FINAL REPORT—SULFATE-REDUCING 

BACTERIA REACTIVE WALL 

DEMONSTRATION

MINE WASTE TECHNOLOGY PROGRAM

ACTIVITY III, PROJECT 12

IAG NO: DW89938870-01-1 

Project Officer 

Mr. Roger Wilmoth

Office of Research and Development

National Risk Management Research Laboratory

Cincinnati, Ohio 45268

MSE 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. 

i 
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

ii 
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. TerraCellTM material, commonly used in landscaping for 

slope stabilization and made of high density polyethylene, was used to form a cellular containment system 

iii 
(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 TerraCellTM 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. 

1 U.S. Patent No. 6,325,923 

iv 
C 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). 

C 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. 

C 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 EC to 

16EC. 

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. 

C 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. 

C 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. 

C For the Calliope site climatic and hydrochemical conditions, the thresholds for the removal of Zn, 

Cd, and Cu were approximately 500 micrograms per liter (μg/L), 5 μg/L, and 80 μg/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. 

C 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. 

v 
C 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. 

C 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

viii 
Tables 

2-1. Acid Mine Drainage Analytical Data and Target Concentrations for Bioreactors ............ 3

4-1a. Flow and Water Level Measurements ............................................ 13

4-1b. 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

6-1a. Total and Dissolved Metals ............................................. 47

6-1b. Iron Speciation ....................................................... 47

6-1c. 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 (k) 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. pH trends ................................................................. 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

6-1a. Total vs. dissolved metals for the cobble chamber of Bioreactor II ...................... 51

6-1b. 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

x 
Acronyms and Abbreviations 

ABA acid-base accounting (analytical method for sulfide)

Al aluminum

AMD acid mine drainage

As arsenic

Ca calcium (also used as the designation for limestone chamber in figures)

CaCO3 calcium carbonate

CCS cellular containment system

Cd cadmium

CO cobble chamber (used only in tables or figures)

Cu copper 

DM dissolved metals

DO dissolved oxygen

DOE U.S. Department of Energy

EH oxidation-reduction potential expressed with reference to standard hydrogen electrode

EPA U.S. Environmental Protection Agency

Fe iron

Fe2+ ferrous iron

Fe3+ ferric iron

Fe(OH)3 ferrihydrite

FeS2 iron disulfide (pyrite)

H2S hydrogen sulfide

HCO3 bicarbonate

HDPE high density polyethylene

IAG Interagency Agreement

ICP inductively coupled argon plasma

Mg magnesium

mil 1/1,000 of an inch

Mn manganese

MCL maximum contaminant level

MPN most probable number

MSE-TA MSE Technology Applications, Inc.

MWTP Mine Waste Technology Program

Or1 organic matter chamber No. 1

Or2 organic matter chamber No. 2

ORP oxidation-reduction potential measured using silver/silver chloride reference electrode

pH measure of hydrogen ion activity

PVC polyvinyl chloride

QA quality assurance

QAPP quality assurance project plan

QC quality control

SHE standard hydrogen electrode

SMCL suggested maximum contaminant level

SRB sulfate-reducing bacteria

TCLP Toxicity Characteristic Leaching Procedure

TOC total organic carbon

TM total metals

VFA volatile fatty acids

xi 
Zn zinc 

xii
Acknowledgment 

Work was conducted through the DOE National Energy Technology Laboratory at the Western 

Environmental Technology Office under DOE Contract Number DE-AC22-96EW96405. 

xiii
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 

in pH. 

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 + 2SO4

2- + 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 + 2HCO3 (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. 

1
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 

NW1⁄4 of SE1⁄4 and SW1⁄4 of NE1⁄4 both of SW1⁄4 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 

(I-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 

2
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 

Concentration (μg/L) Analyte AMD (maximum) AMD (minimum) Target Effluent Comments 

Aluminum 14,100 11.0 1,000 

Cadmium 41.9 3.1 5 

Copper 3,050 2.8 100 

Iron 8,670 8.0 1,000 

Manganese 3,770 690 2,000 

Zinc 11,100 990 4,000 

Arsenic 10.9 1.1 NA 

Sulfate 229,000 69,800 NA 

pH 7.52 3.29 6 to 8 

*Suggested maximum contaminant level (SMCL) 

** Maximum contaminant level (MCL) 

***Secondary maximum contaminant level 

NA = not applicable 

3 

50 to 200 μg/L* 

5 μg/L** 

1,300 μg/L** 

300 μg/L*** 

50 μg/L*** 

5,000 μg/L*** 

50 μg/L** 

250,000 μg/L*** 
4

I-90

I-15

I-90

Figure 2-1. Calliope site location map.
Figure 2-2. Layout of the bioreactors. 

5
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 

1

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, acidresistant 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. 

• TerraCellTM 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 TerraCellTM 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. 

6
• The mixture of cow manure and straw was 

installed in the CCS, which consisted of 10 lifts 

of TerraCellTM material (Figure 3-2) and would 

limit settling (if it occurred) of the organic 

matter to each individual cell. The TerraCellTM 

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 wavyshaped flow line and facilitated the packing of 

each individual cell with the organic matter. 

The TerraCellTM 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 endwalls. 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 

7
8

Figure 3-1. 

required for this reactor as it was constructed

above ground. 

control and monitoring equipment and the intake

sump were like those used for the belowground

bioreactors.

3.2.4 

The bioreactors were equipped with a number of

sampling ports to monitor performance of each

bioreactor. 

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).

Simplified cross-sections of Bioreactors II and IV.

Other features including flow

Sampling Locations and Ports

The ports were either installed at the
9

Figure 3-2. 

Figure 3-3. 

Cellular containment system for organic substrate.

AMD distribution system manifold.
10

Figure 3-4. 

Waste Rock Pile

Water percolating

through Waste Rock Pile

Overflow from Lower Pond

to the gulch

Limestone

Cobbles

Intake

sump

Intake

sump Limestone

End enclosure

Cobbles

End enclosure

Valve box

t

t

t

Overflow from Lower Pond

to the gulch

Overflow from the

supply system

to the gulch

B

B

B

A,L,T

A,L,T

A,L,T F,p

F,p

F,p

F,L

F,L

A,T,F,L,p

Three supply lines

to the reactors

IIIa

IVa

IIa

IVb

IIb

IVc

IIc

IVd

IIId

IId

IIIb IIIc

IVe

IIIe

IIe

ta

B - 

F - 

L - 

p - 

T - 

t - 

 cobbles interface

ta - 

Housing for

ambient

temperature

measurement

Lower Pond

Surface drainage

from Upper Pond

to Lower Pond

Eh

DO

Selected metals

Sulfate

Sulfide

Alkalinity

Volatile Fatty Acids (FAs)

IVa - 

Below-ground bioreactor w/o pre-treatment section (III)

Aboveground bioreactor (IV)

Below-ground bioreactor with pre-treatment section (II)

Organic

carbon

Organic

Carbon

Discharge

line

AMD

supply line to

each reactor

Discharge from adit

Upper Pond

Organic

carbon

Land application

of treated AMD

Sampling and Monitoring Points Locations

Monitored Parameters

A -

Figure 3-5. 

End enclosure and intake sump for Bioreactors II and IV.

Bacteria count

Flow rate

Water level

pH

Termperature of water

Temperature at straw -

Ambient temperature 

Sampling point ID

Sampling and monitoring point locations.
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 yearround. 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 4-1a). 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 4-1a and 4-1b, 

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 recoding interval was set for 

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, TerraCellTM, 

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 (Or1) 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 Or1 and analyzed for SRB population. 

A single organic matter sample was collected from 

Or1 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 Or1 

chamber, including the SRB population count but 

excluding the TCLP. 

Table 4-1a. Flow and Water Level Measurements 

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 (Or1) and the cobble chamber of 

Bioreactors II and IV. 

