EPA/600/R-02/053 June 2002 FINAL REPORT-SULFATE-REDUCING BACTERIA REACTIVE WALL DEMONSTRATION MINE WASTE TECHNOLOGY PROGRAM ACTIVITY III, PROJECT 12 IAGNO: DW89938870-01-1 Project Officer Mr. Roger Wilmoth Office of Research and Development National Risk Management Research Laboratory Cincinnati, Ohio 45268 MSB Technology Applications, Inc. Mike Mansfield Advanced Technology Center 200 Technology Way P.O. Box 4078 Butte, Montana 59702 ------- Disclaimer This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. ------- Foreword The U.S. Environmental Protection Agency is charged by Congress with protecting the Nation's land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to formulate and implement actions leading to a compatible balance between human activities and the ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is providing data and technical support for solving environmental problems today and building a science knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect our health, and prevent or reduce environmental risks in the future. The National Risk Management Research Laboratory is the Agency's center for investigation of technological and management approaches for preventing and reducing risks from pollution that threatens human health and the environment. The focus of the Laboratory's research program is on methods and their cost effectiveness for prevention and control of pollution to air, land, water, and subsurface resources; protection of water quality in public water systems; remediation of contaminated sites, sediments, and ground water; prevention and control of indoor air pollution; and restoration of ecosystems. The NRMRL collaborates with both public and private-sector partners to foster technologies that reduce the cost of compliance and to anticipate emerging problems. NRMRL's research provides solutions to environmental problems by developing and promoting technologies that protect and improve the environment; advancing scientific and engineering information to support regulatory and policy decisions; and providing the technical support and information transfer to ensure implementation of environmental regulations and strategies at the national, state, and community levels. This publication has been produced as part of the Laboratory's strategic long-term research plan. It is published and made available by EPA's Office of Research and Development to assist the user community and to link researchers with their clients. E. Timothy Oppelt, Director National Risk Management Research Laboratory ------- Executive Summary This document is a final report on the performance of sulfate-reducing bacteria (SRB) bioreactors that were constructed and operated for Mine Waste Technology Program (MWTP) Activity III, Project 12, Sulfate-Reducing Bacteria Reactive Wall Demonstration. The MWTP is funded by the U.S. Environmental Protection Agency (EPA) and jointly administered by the EPA and the U.S. Department of Energy (DOE) through an Interagency Agreement (IAG) and under DOE contract number DE-AC22- 96EW96405. Efforts reported in this document focused on the demonstration of a passive technology that could be used for remediation of thousands of abandoned mine sites existing in the Western United States that emanate acid mine drainage (AMD). This passive remedial technology takes advantage of the ability of SRB to increase pH and alkalinity of the water and to immobilize dissolved metals by precipitating them as metal sulfides or hydroxides. The SRB technology was demonstrated by constructing three bioreactors at an abandoned mine site (Calliope Mine) in the vicinity of Butte, Montana. The bioreactors were fed by AMD emanating from a large waste rock pile. The quality of this AMD and its pH are related to the amount of atmospheric water that infiltrates into the waste rock pile and leaches metals. With the exception of the first 8 months of operation, atmospheric precipitation was well below normal. Consequently, the pH of the AMD increased, and the load of metals in the AMD significantly decreased, bringing concentrations of iron, aluminum, and manganese in the influent AMD below the target treatment levels for the project. The bioreactors operated from December 1998 to July 2001 when they were then decommissioned. Two bioreactors were placed below ground (Bioreactors II and III), and one was placed above ground (Bioreactor IV). The aboveground bioreactor was built to evaluate the effect of cold weather and freezing on an SRB system. In addition, Bioreactors II and IV were built with a pretreatment section to evaluate the effect on the efficiency of the SRB of inducing an improved pH and oxidation-reduction potential (EH). Each bioreactor was filled with a combination of organic matter, crushed limestone, and cobbles placed in two or four discrete chambers. The first two chambers of Bioreactors II and IV constituted the pretreatment section and included a chamber filled with organic matter and a chamber filled with crushed limestone. Following the pretreatment section, there was another chamber with organic matter and a chamber filled with cobbles. A pretreatment section was not included in Bioreactor III in order to evaluate its contribution to overall bioreactor efficiency by comparison to Bioreactor II. Bioreactors II and III, 71.5 feet and 61 feet in length respectively, were constructed below ground in 14- foot-wide trapezoidal (4-foot-wide bottom) trenches. Bioreactor IV, 72.5 feet in length, was constructed in a 12-foot-wide metal half-culvert elevated above ground. The chambers filled with organic matter or limestone were each 5 feet in length, whereas the chambers filled with cobbles were 50 feet in length. The organic matter, an electron donor and carbon source for the SRB, was provided as an 80% to 20% by volume mixture of cow manure and cut straw. The cut straw was added to provide secondary porosity to the mix and to prevent settling of the medium. TerraCell™ material, commonly used in landscaping for slope stabilization and made of high density polyethylene, was used to form a cellular containment system in ------- (CCS)1 to house the organic matter. The CCS prevented the organic matter from settling to the bottom of the bioreactor, thus fostering the flow of AMD through the entire cross-sectional area without channeling. Each layer (lift) of TerraCell™ was positioned at 60 degrees off the horizontal plane so that the cells of each lift would be partially offset with respect to the cells of adjacent lifts. Each lift was 6 inches thick (as measured along the horizontal direction of flow) and contained 11-inch by 8.5-inch rhombohedral-shaped cells. The two belowground bioreactors (II and III) were designed to flow year-round. The aboveground bioreactor (IV) was designed to be shut down for winter to let it freeze while full of AMD. The reactors flowed at a rate of 1 gallon per minute for the majority of time. This flow rate corresponded to a calculated 5-1/2 day residence time for the AMD in Bioreactors II and IV and a 4-1/2 day residence time in Bioreactor III. The residence time of the AMD in a single organic matter chamber was approximately 10 hours. Bioreactor performance was monitored monthly by taking pH, EH, dissolved oxygen, and temperature measurements and collecting samples of influent and effluent for chemical analysis. The analytes included SRB population; alkalinity; and concentrations of sulfate, sulfide, dissolved metals, aluminum, arsenic, cadmium, copper, iron, manganese, and zinc. At the end of the project, the bioreactors were decommissioned, and the site was restored to nearly original conditions. The decommissioning activity also included an autopsy of the solid matrix material that was not accessible during the operational time. Autopsy sampling included collection of solid matrix samples for chemical analyses to determine concentrations of total metals [aluminum (Al), arsenic (As), cadmium (Cd), calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn], sulfate, sulfide, nitrogen, phosphorous, and total organic carbon (TOC) in the chambers of organic matter and limestone. Bacteriological analyses were also conducted to determine SRB population in the organic substrate and in the limestone. Because the cobbles did not have a visually discernible film of bacteria or chemical precipitate, no solid matrix samples were collected from these chambers. Aqueous samples were also collected from the previously inaccessible bottom of the crushed limestone and cobble chambers and analyzed for total and dissolved metals. The autopsy on the bioreactors revealed a convoluted biochemical environment that was probably caused by the dramatic change in the AMD chemistry after the first 10 months of operation. The material examined during the autopsy showed the mixed results of processes that were occurring at low pH and a reasonably high load of metals with the subsequent reactions that were characteristic for water of neutral pH laden with much less of the dissolved metals. Interpretation of monthly monitoring results combined with the autopsy findings allowed for the formulation of a number of conclusions and recommendations, the most essential of which are listed below. 'U.S. Patent No. 6,325,923 IV ------- The CCS worked very well in preventing settling of the organic matter and ensuring uniform flow of AMD throughout the entire cross section of the organic carbon with no preferential flow paths (channeling). Configuring the bioreactors to accommodate flow in a horizontal plane (rather than in the vertical direction) was successful. Problems that were experienced with reductions in flow rate turned out to be associated with the AMD distribution system that was plugged by chemical precipitates. This hindrance, however, is common to both configurations. It takes some time for SRB population to be established in the bioreactors. Once established and supplied with organic matter, they maintained a population of E+4 most probable number (MPN)/milliliter or higher in the aqueous phase at temperatures ranging from 2 • C to 16-C. The SRB average population of 2.06E+6 MPN/cubic centimeter in the solid matrix of organic matter was two orders of magnitude greater than the SRB population present in the aqueous phase. The AMD in the bioreactors was notably stratified with respect to oxidation-reduction potential that was up to 400 millivolts lower at the bottom of the bioreactors than at the top. Because maintaining reduced conditions is required for SRB, the bioreactors should have been more carefully isolated from atmospheric air. A plastic liner placed on the top of bioreactors is preferred over the straw bails used for this project. Only Zn, Cu, and Cd were being removed as sulfides due to SRB activities. Changes in concentrations of other metals (Fe, Mn, Al, As), which do not necessarily precipitate as sulfide, seemed to be affected by SRB only in an indirect manner by responding to increased pH caused by SRB activity. For the Calliope site climatic and hydrochemical conditions, the thresholds for the removal of Zn, Cd, and Cu were approximately 500 micrograms per liter ((ig/L), 5 (ig/L, and 80 (ig/L, respectively. These thresholds were slightly lower for Bioreactors II and IV than for Bioreactor III, which did not include a pretreatment cell. This indicates that the removal thresholds were dependent on the configuration of the bioreactor but were not affected by the shutdown and freezing of a bioreactor during winter. Most of the metal sulfides that were formed due to the SRB activity precipitated within the organic matter. The same seems to be true for the rest of the metals that must have formed hydroxides and carbonate compounds. The role of the cobble chamber was limited to a collection sump for a small mass of precipitates that escaped the organic matter chambers. This demonstrated that there was no need for the large cobble chamber, which could have been substituted with a smaller "trap" sump. ------- • The abundance of TOC present (20% by weight) in the organic matter chamber at the end of the project demonstrated that the bioreactors would have worked equally efficiently with a much smaller supply of organic carbon, provided the same residence time of AMD was maintained. Since the organic matter mass inhibits permeability, it is prudent to reduce the ratio of organic carbon to the permeability enhancing component (e.g., gravel, shell, etc.) and have more permeable medium. Since most of the material that caused plugging was found within and adjacent to the outlets of the AMD distribution system, there was a need to devise a system that would allow for occasional breakdown and removal of that material. Such a system might involve only a few outlets rather than the three dozen used in this design. It may include ports extended to the ground surface that would facilitate blowing in combustion engine exhaust to destroy plugging material that would then be removed by bailing. Overall, the project documented that SRB technology, as applied in this demonstration, is effective in removing Zn, Cu, and Cd by precipitating them as sulfides. Removal mechanisms for Fe, Al, Mn, and As were overshadowed by a dramatic change of the quality of the influent AMD. The results of the project have also allowed the formulation of an important recommendation regarding the design and construction of SRB bioreactors. VI ------- Contents Page Executive Summary iii Tables ix Figures x Acronyms and Abbreviations xi Acknowledgment xii 1. INTRODUCTION 1 1.1 Problem Definition 1 1.2 Principles of the Sulfate-Reducing Bacteria Technology and its Application 1 2. SITE DESCRIPTION 2 2.1 Location 2 2.2 Acid Mine Drainage Source 2 2.3 Bioreactor Layout and Configuration 2 3. DESIGN AND CONSTRUCTION 6 3.1 Design Requirements 6 3.2 Construction 6 3.2.1 Materials Used 6 3.2.2 Belowground Bioreactors 7 3.2.3 Aboveground Bioreactor 7 3.2.4 Sampling Locations and Ports 8 4. OPERATION 11 4.1 Flow Rates 11 4.2 Acid Mine Drainage Levels 11 4.3 Sampling and Performance Monitoring 11 4.4 Decommissioning 12 4.4.1 Sampling 12 5. OPERATION PHASE RESULTS 19 5.1 Field Parameters 19 5.1.1 pH, EH, Dissolved Oxygen, and Temperature 19 5.1.2 pH, EH, and Temperature Profiles 20 5.2 Sulfate-Reducing Bacteria Populations 21 5.3 Sulfate and Sulfide 21 5.4 Alkalinity 21 5.5 Metals 22 6. AUTOPSY RESULTS 41 6.1 Aqueous Samples 41 6.1.1 Cobble Chambers 41 vii ------- Contents (Cont'd) Page 6.1.2 Limestone Chambers 42 6.1.3 Sulfate and Sulfide Analyses 42 6.2 Bioreactor Solid Matrix Samples 42 6.2.1 Metals Concentrations in the Bioreactors 42 6.2.2 Sulfate and Sulfide Analyses 44 6.2.3 Total Organic Carbon 45 6.2.4 Plugs in the Outlets of the AMD Distribution System Manifold 45 6.3 Flow Pattern 45 6.4 Sulfate-Reducing Bacteria Population on the Solid Matrix 46 6.5 Toxicity Characteristic Leaching Procedure 46 7. QUALITY ASSURANCE/QUALITY CONTROL 58 7.1 Background 58 7.2 Project Reviews 58 7.2.1 Internal Field Systems Review at the Demonstration Site 58 7.2.2 External Technical Systems Audit 59 7.3 Data Evaluation 59 7.4 Validation Procedures 59 7.4.1 Analytical Evaluation 59 7.5 Program Evaluation 60 7.5.1 Field QC Samples 60 7.5.2 Field Blanks 60 7.5.3 Field Duplicates 60 7.6 Summary 60 8. RECOMMENDED DESIGN IMPROVEMENTS 65 9. CONCLUSIONS 66 10. REFERENCES 69 Vlll ------- Tables 2-1. Acid Mine Drainage Analytical Data and Target Concentrations for Bioreactors 3 4-la. Flow and Water Level Measurements 13 4-lb. Water Level Profile in Bioreactor II 15 4-2. Monitored Parameters and Analytes 15 4-3. Analysis for the Autopsy of the Bioreactors 16 5-1. Field Parameters 23 5-2. EH and pH Profiles for Bioreactors II and III 26 5-3. SRB Populations 27 5-4. Sulfate and Sulfide Concentrations 28 5-5. Alkalinity 29 5-6. Metals Concentrations 30 6-1. Analytical Results of Aqueous Samples Collected During the Autopsy 47 6-la. Total and Dissolved Metals 47 6-lb. Iron Speciation 47 6-lc. Sulfate and Sulfide 47 6-2. Comparison of the Dissolved Metals from the Autopsy and Monthly Sampling 48 6-3. Analytical Results of the Solid Matrix Samples Collected During the Autopsy 48 6-3a. Metal Concentrations 48 6-3b. Nitrogen, Phosphorus, and TOC 49 6-3c. Sulfide Acid-Base Accounting 49 6-3d. Analytical Results for Plugging Material in the Manifold of Bioreactor III 50 6-4. Correlation Coefficients (•) for Total Metals Concentrations in the Solid Matrix of Bioreactors II, III, and IV 50 7-1. Data Quality Indicator Objectives 61 7-2. Summary of QC Checks for Critical Field pH Measurements and Dissolved Metals Analysis .. 61 7-3 Summary of Qualified Data for MWTP Activity III, Project 12 62 IX ------- Figures Page 2-1. Calliope site location map 4 2-2. Layout of the bioreactors 5 3-1. Simplified cross-sections of Bioreactors II and IV 8 3-2. Cellular containment system for organic substrate 9 3-3. AMD distribution system manifold 9 3-4. End enclosure and intake sump for Bioreactors II and IV 10 3-5. Sampling and monitoring point locations 10 4-1. Bioreactor flow 17 4-2. Water level in the bioreactors 17 4-3. Water level profile in Bioreactor II during early operation and the plugging episode of May 1999 18 5-1. pHtrends 32 5-2. EH trends 33 5-3. Dissolved oxygen trends 33 5-4. Temperature trends 34 5-5. Bioreactor II pH and EH profiles for 12/6/99 34 5-6. Bioreactor III pH and EH profiles for 12/6/99 35 5-7. SRB populations 35 5-8. Sulfate concentrations 36 5-9. Soluble sulfide concentrations 36 5-10. Total alkalinity 37 5-11. Zinc concentrations 37 5-12. Copper concentrations 38 5-13. Cadmium concentrations 38 5-14. Aluminum concentrations 39 5-15. Arsenic concentrations 39 5-16. Iron concentrations 40 5-17. Manganese concentrations 40 6-la. Total vs. dissolved metals for the cobble chamber of Bioreactor II 51 6-lb. Suspended metals in the cobble chamber of Bioreactor II 51 6-2a. Total vs. dissolved metals for the cobble chamber of Bioreactor III 52 6-2b. Suspended metals in the cobble chamber of Bioreactor III 52 6-3a. Total vs. dissolved metals for the cobble chamber of Bioreactor IV 53 6-3b. Suspended metals in the cobble chamber of Bioreactor IV 53 6-4. Iron speciation for autopsy aqueous samples 54 6-5. Total metals in the limestone and cobble chambers in Bioreactor II (aqueous samples) 54 6-6. Total metals concentration in the solid matrix of Bioreactor II 55 6-7. Total metals concentration in the solid matrix of chamber 1 in Bioreactor III 55 6-8. Total metals concentration in the solid matrix of Bioreactor IV 56 6-9. Sulfate profiles in the solid matrix of the bioreactors 56 6-10. Sulfide profiles in the solid matrix of the bioreactors 57 6-11. Bioreactor III inlet-plug metals concentrations 57 ------- Acronyms and Abbreviations ABA Al AMD As Ca CaCO3 CCS Cd CO Cu DM DO DOE EH EPA Fe Fe2+ Fe3+ Fe(OH)3 FeS2 H2S HCO3 HOPE IAG ICP Mg mil Mn MCL MPN MSE-TA MWTP Orl Or2 ORP pH PVC QA QAPP QC SHE SMCL SRB TCLP TOC TM VFA acid-base accounting (analytical method for sulfide) aluminum acid mine drainage arsenic calcium (also used as the designation for limestone chamber in figures) calcium carbonate cellular containment system cadmium cobble chamber (used only in tables or figures) copper dissolved metals dissolved oxygen U.S. Department of Energy oxidation-reduction potential expressed with reference to standard hydrogen electrode U.S. Environmental Protection Agency iron ferrous iron ferric iron ferrihydrite iron disulfide (pyrite) hydrogen sulfide bicarbonate high density polyethylene Interagency Agreement inductively coupled argon plasma magnesium 1/1,000 of an inch manganese maximum contaminant level most probable number MSE Technology Applications, Inc. Mine Waste Technology Program organic matter chamber No. 1 organic matter chamber No. 2 oxidation-reduction potential measured using silver/silver chloride reference electrode measure of hydrogen ion activity polyvinyl chloride quality assurance quality assurance project plan quality control standard hydrogen electrode suggested maximum contaminant level sulfate-reducing bacteria Toxicity Characteristic Leaching Procedure total organic carbon total metals volatile fatty acids XI ------- Zn zinc xn ------- Acknowledgment Work was conducted through the DOE National Energy Technology Laboratory at the Western Environmental Technology Office under DOE Contract Number DE-AC22-96EW96405. Xlll ------- 1. Introduction This document is a final report on the performance of sulfate-reducing bacteria (SRB) bioreactors that were constructed and operated for Mine Waste Technology Program (MWTP) Activity III, Project 12, Sulfate-Reducing Bacteria Reactive Wall Demonstration. The MWTP is funded by the U.S. Environmental Protection Agency (EPA) and jointly administered by the EPA and the U.S. Department of Energy (DOE) through Interagency Agreement (IAG) Number DW89938870-01-1 and under DOE contract number DE-AC22- 96EW96405. 