to remove greater than 90% of Cd, Pb and Zn within the first time point of 30 minutes. These are expected results since the solubility product constants (Ksp) of these metals are very small, thus producing a stable form of solid. This also suggests that no inhibitors of metal precipitate formation were carried over with the biogenic sulfide, with the one exception of biogenic sulfide from lactate and zinc removal. It can be seen in Figure 6 that lactate caused zinc to be removed at 95% as opposed to 99%. These exceptions were also observed by Esposito et al which reported a 42% decrease in Zn removal percentage of biogenic sulfide as compared to chemical sulfide and highlighted the need for biogenic sulfide to be tested with actual source water which is subject to natural variations in phosphate, micro-nutrient, acetate and EDTA [Esposito, G., 2006]. These natural variations could explain why there is not 39  always a clear relationship between the sulfate removal extents and metal precipitation. The metals Co, Fe and Mn had more variable results. It is known that the Ksp of iron sulfides are not as small and therefore the formation of this solid required a longer time for equilibrium to be reached. The authors Oh et al reported on the complex chemistry of iron removal and provided data on requirements of up to 20 hours for oxidation of Fe2+ to Fe3+ which is then more reactive in subsequent precipitation reactions [Oh, 2016]. The results in this biogenic sulfide experiment agree with other studies and suggest that there are many different equilibria present at one time for iron species. The poor manganese removal extents are not unexpected, as confirmed by previous reports that have demonstrated requirements of high pH values (8-10) for rapid formation of MnS [Logan, 2005]. Thus, the neutral pH in this experiment was not conducive to Mn precipitation. Notably, the biogenic sulfide from biosolid leachate did actually increase the concentration of Mn by 5% which is expected as pulp & paper mill residues have elevated levels of Ca, Mg and Mn due to thermochemical processes used in this industry [Asemaninejad, 2018]. The results for cobalt are rather unexpected and the large increase in response to the biogenic sulfide from lactate will require further research, as well as the high relative difference in values for all biogenic sulfides suggesting that there is high variability within the natural environment for the speciation of Co.   2.3.4 Microbial Community Analysis In order to explore what types of microorganisms were supported by the different carbon sources, a phylogenetic survey based on 16S rRNA sequencing was performed and distribution of taxonomic groups at the phylum level are presented in Figure 7. Since many different OTUs (operational taxonomic units representing putative bacterial species) were found in the 40  bioreactors and inoculum (1,979 OTUs) the survey was first conducted at the higher taxonomic level of phylum (see further discussion in Section 1.6).   Figure 7. Dominant phyla in the batch bioreactors summarized by carbon source The microbial community profile was analyzed from DNA extracted at day 16 of the experiment. Out of a total of 14 dominant bacterial phyla, it can be seen that Desulfobaterota and Firmicutes are well represented and at least 50% of the population for all carbon sources. These two phyla contain known sulfate reducing OTUs. This finding agrees with another study that reported BCRs and mine sediment containing approximately 50 to 80% of the total number of OTUs 0.000.250.500.751.00Hay and Wood Chip MixLactatePulp Mill BiosolidsSedimentOMFracphylumBacteroidotaCampilobacterotaDesulfobacterotaFirmicutesProteobacteriaSpirochaetota41  belonging to these phyla [Nielsen, 2018]. Another study on BCRs reported that the enrichment of Firmicutes in relation to Bacteroidota coincided with an initiation of zinc sulfide precipitation [Drennan, 2017]. Figure 7 shows that there were two other phyla comprising most of the other 50% of the population, one of those being Bacteroidota and the other Proteobacteria. These phyla are not known for sulfate reducing bacteria and may contain the supporting bacteria needed in anaerobic environments.  For example, a study showed that pulp & paper mill wastewater treatment secondary biosolids when amended to mine tailings promoted the bacteria closely related to the genus Sphingomonas (phylum Proteobacteria), which are likely chemoheterotrophic bacteria that have been previously detected in activated sludge/wastewater treatment plants [Asemaninejad, 2018]. The distribution of phyla in the batch bioreactors and mine inoculum appear to agree with other studies and show promising enrichment of the phylum Firmicutes which is shown to coincide with promoting metal precipitation.   The dominant SRB genera can then be further analyzed by isolating them from the phylum which contain them. Figure 8 shows the types of SRB genera identified in the phylum Desulfobaterota, as well as those SRB genera contained within the orders Desulfitobacteriales and Desulfotomaculales in the phylum Firmicutes. 42   Figure 8. SRB genera represented in the microbial communities summarized by carbon source The microbial community profile was analyzed from DNA extracted at day 16 of the experiment. As of 2008 there were 38 identified SRB genera, and out of these only four main SRB genera were represented at some percentage in the batch bioreactors and inoculum [Muyzer, 2008]. The mine sediment inoculum contained predominantly Desulfovibrio species, which persisted in each bioreactor. The other dominant SRB that were identified but not well represented in the inoculum are Desulfobulbus, Desulfatirhabdium and Desulfosporosinus. As expected, all the bioreactors promoted Desulfobulbus to higher levels than that in the inoculum which has been observed in other enrichments from anaerobic mud samples when cultured with propionate and sulfate [Widdel, 1982]. Notably, Desulfosporosinus is not present in the lactate bioreactors and it is known to be present in alfalfa containing BCRs capable of using varied carbon sources such as formate, pyruvate, fructose, glycerol and yeast extract, and tolerant to high concentrations of 0.000.250.500.751.00Hay and Wood Chip MixLactatePulp Mill BiosolidsSedimentOMfractiongenusDesulfatirhabdiumDesulfobulbusDesulfosporosinusDesulfovibrioNADistribution of SRB Genera in the Microbial Communities43  copper [Drennan, 2017] [Mayeux, 2013][Rezadehbashi, 