Bioreactor II Bioreactor III Bioreactor IV 

Date Flow Water level (ft from 

(gpm) bottom of Bioreactor) Flow Water level (ft from 

bottom of Bioreactor) Flow Water level (ft from bottom of Bioreactor) 

12/11/98 4.15 4.5 4.58 

12/14/98 0.974 0.557 0.798 

12/22/98 0.822 0.633 0 

1/4/99 0.998 1.008 0 

2/3/99 0.827 0.859 0 

2/4/99 4.03 4.41 4.87 

3/3/99 0.996 1.04 0 

4/5/99 0.99 0.696 0 

5/5/99 0.611 0.537 0 

5/7/99 1.63 3.57 4.62 

5/20/99 1.42 4.09 4.66 

6/3/99 0 0.578 0 

6/25/99 3.81 4.38 4.53 

6/27/99 3.55 4.28 4.48 

7/6/99 3.15 4.06 

7/12/99 0.191 0.954 0.861 

7/14/99 2.8 3.88 3.7 

7/15/99 2.66 3.8 

7/19/99 6.43 5.46 

7/21/99 5.36 5.54 

7/30/99 4.62 5.43 4.49 

8/11/99 1.064 4.13 0.802 5.33 0.487 4.31 

8/20/99 3.4 5.38 4.1 

8/29/99 2.63 3.66 3.92 

13
Table 4-1a. Flow and Water Level Measurements 

Bioreactor II Bioreactor III Bioreactor IV 

Date Flow Water level (ft from Flow Water level (ft from Flow Water level (ft from bottom 

(gpm) bottom of Bioreactor) bottom of Bioreactor) of Bioreactor) 

9/1/99 5.41 4.08 4.06 

9/8/99 1.002 5.43 1.063 2.92 1.012 4.69 

9/21/99 4.89 4.51 4.2 

9/24/99 4.92 4.47 4.13 

9/27/99 5.34 

10/7/99 0.947 0.962 1.273 

10/26/99 5.06 3.96 4.37 

11/9/99 0.862 4.97 1.004 3.81 0.537 4.48 

12/8/99 0.868 4.99 0.921 3.74 0 4.12 

1/6/00 0.877 0.763 0 

1/10/00 4.6 3.66 4.45 

2/3/00 0.976 0.91 0 

2/8/00 4.03 3.59 4.49 

3/6/00 0.789 2.66 0.809 3.27 0 4.61 

3/16/00 6 

4/5/00 1.009 5.67 0.602 3.58 0 4.43 

5/8/00 0.863 3.98 1.019 5.57 0 4.68 

5/25/00 2.49 

5/28/00 4.28 

6/7/00 1.071 3.29 1.89 4.05 0 4.61 

6/14/00 2.89 4.51 

6/16/00 1.93 4.35 

6/18/00 0.47 

6/30/00 4.95 

7/5/00 4.63 

7/6/00 1.868 4.46 1.793 4.3 1.964 4.32 

8/10/00 1.324 4.12 1.104 4.21 1.693 4.18 

9/7/00 1.969 2.57 2.116 4.58 2.528 3.28 

9/25/00 1.247 4.38 1.823 4.55 2.048 3.71 

10/31/00 1.428 4.62 1.105 4.59 1.278 4.44 

12/4/00 1.029 5.21 0.715 4.53 0 3.96 

1/8/01 0.891 5.19 0.897 4.63 0 4.26 

2/12/01 0.815 4.3 0.916 4.58 0 3.83 

3/14/01 0.883 0.84 0.754 4.35 0 4.28 

3/27/01 0 0 

4/4/01 0 0 

4/9/01 0.983 0.886 0 

5/9/01 0.854 4.81 0.752 4.06 0 4.41 

6/6/01 0.812 4.61 0.706 3.9 0.917 4.54 

7/9/01 0.919 4.24 0.786 3.75 0.988 4.49 

14
Table 4-1b. Water Level Profile in Bioreactor II 

Water level - feet above bottom of Bioreactor II 

Point 12/11/1998 05/20/1999 

influent sump 5.01 

II fd 4.94 

II gd 4.66 

II hd 4.66 

II bd 4.66 

II cd 4.66 

II id 4.65 

II jd 4.66 

II kd 4.64 

II ld 4.65 

II dd 4.65 

Table 4-2. Monitored Parameters and Analytes 

Measurement Planed Sample Frequency1 Sample Location2 

4.96 

2.93 

2.62 

2.01 

1.92 

1.93 

1.92 

1.93 

1.92 

1.92 

1.91 

Reported frequency and/or comments 

Dissolved metals (Al, As, Cd, days 10, 20, 30, every IIIa, IId, IIId, IVd As planned 

Cu, Fe, Zn, Mn) month thereafter 

Alkalinity, SO4, EH, DO, days 10, 20, 30, every IIIa, IId, IIId, IVd As planned 

Soluble sulfide (HS)- month thereafter 

Volatile fatty acids (VFA) days 10, 30, every 3rd IIIa, IId, IIId, IVd Increased to every month 

month thereafter 

pH every half-hour IIIa, IIe, IIIe, I'VE Monthly; probe got coated 

SRB count days 10, 30; every 3rd IIb, IIIb, IVb Increased to every month 

month thereafter 

Water temperature every half-hour IIIa, IId, IIId, IVd monthly; data logger malfunctioned 

Flow rate every half-hour IIe, IIIe, I'VE monthly; data logger malfunctioned 

Water level every half-hour IId, IIId, IVd monthly; data logger malfunctioned 

Air temperature at straw/rock days 10, 20, 30; every IIc, IIIc, IVc Incomplete data; probes corroded 

interface month thereafter 

Ambient air temperature every half-hour Ambient temperature No records; instrument broke3 

housing 

1

 Samples for Bioreactor IV were not taken during the months in which freezing occurs

2

 See Figure 3-5 for locations

3

Addressed in Section 8, Quality Assurance/Quality Control

15 
Table 4-3. Analysis for the Autopsy of the Bioreactors 

Reactors 

Total No. 

of 

analyses 

Analyzed 

parameter IV (Aboveground) III II 

Chamber 

Total 

Chamber 

Total 

Chamber 

Total 

Or14 Ca5 Or24 CO6 Or1 CO Or1 Ca Or2 CO 

3 TCLP1 110 1 110 1 110 1 

24 TM2 510 211,12 110 111 9 510 111 6 510 211,12 110 111 9 

3 DM3 111 1 111 1 111 1 

24 Fe Spc7 510 211,12 110 111 9 510 111 6 510 211,12 110 111 9 

24 Sulfide8 510 211,12 110 111 9 510 111 6 510 211,12 110 111 9 

24 Sulfate9 510 211,12 110 111 9 510 111 6 510 211,12 110 111 9 

19 N13 510 112 110 7 510 5 510 112 110 7 

19 P14 510 112 110 7 510 5 510 112 110 7 

19 TOC15 510 112 110 7 510 5 510 112 110 7 

10 SRB 210 112 110 4 210 2 210 112 110 4 

169 63 43 63 

Number of Sample 

Locations 8 3 2 1 14 8 1 9 8 3 2 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) Or14 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) 

11)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 

0.0 

0.5 

1.0 

1.5 

2.0 

2.5 

3.0 

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 

Flow (gpm) 

II 

III 

IV 

Figure 4-1. Bioreactor flow. 

Water Level in Bioreactors 

0 

1 

2 

3 

4 

5 

6 

7 

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 

Water level (ft from bottom of bioreactor) 

Bioreactor II 

Bioreator III 

Bioreator IV 

May 1999 

Bioreactor II plugging 

June 2000 

Bioreactor II plugging March 2001 

Bioreactor II inlet valve plugging 

Water levels reported are from 

the cobble chambers 

Figure 4-2. Water level in the bioreactors. 

17
Water Level Profile in Bioreactor II 

During Early Operation and Plugging Episode of May 1999 

0.00 

1.00 

2.00 

3.00 

4.00 

5.00 

6.00 

influent II fd II gd II hd II bd II cd II id II jd II kd II ld II dd 

Piezometer series 

Water level (ft from bottom of bioreator) 

cobble level 

12/11/98 

5/20/99 

Water level on 12/11/98 - one 

week after start up 

Level during May 1999 plugging 

1st 

organic 

carbon 

chamber 

limestone 

chamber 

2nd 

organic 

carbon 

chamber 

cobble 

chamber 

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 EC. 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 EC to 

15.5 EC for the influent and 1 EC to 16 EC for the 

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 EC 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 EC 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 EH, 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 (CO3

2-) 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 μg/L for Reactors 

II and IV and 800 μg/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 μg/L on average for Bioreactors II and IV and, 

again, a slightly higher threshold level of 

approximately 80 μg/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 μg/L to 5 

μg/L, until March 2001 when the Cd concentration 

in the influent dropped to approximately 3 μg/L for 

a 2-month period. Cadmium concentration in the 

effluent in the same time period decreased to less 

than 1 μg/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 μg/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
E

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 abovedescribed 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. 

Table 5-1. Field Parameters 

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. 