1.1 Problem Definition Acid mine drainage (AMD) emanates from many abandoned mines in the Western United States, causing significant environmental problems by contaminating surface waters and groundwater with dissolved metals and raising their acidity. Conventional active treatment of AMD is often not feasible due to the remoteness of the site, the lack of power, and limited site accessibility. Thus, for such sites, there is a need for a passive remedial technology to immobilize metals and increase the pH of the AMD. Sulfate-reducing bacteria have the ability to increase pH and alkalinity of the water and to immobilize dissolved metals by precipitating them as metal sulfides. Some metals [e.g., aluminum (Al)] are removed as hydroxides due to the increase inpH. 1.2 Principles of the Sulfate-Reducing Bacteria Technology and its Application Acid mine drainage is a typical result of mining sulfide-rich ore bodies. Acid mine water is formed when sulfide-bearing minerals, particularly pyrite [iron disulfide (FeS2)], are exposed to oxygen and water as described by the following overall reaction (1-1). FeS2 + 15/4 O2 + 7/2 H2O — > Fe(OH)3 + 2SO42- + 4H+ (1-1) This reaction results in increased acidity of the water (lowered pH), increased metal mobility, and the formation of dissolved sulfate. When provided with an organic carbon source, SRB are capable of reducing the sulfate to soluble sulfide by using sulfate as a terminal electron acceptor; bicarbonate ions are also produced. The soluble sulfide reacts with some metals in the AMD to form insoluble metal sulfides (Reactions 1-2 and 1-3). The bicarbonate ions increase the pH and alkalinity of the water. SO42- + 2CH2O > H2S + 2HCCV (1-2) H2S + M2+ > MS + 2H+, where M = metal (1 -3) The SRB technology was demonstrated in the field by engineering the SRB favorable conditions within three bioreactors that were fed by AMD emanating from an abandoned mine site (Calliope Mine) in the vicinity of Butte, Montana. The bioreactors, on which construction was completed in November 1998, operated from December 1998 to July 2001 (32 months). Performance of the bioreactors was monitored monthly by sampling and conducting chemical and bacteriological analyses of the influent and effluent of the bioreactors. The bioreactors were decommissioned in July 2001, and samples of solid matrix from the bioreactors were collected and analyzed for chemical, physical, and bacteriological parameters. This report includes a description of the site, reactor design and construction, details of the 32 months of monitoring data, data acquired during the decommissioning of the bioreactor, and data interpretation. ------- 2. Site Description 2.1 Location This project was conducted at the Calliope mine (Calliope/Mary Emma Mining Claims) located (Figure 2-1) in Silver Bow County, Montana, in NW% of SE% and SW% of NE% both of SW% of Section 10, T3N, R7W. The majority of the site where the bioreactors were installed is located on the Calliope mining claim, Mineral Survey No. 2972. The Mary Emma mining claim, Mineral Survey No. 5478, which is adjacent to the north of the Calliope, was also used for some installations. 2.2 Acid Mine Drainage Source The abandoned Calliope mine site includes a collapsed adit discharging water into a large waste rock pile. The exposed volume of the waste rock pile is estimated to be 66,000 cubic yards; however, the approximately 50-foot-tall pile that is visible at the present time may not constitute the entire volume of mine waste disposed at the mine. The bottom part of the pile has probably been covered with fill material that was placed there during the construction of Interstate Highway 15 (1-15) and forms a distinct morphological shelf extending westward from the lower pond and the toe of the present waste rock pile. The AMD discharging from the collapsed adit is of relatively good quality with the pH ranging from 6.5 to 7. This AMD flows over the top of the waste rock and accumulates in a small, approximately 50-foot-diameter flow-through pond (Upper Pond). Overflowing the Upper Pond, the AMD forms a surface drainage that, flowing on the surface of the waste rock pile, reaches another flow-through pond (the Lower Pond, which is 35 feet in diameter). In the Lower Pond, the AMD mixes with low pH subsurface seepage that enters the pond along its banks and through its bottom. This subsurface seepage is fed by atmospheric precipitation that infiltrates the waste rock pile and reappears on the surface at the toe of the pile. This seep is enriched in metals with a pH ranging from 2.6 to 3.6. Under natural conditions, the Lower Pond overflows and drains to an adjacent gulch. For this project, approximately 20% of the AMD that flows through the Lower Pond was diverted for treatment in three engineered bioreactors that were built at the site to demonstrate the SRB technology. The quality of the Lower Pond water and its pH are related to the amount of atmospheric precipitation that infiltrates into the waste rock pile and leaches metals. With the exception of the first 8 months of bioreactor operation, atmospheric precipitation was well below normal. Consequently, the quality of the Lower Pond water significantly improved after the first 10 months. Table 2-1 includes analytical information on the water quality of the influent to the bioreactor system. 2.3 Bioreactor Layout and Configuration All three bioreactors (denoted II, III, and IV) were designed and constructed in parallel downstream from the Lower Pond (Ref 1) (Figure 2-2). This allowed the AMD to be piped to and treated by the bioreactors using gravity flow. The bioreactors, constructed in the fall of 1998, were designed to evaluate the SRB technology applied in slightly different environmental conditions. Bioreactors II and III were placed below ground, and Bioreactor IV was placed above ground. The belowground bioreactors were built to minimize temperature changes and to prevent freezing. The aboveground bioreactor was built to evaluate the effect of cold weather and freezing on the system. In addition, Bioreactors II and IV were built with a pretreatment section to evaluate the effect on the efficiency of the SRB to improve pH and oxidation-reduction potential (EH). Due to budget constraints, Bioreactor I was not constructed. Each bioreactor was filled with a combination of organic matter, crushed limestone, and cobbles placed in two or four discrete chambers (Figure 2-2). The first two chambers of ------- Bioreactors II and IV constituted the pretreatment section and included a chamber filled with organic matter and a chamber filled with crushed limestone. Following the pretreatment section was a primary treatment section that included a chamber with organic matter and a chamber filled with cobbles. A pretreatment section was not included in Bioreactor III in order to evaluate its contribution to overall bioreactor efficiency by comparison with Bioreactor II. Each media component was expected to play an important role in the treatment train: - organic matter was the nutrient (the electron donor) for the SRB; - for the pretreatment section, a chamber with organic matter was included to lower the EH of AMD; - crushed limestone provided buffering capacity to increase the alkalinity of AMD in the pretreatment section; and - cobbles placed in the last chamber of each bioreactor were to provide a stable surface for bacterial attachment. Bioreactors II and III, 71.5 feet and 61 feet in length respectively, were constructed below ground in 14-foot-wide trapezoidal (4-foot-wide bottom) trenches. Bioreactor IV, 72.5 feet in length, was constructed in a 12-foot-wide metal half-culvert elevated above ground. The chambers filled with organic matter or limestone were each 5 feet in length, whereas the chambers filled with cobbles were 50 feet in length. Table 2-1. Acid Mine Drainage Analytical Data and Target Concentrations for Bioreactors Analyte Concentration (u,g/L) AMD (maximum) AMD (minimum) Target Effluent *Suggested maximum contaminant level (SMCL) ** Maximum contaminant level (MCL) * ** Secondary maximum contaminant level NA = not applicable Comments Aluminum Cadmium Copper Iron Manganese Zinc Arsenic Sulfate pH 14,100 41.9 3,050 8,670 3,770 11,100 10.9 229,000 7.52 11.0 3.1 2.8 8.0 690 990 1.1 69,800 3.29 1,000 5 100 1,000 2,000 4,000 NA NA 6 to 8 50 to 200 ng/L* 5 ng/L** 1,300 ng/L** 300 |Ig/L*** 50 |Ig/L*** 5,000 |Ig/L*** 50 ng/L** 250,000 |Ig/L*** ------- Figure 2-1. Calliope site location map. ------- Organic Matter Crushed Limeston Organic Matter Cobbles Pretreatment Section Lower Pond, Source of AMD -Treated AMD Discharge Piping Above Ground Bioreactor IV -Below Ground Bioreactors II & III Figure 2-2. Layout of the bio reactors. ------- 3. Design and Construction 3.1 Design Requirements Several functional and operational constraints were identified before the design of the bioreactors began. The most important constraints are listed below with their design solutions. • The entire system needed to be passive. This condition was satisfied allowing for gravity flow by incorporating the site topography and flow control instrumentation into the design. • Construction of the bioreactors had to allow for investigation of the impact of subfreezing temperature on SRB activity. This requirement was satisfied by designing an aboveground bioreactor with features similar to one of the belowground bioreactors. • Construction of the bioreactors had to allow for control of the water level to simulate seasonal droughts, if deemed appropriate. This requirement was achieved by constructing intake sumps where the hydraulic head could be controlled through a system of valves and overflow piping. • The chambers with organic matter had to be designed so they fostered permeation of the AMD through the entire cross-sectional area (without channeling) and prevented settling of the medium. A cellular containment system1 (CCS) was built into the organic carbon chambers to satisfy these requirements. 3.2 Construction Construction activities at the Calliope began in August 1998 and were completed in October 1998. Excavation and grading for construction of all bioreactors and associated piping was completed while maintaining the existing slope grade of 2.5%. This grade was maintained to provide for natural runoff in the SRB construction area and, more importantly, to avoid the influx of surface runoff 'U.S. Patent No. 6,325,923 into the belowground bioreactors. Figure 3-1 is a simplified longitudinal cross section through the bioreactors. Bioreactor III differs from the other two bioreactors by having no pretreatment section (i.e., it consists only of one organic matter chamber and a chamber with cobbles). 3.2.1 Materials Used All bioreactors were constructed with similar materials. Whenever possible, off-the-shelf, acid- resistant building materials were used. These materials and their use are described below. • Schedule 40 polyvinyl chloride (PVC) piping, nontreated finished lumber, 40-mil woven geotextile, and 40-mil PVC liner. The PVC liner, sandwiched between woven geotextile, was used for lining the bottom and the sides of each bioreactor. • A heavy gauge, multiple section, galvanized steel half culvert with fabricated steel end-walls was assembled to form an elevated trough to house the aboveground bioreactor. • Precast reinforced concrete was used for the inlet and outlet walls for the belowground bioreactors. Precast reinforced concrete was also used for the intake sumps, and the end enclosures required for construction of the belowground bioreactors. • TerraCell™ material, commonly used in landscaping for slope stabilization, made of high density polyethylene (HDPE), was used to form a CCS to house and support the organic matter. Each layer (lift) of TerraCell™ was 6 inches high and contained 11-inch by 8.5-inch rhombohedral-shaped cells. • The organic matter was provided as an 80% to 20% by volume mix of cow manure and cut straw. The cut straw was added to provide "secondary" porosity to the mix and prevent settling of the medium. ------- • The mixture of cow manure and straw was installed in the CCS, which consisted of 10 lifts of TerraCell™ material (Figure 3-2) and would limit settling (if it occurred) of the organic matter to each individual cell. The TerraCell™ lifts were positioned at 60 degrees off the horizontal plane so the cells of each lift would be partially offset (only partially overlapping) with respect to the cells of adjacent lifts. Such a configuration promoted migration of AMD along the organic matter chamber in a wavy- shaped flow line and facilitated the packing of each individual cell with the organic matter. The TerraCell™ material (lifts) was individually fastened to a grid of 2-inch by 4-inch lumber positioned at the top of the organic matter chamber. The grid was supported by 6-inch by 6-inch wood beams positioned across the reactor above its top. • The limestone chambers of Bioreactors II and IV contained crushed limestone that was 3 to 5 inches in size. This crushed limestone was placed directly on the last CCS lift of the first organic matter chamber. The front face of the limestone chamber also sloped 60 degrees off the horizontal plane to provide a support surface for the second organic matter chamber. • Cobbles (mostly granodiorite), which were 3 to 5 inches in diameter, filled the remaining portion of the bioreactors. • Two lifts of straw bales, sized 16 inches by 18 inches by 48 inches and placed on top of the belowground bioreactors, were used to create a 32-inch-thick layer to provide thermal insulation. Only one lift of straw bales was used for the aboveground bioreactor. 3.2.2 Belowground Bioreactors The belowground bioreactors, II and III, were constructed in lined trapezoidal cross-section trenches. The liner system, which was the same for the below- and aboveground bioreactors, consisted of a 40-mil PVC liner sandwiched between two layers of a 40-mil woven geotextile. The latter was used to provide additional abrasion resistance for the PVC liner for all subsequent construction activities. Steel-reinforced cement end-walls, with the appropriate piping penetrations, were precast at the site and installed on top and behind the liner system for the AMD inlet and outlet, respectively. Unique to the construction of the belowground reactors was the embedding of a 2-inch inlet manifold for the AMD distribution system (Figure 3-3) into the 60-degree precast inlet end-wall. This distribution system allows the AMD to enter the bioreactors and flow evenly throughout the CCS. A precast reinforced concrete intake sump to control water levels within the bioreactor was installed directly upgradient of each bioreactor (Figure 3-4). Water levels within the bioreactor were controlled via the intake sump. A hose, connected to an overflow drain line, was installed in all intake sumps to control (raising or lowering) the water level in each bioreactor. Water was piped from these intake sumps to the bioreactor manifold distribution systems. Precast reinforced concrete end enclosures (i.e., large manholes) were installed at the end of the belowground bioreactors to control flow rate and to house equipment to monitor bioreactor performance. Effluent from the bioreactors was piped from the end enclosures and applied to the land surface in the gulch (see Figure 3-5). 3.2.3 Aboveground Bioreactor The aboveground bioreactor (IV) was constructed using multiple sections of a heavy gauge, galvanized steel culvert with fabricated steel end- walls. Individual sections were joined together using carriage bolts. The aboveground bioreactor was lined in a manner similar to the belowground bioreactors. Embedding the manifold in this reactor was not possible because the end-wall was fabricated from steel; therefore, the inlet manifold was placed on top of the liner system and supported with wooden blocking material placed on the 60-degree end-wall under the liner system. An end enclosure was not ------- required for this reactor as it was constructed above ground. Other features including flow control and monitoring equipment and the intake sump were like those used for the belowground bioreactors. 3.2.4 Sampling Locations and Ports The bioreactors were equipped with a number of sampling ports to monitor performance of each bioreactor. The ports were either installed at the selected locations of the piping system that was assembled to supply the bioreactors with AMD and to discharge the treated effluent (see Figure 3-5) or were constituted by piezometers that were installed within the body of the bioreactors (Figure 3-1). Figure 3-1. Simplified cross-sections of Bioreactors II and IV. ------- BOTTOM OF BIOREACTORS S DE VEW Figure 3-2. Cellular containment system for organic substrate. 