2018]. The biosolid leachate bioreactors contained the highest fraction of Desulfatirhabdium which was also found to be dominant in microbial community networks with methanogens of BCRs in passive treatment systems [Rezadehbashi, 2018]. This species along with other Desulfovibrio species are able to oxidize lactate to acetate, but only when hydrogen is efficiently removed by hydrogen-consuming methanogens, demonstrating the often cited syntrophic growth of sulfate reducers with methanogens [Muyzer, 2008]. The dissolved carbon molecules in the biosolids leachate could have promoted the growth of incomplete oxidizers, causing an overload of acetate which required methanogenic activity to complete the carbon cycle; therefore, sulfate removal was inhibited. This finding correlates with the percentage of COD utilized in the batch bioreactors. At the tested COD:SO4 ratio, approximately 10% and 20% of COD was utilized in the biosolid leachate and hay leachate containing bioreactors, respectively. The biosolid leachate had less percentage COD utilized and it suggests that not all the dissolved organic carbon molecules in the biosolid leachate were readily utilized by SRB.   As hinted at before in mentioning syntrophic growth of sulfate-reducers and methanogens, there is a vested interest in protecting the biological diversity in natural systems.  The measure of phylogenetic diversity produces different priorities for species conservation, relative to its taxonomic diversity and can be represented as shown in Figure 9 [Faith, 1991].  44   Figure 9. Faith Phylogenetic Diversity of microbial communities by carbon source The microbial community profile was analyzed from DNA extracted at day 16 of the experiment.  Figure 9 shows that the leachates produced more diverse microbial communities, as compared to lactate. Additionally, the diversity intrinsic in the mine sediment used as the inoculum remained constant in the bioreactors with the leachates. This confirms that lactate enrichments select for very specific bacterial types as opposed to the more diverse carbon molecules in the naturally biodegraded leachates.   Another genomic test for detecting the presence of sulfate-reducers was to use quantitative polymerase chain reaction (qPCR) which can target the gene for a key enzyme in the sulfate reduction pathway, the dissimilatory sulfite reductase gene sub-unit A or dsrA. Detection of the dsrA gene infers the metabolic potential for sulfate reduction and could include bacteria other than those detected through taxonomic analysis due to horizontal gene transfer [Nielsen, 2018]. 45  The copies of the dsrA gene relative to the total number of 16S rRNA genes, which is present in all bacteria, gives a measure of the representation of SRB in the community. The number of 16S rRNA and dsrA genes cannot be directly compared since some bacteria can contain multiple 16S rRNA genes therefore making it not a simple one to one comparison [Vetrovsky, 2013].  Figure 10 shows the log10 copy number of the 16S rRNA and dsrA genes in the inoculum and in the bioreactors.  Figure 10. Log copy number of the 16S rRNA and the dsrA genes in the mine sediment inoculum and batch bioreactors summarized by carbon source The microbial community profile was analyzed from DNA extracted at day 16 of the experiment and error bars represent the standard deviation from the mean for triplicate PCR reactions for each sample. The differences between the bioreactors and inoculum in 16S rRNA gene copy numbers were less significant than the differences in the dsrA gene copy numbers. This finding suggests that the copy numbers for the dsrA gene are meaningful in the carbon sources and a trend was found which ranked them as follows: lactate > hay leachate > biosolid leachate > mine sediment. The results of qPCR when compared to the phylogenetic survey of 16S rRNA gene suggest that the 46  relative abundance of certain phyla and genus does not correlate with sulfate removal extents because there is another factor, the quantification of bacteria, that also contributes to the kinetics. The quantification results of qPCR takes into account the absolute abundance of bacteria, not only relative abundance. The 16S rRNA gene surveys do provide valuable information such as identifying the presence of key organisms in the inoculum that can be further enriched and estimating the diversity of the community, yet they do not show directly comparable results to qPCR when attempting to quantify the absolute percentage of SRB to overall microbial population. This work shows that qPCR of the dsrA gene can be a good proxy for estimating sulfate reduction rates.    47  Chapter 3: Continuous Flow Column Study of Hybrid System Treating Real Mine-Influenced Water 3.1 Synopsis The proof-of-concept experiments demonstrated that a 5-day fermentation from the waste organics could promote sulfate reduction to sulfide that reacted with zinc in the real mine-influenced water to remove it to below regulated levels. The next step was to design and test a continuous flow set-up at an actual mine site where required volumes of mine-influenced water were available. The continuous flow set-up consisted of two packed bed reactors or columns which were constructed as a two-stage system including a feedback loop for biogenic sulfide. The two-stage design decoupled the biological sulfate reduction process from the chemical metal precipitation reaction for the purpose of: 1. Enabling metal recovery and potential complete removal from the mine site 2. Removing inhibition of sulfate reduction by metals  3. Preventing precipitates from plugging the bioreactor The biological sulfate reduction column was sized so as to target a hydraulic retention time appropriate for greater than 66% SO42- removal, since this amount of removal would consequently produce biogenic sulfide at a concentration that could react with zinc in an equal mass ratio. The metal precipitation column was sized to target a hydraulic retention time appropriate for not only the quick reaction times for Zn, Cd and Pb removal (minutes) but also the longer durations required for Fe and Mn removal (hours). Three separate two-stage column systems were set up in order to compare the affect that carbon source has on sulfate reduction and metal precipitation. The hay leachate was chosen to continue with due to its performance in 48  previous experiments, which was compared to molasses. Molasses was chosen as a standard as it is often used to support semi-passive treatment systems in the field. The third system had no added carbon source in order to determine how the system performs without promoted biological activity. All three hybrid systems were run for a duration of 96 days in order to determine the effectiveness and reliability of the two stage system at removing metals.   