EPA/600/R-09/158
September 2008
FINAL REPORT— AN INTEGRATED,
PASSIVE BIOLOGICAL TREATMENT SYSTEM
MINE WASTE TECHNOLOGY PROGRAM
ACTIVITY III, PROJECT 16
Prepared by:
MSB Technology Applications, Inc.
200 Technology Way
P.O. Box 4078
Butte, Montana 59702
Prepared for:
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Cincinnati, Ohio 45268
IAG ID No. DW89938870-01-0
and
U.S. Department of Energy
Environmental Management Consolidated Business Center
Cincinnati, Ohio 45202
Contract No. DE-AC09-96EW96405
September 2008
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REVIEWS AND APPROVALS (MWTP-):
Prepared by:
Project Manager
Approved by:
Program Manager
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MWTP-
September 2008
MINE WASTE TECHNOLOGY PROGRAM
Activity III, Project 16
Integrated Passive Biological Treatment System
By:
Suzzann Nordwick
MSB Technology Applications, Inc.
Mike Mansfield Advanced Technology Center
Butte, Montana 59702
Under Contract No. DE-AC09-96EW96405
Through EPA IAG No. DW89938870-01-0
Diana Bless, EPA Program Manager
Sustainable Technology Division
National Risk Management Research Laboratory
Cincinnati, Ohio 45268
This study was conducted in cooperation with
U.S. Department of Energy
Environmental Management Consolidated Business Center
Cincinnati, Ohio 45202
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Disclaimer
This publication is a report of work conducted under the Mine Waste Technology Program that was
funded by the Environmental Protection Agency and managed by the Department of Energy under the
authority of an Interagency Agreement.
Because the Mine Waste Technology Program participated in EPA's Quality Assurance Program, the
project plans, laboratory sampling and analyses, and final report of all projects were reviewed to ensure
adherence to the data quality objectives. The views expressed in this document are solely those of the
performing organization. The views and opinions of authors expressed herein do not necessarily state or
reflect those of the United States Government or any agency thereof.
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 or its contractors or subcontractors.
Neither the United States Government nor any agency thereof, nor any of their employees, nor any of
their contractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes
any legal liability or responsibility for the accuracy, completeness, or any third party's use or the results
of such use of any information, apparatus, product, or process disclosed, or represents that its use would
not infringe privately owned rights.
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Foreword
The U.S. Environmental Protection Agency (EPA) 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 (NRMRL) is the Agency's center for investigation
of technological and management approaches for preventing and reducing risks from pollution that
threaten 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 groundwater; prevention and control of indoor air pollution; and restoration of
ecosystems. 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 project was conducted under the Mine Waste Technology Program. It was funded by the EPA and
administered by the U.S. Department of Energy (DOE) in cooperation with various offices and
laboratories of the DOE and its contractors. It is made available at www.epa.gov/minewastetechnology
by EPA's Office of Research and Development to assist the user community and to link potential users
with the researchers.
Sally Gutierrez, Director
National Risk Management Research Laboratory
in
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Abstract
This report summarizes the results of the Mine Waste Technology Program (MWTP) Activity III,
Project 16, Integrated, Passive Biological Treatment System, funded by the United States Environmental
Protection Agency (EPA) and jointly administered by EPA and the United States Department of Energy
(DOE). This project addressed EPA's technical issue of Mobile Toxic Constituents - Water by
performing a field demonstration of a water treatment technology based on the use of passive biological
treatment at a remote inactive hardrock mine.
Field demonstration work was conducted at the Surething Mine located in the Elliston mining district of
Montana. This mine was never actively mined and has relatively small workings. However, disturbance
of rock at the mine workings resulted in acid rock drainage discharging from the mine portal. This project
was undertaken to demonstrate an integrated passive biological system to treat metal-laden water flowing
from an abandoned mine. The focus was to assess the effectiveness and reliability of the system to
produce a high quality effluent by reducing the level of dissolved sulfate and heavy metals from the acid
rock drainage (ARD) over a 5-year timeframe, between 2001 and 2006. Results and lessons learned for
this technology demonstration project are presented in this report.
The integrated passive biological treatment process installed at the Surething Mine consisted of a multi-
stage reactor system involving sequential treatment of acid rock drainage. Metal laden acidic water
emanating from the mine adit was treated using a series of three anaerobic reactors followed by aerobic
treatment including a final aerobic bioreactor. The anaerobic treatment relied on sulfate-reducing bacteria
(SRB) that converted dissolved sulfate to hydrogen sulfide, which reacted with dissolved metals to form
insoluble metal sulfides. This bacterial metabolism also produced bicarbonates that increased water pH
and limited further dissolution of metals. The SRB bioreactors substantially decreased the concentrations
of six of the seven target metals including aluminum, copper, iron, arsenic, cadmium, and zinc. After
limited success, the seventh target metal, manganese, was eventually removed from the water using an
aerobic bioreactor where manganese removal was consistent with manganese oxidizing bacteria activity.
The bioreactors were commissioned in August 2001 and analytical sampling and field measurements
occurred periodically through October 2005. In general, there was a significant reduction in dissolved
metals concentrations and the discharge pH was increased from near 3 to a more neutral level around 7.
This project showed that this type of integrated passive biological treatment process can offer
comprehensive treatment for many abandoned mine sites.
IV
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Contents
Page
Disclaimer ii
Foreword iii
Abstract iv
Contents v
Figures vi
Tables vii
Acronyms and Abbreviations viii
Acknowledgments ix
Executive Summary ES-1
1. INTRODUCTION 1
1.1 Project Overview 1
1.2 Background 1
1.3 Project Purpose 1
1.4 Scope of the Problem 2
1.5 Site Selection 2
1.5.1 Mine Location 2
1.5.2 Mine Site Geology 2
1.5.3 Mine History 2
1.6 Treatment Technology Background 3
1.7 Project Objectives 3
2. TREATMENT TECHNOLOGY DESCRIPTION 5
2.1 SRB Technology 5
2.1.1 SRB Growth Parameters 5
2.2 MOB Technology 5
2.3 Metals Removal Mechanisms 6
2.3.1 Adsorption by Substrate in Bioreactors 6
2.3.2 Biological Sulfate Reduction in SRB Reactors 7
2.3.3 Hydroxide Precipitation in SRB Reactors 7
3. DEMONSTRATION DESCRIPTION 9
3.1 Laboratory System Design 9
3.2 Laboratory Results 9
3.3 Field Design and Construction 10
3.3.1 Reactor 1 11
3.3.2 Reactor 2 11
3.3.3 Reactor 3 12
3.3.4 Aeration Line 12
3.3.5 Original Reactor 4 12
3.3.6. Improved Reactor 4 13
4. FIELD DEMONSTRATION RESULTS AND DISCUSSION 16
4.1 System Operation 16
4.2 Metals Results 16
4.2.1 Aluminum 16
4.2.2 Copper 16
4.2.3 Cadmium 16
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4.2.4 Zinc 16
4.2.5 Iron 16
4.2.6 Arsenic 17
4.2.7 Manganese 17
4.3 Physical Field Measurement Results 18
4.3.1 ORP 18
4.3.2 pH 19
4.3.3 Temperature 19
4.3.4 Dissolved Oxygen 19
4.3.5 Flow Rate 20
4.5 Other Chemical Measurements 20
4.5.1 Alkalinity 20
4.5.2 Sulfate 20
4.5.3 Sulfide 20
4.5.4 Calcium 21
4.6 Microbiology 21
4.6.1 Manganese Oxidizing Bacteria 21
4.6.2 SRB Counts 21
4.6.3 Microbial Community Analysis 22
5. SUMMARY AND CONCLUSIONS 34
6. LESSONS LEARNED AND RECOMMENDATIONS 35
6.1 Original Reactor 4 Design 35
6.2 Modifications to Original Reactor 4 35
6.3 Construction of New Reactor 4 35
6.4 Aeration System 35
6.5 Aeration Hold Tank 35
6.5 Reactor 2 36
6.6 SRB Reactor Design 36
6.7 Recommendations 36
7. REFERENCES 37
Appendix A: Summary of Quality Assurance Activities A-l
Appendix B: Statistical Analysis B-l
Appendix C: Microbial Analysis Report C-l
Figures
Figure 3-1. Bar graph showing dissolved metals removal through laboratory column test system....Error!
Bookmark not defined.
Figure 3-2. Block-flow of the integrated, passive biological treatment system's unit operations 14
Figure 3-3. Collage showing components of the integrated, passive biological treatment system including
two biomass based anaerobic bioreactors, one limestone based anaerobic bioreactor, and one
limestone based Aerobic bioreactor 15
Figure 4-1. Aluminum concentrations 23
Figure 4-2. Copper concentrations 23
Figure 4-3. Cadmium concentrations 24
vi
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Figure 4-4. Zinc concentrations 24
Figure 4-5. Iron concentrations 25
Figure 4-6. Arsenic concentrations 25
Figure 4-7. Manganese concentrations 26
Figure 4-8. ORP meter readings vs. time 26
Figure 4-9. pH field readings 27
Figure 4-10. Field temperature readings 27
Figure 4-11. Dissolved oxygen field meter readings 28
Figure 4-12. Field flow rate measurements 28
Figure 4-13. Alkalinity concentrations 29
Figure 4-14. Sulfate concentrations 29
Figure 4-15. Plot showing sulfide concentration with time 30
Figure 4-16. Dissolved Calcium concentration 30
Figure 4-17. SRB counts 31
Tables
Table 1-1. Demonstration Goals 3
Table 1-2. Typical Water Chemistry of Surething Mine Drainage 4
Table 3-1. Laboratory system feed water (Calliope Mine) 15
Table 4-1. Sample location descriptions used for retinue sampling events 31
Table 4-2. Dissolved Metals Percent Reduction Between Mine Discharge and the Process Effluent on
Sept. 1,2005 32
Table 4-3. Dissolved Manganese Levels at Progressive Location into Reactor 4 32
Table 4-4. Summary of System Flow Conditions during the Summer of 2002 33
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Acronyms and Abbreviations
ARD acid rock drainage
ATP adenosine triphosphate
BLAST Basic Local Alignment Search Tool
BOD biochemical oxygen demand
COD chemical oxygen demand
COOH carboxylic acid group
DNA deoxyribonucleic acid
DO dissolved oxygen
DOE Department of Energy
EPA Environmental Protection Agency
Fe iron
IAG Interagency Agreement Number
ID identification
Mn manganese
MOB manganese-oxidizing bacteria
MSE MSE Technology Applications, Inc.
mV millivolts
MWTP Mine Waste Technology Program
NPV net present value
NRMRL National Risk Management Research Laboratory
ORP oxidation reduction potential
PCR polymerase chain reaction
pH negative log of hydrogen ion concentration
PMDTS passive mine drainage treatment systems
QA quality assurance
QAPP quality assurance project plan
QC quality control
SRB sulfate-reducing bacteria
TVA Tennessee Valley Authority
VFA volatile fatty acid
Zn zinc
Vlll
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Acknowledgments
This document was prepared by MSB Technology Applications, Inc. (MSB) for the U.S. Environmental
Protection Agency's (EPA) Mine Waste Technology Program (MWTP) and the U.S. Department of
Energy (DOE). For this project, Ms. Diana Bless was EPA's MWTP Program Manager and Mr. Gene
Ashby was DOE's Technical Program Officer. Ms. Helen Joyce was MSB's MWTP Program Manager.