2" ELBOW OUTLET ^CEMENT END Figure 3-3. AMD distribution system manifold. ------- 2" DISCHARGE PIPE GLOBE VALVE^ pH PROBE- 2" HOSE - -4" OVERFLOW PVC PIPE FOR ALL TREAMENT TRENCHES. Figure 3-4. End enclosure and intake sump for Bioreactors H and IV. Sampling and Monitoring Points Locations Discharge from adit Monitored Parameters Upper Pond 1 / CQ3 ^^ / Waste Rock Pile ^•f ~y, -^V^ Water percolatin //\^f~~~"\ \ through Waste Rock / 1 ~-i>«- — N — " NX ^^-- Lower Pond 1 \>Y^^ \ / i \ Th V- L }^ r / — MK/ Surface drainage ^^^ from Upper Pond Valve box ^^ to Lower Pond J ^^ I Overflow from Lower Pond / to the gulch / / AMD ' supply line to *- each reactor Overflow from the supply system \ ) to the gulch ^4^-~^ Int Fh B - Bacteria count g" F- Flow rate 1 Selected metals L' ™aterlevel g A- < Sulfate P_ ^ermperature of ^^ Pile Sulfide t_ Temperature at straw- [CSSTft-yAdd.^, „. --".^ure IVa- Sampling point ID ; supply lines ie reactors Limestone Aboveground bioreactor (IV) |V 1 IVb IVc / IVd IVe ^-^ Intake Organ sump carbo — CA,T,F,L,P") Ilia Below-groun Below-groun "^^4 / / ^ B t A,L,T 4 F,P I t n Cobbles End enclosure \ \ i H B t A,L,T F,P lib He 4 Hid He d bioreactor w/o pre-treatment section (III) d bioreactor with pre-treatment section (II) lib lie \ lid lie •v B t / A,L,T •-, * 7'p — \ / T \ 1 End enclosure Housing for ambient temperature measurement /y Aa\ Discharge line & Land application of treated AMD J( Figure 3-5. Sampling and monitoring point locations. 10 ------- 4. Operation 4.1 Flow Rates The bioreactors operated from December 1998 to July 2001 (32 months). The two belowground bioreactors (II and III) were designed to flow year- round. The aboveground bioreactor (IV) was designed to be shut down for winter to let it freeze while full of AMD. The reactors flowed at a rate of 1 gallon per minute (gpm) for the majority of time (Figure 4-1 and Table 4-la). For 4 months in the summer of 2000, the flow rate was doubled to nearly 2 gpm. Although the flow for Bioreactor IV was shut down for the winter, a center portion of this bioreactor did not freeze due to a small (0.05- gpm) leak through the liner that must have been inadvertently perforated during construction. Although the location of the leak was not defined, the rate of the leak was determined by measuring the water level changes with the valve on the influent closed. A flow rate of 1 gpm corresponds to a 5-1/2 day calculated residence time for the AMD in Bioreactors II and IV and a 4-1/2 day residence time in Bioreactor III. The residence time of the AMD in a single organic matter chamber was approximately 10 hours for the flow rate of 1 gpm. Flow through Bioreactors III and IV was maintained as desired for most of the demonstration. However, the flow rate through Bioreactor II started to decrease in May 1999 and ceased at the beginning of June 1999. The flow rate was restored in July 1999 after the upgradient cell with organic matter was chemically treated and blown out with air using an air compressor to remove biofouling and associated plugging. Similar behavior in Bioreactor II was observed again in May 2000. In this case, the permeability of the upgradient chamber was restored by sparging it with combustion engine exhaust. Cessation of flow in Bioreactor II (indicated in Figure 4-1) in March 2001 was actually caused by sediment that accumulated within the inlet valve. The flow was restored by cleaning the valve. 4.2 Acid Mine Drainage Levels Acid mine drainage levels (or water levels, as it is also referred to in this document) in the bioreactors were controlled by setting their levels in the intake sumps. In general, water level was maintained to just below the top surface of the 5-foot-thick layer of cobbles. A diagram of water level changes in the bioreactors is presented in Figure 4-2, where water level elevations are plotted with reference to the bottom of each bioreactor at its outlet. For Bioreactor II, which experienced two plugging episodes, the water level dropped close to the outlet level, as shown in Figure 4-3, for the episode on June 1999. In each case, most of the water level drop took place between the inlet sump and the first organic matter chamber, indicating plugging within or immediately adjacent to the AMD distribution system. Numerical values of flow and water level measurement for the diagrams in Figures 4-2 and 4-3 are compiled in Table 4-la and 4-lb, respectively. 4.3 Sampling and Performance Monitoring Performance of the bioreactors was monitored monthly by taking measurements manually and collecting samples of influent and effluent for chemical analysis. All aqueous samples were collected and then analyzed by the HKM Analytical Laboratory following the quality assurance project plan (QAPP) (Ref 2). In general, samples were submitted to the laboratory as raw water with the exception of samples for dissolved metals. All samples were preserved as required by the QAPP. In addition, an attempt was made to monitor the pH, temperature, flow rate, and water level of the influent and effluent using stationary transducers or sensors and recording the measurements using data loggers. The receding interval was set for OO O 30 minutes during the first 8 months of operation and for every 4 hours thereafter. However, the reliability of the transducer-generated measurements was unacceptable due to either 11 ------- deterioration of the signal because of organic and/ or chemical coating or repetitive failures of the data loggers. Therefore, the performance reported in this document is based on the records derived through monthly sampling events. The list of measured or analyzed parameters is include in Table 4-2, which also includes references to the sampling locations and monitoring ports shown in Figure 3-5. 4.4 Decommissioning The site was decommissioned beginning in July 2001 in accordance with regulatory guidelines and requirements imposed by the Montana Department of Environmental Quality. The majority of components of the system were either removed from the site or abandoned in place (if they were located in the subsurface), and the site was restored to its predemonstration condition. The only infrastructure remaining at the site are two subsurface inlet sumps and the subsurface piping system to feed the inlet sumps with AMD from the Lower Pond. This infrastructure is currently used for investigations conducted for MWTP Project 24, Improved SRB. The decommissioning process also included autopsy sampling of the interior of each reactor to evaluate how the SRB material was used and if undesired preferred flow paths were developed. The autopsy sampling was neither included in the project work plan (Ref 3) nor in the project QAPP (Ref 2); it was conducted as a value-added investigation to the project to substantiate recommendations for technology improvements and enhance lessons learned conclusions. Autopsy sampling focused on the collection of solid matrix material that was inaccessible during the operational phase of the project. The majority of this visual inspection and sample collection took place within 30 feet of the respective AMD intake to the reactors (i.e., in chambers containing organic matter and limestone). Cobble chambers were inspected adjacent to the respective organic matter chambers. Consistent with the above sampling needs, the cobble chambers for Bioreactors II and III were abandoned in place. Organic matter and limestone was excavated, examined, and reburied in place. All material of aboveground Bioreactor IV was removed from the half-culvert and examined in the same manner as the material from Bioreactor II. After examination and sampling, the material from Bioreactor IV (organic matter, limestone, cobbles) was spread on or near the existing waste rock pile. Other materials (e.g., lumber, TerraCell™, aboveground portions of inlet and outlet enclosures, PVC monitoring pipes, and metal culvert) were salvaged or disposed in a local landfill. Underground pipes that were left in place were plugged. The straw bales covering the bioreactors were used for mulch associated with the final restoration of the site. 4.4.1 Sampling Autopsy sampling included collecting solid matrix samples for chemical analyses to determine concentrations of total metals [aluminum (Al), arsenic (As), cadmium (Cd), calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), zinc (Zn)], sulfate, sulfide, nitrogen, phosphorous, and total organic carbon (TOC) in the organic matter and limestone chambers. The EPA Toxicity Characteristic Leaching Procedure (TCLP), Method 1311 (EPA SW-846) was used for samples of the organic matter retrieved from the upstream organic matter chamber. Bacteriological analyses were conducted to determine the SRB population in the organic matter and in the limestone. Because the cobbles did not have a visually discernible film of bacteria or chemical precipitate, no solid matrix samples were collected from this chamber. Aqueous samples were collected from the bottom of the limestone and cobble chambers and analyzed for the same total metals as those listed for the samples of solid matrix retrieved from the organic matter chamber. The aqueous samples from the cobble chambers were also analyzed for dissolved metals that included Al, As, Cd, Cu, Ca, Fe, Mg, Mn, and Zn. The analytical work performed for the autopsy phase is summarized in Table 4-3. 12 ------- Table 4-3 indicates that the analytical work performed for Bioreactors II and IV was identical. Each upstream organic matter chamber (Orl) of Reactors II and IV had 5 samples of organic matter recovered for chemical analyses from the 10 CCS lifts that made up each chamber. The samples were collected from the center portion of the lifts, starting with lift No. 9 and then every other lift. Two additional organic matter samples (one from lift No. 9 and one from lift No. 2) were recovered from each Orl and analyzed for SRB population. A single organic matter sample was collected from Orl and analyzed by TCLP. Thus, samples from eight locations were collected from each upstream organic matter chamber. A single organic matter sample (lift No.5) was recovered from each downstream organic matter chamber (Or2) and analyzed for the identical constituents as the samples collected from the Orl chamber, including the SRB population count but excluding the TCLP. Each limestone chamber was sampled for a nonaqueous sample (a precipitate coating on the crushed limestone) and an aqueous sample (stagnant water from the base of the chamber). The suite of analyses for the nonaqueous sample was the same as for the samples collected from the Or2 chamber. An aqueous sample collected from each limestone chamber was analyzed for total metals, sulfide, and sulfate only. For the cobble chambers, stagnant water that accumulated near the base of the bioreactor was stirred, and one aqueous sample for each bioreactor was collected and analyzed for total and dissolved metals, sulfide, and sulfate. Analytical work performed for Bioreactor II was identical as that for the upstream chamber of organic matter (Orl) and the cobble chamber of Bioreactors II and IV. Table 4-la. Flow and Water Level Measurements Date Bioreactor II Flow Water level (ft from (gpm) bottom of Bioreactor) Bioreactor III Bioreactor IV Flow Water level (ft from bottom of Bioreactor) Flow Water level (ft from bottom of Bioreactor) 12/11/98 12/14/98 12/22/98 1/4/99 2/3/99 2/4/99 3/3/99 4/5/99 5/5/99 5/7/99 5/20/99 6/3/99 6/25/99 6/27/99 7/6/99 7/12/99 7/14/99 7/15/99 7/19/99 7/21/99 7/30/99 8/11/99 8/20/99 8/29/99 0.974 0.822 0.998 0.827 0.996 0.99 0.611 0 0.191 1.064 4.15 4.03 1.63 1.42 3.81 3.55 3.15 2.8 2.66 6.43 5.36 4.62 4.13 3.4 2.63 0.557 0.633 1.008 0.859 1.04 0.696 0.537 0.578 0.954 0.802 4.5 4.41 3.57 4.09 4.38 4.28 4.06 3.88 3.8 5.46 5.54 5.43 5.33 5.38 3.66 0.798 0 0 0 0 0 0 0 0.861 0.487 4.58 4.87 4.62 4.66 4.53 4.48 3.7 4.49 4.31 4.1 3.92 13 ------- Table 4-la. Flow and Water Level Measurements Bioreactor II Bioreactor III Bioreactor IV Date 9/1/99 9/8/99 9/21/99 9/24/99 9/27/99 10/7/99 10/26/99 11/9/99 12/8/99 1/6/00 1/10/00 2/3/00 2/8/00 3/6/00 3/16/00 4/5/00 5/8/00 5/25/00 5/28/00 6/7/00 6/14/00 6/16/00 6/18/00 6/30/00 7/5/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 3/27/01 4/4/01 4/9/01 5/9/01 6/6/01 7/9/01 Flow Water level (ft from „ Water level (ft from „ Water level (ft from bottom (gpm) bottom of Bioreactor) bottom of Bioreactor) of Bioreactor) 1.002 0.947 0.862 0.868 0.877 0.976 0.789 1.009 0.863 1.071 1.868 1.324 1.969 1.247 1.428 1.029 0.891 0.815 0.883 0 0 0.983 0.854 0.812 0.919 5.41 5.43 4.89 4.92 5.34 5.06 4.97 4.99 4.6 4.03 2.66 6 5.67 3.98 2.49 4.28 3.29 2.89 1.93 0.47 4.95 4.63 4.46 4.12 2.57 4.38 4.62 5.21 5.19 4.3 0.84 4.81 4.61 4.24 1.063 0.962 1.004 0.921 0.763 0.91 0.809 0.602 1.019 1.89 1.793 1.104 2.116 1.823 1.105 0.715 0.897 0.916 0.754 0.886 0.752 0.706 0.786 4.08 2.92 4.51 4.47 3.96 3.81 3.74 3.66 3.59 3.27 3.58 5.57 4.05 4.3 4.21 4.58 4.55 4.59 4.53 4.63 4.58 4.35 4.06 3.9 3.75 1.012 1.273 0.537 0 0 0 0 0 0 0 1.964 1.693 2.528 2.048 1.278 0 0 0 0 0 0 0 0 0.917 0.988 4.06 4.69 4.2 4.13 4.37 4.48 4.12 4.45 4.49 4.61 4.43 4.68 4.61 4.51 4.35 4.32 4.18 3.28 3.71 4.44 3.96 4.26 3.83 4.28 4.41 4.54 4.49 14 ------- Table 4-lb. Water Level Profile in Bioreactor II Point Water level - feet above bottom of Bioreactor 12/11/1998 05/20/1999 influent sump Hfd Hgd II hd II bd Hcd Hid II jd II kd II Id II dd 5.01 4.94 4.66 4.66 4.66 4.66 4.65 4.66 4.64 4.65 4.65 4.96 2.93 2.62 2.01 1.92 1.93 1.92 1.93 1.92 1.92 1.91 Table 4-2. Monitored Parameters and Analytes Measurement Planed Sample Frequency1 Sample Location2 Reported frequency and/or comments Dissolved metals (Al, As, Cd, Cu, Fe, Zn, Mn) Alkalinity, SO4, EH, DO, Soluble sulfide (HS)' Volatile fatty acids (VFA) PH SRB count Water temperature Flow rate Water level Air temperature at straw/rock interface days 10, 20, 30, every month thereafter days 10, 20, 30, every month thereafter days 10, 30, every 3rd month thereafter every half-hour days 10, 30; every 3rd month thereafter every half-hour every half-hour every half-hour days 10, 20, 30; every month thereafter Ilia, lid, Hid, IVd Ilia, lid, Hid, IVd Ilia, lid, Hid, IVd Ilia, He, Hie, I'VE lib, Illb, IVb Ilia, lid, Hid, IVd He, Hie, I'VE lid, Hid, IVd He, IIIc, IVc As planned As planned Increased to every month Monthly; probe got coated Increased to every month monthly; data logger malfunctioned monthly; data logger malfunctioned monthly; data logger malfunctioned Incomplete data; probes corroded Ambient air temperature every half-hour Ambient temperature housing 1 Samples for Bioreactor IV were not taken during the months in which freezing occurs 2 See Figure 3-5 for locations 'Addressed in Section 8, Quality Assurance/Quality Control No records; instrument broke3 15 ------- Table 4-3. Analysis for the Autopsy of the Bioreactors Total No. of analyses 3 24 3 24 24 24 19 19 19 10 169 Number of Locations Analyzed parameter TCLP1 TM2 DM3 Fe Spc7 Sulfide8 Sulfate9 N13 pl4 TOC15 SRB Sample Orl4 I10 5io 5io 5io 5io 5io 5io 5io 210 8 IV (Aboveground) Chamber Ca5 Or24 CO6 211,12 jlO 211,12 jlO 211,12 jlO 211,12 jlO I12 I" I12 I" I12 I" I12 I" 3 2 1" 1" 1" 1" 1" 1 Total 1 9 1 9 9 9 7 7 7 4 63 14 Reactors III Chamber Total Orl CO I10 1 510 1" 6 1" 1 510 1" 6 510 1" 6 510 1" 6 5io 5 5io 5 5io 5 210 2 43 8 1 9 II Chamber Orl Ca Or2 I10 = 10 911'12 1 10 = 10 911'12 1 10 cio 211'12 1 10 cio 211'12 1 10 J10 jl2 jlO clO 112 110 clO 112 110 210 1 12 1 10 832 Total CO 1 1" 9 1" 1 1" 9 1" 9 1" 9 7 7 7 4 63 1 14 1) Toxicity Characteristic Leaching Procedure, EPA Method 1311 (samples of organic matter) 2) Total metals, EPA Method 6010A/Method 3005 3) Dissolved metals, EPA Method 6010A/Method 3005 4) Orl4 and Or24 the upstream and downstream organic matter chambers, respectively (see Figure 2-3) 5) Limestone chambers (see Figure 2-3) 6) Cobble chambers (see Figure 2-3) 7) Fe Spc (Iron Speciation) (Standard Method 3500-Fe); 8) Sulfide, EPA Method 376.1 9) Sulfate, EPA Method 375.2 10) Organic matter (nonaqueous) ll)Aqueous sample (211'12 -limestone/cobble chambers - one of two samples is aqueous) 12) Nonaqueous sample (film, slime, or precipitate coating), (211-12- limestone/cobble chambers-one of two samples is nonaqueous) 13) Total nitrogen 14) Phosphorous 15) Total organic carbon 16 ------- Bioreactors Flow 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 4-1. Bioreactor flow. Water Level in Bioreactors Bioreactor II plugging Bioreactor II plugging I March 2001 Bioreactor II inlet valve plugging 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 4-2. Water level in the bioreactors. 17 ------- Water Level Profile in Bioreactor II During Early Operation and Plugging Episode of May 1999 II bd II cd II id Piezometer series II dd Figure 4-3. Water level profile in Bioreactor II during early operation and the plugging episode of May 1999. 18 ------- 5. Operation Phase Results Results reported in this section include measurements and analyses performed on the aqueous samples. They include parameters measured in the field, bacteriological analyses of the SRB population, and chemical analyses for sulfate, sulfide, alkalinity, and selected dissolved metals. 5.1 Field Parameters Field measurements are compiled in Tables 5-1 and 5-2 and are presented in a graphical form in the figures that are individually referred to in this section of the report. Other field parameters (i.e., the flow rate and water levels in the bioreactors) are presented in Section 4 together with other information regarding operation of the bioreactors. 