3.2 Materials and Methods 3.2.1 Site Description The test site used for testing the continuous flow columns is a closed mine site situated on private land in southeastern British Columbia (BC), where the owner is currently undergoing reclamation efforts. The site currently collects water from both underground and above ground streams and directs them to a water treatment facility for metal removal prior to discharge to the environment. One of these underground sources was investigated as it exceeds BC Water Quality Guidelines (BC WQGs) in the following metals: cadmium (Cd), cobalt (Co), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn). The location of this contamination source was easily accessible for water collection and relatively consistent in metal concentrations over time; therefore, it provided a reliable background for field experiments. Table 15 summarizes the concentrations of metal contaminants in the mine-influenced water and corresponding BC WQGs based on the ambient hardness of the field site studied.       49  Table 15. Source water quality in metals and treatment objectives Parameter Range of Measured Concentrations1 BC WQG 2 (Values based on water hardness* of 142 mg/L) Total Metals (µg/L) Dissolved Metals (µg/L) Cadmium (Cd) 24.1 - 28.4 22.2 – 26.0 Chronic = 0.27 µg/L dissolved Cd Acute = 0.84 µg/L dissolved Cd Cobalt (Co) 17.2 – 17.7 16.3 - 17.0 Chronic = 4 µg/L total Co Acute = 110 µg/L total Co Iron (Fe) 94 – 181 < 50 Total Fe = 1,000 µg/L Fe Dissolved Fe = 350 µg/L Fe Lead (Pb) 117 – 161 72.1 – 86.7 Chronic = 8.3 µg/L total Pb Acute = 127 µg/L total Pb Manganese (Mn) 2,130 – 2,170 2,020 – 2,120 Chronic = 1,200 µg/L total Mn Acute = 2,100 µg/L total Mn Zinc (Zn) 22,700 – 22,800 21,000 – 22,600 Chronic = 292 µg/L total Zn Acute = 1,287 µg/L total Zn Source 1: Measured concentrations of source water from mine site for duration of thesis experiments, Oct 2019 – Feb 2020. Source 2: British Columbia Approved Water Quality Guidelines: Aquatic Life, Wildlife & Agriculture, Summary Report, August 2019. *The value of hardness is relevant for applicability of chronic and acute calculations.  The metals that most exceed BC WQGs are cadmium and zinc, which exceed acute guidelines by up to 20X and chronic guidelines by up to 100X. Other metals such as cobalt, manganese and lead, all exceed the more stringent chronic guidelines by up to 10X, but do not exceed the acute guidelines. The mine-influenced water does not exceed guidelines for iron; however, this metal is prone to re-release in passive treatment systems, and therefore, will be discussed in the results. The metals, cadmium and zinc, will be the focus of this research as they are highest amount over the acceptable freshwater aquatic life guidelines.   When designing a bioprocess for wastewater treatment, the ambient water conditions are important as they create the environment for promoting activity of the desirable microorganisms. Other parameters, such as sulfate and pH, give valuable insight into the biological processes 50  occurring and were measured in the source water during the experiments. Table 16 summarizes the relevant concentrations of design parameters for the researched treatment system and their corresponding guidelines for release into a freshwater ecosystem.  Table 16.  Source water quality in other chemical parameters and treatment objectives Parameter Range of Measured Concentrations1 BC WQG2 Sulfate (SO4) 123 – 177 mg/L Less than 309 mg/L Chemical Oxygen Demand (COD) < 20 mg/L No change to receiving environment pH 6.5 – 7.3 No change to receiving environment Conductivity 449 – 453 µS/cm None Hardness (as CaCO3) 142 mg/L None Dissolved Oxygen (DO) 1.5 – 4.5 mg/L 5 – 11 mg/L Source 1: Measured concentrations in source water for duration of thesis experiments, Oct 2019 – Feb 2020. Source 2: British Columbia Approved Water Quality Guidelines: Aquatic Life, Wildlife & Agriculture, Summary Report, August 2019.  This source water can be classified as neutral mine drainage (rather than acid rock drainage), because it has near neutral to alkaline pH. The other parameters such as sulfate, COD, conductivity and hardness do not pose a problem to the receiving environment and are within the guidelines, except for the need to increase the dissolved oxygen (DO). Sulfate does exist in the mine-influenced water at lower concentrations than those detrimental to a freshwater system; therefore, removal of sulfate is not required. The amount of carbon or chemical oxygen demand (COD) in the influent water is insufficient for promoting the activity of the microorganisms needed for treatment, and therefore, must be supplemented with an external carbon source. As mentioned in Table 6, the recommended COD:SO4 ratio for SRB growth is 0.8 to 1.2 g g-1.   51  3.2.2 Experimental Set-Up The novel hybrid passive system was constructed as a series of two up-flow, packed-bed columns with metering pumps for controlling flow rates and tubing for directing flows.  There were three sets of the column systems:  1) System #1 used a hay and wood chip mix leachate  2) System #2 used molasses  3) System #3 used no carbon source (negative control) The pulp mill biosolids that were tested in Ch. 2 were not used for the field experiments due to the unavailability of fresh biosolids since the neighboring pulp mill was shut down for maintenance. Molasses from Wholesome Organics (unsulfured) was chosen as a standard for comparison to the hay leachate, similar to the lactate standard used in the batch experiments. Molasses is a commonly used liquid carbon source for PTSs, for example, a study reported that molasses has 4.5% nitrogen and a 4.6 C:N ratio, and could increase the SRB population from 8x106 cell/mL to 3.3x107 cell/mL and support high sulfate reduction [Teclu, 2009]. An annotated photo of all three columns systems and associated pumps, tubing, valves and wall-mounting device are shown in Figure 11. 