Location Date pH EH (mV) DO (mg/L) Temperature (EC) 

Influent 12/14/98 3.87 700 6.8 1.6 

12/22/98 6.01 426 8.85 1.1 

1/4/99 4.51 571 10.19 0.9 

2/3/99 5.70 466 11.19 0.6 

3/3/99 5.62 480 8.29 0.5 

5/5/99 3.29 726 6.06 4.6 

6/3/99 3.64 611.4 3.93 9.9 

7/12/99 4.19 315.4 6.39 15.3 

8/11/99 5.14 probe malfunction 14.4 

9/8/99 6.08 403 8.61 9.7 

10/7/99 6.68 309 8.94 7.3 

11/9/99 6.58 357 9.76 4.3 

12/8/99 6.87 294.4 9.94 1.3 

1/6/00 7.14 247.5 10.5 0.7 

2/3/00 6.81 302 8.81 1.2 

3/6/00 6.61 334 8.89 4.7 

23
E

Table 5-1. Field Parameters 

Location Date pH EH (mV) DO (mg/L) Temperature (EC) 

4/5/00 6.07 368 

5/8/00 6.83 363 

6/7/00 5.80 409 

7/6/00 6.24 366 

8/10/00 6.48 342 

9/7/00 6.83 310 

9/25/00 6.92 267 

10/31/00 7.08 267 

12/4/00 7.40 422 

1/8/01 6.94 probe 

malfunction 

2/12/01 7.52 230.9 

3/14/01 7.10 365 

4/9/01 7.33 251 

5/9/01 6.48 32 

6/6/01 4.85 394 

7/9/01 5.01 401 

Bioreactor II 12/14/98 9.95 76 

12/22/98 9.51 252 

1/4/99 8.75 342 

2/3/99 8.42 378 

3/3/99 8.37 353 

5/5/99 8.29 87 

6/3/99 7.87 62.8 

7/12/99 7.61 106.5 

8/11/99 7.68 

9/8/99 7.09 -76 

10/7/99 7.14 -125 

11/9/99 7.20 -36 

12/8/99 7.69 -62 

1/6/00 7.29 -35 

2/3/00 7.46 29 

3/6/00 7.37 42 

4/5/00 6.98 -68 

5/8/00 6.79 -74 

6/7/00 7.25 78 

7/6/00 7.10 16 

8/10/00 6.76 151 

9/7/00 7.31 -14 

9/25/00 6.73 -34 

10/31/00 7.06 133 

12/4/00 7.17 330 

1/8/01 6.98 probe 

8.42 5.2 

8.59 7.5 

7.66 15.4 

7.53 13.3 

7.43 15.5 

7.89 10.5 

8.89 6.6 

9.03 4.3 

13.84 1.6 

11.49 1.2 

10.49 1.5 

9.67 2.7 

9.93 3.8 

8.3 probe malfunction 8.5 

7.1 

0.51 

0.63 

1.09 

0.93 

0.11 

0.04 

0.95 

0.46 

probe malfunction 

malfunction 

2/12/01 7.21 296 

3/14/01 7.74 -83 

4/9/01 6.83 289 

5/9/01 6.61 94 

6/6/01 6.76 98 

7/9/01 6.76 15 

Bioreactor III 12/14/98 9.12 219 

12/22/98 9.32 279 

1/4/99 9.34 403 

2/3/99 8.43 444 

3/3/99 7.78 328 

5/5/99 7.56 273 

6/3/99 7.62 187.2 

7/12/99 7.54 72.9 

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 

0.56 

0.59 

0.48 

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 

2.2 

2.7 

2.9 

4.8 probe malfunction 9.1 

0.47 12.4 

0.05 4.2 

0.18 3.4 

0.44 2.2 

0.76 1.7 

0.17 1.3 

0.04 5.4 

0.08 9.6 

0.15 13.8 

8/11/99 7.03 probe malfunction 15.0 

9/8/99 7.27 -82 0.21 14.8 

10/7/99 7.02 -81 0.24 10.9 

11/9/99 6.75 37 0.27 6.3 

12/8/99 7.21 59 1.18 3.2 

1/6/00 6.98 68 1.43 2.2 

24
E

Table 5-1. Field Parameters 

Location Date pH EH (mV) DO (mg/L) Temperature (EC) 

2/3/00 6.92 259 

3/6/00 7.00 145 

4/5/00 6.82 76 

5/8/00 6.84 -49 

6/7/00 6.63 41 

7/6/00 6.39 114 

8/10/00 6.35 196 

9/7/00 6.45 164 

9/25/00 6.45 241 

10/31/00 6.77 187 

12/4/00 6.73 569 

1/8/01 6.65 probe 

malfunction 

2/12/01 6.79 379.4 

3/14/01 6.64 113 

4/9/01 6.82 334 

5/9/01 6.7 -48 

6/6/01 6.8 161 

7/9/01 6.26 34 

Bioreactor IV 12/14/98 9.98 69 

12/22/98 9.87 339 

1/4/99 9.68 337 

2/3/99 9.19 346 

3/3/99 8.37 135 

5/5/99 8.63 17 

6/3/99 8.27 53.6 

7/12/99 7.59 77.3 

8/11/99 8.12 

9/8/99 7.80 -82 

10/7/99 7.90 -100 

11/9/99 7.91 -191 

12/8/99 8.25 -60 

1/6/00 7.96 -31 

2/3/00 8.17 95 

3/6/00 7.88 53 

4/5/00 7.92 -87 

5/8/00 8.06 -33 

6/7/00 7.49 37 

7/6/00 7.23 37 

8/10/00 6.99 30 

9/7/00 7.81 -53 

9/25/00 7.54 -31 

10/31/00 7.57 98 

12/4/00 7.84 289 

1/8/01 7.70 probe 

0.66 1.6 

2.23 3.6 

0.34 4.1 

0.12 6.7 

0.27 11.8 

0.91 14.1 

0.67 15.7 

2.53 12.9 

1.79 10.0 

0.91 5.8 

5.69 2.8 

5.28 2.5 

3.91 2.1

3.80 2.0

3.22 3.1

4.4 probe malfunction 6.8

0.43 

0.15 

0.40 

0.24 

0.46 

0.06 

0.28 

0.09 

0.15 

probe malfunction 

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 

malfunction 

2/12/01 8.47 242 

3/14/01 7.68 -16 

4/9/01 7.75 254 

5/9/01 7.79 -87 

6/6/01 7.18 39 

7/9/01 6.97 -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 

2.6 probe malfunction 8.2 

0.42 18.1 

25
Table 5-2. EH and pH Profiles for Bioreactors II and III 

Location Piezometer EH 

11/2/99 12/6/99 1/11/00 2/9/00 12/6/99 

pH 

1/11/00 2/9/00 

Influent 418.20 438.8 301.9 302 6.79 6.78 6.67 

Bioreactor II II fd 131.9 53.2 174 243 6.2 5.76 5.79 

II gd -100.7 -135 -38 83 7.01 7.03 7.1 

II hd -63.3 -101.2 -11 22 7.19 7.06 7.36 

II bd 19.5 -91.6 -21 -25 7.46 7.38 7.59 

II cd -10.3 -111.7 -15 0 7.55 7.4 7.43 

II id -56.2 -110.5 -18 -10 7.55 7.45 7.6 

II jd -56.5 -108 -13 -13 7.55 7.45 7.7 

II kd -61.8 -99.9 -1 4 7.6 7.48 7.65 

II ld -57.8 -87.4 12 29 7.53 7.45 7.78 

II fs 328.5 331.7 dry 7.01 dry 

II gs 312.1 284.3 150 214 7.31 6.76 dry 

II hs 380.4 114.7 70 192 7.59 7.43 7.29 

II bs 33.9 105.3 84 106 7.3 7.19 7.13 

II cs -35.2 49.7 52 81 7.08 7.14 7.27 

II is -76.5 33.5 32 24 7.13 7.23 7.34 

II js -80.5 -26.8 32 11 7.13 7.19 7.72 

II ks -82.3 -77.9 1 23 7.25 7.19 7.74 

II ls -76.8 -95.4 -14 42 7.25 7.19 7.6 

Bioreactor III III fd -13.5 -46.5 88 150 6.36 6.19 6.37 

III bd 41.5 -153.2 -23 60 7.61 7.27 7.19 

III gd 81.2 -110.8 -16 71 7.58 7.3 7.12 

III cd 108.2 -72.8 1 98 7.47 7.15 6.74 

III hd 87.6 -55.5 22 114 7.41 7.05 6.81 

III Id 50.4 -36.9 47 141 7.15 6.93 6.71 

III jd 34.9 16 115 259 6.99 6.73 6.55 

III fs dry 

III bs 116.7 97.3 127 202 7 6.85 6.52 

III gs 78.3 47.1 95 227 7 6.82 6.32 

III cs 62.9 56.2 97 229 6.99 6.73 6.14 

III hs 55.3 65.7 110 231 7.04 6.69 6.44 

III Is 41.8 50 96 219 6.65 6.62 6.51 

III js 23.9 53.5 126 245 6.83 6.56 6.59 

26
Table 5-3. SRB Populations 

SRB populations (SRB/mL) 