The Integrated, Passive Biological Treatment System project was the result of contributions by over 40
MSB employees. Of these, Brian Park, Marietta Canty and Creighton Barry made significant
contributions.
Special acknowledgment and thanks are extended to the members of the Newman family - owners of the
Surething Lode and Extension mining claims during this demonstration project.
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Executive Summary
This document is the final report for the Mine Waste Technology Program (MWTP), activity III, Project
16 Integrated, Passive Biological Treatment System. The MWTP is funded by the United States
Environmental Protection Agency (EPA) and is jointly administered by EPA and the United States
Department of Energy (DOE). MSE Technology applications, Inc. (MSE) of Butte, Montana was
responsible for implementing this demonstration project. Both EPA and DOE provided program
administration, demonstration oversight, technical review, and quality assurance and quality control
(QA/QC) oversight. The primary objective of the MWTP is to advance the understanding of engineering
solutions for national environmental issues resulting from past practices in mining and smelting of
metallic ores. The project reported herein addresses treatment of an acidic, metal-laden water draining
from a remote, abandoned mine in Montana through the demonstration of an integrated, passive system
employing both anaerobic and aerobic water treatment biotechnologies. At the demonstration site, a
series of pit-type bioreactors were constructed within waste-rock piles located downhill from the mine
adit. Because of the passive nature of the project, flow through all the bioreactors and the aeration step
was by gravity.
Because of previous MWTP success with passive biological treatment, this technology was again selected
for the Surething site. The new part of this demonstration was using a combination of anaerobic and
aerobic processes to provide a complete treatment system. With in the anaerobic component of the
process, sulfate reducing bacteria (SRB), work by metabolizing sulfates to sulfides. This in turn causes
some metals to precipitate as sulfide complexes, which are typically considerably less soluble than metal
hydroxides. The SRB metabolic system also releases alkalinity, thereby increasing the pH of the effluent.
This is an effective, passive technology that requires little maintenance and can be economical both to
build and operate, and function quite reliably. Anaerobic SRB systems work well to raise pH and remove
contaminants such as arsenic, cadmium, copper, iron, lead, and zinc, while aerobic systems work better to
remove manganese and residual organic nutrients. This portion of the system consisted of aeration of the
water and subsequent treatment with manganese oxidizing bacteria (MOB).
Bench-scale testing was performed in the laboratory to develop parameters for an integrated biological
treatment system prior to building the field system. After successful results in the lab, the field system
was designed and built at the Surething Mine. Because of poor manganese removal initially, numerous
modifications were made to the aerobic portion of the field system. This resulted in a field system that
successfully treated the ARD. Chemical parameters typical of untreated Surething Mine water are shown
in Table ES-1 along with the dissolved metals and the effluent water chemistry for the treated effluent.
This data was collected September 1, 2005 after all system modifications were completed. This
demonstration met its goal to prove that this technology offers a comprehensive passive cleanup method
for numerous remote or abandoned mines that discharge acidic metal-contaminated water.
Table ES-1. Typical Surething Mine influent and effluent dissolved metals and pH
Feed
Effluent
Iron
(mg/L)
15.0
<0.014
Zinc
(mg/L)
22.7
<0.007
Aluminum
(mg/L)
29.5
<0.04
Manganese
(mg/L)
26.7
<0.04
Arsenic
(mg/L)
0.13
<0.01
Cadmium
(mg/L)
0.21
<0.00009
Copper
(mg/L)
2.35
<0.003
pH
2.5
6.9
ES-1
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1.0 Introduction
1.1 Project Overview
This document is the final report for Mine Waste
Technology Program (MWTP), Activity III,
Project 16, Integrated, Passive Biological
Treatment System. The MWTP is a program
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). MSE
Technology Applications, Inc. (MSE) is the
principal contractor for the MWTP. The intent
of this project was to demonstrate the ability of
an integrated anaerobic and aerobic biological
system to treat acid rock drainage (ARD) at the
Surething Mine located in the Elliston Mining
district near Helena, Montana.
This report presents a brief summary of the
bench-scale work and greater detail on the field
results for this project. The bench-scale tests
were performed from 1998-1999. Field results
were gathered during a four plus year field
demonstration occurring from August 2001 to
October 2005. During this time, the ability of the
system to treat acidic, metal-contaminated water
was evaluated.
The integrated passive biological treatment
process consisted of a multi-stage reactor system
with sequential treatment of ARD. Anaerobic
and aerobic bioreactors were constructed near
the portal outside the Surething Mine tunnel.
Metal-laden acidic water emanating from the
mine adit was treated by passing it through a
series of three anaerobic reactors followed by
aeration and a final aerobic bioreactor. The
anaerobic treatment relied on sulfate-reducing
bacteria that reduced dissolved sulfate to
hydrogen sulfide. This, in turn, reacted with
dissolved metals to form insoluble metal
sulfides. The bacterial metabolic system also
produced alkalinity in the form of bicarbonates
that increased water pH and limited further
dissolution of metals. The SRB bioreactors
substantially decreased the concentrations of six
of the seven target metals (copper, iron, arsenic,
cadmium, aluminum and zinc). After limited
success, the seventh-target metal, manganese,
was removed from the water using an aeration
step followed by an aerobic bioreactor. This
reactor was designed to support a self-
establishing population of indigenous
manganese oxidizing bacteria (MOB). And, the
eventual of removal of manganese was
consistent with bacterial oxidation.
For the most part, metal removal was very high.
Initially, manganese removal was poor, but as
the demonstration progressed, design
improvements were made and the removal of
manganese was increased significantly.
1.2 Background
Prior to field testing, bench-scale testing was
initiated in the laboratory in reactors that
simulated conditions at the Surething Mine. The
tests demonstrated the effectiveness of SRB and
MOB technologies to treat ARD. Additionally,
since biological oxidation of manganese was not
well understood, laboratory testing allowed for
proof-of-concept testing for MOB prior to field
implementation of the technology.
1.3 Project Purpose
Congress charges EPA 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 provides data
and technical support for solving environmental
problems today and building a scientific
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 purpose of the Integrated Passive Biological
Treatment System demonstration project was to
test and evaluate a novel bioreactor process and
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determine the capabilities of this process to
remove dissolved metals from ARD emanating
from the Surething Mine. Using SRB
technology to treat ARD was a proven
technology. Therefore, it was proposed that an
integrated biological configuration would allow
for more complete treatment of ARD than would
SRB alone. It was planned that the technical
information gained from this project would
provide technical and economic information on
the capabilities of this innovative application of
biological process to treat ARD and improve
water quality.
1.4 Scope of the Problem
ARD results when metal sulfide minerals,
particularly iron pyrite, come in contact with
oxygen and water and the metal sulfide minerals
are oxidized and then dissolved into the water.
Acid generation occurs when metal sulfide
minerals are oxidized according to the following
general overall reaction equation:
FeS2 + 15/4 O2 + 7/2 H2O <—>
Fe(OH)3 + 2SO42 + 4H+ (1)
This reaction is one of many that results in
increased metal mobility and increased acidity
(lowered pH) of the water.
The oxidation of sulfide minerals is accelerated
by bacterial action. Thiobacillus ferrooxidans is
a naturally occurring bacterium that at pH 3.5 or
less can rapidly accelerate the conversion of
dissolved Fe2+ (ferrous iron) to Fe3+ (ferric iron),
which can act as an oxidant for the oxidation of
FeS2 (Cohen and Staub, 1992). This bacterial
activity may cause up to 80% of the acid
production in ARD (Welch, 1980). Ferric ions,
as well as other metal ions, and sulfuric acid
have a deleterious influence on the biota of
streams receiving ARD (Dugan et al., 1968).
1.5 Site Selection
As an initial step in this project, several mine
sites were screened and prioritized according to
physical accessibility, legal accessibility, and
discharging water characterization. The site
selected was the Surething Mine, a relatively
small mine with a sulfide-based geology that
produced ARD in flooded and near-surface mine
workings. The ARD flowed from the mine
portal into a down-gradient area covered in mine
tailings. The natural slope and large tailings area
provided a good location for construction of a
gravity-fed passive treatment system.
1.5.1 Mine Location
The Surething Mine is located in the Elliston
Mining District of Powell County, Montana is
situated on patented claims in the Helena
National Forest about 11 miles south of Elliston.
The town of Elliston is located about 20 miles
west of Helena and south of the Little Blackfoot
River. The mining district includes the town of
Elliston but is generally in mountainous, heavily
forested terrain. The Surething Mine is located
in the Telegraph Creek drainage at SE1/4 Sec.
15, T. 8 N., R. 6 W at an elevation of
approximately 7,000 feet above mean sea level.
1.5.2 Mine Site Geology
The bedrock geology of the Elliston Mining
District is consistent with the Montana Boulder
Batholith, which is composed of intrusive quartz
monzonite granitic rocks that intruded into older
sedimentary and volcanic rocks such as
limestone, shale, quartzite, and andesite. (Rankin
1950). The Surething Mine's primary ores
contained silver and lead and included abundant
tourmaline.
1.5.3 Mine History
In the first half of the 20th century, the Elliston
District of Powell County, Montana was a small
producer of lead-zinc ores with trace values of
gold and silver.