52   Figure 11. Annotated photo of novel hybrid passive system experimental set-up [tubing was color coded to denote separate flow streams] All columns were made from high density Polyethylene (HDPE) and included built-in ports for tubing connections. Dimensions for both the first-stage and second-stage columns are further detailed in Appendix C. A geosynthetic material was placed at port junctions in order to keep the packed-bed media inside the columns while allowing water to still pass through. Masterflex Quick-Disconnect valves were used at the bottom of each column in order to be able to isolate columns for troubleshooting. The second-stage or biological columns (1B and 2B) were covered with black cloth to avoid photosynthetic biological growth. The mine-influenced water was collected from the field and stored in a 20L container directly below the Influent pump. All pumps were Cole Parmer Masterflex L/S Standard Digital Drives with size small pump cartridges including silicone tubing (outer diameter = 1/16”). Graduated beakers were placed at 53  the effluent tubing of all columns to capture sample water for analyses. Table 17 lists all the directed flows along with corresponding PTFE tubing color and size, flow rate and pump used.  Table 17. Flow descriptions of column systems and corresponding tubing color, pump name and flow rate Flow Description Tubing Size Tubing Color Pump Name Flow Rate Influent Water Mine-influenced water transferred to first-stage columns (1A, 2A and 3A) OD: ¼” Purple Influent 0.5 mL/min Connecting Columns Effluent from first-stage columns (1A, 2A) transferred to second-stage columns (1B and 2B) OD: ¼” Blue Feedback 0.2 mL/min Biogenic Sulfide Feedback Effluent from second-stage columns(1B and 2B) transferred back to first-stage columns (1A and 2A) OD: ¼” Orange Feedback 0.2 mL/min Carbon Sources Carbon mixtures transferred to second-stage columns (1B and 2B) OD: ¼” Clear Carbon 0.25 mL/min OD = outer diameter The media in the second-stage column was chosen to be a rock (red lava rock – pebble sized) with rough edges for increasing surface area available for biofilm growth. The media in the first-stage column was a filter sand of 10/20 mesh (2.0 to 0.84 millimetres) fraction silica sand. Environmental silica sand was used because it does not contribute significant soluble ions to the groundwater [Brown, 2014]. Each system was set up at the same flow rates which resulted in the following parameters shown in Table 18. Table 18. Parameters of chemical and biological columns for hybrid design Parameter First-Stage Column Second-Stage Column Total Volume 1 L 4.2 L Empty Bed Volume 450 mL 2.5 L Media Type Filter sand Lava rock (pebble size) Hydraulic Retention Time 11 hours 4 days 54  The COD dosage will affect the peak SRR and therefore the HRT and thus this parameter will be highly influential in continuous flow experiments.  In order to maintain an anaerobic environment in the biological columns, the effluent from these columns was directed out directly from the top of the column into a fitting which allowed effluent to be flowing into a beaker on the bench. The tubing was always kept submerged in the sample water so as to allow for no oxygen to enter the column through the effluent tubing. Oxygen entering through the silicon tubing at the pump heads was observed to be negligible due to no air bubbles observed throughout the columns and effluent tubing upon start-up.   3.2.3 Sampling Points Samples were taken of the source water influent, effluent of each column, and the carbon source mixtures. Table 19 provides sample point descriptions and Figure 12 places them on a schematic of the column systems. Table 19. Sample descriptions for column effluents in hybrid design Sample Point Description Sample ID Mine-Influenced Water Influent SP00 Carbon Source 1 Feed Hay Leachate SP01 Carbon Source 2 Feed Molasses SP10 Column 1A Effluent Hay Leachate System - Chemical Metal Precipitation Column SP05 Column 1B Effluent Hay Leachate System – Biological Sulfate Reduction Column SP09 Column 2A Effluent Molasses System - Chemical Metal Precipitation Column SP14 Column 2B Effluent Molasses System – Biological Sulfate Reduction Column SP18 Column 3B Effluent Negative Control System – Chemical Metal Precipitation Column SP22 55   Figure 12. Sample point schematic The first-stage column effluents were sampled in order to determine metal removal extents, and the second-stage column effluents were sampled in order to determine sulfate removal percent and sulfide concentration. The carbon source mixtures were sampled in order to determine COD concentration could be metered at the tested COD:SO4 ratio of 1. Samples were taken approximately twice per week and more samples were taken during the inoculation period in order to ensure that the biological columns were becoming active.   3.2.4 Analytical Methods Water samples were sent to ALS labs in Burnaby, BC for analysis. Hardness (also known as Total Hardness) was calculated from the sum of calcium and magnesium concentrations, expressed in CaCO3 equivalents. Total sulfide was measured using a colorimetric method adapted from APHA Method 4500-S2. Sulfate in water was measured by ion chromatography (EPA method 300.1) with conductivity and/or UV detection. Total metals in water was measured by CRC ICPMS (EPA method 200.2/6020A) where samples are digested with nitric acid before analysis. Dissolved metals were measured by CRC ICPMS (APHA method 3030B/6020A) 56  where samples are filtered through 0.45 µm and preserved with nitric acid. COD was measured by colorimetric method using procedures adapted from APHA method 5220.  Upon completion of 96 day experiment, the biological columns were subsampled at 6 different locations for biological matter. These subsamples were filtered through 0.45 µm in order to capture biological solids on the surface of the filter paper, which were then saved and frozen for subsequent DNA extraction. DNA extraction was performed by Power Water Qiagen extraction kit (as per manufacturer’s instructions). Quantitative PCR with 1 ng DNA was performed on Bio-Rad CFX instrument using SsoFast EvaGreen enzyme with both dissimilatory sulfite reductase sub-unit A (dsrA) primer pair (F-ACSCACTGGAAGCACGGCGG/R-GTGGMRCCGTGCAKRTTGG) and a total bacteria (16S) primer pair (F-CGGTGAATACGTTCYCGG/R-GGWTACCTTGTTACGACTT).   