5.1.1 pH, EH, Dissolved Oxygen, and Temperature Diagrams 5-1, 5-2, 5-3, and 5-4 present pH, EH, dissolved oxygen (DO), and temperature, respectively, for the influent and effluent from each bioreactor during the 32-month operation period. These results are also tabulated in Table 5-1. As mentioned in Section 2.2, the quality of water in the Lower Pond improved significantly after the first 10 months of operation. This is also evidenced in the pH trends diagram (Figure 5-1) where pH of the intake water (Lower Pond AMD) increased from a minimum value of 3.29 in May 1999 to 7.14 in January 2000 and then stayed at this or a slightly lower level for the duration of the project, with the exception of the spring months. The initial increase in effluent pH (including June 1999) can largely be attributed to alkalinity present within the organic matter rather than to the presence of limestone. This is indicated by an insignificant pH difference in the effluent from Bioreactors II and III, the latter having no limestone pretreatment chamber. As the SRB became established and the effluent pH from each bioreactor dropped to 8, 7.5, and 7 for Bioreactors IV, II and III respectively, the pH differential between the influent and effluents could be attributed to SRB activity. The slightly lower pH of the effluent from Bioreactors II and IV during this period may have been due to the limestone chambers in these bioreactors. Values of pH of the effluent from Bioreactor III fell below the pH values of its influent during the period from August 2000 to April 2001. However, the subsequent decrease of the influent pH in May and June 2001 to a value of 5 had no effect on the effluent pH, indicating that the bioreactor was still capable of improving the quality of the AMD. This decrease in the influent pH did not impact the pH of the other two reactors. Oxidation reduction potential (ORP) measurements in the field were taken using an ORP electrode with a silver/silver-chloride reference electrode. Since the EH is defined (Ref 4) as a voltage reading with a reference to the standard hydrogen electrode (SHE), these field values were converted to the EH values by adding 200 millivolts (mV) and corrected for temperature to report it for 25 • C. The corrected EH values are presented in Figure 5-2. The EH diagram shows that with the exception of a few time periods, the most noticeable being from late summer 1999 through winter 1999/2000 for Bioreactors II and IV and late summer through fall of 1999 for Bioreactor III, the EH values were positive, indicating an apparent oxidizing environment. There was, however, a significant difference in EH of the influent and the effluent AMD; the former being in the 300-mV to 400-mV range for most of the operating time. This difference indicates that the bioreactors significantly lowered the EH. Moreover, the bioreactors fostered conditions favorable for the SRB population growth, as indicated further in this document. Therefore, it is postulated that the EH measurements of aqueous samples collected at the outlets of the bioreactors (location A in Figure 3-5) may not reflect the EH present in the "pockets" of organic matter where the SRB lived. Additionally, 19 ------- the EH and pH profiling of the bioreactor (see Section 5.1.2) showed that the EH measured at the outlet of the bioreactors was higher than that measured for aqueous samples collected from the organic matter chambers where most of the sulfate reduction took place. The DO diagram (Figure 5-3) indicates that the oxygen level ranging from 4 milligrams per liter (mg/L) to 14 mg/L in the influent decreased in the bioreactors to less than 1 mg/L for the majority of the operation time. However, the 14 mg/L peak in the influent DO concentration in December 2000 was also reflected as a maximum DO concentration in Bioreactor III, which had no limestone chamber and only one organic matter chamber. Moreover, both peak values correlate well with very high EH values recorded for Bioreactor III at the same time (Figure 5-2). The temperature of the influent and effluent are presented in Figure 5-4. The temperatures reflect seasonal variation and change from 0.5 • C to 15.5 -C for the influent and 1 -Cto 16 'Cforthe effluent from both belowground bioreactors. Temperature of the effluent from these two bioreactors was very similar throughout the operating period with the exception of the first 4 months when the effluent from Bioreactor II was up to 2 • C higher. This difference is attributed to the fermentation processes of the organic matter that took place at the beginning of the operation and were more intensive in Bioreactor II, which contained two organic matter chambers. The above-freezing temperatures inside the aboveground bioreactor (IV) during winter were caused by a small (approximately 0.05-gpm) leak from the bioreactor that prevented the bioreactor from freezing solid, as anticipated by the design. The 2 • C higher temperature of the effluent from Bioreactor IV during summer is attributed to its exposure to higher ambient air temperature because of its aboveground location. 5.1.2 pH, EH, and Temperature Profiles Although profiling the bioreactors for pH and EH values was not in the original sampling plan, it was conducted to assist in data interpretation. The pH and EH measurements of the AMD at all deep and shallow piezometers for each bioreactor were taken four times per month beginning in November 1999. The results obtained were similar for all measurement events (Table 5-2). Figures 5-5 and 5-6 present the results of measurements taken in December 1999 for Bioreactors II and III, respectively. Measurements taken in Bioreactor IV are not plotted because they depict nonflowing conditions (the reactor was shut down for winter). The pH profiles document very little change along the flow in both bioreactors with the exception of the first organic matter chamber where pH values dropped by approximately 0.2 units upon the influent entry. This slight decrease in the pH values was probably caused by the release of protons when hydrogen sulfide (H2S) reacted with dissolved metals and precipitated them as metal sulfides as shown in Reaction 1-3. The pH increased approximately 0.5 units in the limestone chamber in Bioreactor II and in the cobble chamber in Bioreactor III. Worth noting is a small difference in the pH measurements taken in the shallow and deep piezometers in the cobble section. The slightly lower values of pH at shallow depth correlates well with the higher EH measurements taken at the same locations. The EH values were more differentiated than the pH readings for both bioreactors. The AMD flow in the bioreactors were notably stratified with respect to Eg, which in Bioreactor II was up to 400 mV higher in the shallow piezometers than in the deep piezometers. This difference, thus also stratification, diminished downgradient and close to the outlet of the bioreactors due to mixing of the water flowing through the cobble section. This mixing process was increased by the placement of the outlet pipe located 6 inches above the bottom of the bioreactors. 20 ------- The water mixing process seems to also be responsible for higher values of EH at the monitoring location at the outlet of the bioreactors in comparison to measurements taken at the bottom of the first organic matter chambers. This difference was approximately 50 mV and 150 mV for Bioreactors II and III, respectively. 5.2 Sulfate-Reducing Bacteria Populations The first 8 months of operation can be described as a period in which the microbial populations were established within the bioreactors. It should be noted that the bioreactors were started in the winter when temperatures were not ideal for microbial growth. As the bioreactor temperatures (Figure 5- 4) began to increase in April and May 1999, an increase in SRB populations (Figure 5-7 and Table 5-3) was also seen. During the second winter of operation, the well-established SRB population was not affected by the low temperatures. The correlation of the SRB population with EH can be seen in Figure 5-2. The EH decreased to -80 mV for Reactor III in September 1999 and -200 mV for Reactor IV in October 1999. During the same time, the SRB populations grew to a level of 2E+5 most probable number (MPN) for Reactor III. There was a small decrease in the SRB population during the winter of 1999-2000, with a subsequent increase in the spring of 2000. This increasing trend ended at the same time as the flow rates were doubled, indicating that SRB might have been flushed out at that flow velocity. The subsequent decrease in the SRB population through the winter of 2000/2001 is considered a delayed effect of doubling the flow rate in the summer of 2000. When the temperature increased in the spring of 2001, the SRB population returned to above the E+4 MPN level. Based on the metals-removal results explained further in this document (Section 5.5), the population of SRB at the E+4 MPN level worked well for the geochemical and climatic conditions present at the Calliope site. It is worth noting, however, that this SRB population should not be considered the optimum and/or recommended population for a SRB bioreactor in general. This is because the activity of the SRB is probably more important than the population size. In other words, a small population of SRB that are very active may be more efficient than a large population of less active cells. Reliable methods for the direct measurement of SRB activity are not currently available for routine sample analysis. Methods have been developed based on the uptake of radioactively labeled sulfate; however, these require specialized equipment and have an inherent safety hazard that makes routine use difficult to justify. Such methods were not included in the monitoring program for the Calliope site. 5.3 Sulfate and Sulfide Sulfate and sulfide concentrations in the influent and effluents of the bioreactors (Figure 5-8 and 5-9, respectively, and Table 5-4) do not give conclusive results regarding sulfate reduction rates. These diagrams are included in this report mainly for documentary purposes. The main reason for the inconclusiveness of the sulfate records could be a high concentration of sulfates in the organic matter built in the reactors, as indicated by often higher concentrations of sulfate in the effluent than in the influent. The use of analytical results for sulfide is limited due to the analytical procedure selected for the project. As this procedure did not require filtering of the sampled material, some of the sulfide detected might have come from suspended metal sulfides. This is especially true for sulfide analyses of the influent AMD that show up to 4 mg/L of sulfide, which should not exist in dissolved form under EH and pH conditions of influent and in the presence of dissolved Cu. Moreover, there was a noticeable H2S odor coming from the influent AMD. 5.4 Alkalinity Analytical data for alkalinity, expressed as milligrams of calcium carbonate (CaCO3), are presented in Figure 5-10 and Table 5-5. Although this diagram shows the total alkalinity, it is actually a bicarbonate (HCO3~) alkalinity for most of the operating time. Only during the first 3 months of operation (2 months for Bioreactor II) did 21 ------- carbonate alkalinity (CO32~) contribute to the total alkalinity, with the hydroxide alkalinity never detected. The alkalinity of a typical AMD is zero, as indicated on Figure 5-10, where, until October 1999, alkalinity of the influent AMD at the intake location was zero. As the AMD at the site improved (due to the change of climatic conditions) and its pH increased to 6.5, the alkalinity of the influent AMD increased to the range of 20 mg/L to 30 mg/L, with the exception of March 2001 when it peaked to 189 mg/L. The alkalinity of the treated AMD is a product of sulfate reduction by SRB that use organic carbon as the electron donor, as described by Reaction 1-2. Therefore, the alkalinity buildup in the effluent from all bioreactors at the Calliope site is a good indication of biochemical reactions taking place in the bioreactors. 5.5 Metals The primary objective of this project was to assess various configurations of the bioreactors and monitor their ability to produce a high-quality effluent (Ref 3). It was the goal of the field demonstration to achieve the effluent characteristics given in Table 2-1. These target concentrations were set arbitrarily at the beginning of the project when the quality of AMD in the Lower Pond was at its worst or close to it. The quality of the AMD improved with time due to climatic conditions, and some of the metals (Al, Zn, and Mn) became irrelevant because their concentrations in the influent were already close to or even below the target concentrations. Analytical results for concentrations of seven dissolved metals (Zn, Cu, Cd, Al, As, Fe, and Mn) in the influent and effluent of the bioreactors are compiled in Table 5-6. These monthly results are also presented in a graphical form in the figures that are individually referred to in this section. Changes in Zn concentrations are presented in Figure 5-11. During the first 7 months of the demonstration, Zn concentrations were rising as the sorptive capacity of the organic matter was being filled. During this period, the percent of Zn removal was as high as 99%. Once the sorptive capacity was filled, Zn concentration stabilized at a threshold of approximately 500 (ig/L for Reactors II and IV and 800 (ig/L for Reactor III. The slightly lower percent of removal in Reactor III is attributed to a smaller total supply of organic carbon, as this reactor has only one chamber with organic matter. Concentrations of Cu are presented in Figure 5-12. Much of the Cu removal observed during the first 7 months of operation can be attributed to adsorption. Once sorption sites fill and the SRB populations become established, Cu was removed through SRB activity to a threshold level of 50 (ig/L on average for Bioreactors II and IV and, again, a slightly higher threshold level of approximately 80 (ig/L for Bioreactor III. Cadmium concentrations are presented in Figure 5- 13 (in linear scale). Similarly to Zn and Cu, the removal of Cd observed during the first 7 months of operation can be attributed to adsorption. Once sorption sites fill and the SRB populations become established, Cd was removed through SRB activity to a threshold level ranging between 4 (ig/L to 5 (ig/L, until March 2001 when the Cd concentration in the influent dropped to approximately 3 (ig/L for a 2-month period. Cadmium concentration in the effluent in the same time period decreased to less than 1 (ig/L. Figure 5-14, which presents the concentration of Al, also shows a reduction of concentration in the effluents, but only for the first 10 months of operation. During this time, Al was removed due to adsorption on the organic matter, similarly to Zn, Cu, and Cd. After September 1999 when the Al concentration in the influent decreased to 100 (ig/L or below level, there is no indication of further removal of Al. Figure 5-15, which presents As concentrations, indicates that As content did not decrease in the bioreactor effluents but rather the effluents were 22 ------- enriched with As for most of the operating time. The reason behind such behavior is two-fold. First, it was found by Canty (Ref 5) that the manure obtained from a similar source but used for another demonstration site, contained elevated (in comparison to the AMD) levels of As. Thus, it is prudent to assume that some As was flushed from the organic matter chambers down to the sampling points. Second, as Robins and Huang (Ref. 6) explain, under oxidized conditions, as present for the source of AMD, Fe3+ (ferric iron) precipitates as ferrihydrite Fe(OH)3, which effectively adsorbs As. However, under reducing conditions, such as in the bioreactors, ferrous iron (Fe2+) becomes the predominant iron species. Because Fe2+ is much more soluble than Fe3+, it is released into solution along with the previously adsorbed As. Because the analytical work for the operation phase of the project did not include speciation of Fe, it is impossible to determine which of the above- described mechanisms was predominantly responsible for the high concentrations of As in the effluent. Iron concentrations, presented in Figure 5-16, show that with exception of the initial 8 months, Fe concentration in the influent was significantly lower than that in the effluents. During the initial 8-month period, Fe seemed to be removed by the bioreactors due to initially high sorptive capacity of the organic matter. Higher dissolved Fe concentration in the effluent than in the influent (beginning in July 1999) can be explained by a very possible, but never measured, difference in dissolved versus total Fe concentrations in the influent. As stated earlier in this document, under oxidizing conditions and nearly neutral pH, such as those present in the Lower Pond, the prevailing form of iron (Fe3+) precipitates as Fe(OH)3. Because the AMD that was piped from the Lower Pond was not filtered, it certainly brought suspended Fe(OH)3 to the bioreactors. It is possible that under reduced conditions in the bioreactors, Fe3+ in Fe(OH)3 was reduced to Fe2+ and released to the aqueous phase as dissolved Fe, increasing its concentration in the effluent. The Mn diagram (Figure 5-17) indicates that the bioreactors were not efficient in lowering concentrations of Mn for most of the demonstration. In fact, reduction of Mn concentration took place only at the beginning of the project and close to its end (for Bioreactor II). At the beginning of the demonstration, Mn was sorbed (like other metals) to the organic matter. At another date in late winter through early spring 2001, Mn was efficiently removed in Bioreactor III. In this case, the efficient removal of Mn coincides with an unexplained increase in DO concentration (Figure 5-3) that was subsequent to the also unexplained peak of EH in the winter of 2000/2001. Table 5-1. Field Parameters Location Date pH £„ (mV) DO (mg/L) Temperature (* O Influent 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 3.87 6.01 4.51 5.70 5.62 3.29 3.64 4.19 5.14 6.08 6.68 6.58 6.87 7.14 6.81 6.61 700 426 571 466 480 726 611.4 315.4 403 309 357 294.4 247.5 302 334 6.8 8.85 10.19 11.19 8.29 6.06 3.93 6.39 probe maliiinction 8.61 8.94 9.76 9.94 10.5 8.81 8.89 1.6 1.1 0.9 0.6 0.5 4.6 9.9 15.3 14.4 9.7 7.3 4.3 1.3 0.7 1.2 4.7 23 ------- Table 5-1. Field Parameters Location Date 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 DH 6.07 6.83 5.80 6.24 6.48 6.83 6.92 7.08 7.40 6.94 £„ CmV) 368 363 409 366 342 310 267 267 422 probe DO Cma/L) 8.42 8.59 7.66 7.53 7.43 7.89 8.89 9.03 13.84 11.49 Temperature (• O 5.2 7.5 15.4 13.3 15.5 10.5 6.6 4.3 1.6 1.2 malfunction 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 Bioreactor II 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 7.52 7.10 7.33 6.48 4.85 5.01 9.95 9.51 8.75 8.42 8.37 8.29 7.87 7.61 7.68 7.09 7.14 7.20 7.69 7.29 7.46 7.37 6.98 6.79 7.25 7.10 6.76 7.31 6.73 7.06 7.17 6.98 230.9 365 251 32 394 401 76 252 342 378 353 87 62.8 106.5 -76 -125 -36 -62 -35 29 42 -68 -74 78 16 151 -14 -34 133 330 probe 10.49 9.67 9.93 probe malfunction 7.1 0.51 0.63 1.09 0.93 0.11 0.04 0.95 0.46 probe malfunction 0.2 0.24 0.53 0.43 0.39 0.48 0.30 0.51 0.21 0.29 0.27 0.31 0.61 0.27 0.46 2.58 0.82 1.5 2.7 3.8 8.3 8.5 16.5 4.9 5.9 4.1 2.4 2.1 6.1 8.9 13.2 15.4 14.1 9.2 6.4 4.4 2.4 1.9 2.9 2.9 5.3 10.6 13.5 15.8 12.9 12.0 6.3 3.7 2.9 malfunction 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 Bioreactor III 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 1 1/9/99 12/8/99 1/6/00 7.21 7.74 6.83 6.61 6.76 6.76 9.12 9.32 9.34 8.43 7.78 7.56 7.62 7.54 7.03 7.27 7.02 6.75 7.21 6.98 296 -83 289 94 98 15 219 279 403 444 328 273 187.2 72.9 -82 -81 37 59 68 0.56 0.59 0.48 probe malfunction 0.47 0.05 0.18 0.44 0.76 0.17 0.04 0.08 0.15 probe malfunction 0.21 0.24 0.27 1.18 1.43 2.2 2.7 2.9 4.8 9.1 12.4 4.2 3.4 2.2 1.7 1.3 5.4 9.6 13.8 15.0 14.8 10.9 6.3 3.2 2.2 24 ------- Table 5-1. Field Parameters Location Date 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 BioreactorlV 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 DH 6.92 7.00 6.82 6.84 6.63 6.39 6.35 6.45 6.45 6.77 6.73 6.65 6.79 6.64 6.82 6.7 6.8 6.26 9.98 9.87 9.68 9.19 8.37 8.63 8.27 7.59 8.12 7.80 7.90 7.91 8.25 7.96 8.17 7.88 7.92 8.06 7.49 7.23 6.99 7.81 7.54 7.57 7.84 7.70 8.47 7.68 7.75 7.79 7.18 6.97 £„ CmV) 259 145 76 -49 41 114 196 164 241 187 569 probe maliunction 379.4 113 334 -48 161 34 69 339 337 346 135 17 53.6 77.3 DO Cma/L) 0.66 2.23 0.34 0.12 0.27 0.91 0.67 2.53 1.79 0.91 5.69 5.28 3.91 3.80 3.22 probe maliunction 0.43 0.15 0.40 0.24 0.46 0.06 0.28 0.09 0.15 probe maliunction -82 -100 -191 -60 -31 95 53 -87 -33 37 37 30 -53 -31 98 289 probe maliunction 242 -16 254 -87 39 -40 0.2 0.25 0.28 0.3 0.31 0.77 0.28 0.37 0.08 