The Surething Mine consists of a near surface
adit into a steep hillside with narrow horizontal
single-layer workings extending a few hundred
feet into the mountain. As with most mines in
the Elliston District, the operations at the
Surething were mostly exploratory and not
extensive. Mine production records show that
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gold, silver, and lead were produced from 1902
to 1947 (McClernan, 1976).
A pile of mine tailings and waste rock was
deposited just downhill from the Surething adit.
The adit historically discharged about 2 gallons
per minute (gpm) of pH 3 ARD associated with
groundwater from the workings. However, the
flow rate of water emanating from the Surething
is greatly influenced by surface precipitation and
snow melt. During spring runoff, the flow
exceeded 10 gpm periodically. The mine
discharge flows toward O'Keefe Creek, which
flow into Telegraph Creek. However, the
discharge infiltrates into the subsurface
approximately 120 feet from the adit and does
not directly affect O'Keefe Creek, which lies
about 1,700 feet northeast of the adit.
1.6 Treatment Technology Background
The technology employed to treat the ARD
discharging from the Surething Mine was
twofold. First, an SRB-based system was
constructed upon the mine tailings pile. This
system consisted of three reactors to biologically
generate sulfide (S~2) and bicarbonate (HCO3~)
that would react with dissolved metals in the
ARD to form metal precipitates and neutralize
the water. This configuration was tested at a
previous MWTP demonstration site which
proved that the design could provide good
conditions for SRB growth. In order to remove
additional metals, passive aeration and aerobic
biological treatment steps were added to the
process. For more detailed information see the
Technology Description (Section 2).
1.7 Project Objectives
The project objective was to develop technical
information on the ability of an integrated
passive biological system to comprehensively
treat ARD. The goal was to improve water
quality at the remote mine waste site by
reducing the amount of acid and metals in the
mine water. The specific purpose of the field
demonstration was to show that SRB and MOB
technology could be integrated together to treat
an acidic aqueous waste by removing toxic
dissolved metallic and anionic constituents and
neutralizing the pH. The goal of the
demonstration was to achieve a 75% reduction
in the dissolved metals data; approximate target
values are shown in Table 1-1.
The project focus was a technology
demonstration, not a remediation project. Since
the purpose of the project was purely scientific,
the objectives did not attempt to address site
remediation considerations. EPA agreed to the
effluent parameters in Table 1-1 by approving
the project quality assurance project plan
(QAPP) (MSB 2001).
The project work plan specified that appropriate
process and environmental information be
collected, such as seasonal effects on system
operation. The project was not limited to
evaluating the effectiveness of integrated
biotechnologies to control acid generation and
treat water, but also focused on the feasibility
and appropriateness of using these technologies
at a remote site that experienced harsh winters.
Successful achievement of the project goals was
quantified by measuring dissolved metals
concentrations, to verify the ability of this
integrated biological system to treat metal
contamination associated with ARD. The
drainage emanating from the Surething Mine
and the effluent from the integrated, passive
biological treatment systems were monitored for
reduction of dissolved sulfate, reduction of
dissolved heavy metals, pH, and ORP. A
detailed discussion of the sampling can be found
in the QAPP (MSB, 2001).
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Table 1-1. Demonstration Goals
Parameter Goal
pH between 6 and 8 S.U.
Dissolved aluminum < 2775 ug/L
Dissolved arsenic < 86 ug/L
Dissolved cadmium < 21 ug/L
Dissolved copper < 204 ug/L
Dissolved iron < 16,000 ug/L
Dissolved zinc < 22.7 ug/L
Dissolved manganese < 3125 ug/L
Table 1-2. Typical Water Chemistry of Surething Mine Drainage
Parameter Value
pH 2.8 S.U.
ORP 531mV
Al 23.7mg/l
As 0.429 mg/1
Cd 0.162 mg/1
Cu 2.02 mg/1
Fe 40.7 mg/1
Mn 22.0 mg/1
Zn 18.3 mg/1
SO4 437 mg/1
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2. Treatment Technology Description
The following section provides descriptions of
anaerobic biotechnology using SRB and aerobic
biotechnology using MOB. In addition,
descriptions are presented for the major
mechanisms that result in metals removal within
bioreactors.
2.1 SRB Technology
SRB are reported to be present in almost all
environments on earth (Young, 1936). For
example, bottom muds of seawater were found to
contain 100 to 10,000 viable SRB cells per gram
(Postgate, 1984). Members of the Desulfovibrio
genus of bacteria are the principal biological
agents that reduce sulfate to sulfide. However,
eight genera of SRB are known to exist:
Desulfovibrio, Desulfomonas, Desulfotomaculum,
Desulfobacter, Desulfobulbus, Desulfococcus,
Desulfosarcina, andDesulfonema (Hunter, 1989).
The dominant species of SRB belong to the genera
Desulfotomaculum and Desulfovibrio (Cohen and
Staub, 1992). Field demonstrations and research
conducted by the MWTP and many others
(Figueroa et al. 2004; Gusek 2002; McGregor
1999; Skousen et al. 2000; Tsukamoto and Miller
2002; Wildeman and Updegraff 1998) have shown
that sulfate-reducing bacteria (SRB) can be used to
effectively treat ARD.
2.1.1 SRB Growth Parameters
Growth rates of SRB are an important parameter
in designing biological reactors, including in situ
applications for the treatment of ARD. The
required amount of substrate for the reaction can
be predicted from experimental growth rates. For
example, growth rates can be used to determine
the necessary reactor residence time (Lee, 1992
and Middleton and Lawrence, 1977).
Postgate (1984) describes Desulfovibrio growth as
linear rather than exponential in many media.
Middleton and Lawrence (1977) reported that
microbial growth of SRB using acetate as the
substrate (single substrate model) could most
closely be modeled by Monod's Equation for the
growth rate of biomass.
Most in situ applications of biological sulfate
reduction can be best modeled by a plug-flow
model and microbial kinetics. In plug-flow
reactors, the fluid retention time in the reactor is
an important parameter since it describes the
contact time the bacteria will have with the
wastewater. A certain portion of the bacteria will
be attached to the substrate; however, another
portion will be free-floating in the water column.
The hydraulic residence time should be at least as
long as the doubling time of the organism; such
duration ensures the SRB are not "washed out" of
the reactor (Lee, 1992). Although in most real
systems wash out would not occur because of cell
adsorption to surfaces. Residence times required
for in situ treatment of ARD have been reported to
range from 20-30 hours to 20-30 days (Cohen and
Staub, 1992).
An in situ application of biological sulfate
reduction would utilize psychrophilic strains of
SRB. SRB are comprised of psychrophilic,
mesophilic, and thermophilic strains. Mesophilic
SRB live in moderate temperatures (30 C), while
thermophilic SRB require higher temperatures (50
to 70 °C) for growth. Psychrophilic SRB (live in
cool temperatures) have been reported in the
literature (Barghoorn and Nichols, 1961), but have
been studied to a very limited degree (Postgate,
1984). In addition, growth of mesophilic SRB is
considered slow in comparison to typical bacterial
growth rates. Postgate (1984) suggested that this
slow growth may be the result of H2S production,
which is intrinsically toxic to living systems.
However, Postgate (1984) also postulated that H2S
reacting with soluble iron to form insoluble iron
sulfide, thus removing iron from availability as a
nutrient, may more likely be the cause of slow
growth.
2.2 MOB Technology
Dissolved manganese is difficult to remove from
mine waters because, unlike other metals, it does
not form a sulfide precipitate at the relatively low
pH levels inherent to treating ARD. Manganese
-------
has a wide solubility range and requires a
relatively high pH to precipitate as an oxide.
Oxidized manganese is extremely slow to
precipitate under neutral pH conditions, however,
oxidized manganese readily precipitates from
solution at pH values above 9.5. Since MOB
induce manganese oxidation, they work to
enhance manganese removal.
Research has identified Leptothrix discophora as
the major manganese-oxidizing bacteria in the
treatment of AMD (Robbins et. al. 1999) and
(Zhang et. al. 2002). As these are ubiquitous
bacteria, the Surething system was designed to
allow for an indigenous population to naturally
establish itself in the MOB reactor. This reactor
was filled with limestone cobble. This media was
chosen due to its proven ability to allow the
growth of a Leptothrix discophora biofilm and for
its ability to gradually add solution alkalinity. The
system was installed with a design that would
allow a viable culture of manganese-oxidizing
bacteria to establish and be continually supported.
In 2000 the original Surething MOB reactor was
designed with a 4-foot depth. However, a system
performance analysis conducted in 2004
determined that a shallower reactor would realize
more effective growth of MOB. Therefore, the
reactor was modified to a more reasonable 1 foot
depth. This rebuild was consistent with other
ongoing research that indicated that MOB reactors
must be shallow to ensure sufficient infiltration of
light and dissolved oxygen (Johnson and Younger
2005).
The basic science of MOB bioreactors is still
being developed and is a topic of much study. It
was beyond the scope of this project to investigate
specific mechanisms of manganese-oxidizing
bacteria.
2.3 Metals Removal Mechanisms
Although one of the purposes of this field testing
was to evaluate the use of SRB to mitigate metal-
contaminated wastewaters, other metal removal
mechanisms are also typically associated with an
organic-based system. Wildeman, et al., (1993)
list removal processes in the following sequence
of decreasing priority: (1) exchange of metals by
an organic-rich substrate; (2) biological sulfate
reduction with precipitation of metal sulfides; (3)
precipitation of metal hydroxides; (4) adsorption
of metals by ferric hydroxides; and (5) metal
uptake by living plants. The last mechanism (5)
can be disregarded for our purposes because plants
were not associated with the design. Each of these
processes is described below.
2.3.1 Adsorption by Substrate in
Bioreactors
The binding of metal ions by organic matter can
play an important role in removing these ions from
solution and most likely some adsorption occurred
at the Surething Mine. Three categories of
macromolecular, colloidal, and particulate matter
are known to be responsible for metal binding at
the solid-solution interface: (1) polymeric organic
substances, most of which contain many
hydrophilic functional groups that are capable of
acting as donor groups for complex formation; (2)
colloidal or particulate organic matter; and (3)
inorganic solids, especially hydrous oxides
(Stumm and Morgan, 1981).