3.2.5 Biological Inoculation In order to conduct the biological inoculation, the second-stage (biological) columns were seeded with microorganisms from sediment at the mine site. This sediment was collected from a nearby seepage marsh on the mine site which was currently located in the flow path of metal contaminated water. It was hypothesized that a sediment taken from this area would contain microorganisms adapted to higher concentrations of metals. Soil was found which was black in color and emitted a rotten egg smell since these are observations indicating sulfate reduction is occurring naturally. Approximately 5 cm of top soil was removed and the soil directly underneath was collected and then stored in an upright plastic (sterile) bottle at room temperature before being used for inoculation. Approximately 250mL of mine sediment was mixed with a 57  mixture of the carbon source and mine-influenced water in order to completely fill the biological columns (10% by volume). The carbon source was added to the mine-influenced water in order to target a COD:SO4 ratio of 2 since this was the ratio in previous batch experiments. The columns were then left to sit in batch mode until biological activity was noted. It was expected that a time period of at least two weeks would be required to achieve sulfate removal in the columns thus denoting that biological activity was established. Measurements of sulfate were taken every week and it required three weeks to achieve 66% SO42- removal. The temperature of the columns was 10 – 15 °C which could account for the slower kinetics. After three weeks of batch inoculation, the mine-influenced water, biogenic sulfide and carbon source flows were all initiated at their pre-determined flow rates (Table 17). This began the continuous flow experiment and all further data is presented from this point as day 0.   3.3 Results and Discussion 3.3.1 Sulfate Removal and Sulfide Production The effluents of the second-stage (biological) columns were sampled and measured for sulfate concentration in order to determine the removal percentages and sulfate reduction rates (SRRs). Since they were not receiving any carbon source, the first-stage (metal precipitation) columns had no observable sulfate reduction and therefore are not presented in this section. Both biological columns initially went through an acclimation period (day 0 through 43) where sulfate removal percentage remained on average below 44% for the hay leachate column and below 17% for the molasses column. The hay leachate column initially had higher removal extents perhaps due to the microorganisms in the mine sediment inoculum being able to readily utilize the carbon molecules, as evidenced in the batch reactor experiments. However, the molasses is a 58  more defined carbon source consisting of carbohydrates, glucose and fructose which required a longer adaptation phase for the microorganisms to readily utilize [Clarke, 2003]. By day 60, both the hay leachate and molasses columns had reached their peak SRRs of 41.2 mgSO4 L-1 day-1 and 28.7 mgSO4 L-1 day-1, respectively. After day 60, these increased SRRs were maintained for both biological columns until the end of the experiment. Thus, the SO42- removal percentage from day 60 onward, once peak SRR was reached, then increased to 85% and 73% for hay leachate and molasses, respectively. Table 20 outlines the observed periods of biological activity and resulting averages of SO42- removal percentages and SRRs.  Table 20. Sulfate removal and reduction rates for second-stage columns in hybrid system Day Range Phase Description Hay Leachate Column Molasses Column SO42- Removal % SRR (mgSO4 L-1 day-1) SO42- Removal % SRR (mgSO4 L-1 day-1) 0 – 43 Acclimation 44% 19.4 17% 6.1 43 – 60 Ramp-Up to Peak SRR 84% 33.9 55% 22.3 60 – 96 Steady-State 85% 28.4 73% 23.8  It should be noted that the operational parameters such as HRT and COD:SO4 ratio were kept constant, and the pH, temperature and conductivity were all relatively constant. The only changes would have been due to natural variations in source water conditions. Thus, the phases of sulfate reduction performance are denoted as biological and not operational phases. These phases are annotated in Figure 13 which depicts the sulfate measurements over time for the biological columns.  59   Figure 13. Sulfate concentration over time in the biological columns’ effluents Once this biological equilibrium was reached, then the operational parameters such as HRT and COD dosage could have been adjusted in order to increase the SRR in the columns. It should be noted that this column study showed approximately 60 days were required to reach equilibrium conditions, with the hay leachate column reaching equilibrium earlier than the molasses column.  The trends found in sulfide production mirrored those observed for sulfate reduction. The concentration of total sulfide in the biological column’s effluent was measured in order to determine percentage product yield and to allow estimation of the amount of biogenic sulfide transferred through the feedback loop to the metal precipitation column. Figure 14 shows the sulfide over time for the biological columns annotated with the biological phases.  60   Figure 14. Sulfide concentration over time in the biological columns’ effluents The percent yield was calculated for each biological column using Equations 4 and 5 (Section 2.3.3). For the hay leachate column, the percent yield was low during the acclimation phase at 40% and then increased throughout the ramp-up phase to achieve 89% by the time steady-state was reached. The molasses column followed a similar trend, the percent yield was initially low at 25% and increased throughout the ramp-up phase to 94%. The percent yields calculated in the column study are overall lower than those observed in the batch studies. Possibly due to the system being open allowing volatilization of gaseous H2S and metal sulfide solids being generated. The percent yield is important because it factors into estimation for the total sulfide produced and therefore the amount of biogenic sulfide available to be fed back into the first-stage column to promote metal sulfide precipitation. The ratio of the mass of total sulfide per mass of total zinc was calculated as a function of the concentration and flow rates of each species. Figure 15 shows the mass ratio of total sulfide to metal precipitation column throughout the duration of the experiment.  61   Figure 15. Biogenic sulfide to zinc mass ratio in the hybrid systems  3.3.2 Metal Removal The metals: Zn, Cd, Co, Mn, Pb and Fe were measured for the duration of the continuous flow experiment and their removal extents follow similar trends as seen in the biological phases outlined in Section 3.3.1. This suggests that the removal of certain metals, notably zinc, does correlate to the ratio of biogenic sulfide (Figure 15). Total zinc as opposed to dissolved zinc is shown in Figure 16 since the British Columbia Water Quality Guidelines regulate the concentrations of the total fraction. The detailed results for total zinc concentration in both first-stage and second-stage columns are in Appendix D.1. 