0.27 0.3 0.30 0.45 0.31 0.24 0.60 1.06 0.73 0.50 0.48 probe maliunction 0.42 Temperature (• O 1.6 3.6 4.1 6.7 11.8 14.1 15.7 12.9 10.0 5.8 2.8 2.5 2.1 2.0 3.1 4.4 6.8 13.7 1.0 0.5 0.3 0.1 0.1 2.0 10.3 16.2 17.0 13.8 7.6 5.6 2.1 0.7 0.0 1.6 3.1 5.0 14.1 14.5 17.3 12.4 7.5 5.5 0.8 1.2 0.5 0.4 0.8 2.6 8.2 18.1 25 ------- Table 5-2. EH and pH Profiles for Bioreactors Location Piezometer Influent Bioreactor II II fd II gd II hd II bd II cd Hid II jd II kd II Id II fs II gs II hs libs lies His II js II ks Ills Bioreactor III III fd Illbd Illgd Hied Illhd HIM Illjd Illfs Hlbs Hlgs IIIcs HIlis mis IIIjs 418.20 131.9 -100.7 -63.3 19.5 -10.3 -56.2 -56.5 -61.8 -57.8 328.5 312.1 380.4 33.9 -35.2 -76.5 -80.5 -82.3 -76.8 -13.5 41.5 81.2 108.2 87.6 50.4 34.9 116.7 78.3 62.9 55.3 41.8 23.9 II and III EH 12/6/99 1/11/00 438.8 53.2 -135 -101.2 -91.6 -111.7 -110.5 -108 -99.9 -87.4 331.7 284.3 114.7 105.3 49.7 33.5 -26.8 -77.9 -95.4 -46.5 -153.2 -110.8 -72.8 -55.5 -36.9 16 97.3 47.1 56.2 65.7 50 53.5 301.9 174 -38 -11 -21 -15 -18 -13 -1 12 150 70 84 52 32 32 1 -14 88 -23 -16 1 22 47 115 127 95 97 110 96 126 2/9/00 302 243 83 22 -25 0 -10 -13 4 29 dry 214 192 106 81 24 11 23 42 150 60 71 98 114 141 259 dry 202 111 229 231 219 245 12/6/99 6.79 6.2 7.01 7.19 7.46 7.55 7.55 7.55 7.6 7.53 7.01 7.31 7.59 7.3 7.08 7.13 7.13 7.25 7.25 6.36 7.61 7.58 7.47 7.41 7.15 6.99 7 7 6.99 7.04 6.65 6.83 PH 1/11/00 6.78 5.76 7.03 7.06 7.38 7.4 7.45 7.45 7.48 7.45 dry 6.76 7.43 7.19 7.14 7.23 7.19 7.19 7.19 6.19 7.27 7.3 7.15 7.05 6.93 6.73 6.85 6.82 6.73 6.69 6.62 6.56 2/9/00 6.67 5.79 7.1 7.36 7.59 7.43 7.6 7.7 7.65 7.78 dry 7.29 7.13 7.27 7.34 7.72 7.74 7.6 6.37 7.19 7.12 6.74 6.81 6.71 6.55 6.52 6.32 6.14 6.44 6.51 6.59 26 ------- Table 5-3. SRB Populations Date Bioreactor II SRB populations (SRB/mL) Bioreactor HI Bioreactor IV 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/12/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/16/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 1.40E+03 2.00E+02 9.20E+00 1.80E+00 1.10E+00 1.70E+01 1.70E+02 2.80E+03 2.80E+04 1.40E+06 4.50E+05 4.50E+04 1.70E+04 1.20E+04 1.10E+04 2.40E+04 1.70E+06 2.00E+03 1.70E+04 1.40E+05 1.10E+05 9.20E+04 4.00E+04 2.80E+02 2.10E+03 4.50E+03 7.80E+03 7.80E+03 2.40E+04 1.40E+05 9.30E+04 1.70E+04 1.10E+04 1.70E+01 1.70E+01 l.OOE-01 1.80E+00 8.10E+00 2.40E+02 7.80E+03 2.00E+03 2.40E+03 2.80E+04 2.00E+05 4.50E+04 4.50E+03 1.10E+04 1.10E+04 1.70E+04 1.10E+05 1.10E+05 1.10E+05 2.00E+04 4.50E+04 1.10E+04 1.40E+04 2.00E+03 1.40E+03 2.40E+03 4.50E+03 2.00E+03 2.10E+03 4.50E+03 1.40E+04 2.80E+04 4.50E+04 7.80E+01 1.40E+02 l.OOE-01 1.80E+00 8.10E+00 2.40E+02 2.00E+03 1.10E+04 1.40E+04 1.40E+04 2.40E+04 4.50E+04 1.40E+04 2.00E+03 2.40E+03 1.10E+04 1.40E+04 7.80E+04 7.80E+04 4.50E+04 6.80E+04 2.00E+04 2.00E+03 2.40E+02 2.40E+03 1.40E+04 1.40E+04 2.10E+04 2.40E+04 7.80E+04 2.00E+04 2.10E+04 4.50E+04 27 ------- Table 5-4. Sulfatt Date 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/12/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/8/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 s and Sulflde Influent 102 115 113 104 143 135 229 200 122 118 109 99 93 88 81 84 83.2 103 88.3 91.6 92.5 78.5 81.4 82.9 69.8 73.3 79.7 72.4 60.6 1,400 81.6 118 101 Concentrations Sulfate (mg/L) II III 250 80 145 111 122 193 281 304 136 92 113 73 87 83 82 82 73.8 100 81.2 82.4 72.7 95.4 88.5 59.2 73.4 65.9 68.1 55.4 8 1 196 131 128 88.7 Values enveloped by lines are considered outliers 172 111 123 115 152 178 223 197 126 80 76 88 100 89 85 87 81.4 95.6 69 84.8 93.5 78.5 83.8 81.5 86.2 79.7 74.9 56.4 59.6 73.7 113 113 102 and were rv 148 114 143 137 186 187 300 457 326 74 92 106 99 98 125 152 114 69.2 127 91.9 64.7 80.3 93.1 86 84.7 163 208 263 251 204 241 121 79.7 not grafted Influent 3.1 2.8 3.7 0.0 0.0 0.0 0.0 0.0 0.0 2.7 1.2 0.0 1.0 1.1 3.1 1.6 1.37 1.5 1.7 2.6 5.1 4.3 3 3.1 3.6 3 3 4.5 5 2.6 2.8 2.2 2.8 Sulflde II 1.2 2.2 6.4 1.2 1.3 0.0 0.0 15.0 0.0 2.8 3.2 0.0 9.6 1.4 4.8 2 1.13 1.5 1.3 2.3 4.8 4.9 3.2 5.2 3.4 3.2 2.6 3 3.7 2.2 1.7 1.3 3.9 (mg/L) III 2.3 2.9 2.4 1.6 2.4 0.0 0.0 2.2 0.0 4.1 3.0 0.0 0.6 3.2 3.9 1.2 1.53 2 3.7 2.9 4.7 7.2 2.3 5.6 2.3 3.3 2 4 4.5 1.7 1.9 1.6 4.1 rv 2.1 3.3 1.9 1.2 0.0 0.0 1.2 1.3 0.0 2.0 1.3 1.2 0.9 0.8 4.2 1.3 1.45 1.2 0.85 2.8 4.7 6.0 3 3.2 2.6 3.2 2.9 4.6 5 2.5 2.6 2 2.4 28 ------- Table 5-5. Alkalinity Date Intake Total Alkalinity (mg/L as CaCO3) II III IV 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/12/99 9/8/99 10/7/99 11/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/8/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14.4 12.4 24.8 19.6 16.2 0.0 14.0 11.0 18.0 19.4 18.2 22.2 20.8 28.0 21 25.4 189 25.2 16 0 0 215.0 111.0 105.0 95.0 95.8 102.0 126.0 474.0 128.0 247.0 238.0 124.0 54.0 53.6 70.4 79.0 90.4 100.0 87.2 74.2 152.0 54.6 45.8 122.0 62.2 63.8 54.0 70.2 95.8 90.4 70.0 83.4 83.8 277.0 192.0 93.8 95.8 94.2 86.0 85.4 99.0 78.2 359.0 248.0 75.8 26.4 35.0 37.0 41.0 35.2 47.0 126.0 38.4 27.2 29.8 23.6 28.0 39.6 26.2 29.2 26.8 24.2 40.2 46.6 75.0 33 294.0 238.0 223.0 203.0 275.0 251.0 241.0 262.0 214.0 290.0 446.0 87.8 90.4 68.2 125.0 129.0 146.0 220.0 426.0 77.2 124.0 81.8 59.6 56.4 65.2 102.0 120 154 25.8 286 347 202 97 29 ------- Table 5-6. Metals Concentration Location Date Al Influent 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 1 1/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 Bioreactorll 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 1 1/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2400.0 1670.0 2080.0 788.0 1160.0 3750.0 14100.0 8770 1580 779 140 26.6 35 93.1 18 11 44.5 134 32 154 35.3 17.3 36.1 18.5 18.9 18.9 73.4 45.5 18 31 40.5 1650 587 39.5 24.6 22.8 30.8 11.2 10.7 13.8 111 49.3 97 128 35.0 15.8 21.3 30.6 16.5 24.6 130 56.5 47.6 55.2 17.3 17.3 98.2 49.6 31.8 18.9 Metals concentration (ng/L) As Cd Cu 2.8 2.5 4.4 5.2 3.6 3.9 1.8 3.1 1.3 2.5 6.5 6.2 6.7 5.5 10.9 5.1 6.1 3.5 7.6 2.1 1.5 1.5 2.5 1.9 2.1 3.3 5.7 4.7 3.4 2 1.6 1.1 1.1 15.6 10.0 7.4 5.1 3.4 4.3 3.0 26.5 5.4 14.9 7.7 5.0 5.6 5 5.4 2.6 1.6 15.6 10 2.8 6.8 3.6 2.3 3.6 3.5 4.7 6.8 15.2 18.1 14.9 16.0 12.1 20.1 41.9 31 16.5 17.9 16.4 12.7 7.3 9.3 10.9 7 8.2 7.3 8.4 8.6 6.3 5.7 3.7 5.7 5.1 4.3 4.3 4.3 2.8 3.1 4.8 8.8 12.1 3.9 3.9 3.9 4.8 4.8 4.8 4.8 2.5 2.5 3.9 4.6 4.9 4.7 5.5 4.7 3.5 3.5 3.5 3.5 3.2 3.2 3.4 3.4 3.9 4.3 4.3 4.3 884.0 737.0 689.0 477.0 518.0 1020.0 3050.0 2090 451 366 200 68.9 78.8 82.8 7.2 53 114 274 145 310 174 81.4 92.7 71.4 44.2 34.1 28.1 2.8 26 30 162 635 459 29.4 11.5 3.3 4.8 4.3 47.5 7.8 75.2 25.6 96.6 95.9 36.8 19.4 23.2 15.8 12.8 21.4 72.2 49.7 21 81.8 17.7 5.9 89.1 57 68.8 31.5 Fe 524.0 377.0 603.0 357.0 265.0 713.0 7220.0 2960 111 281 372 50.6 154 15.7 115 35.9 20.1 124 27.2 73.1 20.5 31.9 15.5 15.5 20.1 19.5 19.5 19.5 8 13 25.6 70.2 195 140.0 77.6 46.9 93.7 35.5 54.2 97.5 1300 686 868 983 545 284 395 352 314 305 1810 1230 111 2110 261 197 926 578 533 234 Mn 1620.0 2020.0 1700.0 1830.0 1620.0 2120.0 3770.0 2860 1600 1950 1950 1580 1320 1170 1420 1130 1220 1360 1270 1230 1170 1070 1090 1140 1110 1000 882 787 690 840 947 1460 1190 35.6 31.1 39.5 117.0 115.0 357.0 551.0 999 964 1040 1340 885 774 765 785 913 982 1000 1140 1000 1440 984 836 1050 564 500 296 Zn 3740.0 4420.0 3610.0 3570.0 3190.0 4870.0 11100.0 7890 3370 3790 3520 2700 2130 2140 2080 1890 2050 2460 2020 2230 1840 1500 1580 1570 1420 1240 1270 1090 990 1000 1570 3180 2720 75.6 18.6 22.3 22.3 43.9 128.0 249.0 388 717 704 1230 1180 750 111 453 176 210 751 467 331 409 372 189 707 517 404 419 30 ------- Table 5-6. Metals Concentration Location Date Al 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 Bioreactor III 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 1 1/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 Bioreactor IV 12/14/98 12/22/98 1/4/99 2/3/99 3/3/99 4/5/99 5/5/99 6/3/99 7/12/99 8/11/99 9/8/99 10/7/99 1 1/9/99 12/8/99 1/6/00 2/3/00 3/6/00 4/5/00 5/8/00 6/7/00 7/6/00 46.5 19 140 130 47.8 118 43.8 40.0 22.8 43.4 16.9 10.7 45.3 23.7 59.6 335 141 52.1 10.1 26.4 19.9 9.4 31.2 68.5 240 50.9 25.3 24.6 49.6 48 83.3 18.9 18.9 18.9 22 85 143 125 80.3 54.2 27.0 29.8 45.2 35.4 18.8 29.5 9.7 57.2 37.7 49.7 14.5 25.3 10.1 13 18.6 21.8 32.9 87.3 43.4 23.4 Metals concentration (ng/L) As Cd Cu 3.3 2.2 4.7 11.4 5.5 4 17.1 11.3 5.0 6.2 4.4 4.6 3.5 3.1 2.9 8.9 12.7 3.5 4.2 3.2 3.5 2.4 1.6 3.6 11.7 3 2.2 1.5 2.8 2.3 3.3 1.9 6.1 3 3.1 2 4.5 4.3 2.7 26.3 21.6 29.1 19.7 19.6 14.6 17.1 26.3 3.4 13.4 28.7 2.3 3.4 2.6 7.8 8.8 10.1 8.8 16.3 9.6 1.6 4.3 0.1 0.3 4.8 4.8 5 3.9 3.9 3.9 4.8 4.8 4.8 4.8 2.5 3.1 7 3.9 3.9 4.7 4.7 4.8 3.5 3.5 3.5 4.7 4.4 3.2 3.4 4.4 3.5 5.6 4.3 4.3 4.3 1 2 9.1 4.8 4.8 3.9 3.9 3.9 4.8 4.8 4.8 4.8 2.5 2.5 3.9 3.9 3.9 4.7 4.7 4.7 3.5 3.5 3.5 3.7 3.2 3.2 23.7 8 100 142 50.6 110 65.5 20.3 8.6 18.1 17.5 6.4 43.4 36.4 28.3 140 69.2 22.5 16.1 14 19.5 18.3 51.8 65.6 114 49.3 96.8 45.8 68.8 69.5 128 24.3 23 20.4 16 65 155 142 122 103.0 79.4 33.0 39.5 44.8 32.3 36.3 36.2 8.0 28.1 32.6 6.4 23.5 17.2 16.9 39.7 47.1 34.6 44.1 47 20.1 Fe 460 110 600 454 638 707 229.0 92.4 57.8 69.8 43.9 49.8 149.0 202 337 1380 875 661 302 163 91.2 95.8 323 1240 3910 1070 115 124 116 115 161 43.9 34.3 25.2 31 150 282 494 638 417.0 155.0 126.0 95.7 230.0 180.0 166.0 301 410 530 782 142 304 141 188 206 325 434 1900 761 448 Mn 871 600 2400 721 849 996 105.0 54.5 33.1 124.0 436.0 768.0 2100.0 1940 1510 3100 1360 1260 1230 1150 1170 1200 1270 1500 1450 1130 1120 886 631 631 493 310 129 73.5 76 280 212 3040 2470 19.2 20.4 19.3 27.5 168.0 215.0 454.0 983 1310 1070 1480 751 883 811 934 1060 1070 1150 2190 1000 1120 Zn 648 48 300 1170 518 899 91.0 34.8 35.8 35.9 60.9 241.0 459.0 1190 1030 1100 847 753 364 659 934 707 682 893 1070 723 956 784 1040 1330 1590 836 871 937 790 850 1720 1590 1010 118.0 53.3 40.6 119.0 72.8 51.3 194.0 421 306 672 595 278 491 544 438 466 471 410 442 463 348 31 ------- Table 5-6. Metals Concentration Location Date Al Metals concentration (ng As Cd Cu Fe Mn Zn 8/10/00 9/7/00 9/25/00 10/31/00 12/4/00 1/8/01 2/12/01 3/14/01 4/9/01 5/9/01 6/6/01 7/9/01 17.3 17.3 17.3 45.4 18.9 51.1 18.9 85 22 55.8 81.7 42.3 1.5 1.8 1.5 2.5 4.1 7.2 5.5 6 8 9.6 4.1 1.1 3.4 3.4 3.4 4.3 4.3 4.3 4.3 0.4 0.7 4.8 4.8 4.8 10.2 9.8 6.3 52.9 16.9 13.6 18.5 530 35 33.9 132 18.2 87.7 67.1 124 506 1090 1590 937 1100 2900 3540 1940 572 837 629 794 942 1320 1810 2000 2300 2300 2610 1400 1270 269 222 145 383 170 203 315 220 250 211 505 292 pH Trends 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-1. pH trends. 32 ------- EH Trends Date Figure 5-2. EH trends. Dissolved Oxygen Trends Date Figure 5-3. Dissolved oxygen trends. 33 ------- Temperature Trends 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-4. Temperature trends. Bioreactor II pH and EH Profiles for 12/6/99 limestone organic carboin , , 0 : chamber chamber " organic carl chamber I cobble chamber •deep Eh •shallow Eh •deep pH 1 shallow pH IIG II H II B IIC III II J Piezometer nest (each piezometer is approximately 10 ft apart) Figure 5-5. Bioreactor II pH and EH profiles for 12/6/99. 34 ------- Bioreactor III pH and EH Profiles for 12/6/99 IIIF IIIB IIIG IIIC IIIH III I Piezometer nest (each piezometer is approximately 10 ft apart) Figure 5-6. Bioreactor III pH and EH profiles for 12/6/99. SRB Populations 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date > Figure 5-7. SRB populations. 35 ------- Sulfate Concentrations 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-8. Sulfate concentrations. Soluble Sulfide Concentrations 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-9. Soluble sulfide concentrations. 36 ------- Total Alkalinity 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-10. Total alkalinity. Zn Concentrations -Intake 'II -III IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-11. Zinc concentrations. 37 ------- Cu Concentrations If1 l.E -Intake 'II -III IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) Date Figure 5-12. Copper concentrations. Cd Concentrations •Intake 'II •III IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autoosv samole (dissolved! 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date i Figure 5-13. Cadmium concentrations. 38 ------- Al Concentrations Intake II III IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) Date Figure 5-14. Aluminum concentrations. I As Concentrations -Intake 'II •III •IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date > Figure 5-15. Arsenic concentrations. 39 ------- Fe Concentrations I -Intake 'II -III IV II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) \ 11/1/98 1/30/99 4/30/99 7/29/99 10/27/99 1/25/00 4/24/00 7/23/00 10/21/00 1/19/01 4/19/01 7/18/01 Date Figure 5-16. Iron concentrations. Mn Concentrations I •5 l.E+02 II autopsy sample (total) III autopsy sample (total) IV autopsy sample (total) II autopsy sample (dissolved) III autopsy sample (dissolved) IV autopsy sample (dissolved) Date Figure 5-17. Manganese concentrations. 40 ------- 6. Autopsy Results Reporting on autopsy sampling in this section is descriptive in its character; it documents the results obtained and includes basic qualitative interpretation. It is expected that a portion of the autopsy data will be used in a quantitative manner to improve and validate a computer program for bioreactor design begun in one of the tasks of another MWTP project (i.e., Project 24, Improved SRB). 6.1 Aqueous Samples Although the autopsy sampling focused on collecting the solid matrix material that was inaccessible during the operational phase of the project, it also included aqueous samples collected from the bottom of the bioreactors within chambers filled with limestone cobbles. To minimize the impact of atmospheric oxygen on the aqueous samples, the samples were collected immediately after the limestone cobbles were removed to provide access to the treated AMD that remained at the bottom of the bioreactors. Aqueous samples collected at the bottom of the cobble chamber were analyzed for total and dissolved metals, iron speciation, sulfate, and sulfide. Samples collected at the bottom of the limestone chambers were analyzed for the same analytes with the exception of dissolved metals. Analytical results are compiled in Table 6-1 (a, b, c). The concentrations of selected analytes are also presented in graphical form in the figures that are individually referred to in this section. 6.1.1 Cobble Chambers Figures 6-la and b, 6-2a and b, and 6-3a and b include diagrams of dissolved versus total and suspended metals for Bioreactors II, III, and IV respectively. Values used for these diagrams are tabulated in Table 6-la. Figures denoted with the letter "a" show total versus dissolved metals, and figures denoted with the letter "b" present suspended metals calculated as the difference between the total and dissolved metals. The autopsy dissolved metal concentrations compare well to the corresponding metals concentrations of the final sampling event as documented in Table 6-2. The differences may be attributed to a 1-week gap between the sampling events and the different sampling locations. In general, the dissolved metal concentrations were higher than during the final monthly sampling event with the biggest difference for As, Al, and Fe. The autopsy dissolved and total metal concentrations are also marked in Figures 5-11 through 5-17. Although not detected by a meter, the smell of H2S was present during sampling of the "soupy" grey water that accumulated at the bottom of the bioreactors. This smell was indicative of reducing conditions and sulfate reduction at these locations. Such judgement was supported by results of speciation analyses for dissolved Fe (Figure 6-4 and Table 6-lb) in the aqueous samples collected from the cobble and limestone chambers. Ferric iron (Fe3+) for those samples was found only in the sample taken from the limestone chamber of Bioreactor II and the limestone and cobble chambers of Bioreactor IV. The maximum concentration of Fe3+ for these locations was 0.17* g/L or 9% of Fe2+ in the limestone chamber of Bioreactor II. However, the analyses for sulfide and sulfate in the dissolved phase (Table 6-lc) show only 3.9 mg/L, 2.2. mg/L, and 4.2 mg/L concentrations of sulfide for Bioreactors II, III, and IV respectively. This indicates that only a small portion of dissolved metals in reduced form were balanced in the solution by sulfides. These were probably Zn, Cu, Cd, and a small portion of Fe. The rest of the metals dissolved in solution, including high concentrations of Ca (Table 6-la), are electronically balanced by a high concentration of sulfate (Table 6-lc), hydroxide, and bicarbonate. Concentrations of the latter two were not analyzed for the autopsy samples. 41 ------- Series "b" diagrams that show a high load of suspended metal compounds in a "soupy" solution attested to the successful operation of the bioreactors. Provided no suspended metals drained from the bioreactors through their outlets, the mass of metal compounds found in the lower portion of the bioreactors together with metal compounds that accumulated within the organic matter and on the limestone should be related to the mass of metals removed from the influent AMD. The analytical data available do not allow for determination of chemical compounds that contained these metals. However, considering the observed (during the autopsy on the bioreactors) orange-color precipitate present on the PVC liner and the HDPE of the CCS, it is possible that solids in the "soupy" water also included significant amounts of ferric hydroxide. It is also hypothesized that Al present as a suspended solid was in the form of hydroxides. 6.1.2 Limestone Chambers Analytical results for total metals for aqueous samples collected at the bottom of the limestone chambers of Bioreactors II and IV resemble those obtained for aqueous samples of the cobble sections, as documented in Figure 6-5 for Bioreactor II. Thus, despite the lack of analytical data for dissolved metals concentration for this section of the bioreactors, it is assumed that the concentrations of suspended metal compounds in the limestone section were similar to those for the cobble section. 6.1.3 Sulfate and Sulfide Analyses Table 6-lc includes analytical data for sulfate and sulfide in the aqueous sample collected during the autopsy of the bioreactors. The table shows that sulfate concentrations were as high as 352 mg/L in Bioreactor III. The highest concentration of sulfide, 4.2 mg/L, was found in Bioreactor IV. 6.2 Bioreactor Solid Matrix Samples Analytical data for solid matrix samples collected during the autopsy of the bioreactors are included in Table 6-3 (a, b, c, d), and they are also presented in graphical form in the figures that are individually referred to in this section. Also presented in this section are analytical data of material that was found plugging the manifold for the AMD distribution system. 