An example of adsorption onto a polymeric
organic substance, such as a humic or fulvic acid,
can be described by the following reactions. In
this example, R represents a complex organic
component and M represents a divalent metal.
RCOOH < >RCOO +H+ (2)
2 RCOO + M2+ < > M(RCOO)2 (3)
Exchange of metals with humic and fulvic acids
(RCOOH) in a substrate such as manure or peat is
a likely mechanism for temporary retention of
metals. Retention in this manner is temporary for
two reasons: (1) Equation 2 is pH-dependent, and
(2) different metals have diverse affinities for
adsorption. The pKa for acid dissociation of
humic materials averages approximately 4.2;
therefore, in mine drainage with a pH of 3, the
dominant species in solution will be carboxylic
-------
acids, which will not complex metal ions.
Therefore, the pH level needs to be at least 4 to
allow metal complexes to form to a significant
degree (Wildeman et al., 1993).
Even if the pH remains sufficiently high,
adsorption is a finite process, dependent on the
quantity of organic material present. As the
amount of organic acids is depleted, more weakly
sorbed metals (such as manganese or zinc) may be
released back into solution in exchange for more
strongly sorbed metals (such as iron or copper).
Consequently, the removal of manganese, zinc,
and cadmium by substrate adsorption is difficult
(Wildeman et al., 1993).
2.3.2 Biological Sulfate Reduction in SRB
Reactors
Biological sulfate reduction requires SRB,
dissolved sulfate as the electron acceptor, and a
carbon source as the electron donor. Certain
environmental conditions, such as a pH between 5
and 8 and a redox potential (EH) below -100
millivolts (mV) (Cohen and Staub, 1992) are also
helpful for optimal growth. Sulfate reduction
generates hydrogen sulfide, which is then
available for reaction with metal ions to form
metal sulfides. Formation of metal sulfides, most
of which are quite insoluble at a low EH and a
neutral pH, is very rapid.
Biological sulfate reduction is defined as the
chemical reduction of dissolved sulfate by the
action of biological processes (Dvorak et al.,
1991). When dealing with the treatment of ARD,
this process is generally limited to the reduction of
dissolved sulfate to hydrogen sulfide and the
concomitant oxidation of organic nutrient
compounds to bicarbonate within the aqueous
solution. Sulfate reduction is accomplished by a
group of heterotrophic, anaerobic bacteria known
as SRB. To thrive, SRB require reducing
conditions. They will not thrive in aerobic
conditions for extended periods. Also, as
heterotrophic bacteria, SRB need a source of
carbon in the form of an organic nutrient.
Biological sulfate reduction improves the quality
of ARD in four ways. First, the hydrogen sulfide
that is produced will react with dissolved metals to
form insoluble metal sulfides that will precipitate
from solution (Equations 4, 5, and 6). Second, the
reaction has a neutralizing effect on the pH of the
ARD because hydronium ions are consumed by
the reduction of sulfate. Third, this reaction
produces alkalinity in the form of bicarbonate
from the oxidation of the organic nutrients.
Finally, sulfate is removed from the aqueous waste
stream to produce hydrogen sulfide.
SO42 + 2CH2O
H7S + 2HCO,
(4)
H2S 2FT + S2' (5)
S2- + M+2 MS, where M = metal (6)
Postgate (1984) reported that lactate, pyruvate,
glycerol, ethanol, and the tricarboxylic acids are
all converted to acetate and carbon dioxide as
major end products by Desulfovibrio (a genus of
SRB). This process is known to involve the
conversion of adenosine triphosphate (ATP) to
adenosine monophosphate, the primary way that
cells transfer energy (Postgate, 1984).
Several studies have been performed in recent
years to research the process by which SRB can
remediate metal-contaminated wastewater. These
studies range from bench-scale experiments, such
as SRB growth in chemostats, to field
applications, such as constructed wetlands. The
use of wetlands, or passive mine drainage
treatment systems (PMDTS), to treat ARD
evolved from the observation that the water
quality of ARD flowing through natural sphagnum
moss bogs improved.
2.3.3 Hydroxide Precipitation SRB
Reactors
Of the metals of interest in the Surething Mine
water (zinc, copper, cadmium, aluminum,
manganese, iron, and arsenic), metal sulfides are
more predominant than hydroxides under the pH
(6-8) and EH (-100 mV) conditions induced on the
system by the technology (assuming sufficient
-------
hydrogen sulfide produced by the SRB). For the only stable hydroxide in this pH and EH range.
example, ferric hydroxide precipitation was Therefore, aluminum removal by hydroxide
viewed as an unlikely occurrence, given the precipitation most likely occurred at the Surething
reducing conditions present in the system, which Mine.
make sulfate reduction and the presence of ferric
ion mutually exclusive. Aluminum hydroxide is
-------
3. Demonstration Description
Demonstration of SRB technology consisted of
two major phases: 1) Laboratory bench-scale
tests, and 2) Field demonstration. The following
sections will discuss the various aspects of the
project. Additionally, other publications have
presented summary information of this
demonstration (Doshi 2006), (Nordwick 2002),
(Nordwick 2005), and (Nordwick 2006).
3.1 Laboratory System Design
Bench-scale testing was performed in MSB's
Resource Recovery Facility in 1998 and 1999.
An MSB internal report was produced
documenting testing. The system consisted of
three sets or arrays of columns with varying
combinations of fill materials. Array #1 most
represented what became the field installation at
the Surething Mine. This set-up consisted of
four anaerobic columns and two aerobic
reactors. The first three columns were 8 inches
in diameter and 2.5 feet high, while the fourth
column was 8 inches in diameter and 3.5 feet
high. All columns operated in an upflow
fashion. The first column contained steer
manure, the second column was filled with
crushed limestone, the third column contained
steer manure, and the fourth column held sized
sand. Sampling Points (SP) were identified as
follows: SP1 was after the first manure column,
SP2 was after the crushed limestone column,
SP3 was after the second manure column, SP4
was after the sand column, and SP5 was after the
aerobic reactor. A plot of some results obtained
by treating mine water using this array are
presented in Figure 3-1.
The aerobic portion of the system consisted of
two sequential reactors. The first aerobic reactor
was intended to aerate the water and precipitate
any remaining iron as ferric hydroxide. The
aeration device consisted of three stacked,
perforated trays placed in a zigzag fashion. A
plastic container (1 foot x 1 foot x 2 inches)
received the aerated water and served as a
settling basin. Water overflowed from this
container into a larger, sand-filled reactor (4 feet
x 2 feet x 8 inches), which was intended to
remove manganese. The manganese-removal
reactor was originally intended to use
technology developed by the Tennessee Valley
Authority (TVA), in which indigenous
manganese-oxidizing bacteria are used to
remove manganese (Vail and Riley 1997).
Acid mine water from the toe of the waste rock
pile at the Calliope Mine near Butte, Montana
was used because a field location had not yet
been identified for this project. The Calliope
Mine was the site of another MWTP field
demonstration and the water was considered
typical of acid mine drainage at other sites. The
composition is shown in Table 3-1.
The laboratory system operated at room
temperature. The feed water tank was open to
the atmosphere, so that system performance
could be evaluated for manganese removal.
However, iron and arsenic removal data would
be compromised due to oxidation in the feed
tank.
Mine water was fed to the anaerobic portion of
the system between early April 1998 and late
August 1999. The aerobic portion of the
laboratory system operated between early
February 1999 and late August 1999. Varying
the flow rate enabled evaluation of the effect of
residence time in the system.
3.2 Laboratory Results
Data taken during the laboratory column tests
included flow measurements, pH, oxidation-
reduction potential (ORP), and analyses of
aluminum, cadmium, copper, iron, manganese,
and zinc.
In general, the bench-scale system was quite
effective in removing metals from the feed
water, with all the analyzed metals in the
discharge being at or near detection limits. After
treating approximately 90 pore volumes of
water, the first column reactor showed decreased
-------
ability to reduce sulfate. This could have been
due to exhaustion of initial alkalinity present in
the manure that acted as a buffer for the bacteria.
SRB can be inhibited by the low pH of ARD. In
addition, ion exchange sites in the substrate may
have filled with cations such as calcium and
magnesium in exchange for protons.
Initially the pH in the effluent from the first
column increased to about 4, but then gradually
decreased to near the feed value by the end of
testing. At the same time, the ORP for the water
leaving this column increased dramatically from
approximately -250 mV to +200 mV apparently
indicating that sulfate reduction was repressed.
If significant sulfide were present, the ORP
value would be much lower. In addition,
aluminum and iron broke through at the same
time. Aluminum was then removed effectively
in the second column (containing limestone),
and iron was removed in the third column
(containing manure). While the aeration step
was intended to remove iron, no iron actually
made it that far in the process, because it was
removed upstream.
There was still a small amount of sulfate
reduction occurring in the first reactor, however,
since the data show complete removal of
cadmium and copper along with the majority of
the zinc throughout testing. This would likely
be due to the presence of small colonies of SRB
still functioning in the column. A portion of the
remaining zinc was removed in the second
column (containing limestone), with the removal
of any remaining zinc occurring in the third
column (containing manure). As described in
the report, the first column probably served to
ensure that no ferric iron was fed to the second
column, avoiding armoring of the limestone with
ferric hydroxide precipitates. Although ORP
conditions were not optimal, this was probably
accomplished by a combination of precipitation
of ferric iron in the feed due to the change in pH
in the first column along with some reduction of
ferric iron to ferrous as carbon in the column
was oxidized.