62   Figure 16. Total zinc concentration over time in the first-stage (metal precipitation) columns’ effluents Zinc was initially removed at approximately 50% during the acclimation phase, and as the biogenic sulfide production increased from the biological columns the zinc removal in the metal precipitation columns increased to 85%. This correlated to an increase in the biogenic sulfide to zinc ratio of approximately 0.5. The BC WQG for acute toxicity is shown on Figure 15 and it can be seen that molasses fed system was able to meet this objective. Upon start-up at day 40, the negative control column removed Zn at approximately 50% but was no longer removing Zn by day 60. It is assumed that the sand media initially absorbed Zn and once the capacity of the media was reached the Zn was able to pass through. This finding proved that the addition of the biogenic sulfide via the feedback loop promoted the precipitation of metal sulfides. The steady-state phase (day 60 – 96) provided consistent results for the achievable metal removal extents in the hybrid design for all metals of interest. Table 21 summarizes the average removal extents in both the first-stage and second-stage columns’ effluents during the steady-state phase.   63  Table 21. Average metal removal extents for first-stage and second-stage columns Metal Hay Leachate Molasses Negative Control First-Stage (Column 1A) Second-Stage (Column 1B) First-Stage (Column 2A) Second-Stage (Column 2B) First-Stage (Column 3A) Cd (Dissolved) 99.8% 99.6% 99.7% 99.9% -27.0% Pb (Total) 99.5% 88.7% 99.5% 98.5% 98.7% Zn (Total) 85.3% 89.8% 90.2% 93.7% 9.4% Co (Total) 63.7% 89.9% 59.1% 85.0% 1.6% Mn (Total) -0.1% 18.7% 11.8% 33.2% 0.3% Fe (Total) -3040% -27.5% -2384% -31.0% 30.6%   The metal precipitation column was first in the process and was intended to remove a majority of the metals; however, the biological column was still able to provide a polishing step for metal removal at the same time that it was actively reducing sulfate. The metals Cd, Pb and Zn were all removed at levels greater than 85% in the metal precipitation columns of the carbon fed systems; however, the metals Co, Mn and Fe had more variable results. This was a similar trend that was observed in the batch experiments (Section 2.3.3). Cadmium was removed to levels below both acute and chronic guidelines. Unlike Zn, as soon as sulfide concentrations were greater than 10 mg/L in the feedback loop the Cd was removed at steady-state values. This is possible because Cd is at much lower concentrations than Zn in this source water and therefore equivalent TDS to metal ratios were reached by day 28 as opposed to day 43. The detailed results for Cd over time for all hybrid systems is in Appendix D.2. Lead was removed to levels below acute guidelines in all systems, even the negative control. This is the only metal that was removed by the sand filter alone. The effluent concentrations for Pb over time are in Appendix D.3. Overall, Cd, Pb, and Zn 64  were removed to below levels regulated for acute toxicity and further testing of the hybrid design could ensure that the chronic guidelines are also met.   For Manganese, it was observed that 0 to 12% removal occurred in the metal precipitation column and 20 to 33% occurred in the biological columns. This suggests that the pH increase (7 to 8) observed in the biological columns allowed for MnS formation. The concentrations of Mn in the effluent of both first and second stage columns is in Appendix D.4. Cobalt was not removed to below chronic guideline levels by the metal precipitation column but was after further treatment in the biological columns (See Appendix D.5). Cobalt sulfide formation appears to be more similar to the mechanism for MnS formation and require increased pH for precipitation. The treatment performance for iron removal was highly variable and it was actually released at concentrations up to 3000% higher than the influent concentrations. This release was only observed in the systems fed carbon sources, thus suggesting that the reductive processes cause iron to release from the media i.e. sand. As shown in Table 5, biological iron reduction occurs at a different redox zone (ORP = -80 mv) than sulfate reduction; therefore, the conditions in the metal precipitation column could be promoting these conditions and reducing any available iron in the sand media. Biological technologies are known to be poor for iron removal, and other studies have outlined using oxidative steps before biological treatment to remove this metal prior to biological processes. The authors Clyde et al and Oh et al both reported on different oxidative technologies in order to achieve 80% to 95% removal of Fe [Clyde, 2016][Oh, 2016]. Prior removal of iron using and oxidation technology would prevent passing through untreated; however, if iron is leaching from the sand or rock media then pre-65  selecting for a completely iron-free media may require more research. Total iron for the first-stage columns is shown in Figure 17 and both stages are shown in Appendix D.6.   Figure 17. Total Iron concentrations over time in the first-stage (metal precipitation) columns’ effluents Future improvements for this testing would be to increase the duration experiment to test the release of iron over longer periods of time. The data points toward the end of the experiment do indicate that the total Fe concentration in the effluents is trending downward.   3.3.3 Quantitative PCR of dsrA Gene As discussed in Section 2.3.4., detection of the dsrA gene is a good proxy for estimating the absolute percentage of SRB in the microbial community. Figure 10 shows the log10 copy number of the 16S rRNA and dsrA genes in the inoculum and in the biological columns. 