6.2.1 Metals Concentrations in the Bioreactors Figures 6-6, 6-7, and 6-8 present the distribution of metals in the solid matrix of the first three chambers of Bioreactors II, III, and IV, respectively. These values are also compiled in Table 6-3a. Concentration of metals within the organic matter (denoted in the figures as organic matter chambers 1 and 2) was determined with respect to the total dry weight of the sample collected. For the limestone chamber, the precipitate present on the limestone was scraped, and metal concentrations were determined with respect to the dry weight, not including the weight of the limestone. As stated in Section 4.4.1, the cobbles that comprised the last chamber of each reactor did not have a visually discernible film of chemical precipitate, thus the metal concentrations in the solid matrix of these chambers were considered null. Concentrations of metals that accumulated within the solid matrix of the bioreactors were measured in thousands of milligrams per kilogram with the exception of those for Cd and As. These high metals concentrations demonstrated that a large load of metals was retained within the organic carbon material, thus indicating the bioreactors were efficient in removing metals from the influent AMD. Unfortunately, the analyses performed using the inductively coupled argon plasma (ICP) method do not allow for distinguishing of metals that were adsorbed by organic matter from those that precipitated as chemical compounds due to SRB activities. 42 ------- Evidence of the biological removal of metals by the SRB comes from other analytical results conducted for the project. First, the high concentrations of sulfide in the organic matter, as reported in Section 6.2.2, need to be stoichiometrically balanced. Metals that form amorphous metal sulfides, a chemical compound that can only be biogenic, probably provided this balance. Secondly, as stated in Section 5.5, Mn that is not biologically removable at the low pH level was efficiently removed only at the beginning of the operation when it was sorbed to the organic matter. After the sorption capacity of the organic matter was exhausted, Mn stayed in the aqueous phase while other metals (Zn, Cu, and Cd) were still being removed. Removal of these metals, with no organic-matter adsorption capacity present, can only be explained by formation of biogenic metal sulfides. Because of a small population of samples collected, the diagrams in Figure 6-6 through 6-8 may be affected by spatial variability of metal concentrations. Nevertheless, the diagrams indicate some general features and trends that might not be explainable and/or clearly conclusive and are worth noting as general observations. • Aluminum concentrations decrease along the flow paths within the first organic matter (Orl) and limestone chambers, then slightly increased in the second organic matter chamber (Or2) for Bioreactors II and IV. • In general, Fe demonstrated a gentle trend of decreasing concentrations, though not without exceptions (mostly in Bioreactor IV). High concentrations of Fe in the first lift for all bioreactors agree with visual observations during the autopsy process when the orange- color precipitate was observed on the HDPE walls of the CCS and within the organic matter itself. This precipitate, iron hydroxide, was most abundant within the first CCS lift. • Zinc seemed to be evenly distributed within Orl of Bioreactor II, but its concentration peaked in the centers of Orl of Bioreactors III and IV. • There is a distinct trend of decreasing concentration for Cu along the flow direction for all three bioreactors. • There seems to be an increasing trend for Mn concentration along the flow path, with the exception of a noticeable decrease in Mn concentration in the limestone chamber of Bioreactor IV. • Cadmium concentrations behaved erratically for Bioreactors III and IV but had a decreasing trend for Bioreactor II. In all three bioreactors, Cd concentrations varied the most of all the metals presented. • There was a decreasing trend for As concentrations within Bioreactors II and III and a similar but less obvious trend for Bioreactor IV. All bioreactors demonstrate much higher concentration of As in the most upgradient CCS lift (No. 1) than in the rest of the solid matrix sampled. For Bioreactor II, these much- elevated As concentrations extend to the second sampling point, lift No.3. • The elevated concentrations of As at the very front portion of the bioreactors correlate very well with high concentrations of Fe at the same location. Statistical correlation coefficients calculated for all possible pairs of the aforementioned metals are presented in Table 6-4. Outlined with heavy lines are three correlation coefficients that because of their high values need to be considered meaningful despite possible spatial variability of the metal concentrations that might have been missed by a small population of samples. These correlations coefficients are for Al and Cu, As and Fe, and Cd andZn. 43 ------- High correlation of Al and Cu may be explained by known observations that they both precipitate quickly when aqueous conditions change to a less acidic and more reducing environment. In such a case, Al precipitates as hydroxides, and Cu precipitates as sulfide. A strong dependancy of As and Fe has already been explained in Section 5.5 by referring to a statement by Robins and Huang (Ref 6) that As effectively adsorbs to Fe(OH)3. As mentioned earlier in this section, observations during the autopsy indicated an abundance of Fe(OH)3 precipitant in the bioreactors. The high correlation coefficient between As and Fe in the bioreactors support findings of Robins and Huang. High correlation coefficient for concentrations of Cd and Zn were expected due to the similarity in chemical behavior of these two elements. As it appears, a two orders of magnitude difference in their concentrations in the aqueous solutions (Figures 5-11 and 5-13) corresponds to their precipitates in the bioreactors where they also differ by two orders of magnitude. 6.2.2 Sulfate and Sulfide Analyses Figures 6-9 and 6-10 present diagrams for sulfate and sulfide profiles in the solid matrix of the bioreactors, respectively. These diagrams were plotted based on values included in Table 6-3c that were obtained from acid base accounting (ABA) analyses. The sulfide diagram (Figure 6-10) was assembled by adding values for insoluble sulfide to values of pyritic sulfides (both present in Table 6-3c) and converting the sum from percent to mg/kg. Figure 6-9 shows different changes of sulfate concentrations within the first organic matter chamber of each bioreactor. However, considering the high value for lift No. 7 in Bioreactor IV as an outlier or a sulfate "nugget," there is some resemblance of concentration of sulfates in Bioreactors II and IV. In both bioreactors, the concentration of sulfates decrease toward the limestone chambers and then rise to a similar level in the second organic matter chamber. Sulfate concentrations in Bioreactor III, which does not include a limestone chamber, shows a general rising trend throughout the organic matter chamber. These features of the sulfate profiles show the advantage of placing a limestone chamber in the bioreactors. Sulfide concentrations (Figure 6-10) within the first organic matter chamber are generally similar, except for CCS lift No. 9 in Bioreactor II. In general, sulfide concentrations in the organic matter chambers are above the 4,000 mg/kg level and are twice as high as concentrations of sulfates. The decrease of sulfide concentration in lift No. 9 of Bioreactor II to 3,300 mg/kg remains a conundrum. Its explanation can be sought in the fact that the relatively small number of samples collected (one every second lift) increases the chance for some samples not being representative of the actual hydrochemical conditions prevailing in the sampled medium. Numerical values for sulfide concentrations in the solid matrix of organic matter are more than three orders of magnitude greater than those for the aqueous phase in the limestone and cobbles chambers (Table 6-lc). The actual difference was even greater because of the conversion of mg/kg for the solid matrix to mg/L for the aqueous phase. This is because 1 cubic decimeter of solid matrix, which equals 1 liter in volume, weighs more than 1 kilogram. Sulfide load in organic matter, together with metal concentrations as addressed in Section 6.2.1, is indicative of metal sulfides precipitating in the organic matter chamber due to SRB activities. This postulate together with the observation of no precipitated metal in the cobble chambers of the bioreactors, indicates again that the role of the cobble section was limited to a sump for a small mass of precipitates that escaped from the organic matter chambers. 44 ------- 6.2.3 Total Organic Carbon Numerical data for TOC concentration in the bioreactors solid matrix are included in Table 6-3b. As expected, they indicate that the lowest TOC concentrations were associated with the limestone chambers: 8.7 % and 1.2% by weight for Bioreactors II and IV respectively. The TOC concentrations within the organic matter chambers ranged from 17% to 24% with the majority of values above 20%. These values of TOC concentrations document that there was plenty of organic carbon remaining in the organic matter chambers after 32 months of bioreactor operation. Although, TOC concentrations for the initial fresh organic matter were not measured, the high TOC values measured during the autopsy may indicate that the depletion of organic carbon would not be a factor for the efficient removal of metals even if these bioreactors were operating for several more years. 6.2.4 Plugs in the Outlets of the AMD Distribution System Manifold Many inlets in all three bioreactors were plugged with chemical precipitates located within the last 1 inch of the L-shaped inlet. Because the outlets (also called bioreactor inlets) were 2 inches in diameter, so were the plugs. The plugs had the appearance and consistency of gel. The upper half of a plug, the one facing the incoming AMD, was black. The lower half, which was in contact with the organic matter, was light brown. Analytical data for a plug collected from one of the outlets in Bioreactor III are tabulated in Table 6-3d and presented in graphical form in Figure 6-11. As implied by a sharp boundary between black and brown portions of the plug, the concentrations of metals in each portion were different. The main differences were the concentrations of Al, Fe, Ca, and Mg (the last two are not shown in the figures). The Al concentration in the black portion of the plug was much higher (approximately 540% higher) than the Al concentration in the brown portion. Conversely, the concentrations of Fe, Ca, and Mg were lower in the black portion (i.e., approximately 45%, 26%, and 15%, respectively, of those in the brown portion). With the exception of Fe, Al, and As, the metal concentrations in the plug were in the same range as those determined for samples collected from the CCS lift. Iron concentration in the brown portion of the plug was nine-fold of that found in the adjacent CCS lift and constituted 12.7% of the weight of the plug. Since it is expected that the majority of the Fe present in that place in the system was associated with Fe(OH)3, this compound made up 24% of weight for the lower portion of the plug. An elevated (five times higher than in adjacent CCS lift) concentration of As was certainly the result of its adsorption to Fe(OH)3, as explained earlier in this report. Aluminum concentration in the upper portion of the plug was more than six-fold of that determined for the adjacent CCS lift. Assuming its presence as a hydroxide, this compound made up 16% of weight for the upper portion of the plug. The high percentage of Fe and Al hydroxides in the plug indicates a high probability that the bioreactor inlets were plugging mainly due to chemical reactions; the sediment carried by the unfiltered AMD was a secondary reason for plugging. 6.3 Flow Pattern Visual observations made during the autopsy of the bioreactors indicated no preferential flow paths developed during the 32-month operation period. This means that the AMD flowed and was treated throughout the entire cross section of the organic matter. Most of the individual cells of the CCS were found full of organic matter. Probably less than 5% of cells were voided of the organic matter in their top portions, with voids never taller than 2 inches. There was no discernible pattern of these voids that seemed to be uniformly distributed throughout each individual CCS lift. 45 ------- 6.4 Sulfate-Reducing Bacteria Population on the Solid Matrix Although the SRB population in the aqueous phase of the bioreactors was measured during the operating period, no measurements of SRB population attached to the solid matrix of the bioreactors were made. To measure the final population of SRB within the solid matrix, samples of both the organic matter and limestone were collected during the autopsy and submitted to a bacteriological laboratory for an SRB count. The results returned were expressed in MPN of SRB per gram (MPN/g) of wet weight of the solid matrix for organic matter and in MPN of SRB per sample for the limestone. Therefore, the results needed to be converted and expressed as a function of the volume of the matrix. This conversion was needed to compare the SRB population in the aqueous and solid phases using the same dimensions (i.e., MPN per volume). To make the aforementioned conversion for the organic matter, an attempt was made to determine its particle density. This presumably simple task was not successful because standard methods used for determination of particle density of solids did not seem to be applicable for organic matter. Therefore, the SRB population per unit volume was determined as a function of particle density. To accomplish this, a geometric mean of MPN/g for SRB populations in eight samples collected from all three bioreactors was calculated. This value, 1.83E+06 MPN/g, and the average volumetric moisture content of 58.5% were used to determine SRB populations as a function of particle density. Thus, the values for SRB populations were expressed as MPN per unit volume of wet organic matter, and they ranged from 1.45E+06 MPN per cubic centimeter (MPN/cc) to 2.63E+6 MPN/cc for the particle density ranging from 0.5 grams per cubic centimeter (g/cc) to 2.05 g/cc, respectively. Assuming particle density of 1.3 g/cc, the MPN for SRB population in 1 cc of wet organic matter was 2.06E+6 MPN/cc. This population of SRB attached to the solid matrix of the organic matter is two orders of magnitude greater than the value of 2.81E+4 MPN/milliliter (mL) calculated as a geometric mean for three aqueous samples collected during the last sampling event on July 9, 2001. To determine SRB population per unit volume of the limestone chamber, an average surface area for the limestone was calculated. This was necessary because the SRB populations on the limestone were determined by the bacteriological laboratory by scraping the biofilm from the sampled limestone and then expressing the population of SRB as MPN per sample. The calculations performed resulted in a determination of SRB population at 8.2E+4 MPN/cc. This SRB population is of the same order of magnitude as for the aqueous samples. The unit of cubic centimeter for the limestone population estimate is used in this case for comparison purposes only. Actually, a more representative unit of volume would have been cubic meter since the limestone ranged in size from 5 centimeters (cm) to 15 cm in diameter. 6.5 Toxicity Characteristic Leaching Procedure The results of TCLP testing demonstrated that the solid matrix of the bioreactors did not exhibit characteristics of toxicity as it was documented by concentrations of Cd and As, the only metals listed in 40 Code of Regulations that might be of concern for the project. Cadmium and As concentrations in leachate from samples of organic carbon matter were 7.6 and 136 times lower, respectively, than the regulatory levels that would classify this matrix as hazardous waste. 46 ------- Tables 6-1. Analytical Results of the Aqueous Samples Collected During the Autopsy Table 6-la. Total and Dissolved Metals Location Al Bioreactor II cobble . ,, OA . . UoU chamber Bioreactor III cobble chamber Bioreactor IV . „ cobble chamber Location Al Bioreactor II ^1QQ limestone chamber Bioreactor II cobble ,_. „„ chamber Bioreactor III *,A~,nn ,,. , , 34300 cobble chamber Bioreactor IV 7^00 limestone chamber Bioreactor IV 9240 cobble chamber Table 6-lb. Iron speciation As Cd 29.3 4.8 16.8 4.8 8.5 4.8 As Cd 251 86.1 53.1 5.3 53.1 5.9 Dissolved metals (• g/L) Ca Cu Fe Mg 13500 86.8 2910 29400 87000 220 2830 20000 124000 38.7 981 33300 Total metals samples (• g/L) Ca Cu Fe Mg 1040000 2450 43000 55100 152000 951 52200 45100 109000 592 42300 36200 30 11.4 539000 506 13200 42800 20 4.8 122000 159 12700 35200 Mn Zn 1760 506 2720 1030 1980 329 Mn Zn 7270 20400 3000 2150 5280 3770 3160 6390 2170 856 Iron Species (mg/L) (nonfiltered samples) Location Bioreactor II limestone chamber Bioreactor II cobble chamber Bioreactor III cobble chamber Bioreactor IV limestone chamber Bioreactor IV cobble chamber Table 6-lc. Sulfate and Sulfide Fell 1.9 3.6 2 0.93 1 Fe III Fe Total 0.17 2.1 <0.05 3.5 <0.05 2.1 0.057 0.99 0.076 1.1 % Fe Recovery 100 100 100 100 100 Sulfate and Sulfide concentrations (mg/L) (nonfiltered samples) Location Bioreactor II limestone chamber Bioreactor II cobble chamber Bioreactor III cobble chamber Bioreactor IV limestone chamber Bioreactor IV cobble chamber Sulfate 3091 2401 352 225 1591 Sulfide 3.2 3.9 2.2 3 4.2 'Corrected values obtained using inductive coupled plasma (ICP) method 47 ------- Table 6-2. Comparison of the Dissolved Metals from the Autopsy and Monthly Sampling Sample Bioreactor , ,. location Date Zn Cu Dissolved metal concentration (ng/L) Mn Cd As Al All1 07/9/01 899 110 996 Bottom2 07/17/01 506 87 1,760 AIII1 07/9/01 1,010 122 2,470 III Bottom2 07/17/01 1,030 220 2,720 AIV1 07/9/01 292 18 1,270 IV Bottom2 07/16/01 329 39 1,980 1 See Figure 3-5 for location 2 Bottom of the cobble chamber adjacent to the organic matter chamber Tables 6-3. Analytical Results of the Solid Matrix Samples Collected During Table 6-3a. Metal Concentrations Location Bioreactor II manure chamber 1 lift 1 Bioreactor II manure chamber 1 lift 3 Bioreactor II manure chamber 1 lift 5 Bioreactor II manure chamber 1 lift 7 Bioreactor II manure chamber 1 lift 9 Bioreactor II limestone chamber Bioreactor II manure chamber 2 lift 5 Bioreactor III intake plug - brown Bioreactor III intake plug - black Bioreactor III manure chamber 1 lift 1 Bioreactor III manure chamber 1 lift 3 Bioreactor III manure chamber 1 lift 5 Bioreactor III manure chamber 1 lift 7 Bioreactor III manure chamber 1 lift 9 Bioreactor IV manure chamber 1 lift 1 Bioreactor IV manure chamber 1 lift 3 Bioreactor IV manure chamber 1 lift 5 Bioreactor IV manure chamber 1 lift 7 Bioreactor IV manure chamber 1 lift 9 Bioreactor IV limestone chamber Al 7830 13700 10900 6880 4700 2650 3730 10300 55800 8600 4020 6060 5500 4550 15100 13000 10900 5680 3890 2400 5 5 6 5 5 5 the Autopsj Metal concentrations