Date Bioreactor II Bioreactor III Bioreactor IV 

12/14/98 1.40E+03 1.70E+01 7.80E+01 

12/22/98 2.00E+02 1.70E+01 1.40E+02 

1/4/99 9.20E+00 1.00E-01 1.00E-01 

2/3/99 1.80E+00 1.80E+00 1.80E+00 

3/3/99 1.10E+00 8.10E+00 8.10E+00 

4/5/99 1.70E+01 2.40E+02 2.40E+02 

5/5/99 1.70E+02 7.80E+03 2.00E+03 

6/3/99 2.80E+03 2.00E+03 1.10E+04 

7/12/99 2.80E+04 2.40E+03 1.40E+04 

8/12/99 1.40E+06 2.80E+04 1.40E+04 

9/8/99 4.50E+05 2.00E+05 2.40E+04 

10/7/99 4.50E+04 4.50E+04 4.50E+04 

11/9/99 1.70E+04 4.50E+03 1.40E+04 

12/8/99 1.20E+04 1.10E+04 2.00E+03 

1/6/00 1.10E+04 1.10E+04 2.40E+03 

2/3/00 2.40E+04 1.70E+04 1.10E+04 

3/6/00 1.70E+06 1.10E+05 1.40E+04 

4/5/00 2.00E+03 1.10E+05 7.80E+04 

5/8/00 1.70E+04 1.10E+05 7.80E+04 

6/7/00 1.40E+05 2.00E+04 4.50E+04 

7/6/00 1.10E+05 4.50E+04 6.80E+04 

8/16/00 9.20E+04 1.10E+04 2.00E+04 

9/7/00 4.00E+04 1.40E+04 2.00E+03 

9/25/00 2.80E+02 2.00E+03 2.40E+02 

10/31/00 2.10E+03 1.40E+03 2.40E+03 

12/4/00 4.50E+03 2.40E+03 1.40E+04 

1/8/01 7.80E+03 4.50E+03 1.40E+04 

2/12/01 7.80E+03 2.00E+03 2.10E+04 

3/14/01 2.40E+04 2.10E+03 2.40E+04 

4/9/01 1.40E+05 4.50E+03 7.80E+04 

5/9/01 9.30E+04 1.40E+04 2.00E+04 

6/6/01 1.70E+04 2.80E+04 2.10E+04 

7/9/01 1.10E+04 4.50E+04 4.50E+04 

27
Table 5-4. Sulfate and Sulfide Concentrations 

Date 

Sulfate (mg/L) Sulfide (mg/L) 

Influent II III IV Influent II III IV 

60.6 8 

1,400 196 

12/14/98 102 250 172 148 3.1 1.2 2.3 2.1 

12/22/98 115 80 111 114 2.8 2.2 2.9 3.3 

1/4/99 113 145 123 143 3.7 6.4 2.4 1.9 

2/3/99 104 111 115 137 0.0 1.2 1.6 1.2 

3/3/99 143 122 152 186 0.0 1.3 2.4 0.0 

4/5/99 135 193 178 187 0.0 0.0 0.0 0.0 

5/5/99 229 281 223 300 0.0 0.0 0.0 1.2 

6/3/99 200 304 197 457 0.0 15.0 2.2 1.3 

7/12/99 122 136 126 326 0.0 0.0 0.0 0.0 

8/12/99 118 92 80 74 2.7 2.8 4.1 2.0 

9/8/99 109 113 76 92 1.2 3.2 3.0 1.3 

10/7/99 99 73 88 106 0.0 0.0 0.0 1.2 

11/9/99 93 87 100 99 1.0 9.6 0.6 0.9 

12/8/99 88 83 89 98 1.1 1.4 3.2 0.8 

1/6/00 81 82 85 125 3.1 4.8 3.9 4.2 

2/3/00 84 82 87 152 1.6 2 1.2 1.3 

3/6/00 83.2 73.8 81.4 114 1.37 1.13 1.53 1.45 

4/5/00 103 100 95.6 69.2 1.5 1.5 2 1.2 

5/8/00 88.3 81.2 69 127 1.7 1.3 3.7 0.85 

6/8/00 91.6 82.4 84.8 91.9 2.6 2.3 2.9 2.8 

7/6/00 92.5 72.7 93.5 64.7 5.1 4.8 4.7 4.7 

8/10/00 78.5 95.4 78.5 80.3 4.3 4.9 7.2 6.0 

9/7/00 81.4 88.5 83.8 93.1 3 3.2 2.3 3 

9/25/00 82.9 59.2 81.5 86 3.1 5.2 5.6 3.2 

10/31/00 69.8 73.4 86.2 84.7 3.6 3.4 2.3 2.6 

12/4/00 73.3 65.9 79.7 163 3 3.2 3.3 3.2 

1/8/01 79.7 68.1 74.9 208 3 2.6 2 2.9 

2/12/01 72.4 55.4 56.4 263 4.5 3 4 4.6 

3/14/01 59.6 251 5 3.7 4.5 5 

4/9/01 73.7 204 2.6 2.2 1.7 2.5 

5/9/01 81.6 131 113 241 2.8 1.7 1.9 2.6 

6/6/01 118 128 113 121 2.2 1.3 1.6 2 

7/9/01 101 88.7 102 79.7 2.8 3.9 4.1 2.4 

Values enveloped by lines are considered outliers and were not grafted 

28
Total Alkalinity (mg/L as CaCO )3

Table 5-5. Alkalinity 

Date Intake II III IV 

12/14/98 0.0 215.0 277.0 294.0 

12/22/98 0.0 111.0 192.0 238.0 

1/4/99 0.0 105.0 93.8 223.0 

2/3/99 0.0 95.0 95.8 203.0 

3/3/99 0.0 95.8 94.2 275.0 

4/5/99 0.0 102.0 86.0 251.0 

5/5/99 0.0 126.0 85.4 241.0 

6/3/99 0.0 474.0 99.0 262.0 

7/12/99 0.0 128.0 78.2 214.0 

8/12/99 0.0 247.0 359.0 290.0 

9/8/99 0.0 238.0 248.0 446.0 

10/7/99 0.0 124.0 75.8 87.8 

11/9/99 14.4 54.0 26.4 90.4 

12/8/99 12.4 53.6 35.0 68.2 

1/6/00 24.8 70.4 37.0 125.0 

2/3/00 19.6 79.0 41.0 129.0 

3/6/00 16.2 90.4 35.2 146.0 

4/5/00 0.0 100.0 47.0 220.0 

5/8/00 14.0 87.2 126.0 426.0 

6/8/00 11.0 74.2 38.4 77.2 

7/6/00 18.0 152.0 27.2 124.0 

8/10/00 19.4 54.6 29.8 81.8 

9/7/00 18.2 45.8 23.6 59.6 

9/25/00 22.2 122.0 28.0 56.4 

10/31/00 20.8 62.2 39.6 65.2 

12/4/00 28.0 63.8 26.2 102.0 

1/8/01 21 54.0 29.2 120 

2/12/01 25.4 70.2 26.8 154 

3/14/01 189 95.8 24.2 25.8 

4/9/01 25.2 90.4 40.2 286 

5/9/01 16 70.0 46.6 347 

6/6/01 0 83.4 75.0 202 

7/9/01 0 83.8 33 97 

29
Table 5-6. Metals Concentration 

Metals concentration (μg/L) 