66   Figure 18. Log copy number of the 16S rRNA and the dsrA genes in the mine sediment inoculum and batch bioreactors summarized by carbon source The microbial community profile was analyzed from DNA extracted at day 96 of the experiment. As reported before in the batch bioreactors, the differences between the biological columns and inoculum in 16S rRNA gene copy numbers were less significant than those in the dsrA gene. A trend was found which ranked them as follows: hay leachate > molasses > mine sediment. This again shows a correlation between the number of dsrA gene copies and the sulfate reduction rates in the columns. More data points establishing the number of dsrA gene copies at different operational set points would help to determine if this correlation is statistically significant in this system.   3.3.4 Comparisons to Hybrid Passive/Active System The hybrid passive/active design was shown to effectively remove sulfate and metals, and its overall performance can be compared to competing technologies. In Table 22, the performance 67  of the biological columns, specifically, was compared to a typical PRB type system and an active system. Table 22. Biological sulfate reduction performance in hybrid passive/active system compared to both a passive and an active system Reference System Type Carbon Source COD:SO4 Ratio SRR Mirjafari, 2016 Passive Up-flow Columns with Solid Organic Material Media • Solid wood • Solid hay • 0.34 and 0.03 • 0.34 and 0.03 • 81 and 51 mgSO4 L-1 d-1 • 272 and 49 mgSO4 L-1 d-1 This Work Hybrid Up-flow Columns with Rock Media • Molasses • Hay leachate • 1 • 1 • 29 mgSO4 L-1 d-1 • 41 mgSO4 L-1 d-1 Bratkova, 2013 Active CSTRs Defined lactate, ethanol, glycerol and citrate 3.5 130 mgSO4 L-1 hr-1  In the study on passive treatment system (PRB type), the author reported that the highest SRR, 272 mgSO4 L-1 d-1, was less than SRR of bioreactors fed a defined carbon source, 3,120 mgSO4 L-1 d-1, but within range of other traditional PRBs [Mirjafari, 2016][Bratkova, 2013]. The low SRRs when using a complex organic material are because the supply of low molecular weight carbon sources preferred by SRB are limited by the rate of hydrolysis of long-chain carbohydrates [Mirjafari, 2016]. It can be seen in Table 22, that as the COD:SO4 ratio decreased over time the HRT required to reduce the same amount of SO42-  increased by 5X in the passive system due to SRR decreasing. Once the available carbon is used up in a PRB type system, there is no current method to increase it other than completely replacing the solid material. However, in the hybrid system, the liquid carbon source can be continually metered in at the desired COD:SO4 ratio. In this work, a COD:SO4 ratio of 1 was able to achieve comparable SRRs to other passive treatment systems and it is hypothesized that higher SRRs are achievable if the HRT is systematically optimized and increased to rates comparable to active systems.  68    In Table 23, the performance of both the biological columns and metal precipitation columns was compared to an active system also studied this year. The authors Sun et al tested a similar metal sulfide precipitation process; however, it consisted of continuous stirred tank reactors (CSTRs). The latter study and this study show variability yet have some common themes. Zinc can be removed at greater than 85%; however, the dosage or ratio of biogenic sulfide to the metal of interest required for the removal extents are variable mainly due to the equilibria of hydrogen sulfide which is highly dependent upon pH of the solution.  Table 23. Metal precipitation performance in hybrid passive/active system compared to an active system Reference System Type Biological Performance Feedback Metal Precipitation Performance This work Hybrid Passive/Active Packed-Bed Columns • SPR: 0.3 mgTDS L-1 hr-1 • COD:SO4 = 1 • HRT = 4 days 30% (v/v)  • TDS:Zn ratio = 0.5 g g-1 • 85% Zn Removal Sun, 2020 Active CSTRs • SPR: 46 mgTDS L-1 hr-1 • COD:SO4 = 0.25 • HRT = 12 hours 33% (v/v)  • TDS:Zn ratio = 0.18 g g-1 • 98% Zn Removal SPR = Sulfide Production Rate According to the comparisons shown in Table 23, the ratio of biogenic sulfide to Zn should be monitored and could require 0.18 to 0.5 mass ratio for up to 85% removal of Zn at concentrations in the range of mg/L. The ratio may vary due to changing environmental conditions which promote other mechanisms for metal removal besides metal sulfide formation. The study by Sun et al also showed that a biogenic sulfide feedback of approximately 30% by volume was required in the system. However, it should be noted that this does depend on the sulfide production in the bioreactor. The hybrid passive/active system in this work showed comparable Zn removal extents as those reported in Sun et al., yet slower sulfate reduction rates 69  (4 days vs 12 hrs). This highlights that the metal removal performance is largely dependent upon the ratio of total dissolved sulfide and not directly tied to sulfate reduction rates of the biological reactor. According to this experiment, a footprint required for hybrid systems would be 8X the size of an active system until further studies can test the upper limits of HRT of this system. Other studies report that a change in HRT which changes the organic loading rate by 0.5 gCOD L-1 d-1 will require a response time of 15 days before the biological conditions equilibrate [Villa-Gomez, 2014]. Therefore, step-wise changes in the organic loading rate for a further experiments should allow for changes up to two weeks later to before establishment of biological equilibrium.   70  Chapter 4: Conclusions and Recommendations A novel hybrid passive/active system was designed and implemented in the field at a mine site, which tested the following hybrid components: 1. External liquid carbon source 2. Feedback loop for biogenic sulfide 3. Separate compartment for metal solids capture The external liquid carbon source and feedback loop for biogenic sulfide were first tested at the lab-scale (before field implementation) by a series of proof-of-concept experiments. Dissolved organic carbon was produced by a 5-day fermentation of waste solid organics from neighboring industries, and the solids chosen were a hay & wood chip mix and pulp mill biosolids. The soluble liquid products or leachates from these materials were tested in batch bioreactors and achieved peak sulfate reduction rates (SRRs) of 1.13 g L-1 day-1 gCOD-1 and 1.20 g L-1 day-1 gCOD-1 for the hay leachate and biosolid leachate, respectively. The peak SRRs were 1000X faster than rates reported for batch bioreactors using a solid organic carbon source as opposed to liquid. Additionally, the leachates promoted the growth of SRB genera Desulfovibrio, Desulfobulbus, Desulfosporosinus and Desulfatirhabdium, which were all present in the mine sediment inoculum. The relative abundance of SRB genera types showed no correlation to the predicting the peak SRRs achievable in the carbon sources; however, the qPCR results of the number of copies of the dsrA gene did show significant trends. The quantification of the dsrA gene copy numbers did correlate to higher SRRs and should be further explored in its ability to predict biological sulfate reduction. The sulfide produced from a batch bioreactors was proven to precipitate dissolved metals in real mine-influenced water from the mine site. Cd, Pb and Zn 71  were removed at 90% and above; whereas, other metals such as Fe were increased above influent concentrations by up to 10% with the addition of biogenic sulfide from the leachates.   