of the solid matrix samples (ing/Kg) As Cd Ca Cu Fe 42.1 31.4 23.6 22.8 15.4 8.3 10.1 400 485 76.1 24 19.1 17.8 15.8 109 18.2 14.9 22 14.4 4.2 23.4 31.9 21.6 28.6 12.8 3.8 2.4 25.7 31.2 24.3 10.1 35.5 5 18.7 10.5 10.8 59.3 19.4 0.92 0.91 8490 10400 6350 12300 38800 208000 11500 5700 1480 9560 13100 10500 15400 12600 2740 6130 9460 16600 19100 274000 1470 2970 2260 975 438 163 147 7340 8560 1480 241 596 579 203 2400 2340 1670 562 107 58.9 11400 9960 6910 9040 7010 4150 7280 127000 57800 14000 7710 8280 8970 8370 22900 7390 6580 7830 9210 4050 4 29 3 17 1 9 Mg 5170 9890 6990 7640 6480 5110 5920 3980 613 6180 6670 7130 6680 5210 4750 6130 7600 5290 4930 4800 118 1,380 80 353 42 162 Mn 931 902 387 1200 884 740 866 25200 35100 637 940 783 912 1180 263 402 668 655 800 185 Fe 707 2,910 638 2,838 572 981 Zn 4450 3790 3470 4270 2720 1000 3010 2530 2100 3630 3820 5790 2900 4880 1660 1600 7690 6400 1610 566 48 ------- Table 6-3a. Metal Concentrations Location Al Metal concentrations of the solid matrix samples (ing/Kg) As Cd Ca Cu Fe Mg Mn Zn Bioreactor IV manure chamber 2 lift 5 4700 19.9 0.92 18500 178 10300 7110 1500 583 Table 6-3b. Nitrogen, Phosphorus, and TOC Analytical chemistry for solid matrix samples (mg/Kg) Location Total Nitrogen Phosphorous TOC (%) Bioreactor II manure chamber 1 lift 1 Bioreactor II manure chamber 1 lift 3 Bioreactor II manure chamber 1 lift 5 Bioreactor II manure chamber 1 lift 7 Bioreactor II manure chamber 1 lift 9 Bioreactor II limestone chamber Bioreactor II manure chamber 2 lift 5 Bioreactor III manure chamber 1 lift 1 Bioreactor III manure chamber 1 lift 3 Bioreactor III manure chamber 1 lift 5 Bioreactor III manure chamber 1 lift 7 Bioreactor III manure chamber 1 lift 9 Bioreactor IV manure chamber 1 lift 1 Bioreactor IV manure chamber 1 lift 3 Bioreactor IV manure chamber 1 lift 5 Bioreactor IV manure chamber 1 lift 7 Bioreactor IV manure chamber 1 lift 9 Bioreactor IV limestone chamber Bioreactor IV manure chamber 2 lift 5 10251 14623 12259 12141 12983 3056 13086 10917 15533 10406 11074 6689 8308 13528 11063 10501 9920 3963 10682 58.2 86.1 53 62.1 124 144 65.9 74.4 104 44.1 119 72.4 60.4 90.4 78.9 66.1 68.4 22 168 20.0 20.0 22.3 23.4 17.2 8.7 23.3 22.6 21.6 20.7 21.2 21.4 24 22.1 23.5 21.6 23.9 1.2 16.9 Table 6-3c. Sulflde Acid-Base Accounting Sulfide Analysis by Acid-Base accounting Location Bioreactor II manure chamber lliftl Bioreactor II manure chamber 1 lift 3 Bioreactor II manure chamber 1 lift 5 Bioreactor II manure chamber 1 lift 7 Bioreactor II manure chamber 1 lift 9 Bioreactor II limestone chamber Bioreactor II manure chamber 2 lift 5 Bioreactor III manure chamber 1 lift 1 Bioreactor III manure chamber 1 lift 3 Bioreactor III manure chamber 1 lift 5 Bioreactor III manure chamber 1 lift 7 Bioreactor III manure chamber 1 lift 9 Total % Sulfur 0.79 0.70 0.63 0.69 0.46 0.15 0.87 0.76 0.82 1.12 1.02 1.10 J.J.UI »» tHC» Extractable S % Sulfate 0.20 0.19 0.03 0.18 0.07 0.07 0.25 0.23 0.28 0.44 0.33 0.52 HQ Extractable S % Insoluble Sulfide 0.04 0.03 0.14 0.05 <0.01 <0.01 0.06 0.04 0.08 0.13 0.11 <0.01 HNO3 Extractable S % Pyritic Sulflde 0.48 0.44 0.41 0.41 0.33 0.17 0.50 0.37 0.32 0.41 0.41 0.56 Residual S % Organic 0.08 0.05 0.06 0.05 0.07 0.02 0.06 0.12 0.14 0.14 0.17 0.06 49 ------- Table 6-3c. Sulflde Acid-Base Accounting Sulflde Analysis by Acid-Base accounting Location Bioreactor IV manure chamber 1 lift 1 Bioreactor IV manure chamber 1 lift 3 Bioreactor IV manure chamber 1 lift 5 Bioreactor IV manure chamber 1 lift 7 Bioreactor IV manure chamber 1 lift 9 Bioreactor IV limestone chamber Bioreactor IV manure chamber 2 lift 5 Total % Sulfur 0.68 0.72 0.84 1.19 0.90 <0.01 0.69 AAJJl »» illCl Extractable S % Sulfate 0.21 0.23 0.27 0.81 0.29 <0.01 0.29 HQ Extractable S % Insoluble Sulflde 0.06 0.02 0.03 <0.01 0.06 <0.01 <0.01 HNO, Extractable S % Pyritic Sulflde 0.32 0.42 0.46 0.47 0.49 <0.01 0.32 Residual S % Organic 0.10 0.05 0.08 0.14 0.07 <0.01 0.08 Table 6-3d. Analytical Results for Plugging Material in the Manifold of Bioreactor HI Total metals in plugging material (ing/Kg) Location Al As Cd Ca Cu Fe Mg Mn Zn Reactor III intake plug - brown Reactor III intake plug - black 10300 55800 400 485 25.7 31.2 5700 1480 7340 8560 127000 57800 3980 613 25200 35100 2530 2100 Table 6-4. Correlation Coefficients (•) for Total Metals Concentrations in the Solid Matrix of Bio reactors H, HI, and TV Al Al 1 As Cd Cu Fe Mn Zn As 0.602 1 Cd 0.475 0.095 1 Cu 0.9703 0.5278 0.476 1 Fe 0.5622 0.9434 0.0175 0.4464 1 Mn -0.376 -0.281 0.0003 -0.39 -0.096 1 Zn 0.1745 -0.0387 0.8375 0.1559 -0.0747 0.1254 1 50 ------- Total vs Dissolved Metals for the Cobble Chamber of Bio reactor II Fe Cu Metals concentration in ug/L # Mn D Total metals D Dissolved metals Cd Figure 6-la. Total vs. dissolved metals for the cobble chamber of Bioreactor II. Suspended Metals in the Cobble Chamber of Bioreactor II Metals concentration in ug/L Cu # Mn D Total minus Dissolved Figure 6-lb. Suspended metals in the cobble chamber of Bioreactor IL 51 ------- Total vs Dissolved Metals for the Cobble Chamber of Bio reactor III Metals concentration are in ug/L Cu Mn D Total metals D Dissolved metals Figure 6-2a. Total vs. dissolved metals for the cobble chamber of Bioreactor IIL Suspended Metals in the Cobble Chamber of Bioreactor III Metals concentration in ug/L Fe -? Mn D Total minus Dissolved Cd Figure 6-2b. Suspended metals in the cobble chamber of Bioreactor IIL 52 ------- Total vs Dissolved Metals for the Cobble Chamber of Bioreactor IV Metals concentration are ug/L D Total metals D Dissolved metals Cd Figure 6-3a. Total vs. dissolved metals for the cobble chamber of Bioreactor IV. Suspended Metals in the Cobble Chamber for Bioreactor IV Metals concentration in ug/L s Mn D Total minus Dissolved Figure 6-3b. Suspended metals in the cobble chamber of Bioreactor IV. 53 ------- Iron Speciation for Autopsy Aqueous Samples Bioreactor II Bioreactor II cobble Bioreactor III cobble Bioreactor IV Bioreactor IV cobble limestone chamber chamber chamber limestone chamber chamber Location Figure 6-4. Iron speciation for autopsy aqueous samples. Total Metals in the Limestone and Cobble Chambers in Bioreactor II (Aqueous Samples) Figure 6-5. Total metals in the limestone and cobble chambers in Bioreactor II (aqueous samples). 54 ------- Total Metals Concentration in Solid Matrix of Bioreactor II Limestone Chamber 2 Sample location Figure 6-6. Total metals concentration in the solid matrix of Bioreactor IL Total Metals Concentration in Solid Matrix of Chamber 1 in Bioreactor III 1II/5/1 Sample location Figure 6-7. Total metals concentration in the solid matrix of Chamber 1 in Bioreactor III. 55 ------- Total Metals Concentration in Solid Matrix of Bioreactor IV Limestone Chamber 2 Sample location Figure 6-8. Total metals concentration in the solid matrix of Bioreactor IV. Sulfate Profiles in Solid Matrix of Bioreactors .2 5000 „. 1st organic chamber Flow ^. & ^~ •!! solid matrix ^^^^III solid matrix • • >IV solid matrix • II aqueous matrix (mg/L) • III aqueous matrix (mg/L) A IV aqueous matrix (mg/L) / i \ i \ f \ g i i i S t ^r / /^^/ l ^^\" '"' Ss x^x^ ^^' "~H limestone chamber ~^A'' 2nd i cobble organic ! chamber chamber i i | i f \ V I / \ i 7th lift Location 9th lift Figure 6-9. Sulfate profiles in the solid matrix of the bioreactors. 56 ------- Sulflde Profiles in Solid Matrix of Bioreactors -a Flow _ "X ^M ^ 1st organic chamber x/"s-r- «• „. _S / ^ ^/'"" + ^~ "II solid matrix ^^^^III solid matrix ~ • 'IV solid matrix • II aqueous matrix (mg/L) III aqueous matrix (mg/L) A IV aqueous matrix (mg/L) ^***7* * ' » X ' X t \ 1 \ • . 1 \ • \ ' limestone chamber / / V / 1 / 1 J g • , * 1 « f « 1 2nd organic ch^nber / / f g i r g g g cobble chamber 5th lift 7th lift 9th lift Location Figure 6-10. Sulfide profiles in the solid matrix of the bioreactors. I Bioreactor III Inlet-Plug Metals Concentration D Reactor III intake plug - lower (brown) portion • Reactor III intake plug - upper (black) portion Figure 6-11. Bioreactor III inlet-plug metals concentrations. 57 ------- 7. Quality Assurance/Quality Control 7.1 Background Following is a summary of the Quality Assurance (QA) activities associated with the project. Samples were collected according to the schedule outlined in the approved project-specific QAPP document. Performance data were collected monthly for 32 months. All field and laboratory data available had been evaluated to determine the usability of the data. Dissolved metals (Al, Cd, Cu, Fe, Mn, and Zn) analysis and field pH measurements were classified as critical analyses for this project. A critical analysis is an analysis that must be performed in order to determine if project objectives were achieved. Data from noncritical analyses were also evaluated. 7.2 Project Reviews During the project, the following evaluations were performed: - internal field systems review at the demonstration site; and - external Technical Systems Audit (TSA). 7.2.1 Internal Field Systems Review at the Demonstration Site A field systems review was performed on October 10, 1999, at the Calliope Mine. The field systems review included a review of the following items: - personnel, facilities, and equipment; - documentation (chain-of-custody, logbooks); - calibration of equipment; and - sampling procedures. No concerns were identified during the audit. 7.2.1.1 Personnel, Facilities, and Equipment Personnel present during the audit included John Trudnowski, MSB Project Engineer; Rod Schwab, MSB Sampler; and Ken Reick, MSB Project QA Officer. The Project Engineer and Sampler were knowledgeable about the demonstration and their duties and responsibilities at the demonstration site. 7.2.1.2 Documentation Chain-of-custodies (COC) were reviewed at the demonstration site, and all COC procedures were being followed. The project logbooks were also reviewed. The sampling logbook was very thorough and included spaces where specific information was required. Sampling personnel were familiar with the logbook format and COC procedures. The sampling logbook did not conform to the Standard Operating Procedures (SOP) because the pages of the logbook were not numbered consecutively as stated in the SOP. 7.2.1.3 Calibration of Equipment Field equipment was used to measure pH, dissolved oxygen, temperature, and EH potential. This information was recorded in the project logbooks. All meters were properly calibrated prior to performing measurements. Standard operating procedures were available at the demonstration site and explained how to calibrate/operate the meters. Sampling personnel were familiar with the SOPs and requirements for routine calibration of the various meters. Measurement of pH in the water samples is a critical measurement and, therefore, has had quality control (QC) objectives already assigned. The quantitative objectives are accuracy, precision, and completeness. The absolute difference between the measurement and the buffer pH is reported as accuracy. Precision is based on consecutive determinations of accuracy. During sampling, accuracy was determined by measuring the appropriate buffer; however, it was performed only once (this QC check was well within the required limits). Because consecutive determinations were not performed, precision could not be determined. For future sampling events, the samplers were 58 ------- required to determine the accuracy and precision of the pH measurements. As a corrective action for the audit observations, an amended QAPP was prepared. The need for accuracy and precision measurements associated with critical field measurements were reiterated in annual MSB internal sampling refresher training. 7.2.1.4 Sampling Procedures A review of sampling activities was also performed during the systems review. All sample collection procedures and equipment decontamination procedures were followed by sampling personnel. 7.2.2 External Technical Systems Audit In addition to the internal field systems review by MSB, an external TSA of both the project and the HKM Laboratory was performed by Joe Evans of Science Applications International Corporation (subcontractor to EPA) during the week of September 25, 2000. There were no findings resulting from the audit; however, there were three observations. All three observations related to making minor changes to the QAPP to more accurately reflect field procedures. An amended project-specific QAPP was prepared as a result of the audit. 7.3 Data Evaluation The data quality indicator objectives for field pH measurements and dissolved metals analysis were outlined in the QAPP and were compatible with project objectives and the methods of determination being used. The data quality indicator objectives were method detection limits (MDLs) for accuracy, precision, and completeness. Control limits for each of these objectives are summarized in Table 7-1. In addition to the data quality indicators listed in Table 7-1, HKM also analyzed internal QC checks, including calibration, calibration verification checks, calibration blanks, method blanks, and laboratory control samples. These QC checks have also been evaluated for the purpose of this data review. 7.4 Validation Procedures Data that were generated throughout the project were validated. The purpose of data validation is to determine the usability of data generated during a project. Data validation consists of two separate evaluations: 1) an analytical evaluation and 2) a program evaluation. 7.4.1 Analytical Evaluation An analytical evaluation is performed to determine the following: - that all analyses were performed within specified holding times; - that calibration procedures were followed correctly by field and laboratory personnel; - that laboratory analytical blanks contain no significant contamination; - that all necessary independent check standards were prepared and analyzed at the proper frequency and that all remained within control limits; - that duplicate sample analysis was performed at the proper frequency and that all Relative Percent Differences (RPD) were within specified control limits; - that matrix spike sample analysis was performed at the proper frequency; and - that all spike recoveries (%R) were within specified control limits. Measurements that fall outside of the control limits specified in the QAPP, or for other reasons were judged to be outlier, were flagged appropriately to indicate that the data were judged to be estimated or unusable. An analytical evaluation was performed to determine the usability of data that were generated by the HKM Laboratory for the project. Laboratory data validation was performed using 59 ------- Reference 7 as a guide. The QC criteria outlined in the QAPP were also used to identify outlier data and to determine the usability of the data for each analysis. A summary of QC check results for the critical dissolved metals and pH analyses are presented in Table 7-2. All data requiring flags are summarized in Table 7-3. In addition to the analytical evaluation, a program evaluation was performed. 7.5 Program Evaluation Program evaluations include an examination of data generated during the project to determine: - that all samples, including field QC samples, were collected, sent to the appropriate laboratory for analysis, and were analyzed and reported by the laboratory for the appropriate analyses; - that all field blanks contained no significant contamination; and - that all field duplicate samples demonstrated precision of field as well as laboratory procedures by remaining within control limits established for RPD. Program data that were inconsistent or incomplete and did not meet the QC objectives outlined in the QAPP were viewed as program outliers and were flagged appropriately to indicate the usability of the data. 7.5.1 Field QC Samples In addition to internal laboratory checks, field QC samples were collected to determine overall program performance. 7.5.2 Field Blanks None of the field blanks collected for the project showed significant contamination for dissolved metals analysis, with two exceptions. The field blank collected on June 3, 1999, showed significant contamination (greater than the Contract Required Detection Limit under the Contract Laboratory Program). Iron samples with concentrations less than 10 times the contamination concentration in the field blank were flagged "U." A "U" flag indicates the data are undetected below the associated value. Another field blank collected on August 11, 1999, showed significant contamination for manganese analysis; however, all of the sample concentrations were greater than 10 times the contamination, so no samples required a flag. Early in the project, several field blanks showed significant contamination for cadmium and zinc. The problem was investigated and traced to the holding tank being used to store deionized (DI) water. A clean tank solved the problem. Samples were not flagged because the contamination source was not linked to contamination problems resulting from sampling procedures. 7.5.3 Field Duplicates All field duplicates collected were within control limits for all analyses, with the four exceptions. A field duplicate collected on January 8, 2001, was out of control for aluminum analysis. While EPA does not specify control limits for field duplicates, the data reviewer is allowed discretion when evaluating field duplicates. For this project, precision control limits of • 35% RPD were used for field duplicates. As a result, the samples from the January 8, 2001, event were flagged "J." A "J" flag indicates that the associated value is estimated. Field duplicates samples collected on April 5, 1999, and February 12, 2001, were out of control for copper analyses, resulting in samples from these events being flagged "J" for copper. A field blank collected on February 3, 1999, was out of control for iron analysis, and associated samples were flagged "J." 7.6 Summary All data from the HKM Laboratory were validated according to EPA guidelines and the project- specific QAPP. Some of the data were flagged for various reasons and are summarized in Table 7-3. 60 ------- The importance of calibration of field meters should be reiterated to sampling personnel because the lack of calibration resulted in critical pH data from two sampling events being discarded. On a positive note, the data were very organized, which made the data evaluation process much easier. MWTP, Activity III, Project 12 presented unique challenges for the sampling and analytical team. While several of the data points were qualified for various reasons, this multi-year project produced high quality data. Table 7-1. Data Quality Indicator Objectives Parameter Matrix Unit MDLa Precision1" Accuracyc0 Completeness11 pH Aqueous SU N/A ±0.1e ±0.1f 95% Dissolved Metals Aqueous jjg/L 5 -20% 75-125% 95% aMDLs were based on what is achievable by the methods and what is necessary to achieve project objectives and account for anticipated dilutions to eliminate matrix interferences. MDLs were adjusted as necessary when dilutions of concentrated samples are required. bRelative percent difference of analytical sample duplicates. 