Manganese was effectively removed in the
aerobic portion of the system. However, it
cannot be conclusively said that this was due to
bacterial activity, since no MOB counts or other
confirmatory analyses were performed. It is
assumed that manganese removal was due to
oxidation and precipitation, either biotic or
abiotic. The only other plausible explanation
would be the removal of manganese as
rhodochrosite (MnCO3); this cannot be
evaluated further since no alkalinity analyses are
available. A noticeable aspect of the data, which
is extremely important, is the relatively high pH
of the water entering the manganese-removal
step. The values were over 8 throughout testing,
and ranged as high as 8.6. pH is very important
in the kinetics of manganese oxidation, both for
biotic and abiotic oxidation. In addition, the
manganese-removal reactor, according to the
TVA design criteria, was very oversized for the
application. The TVA design criteria is 0.5
gram (g) manganese (Mn) per square meter (m2)
per day. At a flow rate of 2.5 milliliters per
minute (ml/min) and a manganese concentration
of 12 milligrams per liter (mg/1), an area of
0.086 m2 or 0.92 square feet (ft2) would be
required, at a flow rate of 5 ml/min and the same
manganese concentration, an area of 0.172 m2 or
1.85 ft2 would be required. Our reactor had an
area of 8 ft2. This very large reactor size, along
with operation at room temperature, may also
explain the effective system performance. The
12 mg/1 manganese concentration in this
calculation is based on the typical value fed to
the manganese-removal step, different from the
typical feed value of about 7 mg/1. It is
uncertain why these were different; possibly
manganese loaded into the system early in the
testing phase and was released later, resulting in
higher downstream concentrations.
3.3 Field Design and Construction
The bench-scale data was drawn upon to size the
field system. A design flow rate of 2 gallons per
minute (gpm) was used for design of the field
system. One change from the bench-scale
system was that the field system did not contain
10
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a sand filter immediately before the final
bioreactor.
Prior to system construction details of the
process flows and proposed system analysis
were documented and approved in the project
specific quality assurance project plan (QAPP).
The specific design factors of each component
of the integrated, passive biological treatment
system are discussed in the following sections.
Additionally, it may be helpful to review Figures
3-2 and 3-3, which are a schematic of the
process flows through the treatment system and
a photographic collage of the field treatment
system, respectively.
3.3.1 Reactor 1
The first SRB reactor, referred to as Reactor 1,
was sized for a 3.5-day residence time at 2 gpm.
This residence time was based on results from
the bench-scale testing and from knowledge
gained from past MWPT demonstrations.
The reactor contained a 50%-50% mixture (by
volume) of cow manure and walnut shells. This
mixture formula was a preliminary result of
MWTP Activity III, Project 24, and was
anticipated to provide better permeability than
the originally planned mixture of cow manure,
straw, and an inert ceramic material. Final
results of Project 24 indicated that a mixture of
80% walnut shells and 20% cow manure may
have worked better. Excess substrate was used
to allow for settling and to help avoid freeboard
of water on the top of the reactor.
Reactor 1 was designed to hold 146 cubic yards
of organic substrate with a liquid residence time
of 3.5 days at 2 gpm. Following construction,
field measurements indicated a reactor volume
of 134 yd3. Assuming a porosity of 0.34 gave a
pore volume of 1230 ft3, which resulted in a
reactor residence time of 3.2 days.
The original reactor was designed to be fed
horizontally by a series of manifolds near the
bottom of the reactor. The flow was intended to
proceed upward and across the reactor, and exit
through a manifold near the top of the reactor on
the downstream side. The reactor was covered
with a 6-inch layer of alfalfa to provide thermal
insulation above the substrate.
The original feed distribution system failed due
to plugging as precipitates built up on the
bottom of the reactor. So, the feed distribution
was modified in the summer of 2003 to allow for
vertical distribution of the feed at three locations
to depth of 6 feet near the front end of the
reactor. Additionally, these new pipes allowed
for easier cleanout
3.3.2 Reactor 2
According to the project QAPP, the limestone
reactor, referred to as Reactor 2, was sized for a
1.25-day residence time, based on good bench-
scale test results at 5 ml/min. Also according to
the project QAPP, Reactor 2 contains about 53
yd3 of crushed limestone. Therefore, at an
assumed porosity of 0.34, the reactor contains
about 481 ft3 of pore volume, resulting in a 1.25-
day residence time at 2 gpm. Following
construction, field measurements indicated a
slightly larger reactor volume of 55 cubic yards,
resulting in an actual residence time of 1.3 days.
Similar to Reactor 1, Reactor 2 was originally
fed using a manifold near the bottom of the
reactor, with the flow proceeding upward and
across the reactor The water exited the reactor
through another manifold near the top on the
downstream side. Reactor 2 was also covered
with 6-inch layer of alfalfa to provide thermal
insulation.
Like Reactor 1, the original feed distribution
system in Reactor 2 suffered from precipitate
plugging. Precipitates carried over from Reactor
1 plugged the feed distribution line into Reactor
2. The feed distribution system of Reactor 2
was modified in the summer of 2004. Similar to
Reactor 1, the new plumbing allowed for
vertical distribution of the feed near the front of
the reactor and was designed to allow for
cleanout of future precipitates.
11
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Two additional modifications were made to
Reactor 2. First, a 250 gallon hold tank was
inserted into the top front end of Reactor 2. This
allowed for even feed distribution in the new
system and served as a collection settler for
precipitates carried over from Reactor 1. The
second modification was to place liner material
over the top of Reactor 2. This was done to help
maintain anaerobic conditions between Reactor
1 and Reactor 3. The cover was not air-tight but
provided some inhibition of oxidation in the
reactor.
3.3.3 ReactorS
According to the project QAPP, the second SRB
reactor, referred to here as Reactor 3, was sized
for a 1.25-day residence time based on good
bench-scale results at 5 ml/min. Similar to
Reactor 1, Reactor 3 contained a 50%-50% (by
volume) mixture of cow manure with empty
walnut shells.
Reactor 3 field measurements indicated a total
volume of about 85 yd3. Assuming a porosity of
0.34, this would result in a pore volume of 780
ft3 with residence time of about 2.02 days at 2
gpm.
Similar to Reactor 1, Reactor 3 was fed by a
series of manifolds near the bottom of the
reactor, with the flow proceeding upward and
across the reactor, and leaving through another
manifold near the top on the downstream side.
Reactor 3 was also covered with a 6-inch layer
of alfalfa to provide thermal insulation.
Reactor 3 was not modified over the course of
the demonstration. However, if the
demonstration continued much longer, Reactor 3
would have likely required a vertical feed
distribution system to continue flowing.
3.3.4 A eration Line
The line connecting Reactor 3 with Reactor 4 is
called the aeration line. There is a 40-foot
vertical drop between these two reactors and
about 100 foot horizontal distance. This
topography was at the toe of the waste rock pile
on which the first three reactors were
constructed.
The original design consisted of 165 feet of 8
inch corrugated pipe which was snaked down
the hillside. Three vertical air vents were placed
along the length of the pipe to provide air to the
line.
Analysis of the system indicated that the original
design did not sufficiently oxidize the water.
Therefore, in August 2003, two design
modifications were made to the aeration line.
First, the length was increased to 300 feet. And,
second, small slits were made on the top of the
line at two-foot intervals. These slits were made
to allow for the placement of wooden weirs. The
weirs served to increase retention time and to
provide turbulence for internal mixing of oxygen
which was available through the slits.
3.3.5 Original Reactor 4
The original Reactor 4 was sized for a 10-day
residence time and was designed to be filled
with 380 yd3 of crushed limestone. Reactor 4
was designed based on TVA criteria for
manganese removal, which specified 0.5 g
Mn/m2/day. Calculations used to size Reactor 4
used a 2 gpm flow with a manganese
concentration of 13 mg/1. This resulted in a
daily manganese loading of 141.5 grams. Use of
the TVA formula, results in a required reactor
surface area of 283 m2 or 3,045 ft2. Actual
reactor dimensions were about 100 feet wide, 30
feet long and about 4 feet deep (total surface
area of 3000 ft2).
Field calculations using the reactor geometry
and the actual water level as set by the exit
manifold indicate a reactor volume of about 357
yd3. Assuming a limestone porosity of 0.34
results in a pore volume of 3,277 ft3 and a
reactor residence time of about 8.51 days at 2
gpm rather than the stated 2 day residence time.
Note that this calculated residence time can be
considered theoretical since is not likely that the
full reactor volume was used.
12
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The limestone in Reactor 4 was piled above the
water level, as is customarily done in these
systems, to avoid outlet plugging by leaves, pine
needles, etc.
3.3.6. Improved Reactor 4
Since a primary objective of this project was to
investigate manganese removal, the function of
Reactor 4 was critical After three year's of
attempting to make the original design work,
Reactor 4 was finally re-designed and rebuilt.
In June of 2004, a new reactor was constructed
on top of the existing Reactor 4. New liner
material was placed over the old reactor to
create a much shallower reactor of about 1 foot
deep. This was filled with limestone and
additional liner material was used to create
vertical baffles at 10-foot intervals to force water
to the surface as it flowed along the long, 100
foot, dimension of the reactor.
Column Study
DCopper DCadmium DArsenic DAIuminum DZinc •Iron •Manganese
90%
SP1
SP2
SP3
Sample Point
SP4
SP5
°(>Per
Figure 3-1. Bar graph showing dissolved metals removal through laboratory column test
system. (Note, Sampling Points were as follows: SP1 was after the first manure column, SP2 was
after the crushed limestone column, SP3 was after the second manure column, SP4 was after the sand
column, and SP5 was after the aerobic reactor.)
13
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ARD
Reactor #1
Primary
Anaerobic
Reactor
Aeration
Holding
Tank
Reactor #2
Passive
Alkalinity
Addition
Passive
Aeration
Reactor #4
Aerobic
Reactor
Reactor #3
Secondary
Anaerobic
Reactor
Treated
Effluent
Figure 3-2. Block-flow of the integrated, passive biological treatment system's unit operations.
(Sample points are indicated and located as follows: INF or Influent indicates the Surething adit
discharge, SP1 or sample port #1 is located between Reactor #1 and Reactor #2, SRB1 or sulfate-
reducing bacteria #1 indicates within Reactor #1, SP2 or sample port #2 is located between Reactor
#2 and Reactor #3, SRB3 or sulfate-reducing bacteria #3 indicates within Reactor #3, SP3 or sample
port #3 is located between Reactor #3 and the aeration system, SP4 or sample port #4 is located
between the aeration system and Reactor #4, and ENF or effluent indicates the treatment system
discharge stream.)
14
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Gravity
Aeration Line
3 Anaerobic
Bioreactors
Figure 3-3. Collage showing components of the integrated, passive biological treatment system
including two biomass based anaerobic bioreactors, one limestone based anaerobic bioreactor,
and one limestone based aerobic bioreactor.
Table 3-1. Laboratory system feed water (Calliope Mine).
Parameter
pH
ORP
Al
As
Cd
Cu
Fe
Mn
Zn
SO4
Value
2.6 standard unit
(S.U.)