Location Date Al As Cd Cu Fe Mn Zn 

Influent 12/14/98 2400.0 2.8 15.2 884.0 524.0 1620.0 3740.0 

12/22/98 1670.0 2.5 18.1 737.0 377.0 2020.0 4420.0 

1/4/99 2080.0 4.4 14.9 689.0 603.0 1700.0 3610.0 

2/3/99 788.0 5.2 16.0 477.0 357.0 1830.0 3570.0 

3/3/99 1160.0 3.6 12.1 518.0 265.0 1620.0 3190.0 

4/5/99 3750.0 3.9 20.1 1020.0 713.0 2120.0 4870.0 

5/5/99 14100.0 1.8 41.9 3050.0 7220.0 3770.0 11100.0 

6/3/99 8770 3.1 31 2090 2960 2860 7890 

7/12/99 1580 1.3 16.5 451 272 1600 3370 

8/11/99 779 2.5 17.9 366 281 1950 3790 

9/8/99 140 6.5 16.4 200 372 1950 3520 

10/7/99 26.6 6.2 12.7 68.9 50.6 1580 2700 

11/9/99 35 6.7 7.3 78.8 154 1320 2130 

12/8/99 93.1 5.5 9.3 82.8 15.7 1170 2140 

1/6/00 18 10.9 10.9 7.2 115 1420 2080 

2/3/00 11 5.1 7 53 35.9 1130 1890 

3/6/00 44.5 6.1 8.2 114 20.1 1220 2050 

4/5/00 134 3.5 7.3 274 124 1360 2460 

5/8/00 32 7.6 8.4 145 27.2 1270 2020 

6/7/00 154 2.1 8.6 310 73.1 1230 2230 

7/6/00 35.3 1.5 6.3 174 20.5 1170 1840 

8/10/00 17.3 1.5 5.7 81.4 31.9 1070 1500 

9/7/00 36.1 2.5 3.7 92.7 15.5 1090 1580 

9/25/00 18.5 1.9 5.7 71.4 15.5 1140 1570 

10/31/00 18.9 2.1 5.1 44.2 20.1 1110 1420 

12/4/00 18.9 3.3 4.3 34.1 19.5 1000 1240 

1/8/01 73.4 5.7 4.3 28.1 19.5 882 1270 

2/12/01 45.5 4.7 4.3 2.8 19.5 787 1090 

3/14/01 18 3.4 2.8 26 8 690 990 

4/9/01 31 2 3.1 30 13 840 1000 

5/9/01 40.5 1.6 4.8 162 25.6 947 1570 

6/6/01 1650 1.1 8.8 635 70.2 1460 3180 

7/9/01 587 1.1 12.1 459 195 1190 2720 

Bioreactor II 12/14/98 39.5 15.6 3.9 29.4 140.0 35.6 75.6 

12/22/98 24.6 10.0 3.9 11.5 77.6 31.1 18.6 

1/4/99 22.8 7.4 3.9 3.3 46.9 39.5 22.3 

2/3/99 30.8 5.1 4.8 4.8 93.7 117.0 22.3 

3/3/99 11.2 3.4 4.8 4.3 35.5 115.0 43.9 

4/5/99 10.7 4.3 4.8 47.5 54.2 357.0 128.0 

5/5/99 13.8 3.0 4.8 7.8 97.5 551.0 249.0 

6/3/99 111 26.5 2.5 75.2 1300 999 388 

7/12/99 49.3 5.4 2.5 25.6 686 964 717 

8/11/99 97 14.9 3.9 96.6 868 1040 704 

9/8/99 128 7.7 4.6 95.9 983 1340 1230 

10/7/99 35.0 5.0 4.9 36.8 545 885 1180 

11/9/99 15.8 5.6 4.7 19.4 284 774 750 

12/8/99 21.3 5 5.5 23.2 395 765 772 

1/6/00 30.6 5.4 4.7 15.8 352 785 453 

2/3/00 16.5 2.6 3.5 12.8 314 913 176 

3/6/00 24.6 1.6 3.5 21.4 305 982 210 

4/5/00 130 15.6 3.5 72.2 1810 1000 751 

5/8/00 56.5 10 3.5 49.7 1230 1140 467 

6/7/00 47.6 2.8 3.2 21 772 1000 331 

7/6/00 55.2 6.8 3.2 81.8 2110 1440 409 

8/10/00 17.3 3.6 3.4 17.7 261 984 372 

9/7/00 17.3 2.3 3.4 5.9 197 836 189 

9/25/00 98.2 3.6 3.9 89.1 926 1050 707 

10/31/00 49.6 3.5 4.3 57 578 564 517 

12/4/00 31.8 4.7 4.3 68.8 533 500 404 

1/8/01 18.9 6.8 4.3 31.5 234 296 419 

30
Table 5-6. Metals Concentration 

Metals concentration (μg/L) 

Location Date Al As Cd Cu Fe Mn Zn 

2/12/01 46.5 3.3 4.3 23.7 460 871 648 

3/14/01 19 2.2 0.1 8 110 600 48 

4/9/01 140 4.7 0.3 100 600 2400 300 

5/9/01 130 11.4 4.8 142 454 721 1170 

6/6/01 47.8 5.5 4.8 50.6 638 849 518 

7/9/01 118 4 5 110 707 996 899 

Bioreactor III 12/14/98 43.8 17.1 3.9 65.5 229.0 105.0 91.0 

12/22/98 40.0 11.3 3.9 20.3 92.4 54.5 34.8 

1/4/99 22.8 5.0 3.9 8.6 57.8 33.1 35.8 

2/3/99 43.4 6.2 4.8 18.1 69.8 124.0 35.9 

3/3/99 16.9 4.4 4.8 17.5 43.9 436.0 60.9 

4/5/99 10.7 4.6 4.8 6.4 49.8 768.0 241.0 

5/5/99 45.3 3.5 4.8 43.4 149.0 2100.0 459.0 

6/3/99 23.7 3.1 2.5 36.4 202 1940 1190 

7/12/99 59.6 2.9 3.1 28.3 337 1510 1030 

8/11/99 335 8.9 7 140 1380 3100 1100 

9/8/99 141 12.7 3.9 69.2 875 1360 847 

10/7/99 52.1 3.5 3.9 22.5 661 1260 753 

11/9/99 10.1 4.2 4.7 16.1 302 1230 364 

12/8/99 26.4 3.2 4.7 14 163 1150 659 

1/6/00 19.9 3.5 4.8 19.5 91.2 1170 934 

2/3/00 9.4 2.4 3.5 18.3 95.8 1200 707 

3/6/00 31.2 1.6 3.5 51.8 323 1270 682 

4/5/00 68.5 3.6 3.5 65.6 1240 1500 893 

5/8/00 240 11.7 4.7 114 3910 1450 1070 

6/7/00 50.9 3 4.4 49.3 1070 1130 723 

7/6/00 25.3 2.2 3.2 96.8 115 1120 956 

8/10/00 24.6 1.5 3.4 45.8 124 886 784 

9/7/00 49.6 2.8 4.4 68.8 116 631 1040 

9/25/00 48 2.3 3.5 69.5 115 631 1330 

10/31/00 83.3 3.3 5.6 128 161 493 1590 

12/4/00 18.9 1.9 4.3 24.3 43.9 310 836 

1/8/01 18.9 6.1 4.3 23 34.3 129 871 

2/12/01 18.9 3 4.3 20.4 25.2 73.5 937 

3/14/01 22 3.1 1 16 31 76 790 

4/9/01 85 2 2 65 150 280 850 

5/9/01 143 4.5 9.1 155 282 212 1720 

6/6/01 125 4.3 4.8 142 494 3040 1590 

7/9/01 80.3 2.7 4.8 122 638 2470 1010 

Bioreactor IV 12/14/98 54.2 26.3 3.9 103.0 417.0 19.2 118.0 

12/22/98 27.0 21.6 3.9 79.4 155.0 20.4 53.3 

1/4/99 29.8 29.1 3.9 33.0 126.0 19.3 40.6 

2/3/99 45.2 19.7 4.8 39.5 95.7 27.5 119.0 

3/3/99 35.4 19.6 4.8 44.8 230.0 168.0 72.8 

4/5/99 18.8 14.6 4.8 32.3 180.0 215.0 51.3 

5/5/99 29.5 17.1 4.8 36.3 166.0 454.0 194.0 

6/3/99 9.7 26.3 2.5 36.2 301 983 421 

7/12/99 57.2 3.4 2.5 8.0 410 1310 306 

8/11/99 37.7 13.4 3.9 28.1 530 1070 672 

9/8/99 49.7 28.7 3.9 32.6 782 1480 595 

10/7/99 14.5 2.3 3.9 6.4 142 751 278 

11/9/99 25.3 3.4 4.7 23.5 304 883 491 

12/8/99 10.1 2.6 4.7 17.2 141 811 544 

1/6/00 13 7.8 4.7 16.9 188 934 438 

2/3/00 18.6 8.8 3.5 39.7 206 1060 466 

3/6/00 21.8 10.1 3.5 47.1 325 1070 471 

4/5/00 32.9 8.8 3.5 34.6 434 1150 410 

5/8/00 87.3 16.3 3.7 44.1 1900 2190 442 

6/7/00 43.4 9.6 3.2 47 761 1000 463 

7/6/00 23.4 1.6 3.2 20.1 448 1120 348 

31
Table 5-6. Metals Concentration 

Metals concentration (μg/L) 

Location Date Al As Cd Cu Fe Mn Zn 

8/10/00 17.3 1.5 3.4 10.2 87.7 837 269 

9/7/00 17.3 1.8 3.4 9.8 67.1 629 222 

9/25/00 17.3 1.5 3.4 6.3 124 794 145 

10/31/00 45.4 2.5 4.3 52.9 506 942 383 

12/4/00 18.9 4.1 4.3 16.9 1090 1320 170 

1/8/01 51.1 7.2 4.3 13.6 1590 1810 203 

2/12/01 18.9 5.5 4.3 18.5 937 2000 315 

3/14/01 85 6 0.4 530 1100 2300 220 

4/9/01 22 8 0.7 35 2900 2300 250 

5/9/01 55.8 9.6 4.8 33.9 3540 2610 211 

6/6/01 81.7 4.1 4.8 132 1940 1400 505 

7/9/01 42.3 1.1 4.8 18.2 572 1270 292 

pH Trends 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

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 

pH units 

Intake 

II 

III 

IV 

5 

Figure 5-1. pH trends. 