'Percent recovery of matrix spike, unless otherwise indicated. dBased on number of valid measurements compared to the total number of samples. Trecision of pH measurements was based on consecutive determinations of accuracy. 'Accuracy of pH measurements was based on the absolute difference between accepted value of the buffer and the measured value of the buffer. Table 7-2. Summary of QC Checks for Critical Field pH Measurements and Dissolved Metals Analysis Analysis Field pH Measurements Mean Absolute Difference (Precision) 0.02 SU Range of Absolute Differences (Precision) 0-0.09 SU Analysis Dissolved Al Dissolved As Dissolved Cd Dissolved Cu Dissolved Fe Dissolved Mn Dissolved Zn Analysis Field pH Measurements Mean RPD for Sample Duplicates 9.9% 7.4% 2.8% 8.4% 4.6% 2.6% 8.2% Mean Absolute Difference (Accuracy) 0.02 SU Range of RPDs for Sample Duplicates 0-28.3% 1.3-18.8% 0.9-8% 0-50.6% 0-12.6% 0-8.4% 0-70.3% Range of Absolute Differences (Accuracy) 0-0.09 SU Analysis Dissolved Al Dissolved As Dissolved Cd Dissolved Cu Dissolved Fe Dissolved Mn Dissolved Zn Mean Matrix Spike Recovery 101% 95.5% 99.6% 100.4% 101.1% 102.2% 98.2% Range of Matrix Spike Recoveries 82-131% 58.9-114% 88-110% 84-115% 82.2-134% 79.3%-127% 69.6-114.8% 61 ------- Table 7-3. Date1 12/23/98 01/04/99 02/03/99 Summary of Qualified Data for MWTP Activity IE, Project 12 Sample Analysis QC ID Criteria BDRB Dissolved Analytical D- Cu Duplicate Blank IAD IILD IIIA IVKD IVKDD IVKDS HID Alkalinity Initial IIIJD (forms) Calibration IVKD Verification IIIA ID Dissolved Field IIIA Fe Duplicate IIIAFC IIIJD IILD IVKD Control Result Flag2 Comment Limit • 20% RPD 50.6 J Samples were flagged "J" due to an out-of-control field duplicate. 90- 110% recovery 63% R Initial calibration recovery verification had <75% recovery; therefore, the data should be removed from consideration. • 35% RPD 47.4% RPD J Flag samples "J" for out- of-control field duplicate. 04/05/99 Field pH Holding Time Analyze immediately Hours elapsed 04/05/99 ID IIIA IIIAFC IIIJD IILD IVKD 06/03/99 ID IIIA IIIAFC IIIJD IILD IVKD Dissolved Cu Field Duplicate • 35%RPD 165% RPD Because pH readings were not performed immediately in the field but instead analyzed in the laboratory following transport to Butte, the pH readings from this sampling event should be considered estimated and flagged with a "J." Flag samples "J" for out- of-control field duplicate. Dissolved Fe Field Blank 100 ppb 128 ppb U Flag samples with concentrations less than 10 times the contamination concentration "U" due to out-of-control field blank. 62 ------- Date1 7/12/99 08/11/99 08/11/99 04/05/00 06/07/00 12/04/00 Sample ID BDRB FB ID IIIA IIIAFC IIIJD IILD IVKD BDRB FB ID IIIA IIIAFC IIIJD IILD IVKD BDRB FB ID IIIA IIIAFC IIIJD IILD IVKD BDRB FB ID IIIA IIIAFC IIIJD IILD IVKD Intake IIBD IIIBD IVB IILD IIIJD IVKD Analysis QC Criteria Dissolved Matrix Spike Zn Analytical Duplicate Dissolved Matrix Spike Zn Field pH Calibration Dissolved Matrix Spike As Dissolved Serial Dilution Zn Field pH Calibration Field ORP Control Result Limit 75- 125% recovery 69.6% • 20% RPD 70.3% 75- 125% recovery 72.8 Required each Not sampling event performed 75- 125% recovery 58.9 • 10% difference 15.7% Required each Not sampling event performed Required each Not sampling event performed Flag2 Comment J Zinc results should be flagged "J" as estimated for out-of-control spike and duplicate. J Zinc results should be flagged "J" as estimated for out-of-control matrix spike. R Flag samples "R" as unusable due to lack of calibration documentation. J Arsenic results should be flagged "J" as estimated for out-of-control matrix spike. J Zinc results should be flagged "J" as estimated due to out-of-control serial dilution analysis. R Flag samples "R" as unusable due to lack of calibration documentation. 63 ------- Date1 Sample Analysis QC ID Criteria 1/8/01 Intake Dissolved Field IIBD Al Duplicate IIIBD IVB IILD IIIJD IVKD 2/12/01 Intake Dissolved Field IIBD Cu Duplicate IIIBD IVB IILD IIIJD IVKD 3/14/01 IIBD Sulfate Outlier 4/09/01 Intake Sulfate Outlier 7/20/01 RCIIC Dissolved Matrix Spike OB Al RCIIIC OB Dissolved RCIVC Fe OB Dissolved Mn Control Result Flag2 Comment Limit • 35% RPD 40. 1% RPD J Flag samples " J" for out- of-control field duplicate. • 35% RPD 40. 1% RPD J Flag samples " J" for out- of-control field duplicate. Rosner's Test for l,400mg/L R Sample is outlier Outliers is an outlier according to Rosner' s test. at a 5% Data were transformed to significance achieve normal level distribution prior to performing outlier test. The data should be removed from further consideration. Rosner's Test for 8 mg/L is an R Sample is outlier Outliers outlier at a according to Rosner' s test. 5% Data were transformed to significance achieve normal level distribution prior to performing outlier test. The data should be removed from further consideration. 75-125% recovery 130.7% J Flag samples "J" for out- of-control matrix spike. 134.5% 127.5% 1 Date that the samples were collected. 2 Data Qualifier Definitions: U-The material was analyzed for but was not detected above the level of the associated value (quantitation or detection limit). J-The sample results are estimated. R-The sample results are unusable. UJ-The material was analyzed for but was not detected, and the associated value is estimated 64 ------- 8. Recommended Design Improvements To minimize AMD stratification with respect to ORP and thus increasing bioreactors efficiency, the bioreactors need to be covered with a plastic liner that would minimize oxygen intrusion either directly from the atmosphere or through atmospheric precipitation. The layer of two lifts of straw bails used at the demonstration site was not sufficient. Because the biochemical reactions take place within the organic matter where they also precipitate, there is no need for a large cobble chamber. If a preventive "trap sump" to collect a small mass of precipitates that might escape from the organic matter chamber needs to be included in the design, it should have its outlet placed high above its bottom. A trap sump could be designed as a flow-through container or a small lined retention pond filled with cobbles supporting a plastic liner covering the pond. Other designs could also be considered, provided they would minimize the possibility of agitation of the collected effluent of the bioreactor by atmospheric conditions (wind and precipitation) or human and animal encounters. The abundance of TOC present in the organic matter chamber at the end of the project demonstrates that the bioreactors would have worked equally efficiently with a much smaller supply of organic carbon, provided the same residence time of AMD was maintained. Since most of the organic matter mass inhibits permeability, it is prudent to reduce the ratio of organic matter and the permeability enhancing component (e.g., gravel, shell, etc.) and have a more permeable medium. Although not explicitly indicated by the demonstration project results, the straw added to the organic matter does not seem to be an appropriate material to increase the permeability of the medium. More rigid material like walnut shells or even gravel, added in high proportion to the organic matter, is needed to effectively increase permeability. Since most of the plugging material that restricted the flow was found within and adjacent to the outlets of the AMD distribution system, there is a need to devise a system that would allow for occasional breakdown and removal of the plugging material. Such a system may need to involve only a few outlets rather than the three dozen used in this demonstration. It might include ports extended to the ground surface that would facilitate blowing in combustion engine exhaust to destroy plugging material that would then be removed by bailing. If any instrumentation for automatic measurements of the AMD quality and quantity is used, it is recommended that a filter be installed upstream of the sensor. 65 ------- 9. Conclusions A CCS built in the bioreactors worked very well in preventing settling of organic matter and ensuring uniform flow of AMD throughout the entire cross section of the organic matter with no preferential flow paths (channeling). Most of the individual cells of the CCS were found full of organic matter, with probably less than 5% of the cells void of the organic matter in their top 2-inch portions. This indicates that the organic matter could have been packed less tightly and still conform to the design parameters that allowed approximately 3 inches of settling. Configuring the bioreactors to accommodate flow in a horizontal plane (rather than in the vertical direction) was successful. Problems that were experienced with reduction in the flow rate turned out to be associated with the AMD distribution system that plugged with chemical J r OO precipitates. However, this hindrance is common to both configurations. It takes some time for the SRB to be established in a new bioreactor. Once established and supplied with organic carbon, they maintained a population of E+4 MPN/mL or higher in an aqueous phase at temperatures ranging from 2 •Cto 16-C. Winter freezing of a well-established SRB population has little or no effect on their activity for the remainder of the year. The SRB average population of 2.06E+6 MPN/cc attached to solid matrix of organic matter was two orders of magnitude greater than the value of 2.81 E+4 MPN/mL calculated as an average SRB population in the aqueous samples collected during the last sampling event on July 9, 2001. The SRB population attached to the limestone was of the same order of magnitude (E+4) as for the aqueous samples. High sulfide load in the organic matter, together with high concentrations of metals, is indicative of metal sulfides precipitating in the organic matter chamber due to SRB activities. A drop in the SRB population in July of 2000, which paralleled the 100% increase of the flow rates, might indicate flushing out of the bacteria at that flow velocity. Although it appeared a limestone chamber slightly increased effluent pH, its role was not dominant for the overall performance of the bioreactors. The EH values measured in the most downgradient piezometer show that with the exception of a few periods, the EH values were positive. However, data acquired during the EH profiling of the bioreactor document that the EH values in the deep portion of the organic matter were approximately 50 mV and 150 mV lower for Bioreactors II and III respectively. This is an important observation because it confirms that reduced conditions, which are required for the SRB activity, were present within the portions of the bioreactors where most of the biochemical reactions took place and where most metals precipitated. The bioreactors were notably stratified with respect to EH. In Bioreactor II, it was up to 400 mV lower at the bottom of the bioreactor than close to the surface. This difference, thus also stratification, diminished downgradient and close to the outlet from the bioreactors due to mixing of water flowing through the cobble section. It is postulated that the EH 66 ------- stratification was caused by an inadequate isolation of atmospheric air at the top of the bioreactors. The alkalinity buildup in the effluents is a good indication of biochemical reactions (i.e., sulfate reduction) taking place in the bioreactors. Data acquired from the project indicate that only Zn, Cu, and Cd were being removed as sulfides due to SRB activities. This statement is based on the stoichiometric balance that includes analytical data for metals and sulfide and is also supported by Mn data that indicate that the adsorptive capacity of the organic matter was exhausted after 8 months of operation. Changes in concentrations of other metals, which precipitated not necessarily as sulfides, seem to be affected by the SRB only in an indirect manner, by responding to changes of chemical conditions caused by the SRB. Zinc removal thresholds of 500 (ig/L for Bioreactors II and IV and 800 (ig/L for Bioreactor III seem to be independent of influent concentrations. Copper removal thresholds of 50 (ig/L for Bioreactors II and IV and 80 (ig/L for Bioreactor III also seem to be independent of influent concentrations. Cadmium removal thresholds of 5 (ig/L that prevailed for most of the operating time decreased when the Cd concentration of the influent dropped to 1 • g/L. Different Zn and Cu removal thresholds for Bioreactor III than the respective removal thresholds for Bioreactors II and IV indicate that the thresholds depend on the configuration of the bioreactors but are not affected by the closing up and freezing of a bioreactor during winter. A slightly lower metal removal efficiency in Bioreactor III that contained only one chamber with organic matter may indicate that the residence time of 10 hours within the organic carbon matter is close to minimal. This residence time may vary for different climates. Bioreactor III, with only one organic matter chamber and no limestone chamber, was noticeably less efficient in creating a reducing environment and also less efficient in removing dissolved metals. It is precarious to discriminate whether Bioreactor III was less efficient due to the absence of a limestone chamber or a second organic matter chamber. Evidence of metal sulfides precipitating in the organic matter chambers, together with the observation of no precipitate in the cobble chambers, indicates that the cobble chamber was not essential for biochemical reaction. For this demonstration, the role of the cobble chamber was limited to a collection sump for a small mass of precipitates that escaped from the organic matter chambers. The autopsy on the bioreactors revealed a convoluted biochemical environment that was probably caused by a dramatic change in the AMD chemistry after the first 10 months of operation. The environment examined during the autopsy included mixed results of processes that were occurring first at a low pH and a reasonably high load of metals with the subsequent reactions that were characteristic for water with a neutral pH laden with much less dissolved metals. Aqueous samples collected during the autopsy indicated that only a small portion of the dissolved metals in reduced form were balanced in the solution by sulfides. These were probably Zn, Cu, Cd, and a small portion of Fe. Other metals dissolved in solution, including high 67 ------- concentrations of Ca, were electronically balanced by high concentration of sulfate, bicarbonate, and assumed-present hydroxide. • It is hypothesized, based on the analytical data and visual observation during the autopsy on the bioreactors, that water accumulated at the bottom of the bioreactors contained large amounts of suspended ferric and Al hydroxides. • The high concentration of metals that accumulated with the solid matrix of the bioreactors demonstrated that a large load of metals was retained within the organic matter material, thus indicating the bioreactors were efficient in removing metals from the influent AMD. • For the autopsy data, there was a trend of decreasing concentrations for some metals as they were retained along the flow path within the first organic matter chamber. These metals include Al, Fe, Cu, and As. The reason behind such a behavior remains a conundrum. • A high correlation coefficient (• = 0.9434) for concentration trends for As and Fe in the solid matrix of the bioreactors seem to support the inference that the higher As concentration in the effluents rather than influent was controlled by sorption processes of As to Fe(OH)3. • In general, sulfide concentrations in the organic matter chambers were above the 4,000 mg/kg level and were twice as high as concentrations of sulfates. This large sulfide load together with high metal concentrations is indicative of metal sulfides precipitating in the organic matter chamber due to SRB activities. Although TOC concentrations for the initial fresh organic matter were not measured, the high TOC values measured during the autopsy strongly indicate that the depletion of organic carbon would not be a factor for the efficient removal of metals even if these bioreactors were operating for several more years. Plugging of the bioreactors took place within or immediately adjacent to the AMD distribution system. This statement is supported by the water level decrease between the inlet sump and first organic matter chamber. Also supporting this conclusion were the observations during the autopsy that more chemical precipitate was present in the front portion of the bioreactors than downstream. The high percentage of Fe and Al hydroxides in the plug removed from the AMD inlet to Bioreactor III indicates a high probability that bioreactor inlets were plugging mainly due to chemical reactions; the sediment carried by the AMD was a secondary reason for plugging. The reliability of transducer-/sensor-generated measurements was poor especially for the pH sensors that were coated with precipitate within several weeks. Part of the problem was a very slow flow that did not allow for dynamic cleaning of the sensor. The low flow rate also inhibited measurements because of the necessity of using a small diameter vortex that was prone to plugging. If sulfide concentrations in aqueous samples from the influent and effluent are to be indicative of the bioreactor performance, they need to be filtered prior to laboratory analysis. In addition, the initial and final concentrations of sulfate and sulfide in the organic matter need to be known. 68 ------- 10. References 1. Zaluski, M., M. Foote, K. Manchester, M. Canty, M. Willis, J. Consort, J. Trudnowski, M. Johnson, and M.A. Harrington-Baker, "Design and Construction of Bioreactors with Sulfate-Reducing Bacteria for Acid Mine Drainage Control," Proceedings of The Fifth International In Situ and On-Site Bioremediation Symposium, Battelle Press, San Diego, CA, April 19-22, 1999. 2. MSB Technology Applications, Inc., Quality Assurance Project Plan for Sulfate-Reducing Bacteria Reactive Wall Demonstration Project, Mine Waste Technology Program; Activity III, Project 12, Phase 2, 1998. 3. MSB Technology Applications, Inc., Work Plan-Sulfate'-Reducing Bacteria Reductive Wall Demonstration, Mine Waste Technology Program; Activity III, Project 12, Phase 2, 1998. 4. Greenberg, A.F., L.S. Clesceri, A.D. Eaton, "Standard Methods for Examination of Water and Wastewater," American Public Health Association, Section 2580 B, 1992. 5. Canty, M., "Overview of the Sulfate-Reducing Bacteria Demonstration Project under the Mine Waste Technology Program," Mining Engineering, June 1999. 6. Robins, RG. and J.C.Y. Huang, "The Adsorption of Arsenate Ion by Ferric Hydroxide," Proceedings of the Arsenic Metallurgy Symposium, TMS/AIME Annual Conference, Phoenix, AZ, January 1988. 7. EPA, USEPA Contract Laboratory Program National Functional Guidelines for Inorganics Data Review, 1994. 69 ------- |