520 mV
40 mg/1
—
0.1 mg/1
10 mg/1
27 mg/1
7 mg/1
28 mg/1
675 mg/1
15
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4. Field Demonstration Results and Discussion
4.1 System Operation
The field system design was completed in July
2000. However, construction was postponed until
the summer of 2001 because of closure of the
national forests due to extreme fire danger in the
summer of 2000.
The field system was commissioned in July 2001.
The Mine Waste Technology Program continued
to operate the system through October 2005.
During most of that period the project goal of 75%
reduction was being met for all monitored
dissolved metals and the pH of the water returned
to a neutral range. During 4.25 years of operation,
the system treated approximately 4 million gallons
of ARD. A summary of the quality assurance
(QA) activities from the project specific QAPP are
contained in Appendix A.
As with most acidic mine effluents, the water
emanating from the Surething Mine contains
significant quantities of metals - both dissolved
and contained in particulate matter. The portion of
a sample, which passes through a 0.45-micron
filter, is considered to be dissolved. Samples were
collected from up to eight locations and were
analyzed for dissolved aluminum, arsenic,
cadmium, copper, iron, manganese, and zinc.
These metals were chosen because of their
presence in the Surething Mine ARD. The sample
location descriptions are summarized in Table 4-1.
Chemical parameters typical of untreated
Surething Mine water are shown in Table 4-2
along with the dissolved metals and the effluent
water chemistry for the final treated effluent. This
data was collected during the second to last
sampling event on September 1, 2005 after all
system modifications were completed.
Initially, samples and physical measurements were
taken almost monthly. Exceptions to this schedule
resulted from equipment failure and the system
freezing over during several of the winters.
4.2 Metals Results
Plots showing laboratory analytical data for
dissolved metals concentrations throughout the
demonstration are presented in Figures 4-1 to 4-7.
The statistical analysis of the metals analysis data
is contained in Appendix B along with the QA
data summary table. Specific results for each
parameter are discussed in the following sections.
4.2.1 Aluminum
Dissolved aluminum data are presented in Figure
4-1. Until June 2003, aluminum was essentially
removed in Reactor 1. The last few data points
from 2002 indicated increased aluminum
concentration, which showed that the reactor was
in the process of failing from a sulfate reduction
perspective. This was seen in the bench-scale
testing and was expected to occur in the field.
However, Reactor 3 continued to effectively
remove aluminum for the reminder of the
demonstration.
4.2.2 Copper
Throughout the demonstration, copper was very
effectively removed in Reactor 1. Analytical
results are presented in Figure 4-2.
4.2.3 Cadmium
Very similar to copper, cadmium was very
effectively removed in Reactor 1. Analytical
results are presented in Figure 4-3.
4.2.4 Zinc
Similar to cadmium and copper, zinc was very
effectively removed in Reactor 1. Analytical
results are presented in Figure 4-4.
4.2.5 Iron
Analytical results for iron are presented in Figure
4-5. For the first year, iron was effectively
removed in Reactor 1 until the summer of 2002,
when it began breaking through. After that it was
partially removed in Reactor 2 and completely
removed in Reactor 3. This is the same trend that
was seen in the bench-scale testing.
16
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The inability of Reactor 1 to completely remove
iron after the first year of operation indicates a loss
of sulfate reduction ability. This is the same
situation experienced in the laboratory system and
it was expected in the field system. Effective iron
removal was one of the reasons the additional SRB
reactor was installed.
Iron speciation analyses were completed on
several samples. These analyses snowed that the
feed was 92% ferric iron and 8% ferrous iron,
indicating oxidizing conditions were present in the
ARD. However, Reactor 1 discharge showed 93%
ferrous and 7% ferric. So, most of the iron that
was not removed in Reactor 1 was in the reduced
form.
4.2.6 Arsenic
Dissolved arsenic data are presented in Figure 4-6.
In general, throughout the demonstration, arsenic
was effectively removed in Reactor 1.
However, there is a peculiarity in the data as
dissolved arsenic concentration show a slight
increase as the water passed through Reactor 4. It
is not likely that arsenic leached from the
limestone in Reactor 4, for two reasons: 1) the
same limestone was used in Reactor 2, which did
not show an increase in arsenic concentration; and
2) analysis of limestone obtained from the supplier
indicates nondetectable arsenic content (less than 2
mg/kilogram).
One likely source of the slight increase in
dissolved arsenic concentration in Reactor 4 would
be the resolubilization of a suspended form or
arsenic such as arsenic adsorbed to a ferric iron
precipitate.
4.2.7 Manganese
Dissolved manganese data are presented in Figure
4-7. Inspection of the manganese data shows that
during the first year of operation, manganese was
being removed sequentially through the system.
Since the bulk of this early manganese removal
occurred in the SRB reactors, it has been attributed
to adsorption and ion exchange between the
dissolved manganese and the reactor materials.
Also, during the first year of operation, Reactor 4
indicated that it was somewhat effective in
removing manganese. About 30% of the dissolved
manganese that entered Reactor 4 was removed.
However, this 30% only amounted to about 2 or 3
ppm.
At the time, it was assumed that the manganese
removal mechanism was MOB activity. But, after
the reactor failed to increase its manganese
removal efficiency over time, it was suggested that
J ' OO
the initial manganese removal mechanism may
have been carbonate precipitation due to high
alkalinity in the system. The form of the
precipitated manganese was not investigated.
Lending evidence that the initial manganese
removal mechanism in Reactor 4 was not MOB
activity was the low ORP values (-150 mV) in
Reactor 4. This value indicates that the conditions
in Reactor 4 were not sufficiently aerobic.
As the system started its second year of operation,
manganese removal performance decreased
steadily. In the fall of 2002, the dissolved
manganese concentrations leaving Reactor 1 and
Reactor 2 were actually higher than the feed
concentration. This indicated that manganese
previously removed by adsorption was
resolubilized. For a short period of time, Reactor
3 was able to remove this additional manganese.
However, by the winter of 2002, the dissolved
manganese concentration throughout the system
showed little change, indicating essentially no
manganese removal taking place.
At this point, a comprehensive analysis of the
system operation was conducted. It was concluded
that the original system design was unable to
remove manganese due to insufficient oxidation
that was required to turn the water chemistry from
sulfate-reducing conditions to oxidizing
conditions. Reactor 4 would work better under
more favorable oxidizing conditions.
To increase the oxidizing conditions in Reactor 4,
three major modifications were made in the
summer of 2003. First, the length of the aeration
17
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line running between Reactor 3 and Reactor 4 was
increased from 100 feet to 300 feet. Second, 3-
inch diameter wooden weirs were placed at two-
foot intervals within the new line. And third, the
direction of flow through Reactor 4 was altered
from the original design of cross flow to allow for
lengthwise flow.
The above modifications produced significant
favorable changes in the chemical conditions of
Reactor 4. However, little change was noted in
manganese removal performance for the
remainder of the 2003 operation season. So for
the 2004 operation season three additional system
modifications were made. First, Reactor 4 was
rebuilt using a much shallower depth (1 foot vs. 4
foot) constructed on top of the original reactor.
Second, wiers were placed within the reactor to
increase the residence time as the water flowed
through the reactor. And, third, the aeration
retention time was increased by placing a 500
gallon hold tank immediately prior to Reactor 4 to
allow more oxygen picked up in the line to
dissolve into the water.
During the 2004 operating season, after these
modifications were made, a slight increase in
manganese removal was seen in Reactor 4.
Because the manganese removal was consistent
with bacterial oxidation, it was assumed to be due
to the establishment of an indigenous MOB
population. During the 2005 operating season, an
active population of MOB was indicated by the
presence of a black colored slime that had formed
on the limestone near the surface - more
concentrated in the inlet area and dissipating over
the next 20 feet. The accumulation of a black
slime and the increased removal of manganese
from the Surething water were consistent with and
indicate the establishment of a population of
MOB.
During the final sampling event on October 25,
2004, a series of 12 samples was taken along the
length of Reactor 4. This was to see how quickly
the manganese was being removed. Results
presented in Table 4-3 indicate the presents of an
active treatment zone in the first part of the
reactor, which is followed by a fairly even
manganese removal rate across the remaining two-
thirds of the reactor.
In order to determine MOB activity, limestone
rock samples from Reactor 4 were also collected.
Specimens with dark deposits (presumed to be
manganese compounds) were chosen. These were
submitted for DNA extraction and microbial
community analysis. Results were inconclusive
and are presented in the microbiology section
below.
4.3 Physical Field Measurement Results
Plots showing physical measurements are
presented in Figures 4-8 to 4-12. Specific results
for each parameter are discussed in the following
sections.
4.3.1 ORP
ORP field readings are presented in the graph in
Figure 4-8. There are several noteworthy trends in
the ORP data graph.
In general, ORP values in the reactors trended
upward starting in the second year of operation.
This was not a desirable trend for the anaerobic
portion of the system, where low ORP values are
preferred. The ORP values were apparently still
low enough for sulfate reduction to occur. The
increase in ORP seen at Reactor 1 was more
significant and indicated poor reactor performance
from an SRB standpoint.
Following the major system modifications made
during the summers of 2003 and 2004, ORP
values in Reactors 1, 2, and 3 dropped back down.
These modifications had the desired impact of
improving anaerobic conditions in these reactors.
Initially, the ORP values in the aerobic portion of
the system (Reactor 4 and effluent) were lower
than those seen in bench-scale testing by 100 to
200 mV. This data was used in conjunction with
the manganese removal data to indicate that
system modifications were required.
For the last two years of the demonstration, the
ORP data correlated well with the dissolved
manganese data and indicated that the system
18
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modifications were effective in creating favorable
aerobic conditions in which MOB could self
establish. But, in general the ORP values finally
realized in Reactor 4 (200 mV) were still lower
than the ORP readings in the feed. This indicated
that there was still room for improvement in the
design of the aeration system and Reactor 4.
4.3.2 pH
pH field readings are presented in the graph in
Figure 4-9. There are several noteworthy items in
the pH data.
The first trend corresponds to the ORP trend
identified at the SRB1 sample point described
above. The pH steadily decreased starting in
March 2002. This was another indication that
Reactor 1 was in the process of failing from a
sulfate reduction perspective. Along with this, in
the summer of 2003, the pH in Reactor 1 started to
follow the same downward trend.