32
EH Trends 

-400 

-200 

0 

200 

400 

600 

800 

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 

EH (mV) 

Intake 

II 

III 

IV 

Figure 5-2. EH trends. 

Dissolved Oxygen Trends 

0 

2 

4 

6 

8 

10 

12 

14 

16 

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 

DO (mg/L) 

Intake 

II 

III 

IV 

Figure 5-3. Dissolved oxygen trends. 

33
34

Temperature Trends

0

2

4

6

8

10

12

14

16

18

20

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

Temperature (oC)

Intake

II

III

IV

Figure 5-4. 

Bioreactor II pH and EH Profiles for 12/6/99

-200

-100

0

100

200

300

400

500

intake II F II G II H II B II C II I II J II K II L

Piezometer nest (each piezometer is approximately 10 ft apart)

EH (mV)

0

2

4

6

8

10

12

14

pH

deep Eh

shallow Eh

deep pH

shallow pH

1st 

organic carbon

chamber

limestone

chamber

2nd

organic carbon 

chamber

cobble

chamber

Figure 5-5. H profiles for 12/6/99.

Temperature trends.

Bioreactor II pH and E
Bioreactor III pH and EH Profiles for 12/6/99 

-200 

-100 

0 

100 

200 

300 

400 

500 

intake III F III B III G III C III H III I III J 

Piezometer nest (each piezometer is approximately 10 ft apart) 

EH (mV) 

0 

2 

4 

6 

8 

10 

12 

14 

pH 

deep Eh 

shallow Eh 

deep pH 

shallow pH III F shallow was dry 

organic 

carbon 

chamber 

cobble 

chamber 

Figure 5-6. Bioreactor III pH and EH profiles for 12/6/99. 

SRB Populations 

1.E-01 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

1.E+05 

1.E+06 

1.E+07 

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 

SRB/mL 

II 

III 

IV 

Figure 5-7. SRB populations. 

35
Sulfate Concentrations 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

500 

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 

Sulfate (mg/L)

Intake 

II 

III 

IV 

Figure 5-8. Sulfate concentrations. 

Soluble Sulfide Concentrations 

0 

2 

4 

6 

8 

10 

12 

14 

16 

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 

Sulfide (mg/L) 

Intake 

II 

III 

IV 

Figure 5-9. Soluble sulfide concentrations. 

36
Total Alkalinity 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

500 

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 

Alkalinity [CaCO3(mg/L)]

Intake 

II 

III 

IV 

Figure 5-10. Total alkalinity. 

Zn Concentrations 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

1.E+05 

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 

Concentration (ug/L) 

Intake 

II 

III 

IV 

II autopsy sample (total) 

III autopsy sample (total) 

IV autopsy sample 

II autopsy sample (dissolved) 

III autopsy sample (dissolved) 

IV autopsy sample (dissolved) 

(total) 

Figure 5-11. Zinc concentrations. 

37
Cu Concentrations 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

1.E+05 

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 

Concentration (ug/L) 

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) 

Figure 5-12. Copper concentrations. 

Cd Concentrations 

0 

5 

10 

15 

20 

25 

30 

35 

40 

45 

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 

Concentration (ug/L) 

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) 

Figure 5-13. Cadmium concentrations. 

38
Al Concentrations 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

1.E+05 

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 

Concentration (ug/L) 

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) 

Figure 5-14. Aluminum concentrations. 

As Concentrations 

0 

10 

20 

30 

40 

50 

60 

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 

Concentration (ug/L) 

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) 

Figure 5-15. Arsenic concentrations. 

39
Fe Concentrations 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

1.E+05 

1.E+06 

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 

Concentration (ug/L) 

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) 

Figure 5-16. Iron concentrations. 

Mn Concentrations 

1.E+00 

1.E+01 

1.E+02 

1.E+03 

1.E+04 

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 

Concentration (ug/L) 

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) 

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 6-1a 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 6-1a. 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 6-1b) 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 Fg/L or 9% of Fe2+ in the limestone chamber 

of Bioreactor II. However, the analyses for sulfide 

and sulfate in the dissolved phase (Table 6-1c) 

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 6-1a), 

are electronically balanced by a high concentration 

of sulfate (Table 6-1c), 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 6-1c 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 (Or1) 

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 orangecolor 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 Or1 

of Bioreactor II, but its concentration peaked in 

the centers of Or1 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 muchelevated 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 

and Zn. 

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 6-1c). 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
F

F

Tables 6-1. Analytical Results of the Aqueous Samples Collected During the Autopsy 

Table 6-1a. Total and Dissolved Metals 

Dissolved metals (Fg/L) 

Location Al As Cd Ca Cu Fe Mg Mn Zn 

Bioreactor II cobble 1380 29.3 4.8 13500 86.8 2910 29400 1760 506 chamber 

Bioreactor III 353 16.8 4.8 87000 220 2830 20000 2720 1030 cobble chamber 

Bioreactor IV 162 8.5 4.8 124000 38.7 981 33300 1980 329 cobble chamber 

Total metals samples (Fg/L) 

Location Al As Cd Ca Cu Fe Mg Mn Zn 

Bioreactor II 34700 251 86.1 1040000 2450 43000 55100 7270 20400 limestone chamber 

Bioreactor II cobble 37100 53.1 5.3 152000 951 52200 45100 3000 2150 chamber 

Bioreactor III 34300 53.1 5.9 109000 592 42300 36200 5280 3770 cobble chamber 

Bioreactor IV 7300 30 11.4 539000 506 13200 42800 3160 6390 limestone chamber 

Bioreactor IV 9240 20 4.8 122000 159 12700 35200 2170 856 cobble chamber 

Table 6-1b. Iron speciation 

Iron Species (mg/L) (nonfiltered samples) 

Location Fe II Fe III Fe Total % Fe Recovery 

Bioreactor II limestone chamber 1.9 0.17 2.1 100 

Bioreactor II cobble chamber 3.6 <0.05 3.5 100 

Bioreactor III cobble chamber 2 <0.05 2.1 100 

Bioreactor IV limestone chamber 0.93 0.057 0.99 100 

Bioreactor IV cobble chamber 1 0.076 1.1 100 

Table 6-1c. Sulfate and Sulfide 

Sulfate and Sulfide concentrations (mg/L) (nonfiltered samples) 

Location Sulfate Sulfide 

Bioreactor II limestone chamber 3091 3.2 

Bioreactor II cobble chamber 2401 3.9 

Bioreactor III cobble chamber 352 2.2 

Bioreactor IV limestone chamber 225 3 

Bioreactor IV cobble chamber 1591 4.2 1

Corrected values obtained using inductive coupled plasma (ICP) method 

47
Table 6-2. Comparison of the Dissolved Metals from the Autopsy and Monthly Sampling 

Bioreactor location Date 

Zn Cu Mn Cd As Al Fe 

Sample Dissolved metal concentration (μg/L) 

AII1 07/9/01 899 110 996 5 4 118 707 

II 

Bottom2 07/17/01 506 87 1,760 5 29 1,380 2,910 

AIII1 07/9/01 1,010 122 2,470 6 3 80 638 

III 

Bottom2 07/17/01 1,030 220 2,720 5 17 353 2,838 

AIV1 07/9/01 292 18 1,270 5 1 42 572 

IV 

Bottom2 07/16/01 329 39 1,980 5 9 162 981 

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 the Autopsy 

Table 6-3a. Metal Concentrations 

Metal concentrations of the solid matrix samples (mg/Kg) 

Location Al As Cd Ca Cu Fe Mg Mn Zn 

Bioreactor II manure 7830 42.1 23.4 8490 1470 11400 5170 931 4450 chamber 1 lift 1 

Bioreactor II manure 13700 31.4 31.9 10400 2970 9960 9890 902 3790 chamber 1 lift 3 

Bioreactor II manure 10900 23.6 21.6 6350 2260 6910 6990 387 3470 chamber 1 lift 5 

Bioreactor II manure 6880 22.8 28.6 12300 975 9040 7640 1200 4270 chamber 1 lift 7 

Bioreactor II manure 4700 15.4 12.8 38800 438 7010 6480 884 2720 chamber 1 lift 9 

Bioreactor II limestone 2650 8.3 3.8 208000 163 4150 5110 740 1000 chamber 

Bioreactor II manure 3730 10.1 2.4 11500 147 7280 5920 866 3010 chamber 2 lift 5 

Bioreactor III intake 10300 400 25.7 5700 7340 127000 3980 25200 2530 plug - brown 