After the proof-of-concept experiments, the proposed two-stage design with its added hybrid components was constructed and tested at the bench-scale on a mine site for a duration of 96 days. The first-stage (metal precipitation) was a packed-bed column with sand media operated at an 11 hour HRT and the second-stage (biological) was a packed-bed column with porous rock media operated at a 4 day HRT. By day 60, both the hay leachate and molasses fed columns had reached their peak SRRs of 41.2 mgSO4 L-1 day-1 and 28.7 mgSO4 L-1 day-1, respectively. The copy numbers for the dsrA gene were higher in the hay leachate column than in the molasses column, suggesting that qPCR could be a useful field tool for predicting SRRs. The metal precipitation columns were able to remove Cd, Pb and Zn at 85% and above, regardless of the carbon source used to produce the biogenic sulfide. The two-stage design was validated by a successful 96 day continuous flow experiment, which prove its applicability for capturing and removing Cd, Pb and Zn below British Columbia Water Quality Guidelines.  The metals Co, Fe and Mn were either re-released at greater concentrations or not removed below BC WQGs and their removal mechanisms should be further researched before full-scale implementation of the proposed hybrid design. 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Chemosphere 154: 215-223, DOI: 10.1016/j.chemosphere.2016.03.103. 60. Zhao, Y., Ren, N. & Wang, A. (2008) Contributions of Fermentative Acidogenic Bacteria and Sulfate-Reducing Bacteria to Lactate Degradation and Sulfate Reduction. Chemosphere 72: 233-242, DOI: 10.1016/j.chemosphere.2008.01.046.   82  Appendices  Appendix A   Table 24. Metals in soil results for solid organic materials Metals	(Soil)	Lower	Detection	Limit	Units	Pulp	Mill		Wastewater	Treatment	Primary	Solids	Pulp	Mill	Wastewater	Treatment	Secondary	Biosolids	Municipal	Landfill	Compost	Spent	Brewery	Grain	Hay	and	Grass	50/50	Mix	SPF	Wood	Chips	Mine	Sediment	Aluminum	(Al)	 50	 mg/kg	 231	 1630	 7890	 <50	 107	 59	 47700	Antimony	(Sb)	 0.10	 mg/kg	 <0.10	 0.18	 3.90	 <0.10	 <0.10	 <0.10	 1.24	Arsenic	(As)	 0.10	 mg/kg	 0.12	 1.31	 3.86	 <0.10	 <0.10	 <0.10	 20.7	Barium	(Ba)	 0.50	 mg/kg	 48.0	 291	 106	 15.3	 28.5	 13.9	 91.7	Beryllium	(Be)	 0.10	 mg/kg	 <0.10	 <0.10	 0.11	 <0.10	 <0.10	 <0.10	 6.15	Bismuth	(Bi)	 0.20	 mg/kg	 <0.20	 <0.20	 0.22	 <0.20	 <0.20	 <0.20	 0.33	Boron	(B)	 5.0	 mg/kg	 <5.0	 55.3	 20.4	 <5.0	 6.9	 <5.0	 8.2	Cadmium	(Cd)	 0.020	 mg/kg	 0.362	 8.26	 0.537	 0.066	 0.151	 0.078	 26.7	Calcium	(Ca)	 50	 mg/kg	 81000	 49200	 20100	 3490	 3500	 858	 16100	Chromium	(Cr)	 0.50	 mg/kg	 7.80	 16.0	 18.0	 <0.50	 <0.50	 <0.50	 4.75	Cobalt	(Co)	 0.10	 mg/kg	 0.26	 0.72	 4.60	 <0.10	 0.14	 <0.10	 6.81	Copper	(Cu)	 0.50	 mg/kg	 3.60	 29.1	 135	 17.0	 8.00	 2.34	 94.9	Iron	(Fe)	 50	 mg/kg	 597	 1490	 11900	 216	 216	 96	 9380	Lead	(Pb)	 0.50	 mg/kg	 0.90	 7.49	 59.9	 <0.50	 <0.50	 <0.50	 123	Lithium	(Li)	 2.0	 mg/kg	 <2.0	 <2.0	 4.8	 <2.0	 <2.0	 <2.0	 5.0	Magnesium	(Mg)	 20	 mg/kg	 1140	 1300	 4200	 3420	 2030	 176	 3060	Manganese	(Mn)	 1.0	 mg/kg	 90.5	 2210	 314	 60.2	 109	 61.5	 1560	Mercury	(Hg)	 0.050	 mg/kg	 <0.050	 0.573	 0.125	 <0.050	 <0.050	 <0.050	 0.105	Molybdenum	(Mo)	 0.10	 mg/kg	 0.32	 2.12	 1.50	 1.98	 1.91	 <0.10	 0.51	Nickel	(Ni)	 0.50	 mg/kg	 4.14	 9.51	 20.6	 <0.50	 1.81	 1.43	 36.1	Phosphorus	(P)	 50	 mg/kg	 101	 3130	 1470	 8830	 3720	 70	 532	Potassium	(K)	 100	 mg/kg	 <100	 460	 2030	 1640	 41700	 640	 300	Selenium	(Se)	 0.20	 mg/kg	 <0.20	 0.57	 <0.20	 0.49	 <0.20	 <0.20	 0.43	Silver	(Ag)	 0.10	 mg/kg	 <0.10	 1.29	 0.27	 <0.10	 <0.10	 <0.10	 0.49	Sodium	(Na)	 50	 mg/kg	 2440	 4640	 536	 66	 152	 <50	 169	Strontium	(Sr)	 0.50	 mg/kg	 55.1	 86.3	 84.7	 23.4	 13.1	 5.22	 26.1	Sulfur	(S)	 1000	 mg/kg	 <1000	 12600	 1100	 3100	 2900	 <1000	 8800	Thallium	(Tl)	 0.050	 mg/kg	 <0.050	 0.084	 <0.050	 <0.050	 <0.050	 <0.050	 0.054	Tin	(Sn)	 2.0	 mg/kg	 <2.0	 15.3	 13.8	 <2.0	 <2.0	 <2.0	 <2.0	Titanium	(Ti)	 1.0	 mg/kg	 19.4	 44.5	 394	 1.1	 5.7	 2.5	 62.0	83  Tungsten	(W)	 0.50	 mg/kg	 <0.50	 1.07	 1.89	 <0.50	 <0.50	 <0.50	 <0.50	Uranium	(U)	 0.050	 mg/kg	 0.081	 1.36	 0.347	 <0.050	 <0.050	 <0.050	 6.76	Vanadium	(V)	 0.20	 mg/kg	 0.85	 2.39	 24.9	 <0.20	 0.42	 <0.20	 5.29	Zinc	(Zn)	 2.0	 mg/kg	 35.4	 331	 253	 128	 27.2	 8.9	 7490	Zirconium	(Zr)	 1.0	 mg/kg	 <1.0	 1.3	 1.9	 <1.0	 <1.0	 <1.0	 2.7	  Appendix B     Figure 19. Dissolved cadmium over time in metal precipitation batch reactors by carbon source  84   Figure 20. Dissolved cobalt over time in metal precipitation batch reactors by carbon source   Figure 21. Dissolved iron over time in metal precipitation batch reactors by carbon source  85   Figure 22. Dissolved lead over time in metal precipitation batch reactors by carbon source   Figure 23. Dissolved manganese over time in metal precipitation batch reactors by carbon source    86  Appendix C     Figure 24. Dimension for the columns used in the hybrid design. LHS is first-stage column and RHS is second-stage column.   87   Appendix D   D.1 Zinc  Figure 25. Total zinc over time for first and second stage columns in hybrid design D.2 Cadmium  Figure 26. Dissolved cadmium over time for the first and second stage columns in hybrid design 88  D.3 Lead  Figure 27. Total lead over time for first and second stage columns in hybrid design D.4 Manganese  Figure 28. Total manganese over time for first and second stage columns in hybrid design 89  D.5 Cobalt  Figure 29. Total cobalt over time for both first and second stage columns in hybrid design  D.6 Iron  Figure 30. Total iron over time for the first and second stage columns in hybrid design 90   Figure 31. Dissolved iron over time for the first and second stage columns in hybrid design 

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