The second noteworthy item is that the pH values
at SP3 trended downward until Spring of 2004
where a significant rebound is noted. The
coincides with the field modifications made at that
time.
The third noteworthy item is the pH values at SP4
and the effluent dropped about 1.0 to 0.5 during
the second year of operation. This correlated with
the time frame in which the system was
experiencing poor manganese removal
performance. Since manganese oxidation rates are
proportional to the hydroxide ion concentration,
this also served as an indication that the system
required modification.
A forth noteworthy item in the pH data is that after
the final modifications were made to Reactor 4,
the pH of both SP4 and the effluent showed
significant increases that correlated well to the
improvement seen in manganese removal.
4.3.3 Temperature
The temperature data are presented in Figure 4-10.
For the most part, the plot shows annual
fluctuations with seasonal weather changes, as
expected. One notable data point is the high
temperature seen in Reactor 4 during the final
summer of operation. This is indicative of the
shallow nature of the reactor as it absorbed the
summer heat.
Overall system temperatures were well below the
room temperature at which bench-scale testing
was conducted indicating that this integrated
system performed well in the harsh environment
for which it was designed.
4.3.4 Dissolved Oxygen
The dissolved oxygen (DO) concentrations field
readings are presented in Figure 4-11. The DO
probe responded much slower at low oxygen
levels, such as those at the anaerobic sample
points, than in oxygenated environments. Since
the DO probe took a long time to stabilize,
readings at low levels are less accurate.
Data showed that throughout the system, DO
values were higher during the colder seasons.
This is what would be expected since oxygen
solubility in water increases at lower temperatures.
And, as would be expected, DO levels were very
low in the anaerobic portion of the system.
When using the original design, the aerobic
portion of the system had higher DO. Values
entering Reactor 4 were around 5 ppm, indicating
that some aeration had occurred in the original
aeration system. But 5 ppm did not represent a
highly aerated condition and with Reactor 4 DO
values trending downward and correlating with the
systems failure to effectively remove manganese,
the need to modify the aeration design was
indicated.
Figure 4-11 also shows significant increases in DO
in Reactor 4 and the effluent after aeration
efficiency modifications were completed. There is
a direct correlation with increased DO and
increased removal of manganese.
Other work has stated that DO levels need to
exceed about 30% saturation in order for DO not
to be the limiting reactant in MOB reactors
(Marble et. al. 1999). There was typically more
than that entering Reactor 4, though probably not
in late summer 2002.
19
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4.3.5 Flow Rate
The field flow rate readings are presented in the
graph in Figure 4-12. Initially, the flume
indicating the feed flow rate was not accurate at
the low flow rates being fed due to its orientation.
As such, the feed end flume was supplemented in
August 2002 by a modifying the feed system to
allow for bucket checks of the feed flow rate. Prior
to this change, the effluent flow would serve as a
more reliable indicator of the feed flow through
the system. As seen in the plot, after the
modification was made, the feed and discharge
flow rates matched quite well.
In general, the system operated at lower flow rates
than the design flow rate of 2 gpm. This resulted
in longer residence times than designed. In the fall
time frames, there was insufficient water flowing
in the adit to feed the system at its design basis of
2 gpm.
There are also lower flows through the system at
the time the system started effectively removing
manganese. This low flow was a direct result of
the seasonal flows seen under summer and fall
conditions. It is assumed that the system would
still have effectively removed manganese at the 2
gpm design flow.
Throughout the demonstration, achieving a
consistent flow rate was an operational problem.
Starting in the summer of 2002, the system
suffered several flow disruptions. As a result,
modifications were made that helped maintain
flow through the reactor feed systems during the
spring and summer of 2003.
For lessons learned aspects, several of the flow
related issues experienced in 2002 at the Surething
are described in Table 4-4.
4.5 Other Chemical Measurements
In addition to metals analysis and the reported
field measurements, several other parameters were
measured. This included alkalinity, sulfate,
sulfide, and dissolved calcium. Plots showing
these results are presented in Figures 4-13 to 4-16.
Every sampling event is shown versus time which
allows for analytical trends to be distinguished.
The specific results for each parameter are
discussed in the following sections.
4.5.1 Alkalinity
Alkalinity analysis results are presented in Figure
4-13. For the first year of the demonstration,
alkalinity showed an expected increase across
Reactor 1. This increase became much smaller in
the latter part of 2002. This downward trend in
the alkalinity data is yet another indicator that
Reactor 1 was realizing less SRB activity.
Similarly to the alkalinity trend seen across
Reactor 1, Reactor 2 also showed a downward
trend in alkalinity concentration. However,
throughout the demonstration, Reactor 3 remained
effective in generating alkalinity indicating an
active SRB population.
4.5.2 Sulfate
Sulfate analysis results are presented in Figure 4-
14. The plot shows that Reactor 1 was providing
the bulk of the sulfate reduction until the spring of
2002. In the ensuing months, sulfate levels within
Reactor 1 showed a significant increase. This was
another indication of diminished sulfate reduction
occurring in Reactor 1. For the rest of the
demonstration, Reactor 3 provided the bulk of the
sulfate reduction.
The sulfate plot also indicates that between June
2002 and June 2003, sulfate reduction was
occurring within Reactor 4. Given all of the other
indicators that Reactor 4 was not effectively
removing manganese, this finding is not surprising
and is evidence that there were undesirable
anaerobic conditions present in Reactor 4.
Sometime after this reactor was modified to
produce better aerobic conditions, the sulfate data
improved. This is most notable beginning in the
spring of 2005.
4.5.3 Sulfide
Sulfide analysis results are presented in Figure 4-
15. Inspection of the sulfide plot shows similar,
but opposite, trends to those discussed above in
the sulfate section.
20
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Reactor 1 produced more sulfide early in its
operation than was produced as the demonstration
progressed. During the first half of 2002, this
dropped off considerably, with more sulfide being
produced by Reactor 3. This serves as yet another
indicator that Reactor 1 was losing its sulfate
reduction capability.
Although sulfide levels decreased greatly in
Reactor 1 over the course of the demonstration,
levels remained sufficiently high enough to allow
Reactor 1 to continue to effectively remove
copper, cadmium, zinc, and arsenic from the feed
water.
As with the sulfate data, the sulfide data indicated
that Reactor 4 was reducing sulfate to sulfide. As
there was a slight increase in sulfide concentration
through the reactor indicating sulfate reduction
was occurring.
Another noteworthy aspect of the sulfide data is
notably high concentrations of sulfide that
continuously exited Reactor 3. While the aeration
step was effective in decreasing the sulfide
concentration, a dramatic improvement was
realized with each modification to the Reactor 4
and the aeration system.
4.5.4 Calcium
Following the system modifications made in 2003,
samples were analyzed for dissolved calcium to
help determine the effectiveness of the limestone
used in Reactor2 and Reactor 4. Laboratory
calcium analysis results are presented in Figure 4-
16.
The plot if dissolved calcium concentrations show
that Reactor 2 added the majority of calcium to the
water. There was a slight increase in calcium
concentration through Reactor 1 with no major
increase seen following Reactor 2.
4.6 Microbiology
4.6.1 Manganese Oxidizing Bacteria
The MOB that this project sought to employ were
assumed to be heterotrophic microorganisms, i.e.
they required organic carbon for growth.
Manganese oxidation by heterotrophic MOB is a
reaction that occurs co-metabolically as part of the
MOB metabolism, i.e., carbon and manganese are
oxidized while oxygen is reduced. The amount of
carbon consumed relative to manganese oxidized
was not measured. Organic material was not
likely a limiting reactant because of its abundance
in the Reactor 3 effluent. In evaluating the
presence of nutrients, it should be noted that
generally the relative ratios of carbon, nitrogen,
and phosphorus required for bacterial growth are
on the order of 100:10:1. Analyses of dissolved
organic carbon, nitrogen forms, and phosphorus
have not been performed throughout the system.
Ammonia and nitrate/nitrite have been analyzed in
the discharge water, and these have generally
shown on the order of 5 to 15 mg/1 of nitrogen
present in the discharge. Analyses of dissolved
organic carbon (4.3 mg/1), total Kjeldahl nitrogen
(3.4 mg/1), and total phosphorus (6.0 mg/1) were
performed on the Reactor 4 effluent water in
November 2002. Note that these were
downstream of the reactor, and note also that there
was probably some SRB activity occurring in the
reactor. This limited data would indicate that
there is plenty of phosphorus and nitrogen present
to support MOB activity, and a possibility of a
carbon deficiency. The reactor being deficient in
carbon would be very surprising, since it is
immediately downstream of many tons of cow
manure and walnut shells.
4.6.2 SRB Counts
The SRB count data are presented in figure 4-17.
Initially, both Reactor 1 and Reactor 3 appear to
be establishing very active SRB populations.
However the plot shows a striking decrease
between the June 2002 and the July 2002 sampling
events. This correlates with when the feed flow to
the system had plugged and resulted in little or no
flow through the system for an unknown amount
of time. This may have caused the biofilms
containing SRB to detach, become dislodged and
be flushed downstream when flow was
reestablished. It is also possible that these SRB
biofilms became active again once they reached
Reactor 4. As this coincides with the timeframe in
which Reactor 4 started to show anaerobic
tendencies.
21
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Another possibility for the decrease seen in the
SRB populations between June and July 2002
could have been due to significantly high levels of
sulfide building up in Reactor 1 and Reactor 3 to
the point of being toxic to the SRB populations
present.
Despite the drop in SRB population seen in July
2002, Reactor 3 was able to hold a steady SRB
population. However, Reactor 1 was never able to
significantly reestablish a highly active SRB
population. This was most likely the result of the
numerous flow disruptions seen during the
summer of 2002 which could have been a factor in
how fast Reactor 1 lost its ability to effectively
reduce sulfate.
4.6.3 Microbial Community Analysis
At the conclusion of the project, samples were
collected from the reactors and examined at
Montana State University's Center for Biofilm
Engineering using molecular community
analytical techniques. Due to project budget
constraints, analyses were not performed on fresh
samples. The samples were six months old before
analysis was conducted; so, aging may have
affected the results.