Bioreactor III intake 55800 485 31.2 1480 8560 57800 613 35100 2100 plug - black 

Bioreactor III manure 8600 76.1 24.3 9560 1480 14000 6180 637 3630 chamber 1 lift 1 

Bioreactor III manure 4020 24 10.1 13100 241 7710 6670 940 3820 chamber 1 lift 3 

Bioreactor III manure 6060 19.1 35.5 10500 596 8280 7130 783 5790 chamber 1 lift 5 

Bioreactor III manure 5500 17.8 5 15400 579 8970 6680 912 2900 chamber 1 lift 7 

Bioreactor III manure 4550 15.8 18.7 12600 203 8370 5210 1180 4880 chamber 1 lift 9 

Bioreactor IV manure 15100 109 10.5 2740 2400 22900 4750 263 1660 chamber 1 lift 1 

Bioreactor IV manure 13000 18.2 10.8 6130 2340 7390 6130 402 1600 chamber 1 lift 3 

Bioreactor IV manure 10900 14.9 59.3 9460 1670 6580 7600 668 7690 chamber 1 lift 5 

Bioreactor IV manure 5680 22 19.4 16600 562 7830 5290 655 6400 chamber 1 lift 7 

Bioreactor IV manure 3890 14.4 0.92 19100 107 9210 4930 800 1610 chamber 1 lift 9 

Bioreactor IV limestone 2400 4.2 0.91 274000 58.9 4050 4800 185 566 chamber 

48
Table 6-3a. Metal Concentrations 

Metal concentrations of the solid matrix samples (mg/Kg) 

Location Al As Cd Ca Cu Fe Mg Mn Zn 

Bioreactor IV manure 4700 19.9 0.92 18500 178 10300 7110 1500 583 chamber 2 lift 5 

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 

Table 6-3c. Sulfide Acid-Base Accounting 

10251 58.2 20.0 

14623 86.1 20.0 

12259 53 22.3 

12141 62.1 23.4 

12983 124 17.2 

3056 144 8.7 

13086 65.9 23.3 

10917 74.4 22.6 

15533 104 21.6 

10406 44.1 20.7 

11074 119 21.2 

6689 72.4 21.4 

8308 60.4 24 

13528 90.4 22.1 

11063 78.9 23.5 

10501 66.1 21.6 

9920 68.4 23.9 

3963 22 1.2 

10682 168 16.9 

Sulfide Analysis by Acid-Base accounting 

Hot Water 

Location Total Extractable S HCl Extractable S HNO3 Extractable S Residual S 

% Sulfur % Sulfate % Insoluble Sulfide % Pyritic Sulfide % Organic 

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

0.79 0.20 0.04 0.48 0.08 

0.70 0.19 0.03 0.44 0.05 

0.63 0.03 0.14 0.41 0.06 

0.69 0.18 0.05 0.41 0.05 

0.46 0.07 <0.01 0.33 0.07 

0.15 0.07 <0.01 0.17 0.02 

0.87 0.25 0.06 0.50 0.06 

0.76 0.23 0.04 0.37 0.12 

0.82 0.28 0.08 0.32 0.14 

1.12 0.44 0.13 0.41 0.14 

1.02 0.33 0.11 0.41 0.17 

1.10 0.52 <0.01 0.56 0.06 

49
k

Table 6-3c. Sulfide Acid-Base Accounting 

Sulfide Analysis by Acid-Base accounting 

Total Hot Water HCl Extractable S HNO3 Extractable S Residual S Location % Sulfur Extractable S % Insoluble Sulfide % Pyritic Sulfide % Sulfate % Organic 

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 

0.68 0.21 0.06 0.32 0.10 

0.72 0.23 0.02 0.42 0.05 

0.84 0.27 0.03 0.46 0.08 

1.19 0.81 <0.01 0.47 0.14 

0.90 0.29 0.06 0.49 0.07 

<0.01 <0.01 <0.01 <0.01 <0.01 

0.69 0.29 <0.01 0.32 0.08 

Table 6-3d. Analytical Results for Plugging Material in the Manifold of Bioreactor III 

Total metals in plugging material (mg/Kg) 

Mg 

Reactor III intake plug -

brown 10300 400 25.7 5700 7340 127000 3980 25200 2530 

Reactor III intake plug -

black 55800 485 31.2 1480 8560 57800 613 35100 2100 

Location Al As Cd Ca Cu Fe Mn Zn 

Table 6-4. Correlation Coefficients (k) for Total Metals Concentrations in the Solid Matrix of Bioreactors II, III, and 

Al As Cd Cu Fe Mn Zn 

Al 1 0.602 0.475 0.9703 0.5622 -0.376 0.1745 

As 1 0.095 0.5278 0.9434 -0.281 -0.0387 

Cd 1 0.476 0.0175 0.0003 0.8375 

Cu 1 0.4464 -0.39 0.1559 

Fe 1 -0.096 -0.0747 

Mn 1 0.1254 

Zn 1 

50

IV 
Total vs Dissolved Metals 

for the Cobble Chamber of Bioreactor II 

52200 

2150 

951 

3000 5.3 

53.1 

37100 

2910 

506 

86.8 

1760 4.8 

29.3 

1380 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total metals 

Dissolved metals 

Metals concentration in ug/L 

Figure 6-1a. Total vs. dissolved metals for the cobble chamber of Bioreactor II. 

Suspended Metals 

in the Cobble Chamber of Bioreactor II 

1644 

864.2 

1240 0.5 

23.8 

35720 

49290 

0.1 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total minus Dissolved 

Metals concentration in ug/L 

Figure 6-1b. Suspended metals in the cobble chamber of Bioreactor II. 

51
Total vs Dissolved Metals 

for the Cobble Chamber of Bioreactor III 

42300 

3770 

592 

5.9 5280

53.1 

34300 

2830 

1030 

220 

4.8 2720

16.8 

353 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total metals 

Dissolved metals 

Metals concentration are in ug/L 

Figure 6-2a. Total vs. dissolved metals for the cobble chamber of Bioreactor III. 

Suspended Metals 

in the Cobble Chamber of Bioreactor III 

2740 

372 

2560 

1.1 

36.3 

33947 

39470 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total minus Dissolved 

Metals concentration in ug/L 

Figure 6-2b. Suspended metals in the cobble chamber of Bioreactor III. 

52
Total vs Dissolved Metals 

for the Cobble Chamber of Bioreactor IV 

12700 

856 

159 

4.8 2170

20 

9240 

981 

329 

38.7 

8.5 4.8 1980 162 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total metals 

Dissolved metals 

Metals concentration are ug/L 

Figure 6-3a. Total vs. dissolved metals for the cobble chamber of Bioreactor IV. 

Suspended Metals 

in the Cobble Chamber for Bioreactor IV 

527 

120.3 

190 0 

11.5 

9078 

11719 

1 

10 

100 

1000 

10000 

100000 

Zn 

Cu 

Mn 

As Cd

Al 

Fe 

Total minus Dissolved 

Metals concentration in ug/L 

Figure 6-3b. Suspended metals in the cobble chamber of Bioreactor IV. 

53
0 

500 

1000 

1500 

2000 

2500 

3000 

3500 

4000 

Concentration (ug/L) 

Bioreactor II 

limestone chamber 

Bioreactor II cobble 

chamber 

Bioreactor III cobble 

chamber 

Bioreactor IV 

limestone chamber 

Bioreactor IV cobble 

chamber 

Location 

Iron Speciation for Autopsy Aqueous Samples 

Fe II 

Fe III 

Figure 6-4. Iron speciation for autopsy aqueous samples. 

1 

10 

100 

1000 

10000 

100000 

Concentration (ug/L) 

Al As Cd Cu Fe Mn Zn 

Metal 

Total Metals in the Limestone and Cobble Chambers 

in Bioreactor II (Aqueous Samples) 

Bioreactor II 

limestone chamber 

Bioreactor II cobble 

chamber 

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 

0.1 

1 

10 

100 

1000 

10000 

100000 

II/1/1 II/3/1 lI/5/1 II/7/1 II/9/1 Limestone Chamber 2 

Sample location 

Concentration (mg/kg) 

As 

Cd 

Mn 

Cu 

Zn 

Fe 

Al 

Chamber 1 

Flow direction 

Figure 6-6. Total metals concentration in the solid matrix of Bioreactor II. 

Total Metals Concentration in Solid Matrix 

of Chamber 1 in Bioreactor III 

0.1 

1 

10 

100 

1000 

10000 

100000 

III/1/1 III/3/1 lII/5/1 III/7/1 III/9/1 

Sample location 

Concentration (mg/kg) 

As 

Cd 

Mn 

Cu 

Zn 

Fe 

Al 

Chamber 1 

Flow direction 

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 

0.1 

1 

10 

100 

1000 

10000 

100000 

IV/1/1 IV/3/1 lV/5/1 IV/7/1 IV/9/1 Limestone Chamber 2 

Sample location 

Concentration (mg/kg) 

As 

Cd 

Mn 

Cu 

Zn 

Fe 

Al 

Chamber 1 

Flow direction 

Figure 6-8. Total metals concentration in the solid matrix of Bioreactor IV. 

Sulfate Profiles in Solid Matrix of Bioreactors 

0 

1000 

2000 

3000 

4000 

5000 

6000 

7000 

8000 

9000 

1st lift 3rd lift 5th lift 7th lift 9th lift 5th lift 

Location 

Sulfate concentration (mg/kg) 

II solid matrix 

III solid matrix 

IV solid matrix 

II aqueous matrix (mg/L) 

III aqueous matrix (mg/L) 

IV aqueous matrix (mg/L) 

Flow 1st organic chamber limestone 

chamber 

2nd 

organic 

chamber 

cobble 

chamber 

Figure 6-9. Sulfate profiles in the solid matrix of the bioreactors. 

56
Sulfide Profiles in Solid Matrix of Bioreactors 

0 

1000 

2000 

3000 

4000 

5000 

6000 

1st lift 3rd lift 5th lift 7th lift 9th lift 5th lift 

Location 

Sulfide concentration (mg/kg) 

II solid matrix 

III solid matrix 

IV solid matrix 

II aqueous matrix (mg/L) 

III aqueous matrix (mg/L) 

IV aqueous matrix (mg/L) 

Flow 1st organic chamber limestone 

chamber 

2nd 

organic 

chamber 

cobble 

chamber 

Figure 6-10. Sulfide profiles in the solid matrix of the bioreactors. 

1 

10 

100 

1,000 

10,000 

100,000 

1,000,000 

Metal concentration (ug/kg) 

Al As Cd Ca Cu Fe Mg Mn Zn 

Metal 

Bioreactor III Inlet-Plug Metals Concentration 

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, MSE Project Engineer; Rod Schwab, 

MSE Sampler; and Ken Reick, MSE 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 MSE 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 

MSE, 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 projectspecific 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 Precisionb Accuracycc Completenessd 

pH Aqueous SU N/A ±0.1e ±0.1f 95% 

Dissolved Metals Aqueous μg/L 5 #20% 75-125% 95% 

a

MDLs 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.

b

Relative percent difference of analytical sample duplicates.

c

Percent recovery of matrix spike, unless otherwise indicated.

d

Based on number of valid measurements compared to the total number of samples.

e

Precision of pH measurements was based on consecutive determinations of accuracy.

f

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 Mean Absolute Difference (Precision) Range of Absolute Differences (Precision)

Field pH Measurements 0.02 SU 0–0.09 SU 

Analysis Mean RPD for Sample Duplicates Range of RPDs for Sample Duplicates 

Dissolved Al 9.9% 0–28.3% 

Dissolved As 7.4% 1.3–18.8% 

Dissolved Cd 2.8% 0.9–8% 

Dissolved Cu 8.4% 0–50.6% 

Dissolved Fe 4.6% 0–12.6% 

Dissolved Mn 2.6% 0–8.4% 

Dissolved Zn 8.2% 0–70.3% 

Analysis Mean Absolute Difference (Accuracy) Range of Absolute Differences (Accuracy) 

Field pH Measurements 0.02 SU 0–0.09 SU 

Analysis Mean Matrix Spike Recovery

Dissolved Al 101%

Dissolved As 95.5%

Dissolved Cd 99.6%

Dissolved Cu 100.4%

Dissolved Fe 101.1%

Dissolved Mn 102.2%

Dissolved Zn 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. Summary of Qualified Data for MWTP Activity III, Project 12 

Date1 Sample Analysis QC Control Result Flag2 Comment 

ID Criteria Limit 

12/23/98 BDRB 

DBlank 

IAD 

IILD 

IIIA 

IVKD 

IVKDD 

IVKDS 

01/04/99 II1D 

IIIJD 

IVKD 

IIIA 

02/03/99 ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

04/05/99 

04/05/99 ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

06/03/99 ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

Dissolved Analytical #20% RPD 50.6 J Samples were flagged "J" 

Cu Duplicate due to an out-of-control 

field duplicate. 

Alkalinity Initial 90-110% recovery 63% R Initial calibration 

(forms) Calibration recovery verification had <75% 

Verification recovery; therefore, the 

data should be removed 

from consideration. 

Dissolved Field #35% RPD 47.4% RPD J Flag samples "J" for outFe Duplicate 

Field pH Holding Time Analyze Hours 

immediately elapsed 

of-control field duplicate. 

J 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." 

Dissolved Field #35%RPD 165% RPD J Flag samples "J" for outCu Duplicate of-control field duplicate. 

Dissolved Field Blank #100 ppb 128 ppb U Flag samples with 

Fe concentrations less than 10 

times the contamination 

concentration "U" due to 

out-of-control field blank. 

62
Date1 Sample Analysis QC Control Result Flag2 Comment 

ID Criteria Limit 

7/12/99 BDRB 

FB 

ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

08/11/99 BDRB 

FB 

ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

08/11/99 

04/05/00 BDRB 

FB 

ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

06/07/00 BDRB 

FB 

ID 

IIIA 

IIIAFC 

IIIJD 

IILD 

IVKD 

12/04/00 Intake 

IIBD 

IIIBD 

IVB 

IILD 

IIIJD 

IVKD 

Dissolved Matrix Spike 75-125% recovery 69.6% J Zinc results should be 

Zn flagged "J" as estimated 

Analytical #20% RPD 70.3% for out-of-control spike 

Duplicate and duplicate. 

Dissolved Matrix Spike 75-125% recovery 72.8 J Zinc results should be 

Zn flagged "J" as estimated 

for out-of-control matrix 

spike. 

Field pH Calibration Required each Not R Flag samples "R" as 

sampling event performed unusable due to lack of 

calibration documentation. 

Dissolved Matrix Spike 75-125% recovery 58.9 J Arsenic results should be 

As flagged "J" as estimated 

for out-of-control matrix 

spike. 

Dissolved Serial Dilution #10% difference 15.7% J Zinc results should be 

Zn flagged "J" as estimated 

due to out-of-control serial 

dilution analysis. 

Field pH Calibration Required each Not R Flag samples "R" as 

sampling event performed unusable due to lack of 

calibration documentation. 

Field ORP Required each Not 

sampling event performed 

63
Date1 Sample Analysis QC Control Result Flag2 Comment 

ID Criteria Limit 

1/8/01 Intake Dissolved Field #35% RPD 40.1% RPD 

IIBD Al Duplicate 

IIIBD 

IVB 

IILD 

IIIJD 

IVKD 

2/12/01 Intake Dissolved Field #35% RPD 40.1% RPD 

IIBD Cu Duplicate 

IIIBD 

IVB 

IILD 

IIIJD 

IVKD 

3/14/01 IIBD Sulfate Outlier Rosner’s Test for 1,400 mg/L 

Outliers is an outlier 

at a 5% 

significance 

level 

4/09/01 Intake Sulfate Outlier Rosner’s Test for 8 mg/L is an 

Outliers outlier at a 

5% 

significance 

level 

7/20/01 RCIIC Dissolved Matrix Spike 75-125% recovery 130.7% 

OB Al 

RCIIIC 

OB Dissolved 134.5% 

RCIVC Fe 

OB 

Dissolved 127.5% 

Mn 

J Flag samples "J" for outof-control field duplicate. 

J Flag samples "J" for outof-control field duplicate. 

R Sample is outlier 

according to Rosner’s test. 

Data were transformed to 

achieve normal 

distribution prior to 

performing outlier test. 

The data should be 

removed from further 

consideration. 

R Sample is outlier 

according to Rosner’s test. 

Data were transformed to 

achieve normal 

distribution prior to 

performing outlier test. 

The data should be 

removed from further 

consideration. 

J Flag samples "J" for outof-control matrix spike. 

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 

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 

EC to 16 EC. 

• 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 2.81E+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 μg/L for 

Bioreactors II and IV and 800 μg/L for 

Bioreactor III seem to be independent of influent 

concentrations. 

• Copper removal thresholds of 50 μg/L for 

Bioreactors II and IV and 80 μg/L for 

Bioreactor III also seem to be independent of 

influent concentrations. 

• Cadmium removal thresholds of 5 μg/L that 

prevailed for most of the operating time 

decreased when the Cd concentration of the 

influent dropped to 1 Fg/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 (k= 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. MSE 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. MSE 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, R.G. 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