DNA was extracted from samples using a
laboratory kit. The DNA was then purified
according to kit protocols and subsequently used
as a template for polymerase chain reaction (PCR).
PCR was performed on the extracted DNA using
primers specific for SRB and MOB. The SRB
primers were specific for a sulfate reductase that is
fairly well conserved among the various SRB
species. The MOB primers were specific for a Mn
(II) oxidase found in Bacillus spores.
In samples taken from Reactor 1, both sulfate
reducing bacteria and manganese oxidizing
bacteria appear to have been present. And, in
samples taken from Reactor 4, several bands were
detected that indicated the possible presence of
manganese oxidizing bacteria. See Appendix C
for the full report by the Center for Biofilm
Engineering.
22
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Dissolved Aluminum vs. Time
(Log scale parts per billion)
100000 -i
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-1. Aluminum concentrations.
Dissolved Copper vs. Time
(Parts per Billion)
a-eee-e—a—nan anno—e—a—no n n
-250
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-2. Copper concentrations.
23
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Dissolved Cadmium vs. Time
(parts per billion)
400
350 -
•a—ana nann a—a-=o-o-©-o
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-3. Cadmium concentrations.
Dissolved Zinc vs. Time
(Parts per Billion)
40000
o-oo-o-
e-ooo-®-^©—e o o ooo» A—a 00-6 a
5000 -
ol§
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-4. Zinc concentrations.
-Feed
SP1
-SP2
-SP3
-SP4
•Effluent
24
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Dissolved Iron vs. Time
(Parts per Billion)
60,000
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-5. Iron concentrations.
Dissolved Arsenic vs. Time
(Parts per billion)
1300
1200
1100 -
1000
900
Ju-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-6. Arsenic concentrations.
25
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Dissolved Manganese vs. Time
(Parts per Billion)
70,000
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-7. Manganese concentrations.
ORP vs. Time
•Feed - +- SRB1
SP1
SP2 - X- SRB3
-SP3
SP4
•Effluent
-300
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-8. ORP meter readings vs. time.
26
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Effluent —e— SP4 —e— SP3 - X- SRB3 —O— SP2 SP1 - +- SRB1 —0—Feed
Jul-01 Jan-02 Aug-02 Feb-03 Sep-03 Mar-04 Oct-04 May-05 Nov-05
Figure 4-9. pH field readings.
Temperature vs. Time
Feed -- + --SRB1 SP1 A SP2 -- + --SRB3 O SP3 0 SP4 O Effluent
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-10. Field temperature readings.
27
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Dissolved Oxygen (mg/L) vs. Time
Feed -- + --SRB1 SP1 —A— SP2 ---X--SRB3 —9— SP3 —0—SP4 —6—Effluent
12 n
0
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-11. Dissolved oxygen field meter readings.
Flow (GPM) vs. Time
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03
Figure 4-12. Field flow rate measurements.
Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
28
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Alkalinity (mg/L) vs. Time
800
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-13. Alkalinity concentrations.
Sulfate (mg/L) vs. Time
1000
900 -
Dec-01
Jun-02
Dec-02 Jun-03 Dec-03 Jun-04
Dec-04 Jun-05
Dec-05
Figure 4-14. Sulfate concentrations.
29
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Sulfide (mg/L) vs. Tim
160
Feed SP1 SP2 -G-SP3
Jul-01 Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-15. Plot showing sulfide concentration with time.
Dissolved Calcium (mg/L) - vs. Time
Feed SP1 SP2 -9-SP3 -0-SP4 -e-Effuent
Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
-16. Dissolved Calcium concentration.
30
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10,000,000
1,000,000 -
SRB Counts vs. Time
(MPN, cells/ml)
SRB1 SP1 SP2 -I-SRB3 -0-SP3 -G- Effluent
Dec-01 Jun-02 Dec-02 Jun-03 Dec-03 Jun-04 Dec-04 Jun-05 Dec-05
Figure 4-17. SRB counts.
Table 4-1. Sample location descriptions used for retinue sampling events.
Sample
Location
Influent
Sample Port #1
Sample Port #2
Sample Port #3
Sample Port #4
Effluent
Simple
ID
INF
SP1
SRB1
SP2
SP3
SRB3
SP4
ENF
Location Description
Surething Adit Discharge
Between Reactor # 1 and Reactor #2
Reactor # 1 Compartment
Between Reactor #2 and Reactor #3
Between Reactor #3 and Aeration Line
Reactor #3 Compartment
Between Aeration Line and Reactor #4
System Effluent Following Reactor #4
31
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Table 4-2. Dissolved Metals Percent Reduction Between Mine Discharge and the Process Effluent on Sept. 1, 2005
Metal
Aluminum
Arsenic
Cadmium
Copper
Iron
Lead
Manganese
Zinc
Feed Concentration
(mg/L)
29.5
0.127
0.208
2.35
15.0
0.151
26.7
22.7
Discharge Concentration
(mg/L)
<0.04
<0.01
<0. 00009
O.003
<0.014
0.004
0.037
<0.007
% Reduction
>99.86%
>92.13%
>99.96%
>99.87%
>99.91%
97.35%
99.86%
>99.97%
Table 4-3. Dissolved Manganese Levels at Progressive Location into Reactor 4
Location in Reactor 4
1
2
3
4
5
6
7
8
9
10
11
12
Dissolved Mn (u,g/L)
20,100
8,430
10,500
20,200
16,300
14,100
10,300
7,090
5,410
2,310
1,990
747
32
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Table 4-4. Summary of System Flow Conditions during the Summer of 2002
Date Notes
May 30, 2002 Reactor 1 was found to be overflowing due to the feed valve being left wide open over the winter from
an effort to maximize flow the previous fall. The flume flow rate was estimated to be 281 gpm. It was
reset to 2 gpm.
Jun 11,2002 Reactor 1 was no longer overflowing. Feed flow was measured to be 5.5 gpm. This was reset to 2 gpm.
The corresponding discharge flow was measured to be .5 gpm.
Jul 11,2002 The system was found not to be flowing at all. The feed valve to the system was found to be plugged by
ferric iron precipitate. It had been left in an almost closed condition due to the high level of winter runoff
exiting the mine. The feed valve was cleaned and reset to deliver 2.0 gpm.
Aug 1, 2002 Reactor 1 was found to be overflowing into Reactor 2. It was determined that the feed system to Reactor
2 was plugged, causing water to back into Reactor 1.
Aug 8, 2002 The feed line leading to the Reactor 2 feed distribution system was inadvertently broken when
investigating the plug situation. Water rushed into Reactor 2 and the entire system began flowing
properly. Black precipitate was observed in the flowing water.
Aug 21, 2002 Modifications were made to allow the feed flow rate to be checked by timer and bucket. Feed flow was
set at 2 gpm.
Sep 11, 2002 Both the feed and effluent flows agreed at about 1.5 gpm. This was the entire flow from the Surething
adit.
Oct 10, 2002 Both the feed and effluent flows agreed at about 1.0 gpm.
Nov 12, 2002 The feed flow had dropped to 0.83 gpm, while the effluent flow could not be measured due to the
effluent being frozen.
33
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5. Summary and Conclusions
A 4-year technology demonstration of an
integrated passive treatment process was
conducted between July 2001 and October 2005.
This field demonstration was initiated following
two years of successful operation of a laboratory
scale system. This project was conducted at the
Surething Mine near Elliston, Montana under the
EPA's Mine Waste Technology Program.
The integrated passive biological treatment
process installed at the Surething Mine consisted
of a multi-stage reactor system involving
sequential treatment of acid rock drainage.
Metal-laden acidic water emanating from the
mine adit was treated through a series of three
anaerobic reactors followed by a final aerobic
bioreactor. The anaerobic treatment relied on
sulfate-reducing bacteria that reduced dissolved
sulfate to hydrogen sulfide, which reacted with
dissolved metals to form insoluble metal
sulfides. This bacterial metabolism also
produced bicarbonates that increased water pH
and limited dissolution of metals. Six of the
seven target metals including copper, iron,
arsenic, cadmium, and zinc were addressed
through anaerobic sulfate reducing bacteria
bioreactors. The last target metal - manganese,
was addressed in an aerobic bioreactor.
Overall, this demonstration project proved that
this technology, once functioning optimally,
could offer advantages over many ARD
treatment systems because it does not require a
power source and should not require frequent
operator attention. Final results indicated that
this integrated, passive system offer a promising
ARD treatment method at remote locations.
Additionally, the following conclusions were
made based on the data presented in this report.
• Though difficult to do, it is possible to
design a passive biological based system
that changes from anaerobic to aerobic
treatment conditions. In other words, passive
aeration was sufficient to change from an
anaerobic to an aerobic environment.
• SRB populations appeared immediately in
Reactor 1 and Reactor 3.
• Initially, manganese removal was not noted.
This was through to be due to the slow self-
establishment of an indigenous MOB
population. However, the most likely cause
of lack of manganese removal was due to
faulty reactor development. As the
demonstration progressed and design
improvements were made, the removal of
manganese increased significantly.
• Overall dissolved metals data showed that
metal removal was very high once operation
of the aeration section was modified.
• The data indicates that Reactor 1 started to
fail from a sulfate reduction standpoint as
the demonstration entered its second year of
operation. However, Reactor 1 continued to
effectively remove copper, cadmium, zinc,
and arsenic throughout the demonstration.
• The system was very susceptible to flow
disruptions from problems with being able
to supply feed at a consistent rate and
significant plugging opportunities being
built into the initial reactor designs.
• Once the conditions and design of Reactor 4
were optimized, manganese removal was
consistent with bacterial oxidation and
visual inspection indicated the presence of
an active population of manganese-oxidizing
bacteria.
• An increase in arsenic concentrations was
observed in Reactor 4 during the first year of
operation. This was attributed to the
resolubilization of arsenic associated with
precipitated iron hydroxide, which had been
carried into Reactor 4.
• Although of sufficient capacity to provide
significant water treatment much of the year,
the system was undersized for being able to
treat high flow, spring run-off conditions at
the Surething.
34
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6. Lessons Learned and Recommendations
Recommendations for future similar technology
demonstrations and the major lessons learned
from this technology demonstration project are
presented in this section.
6.1 Original Reactor 4 Design
Reactor 4 did not remove manganese as
efficiently as was seen in the bench-scale
aerobic reactor. This was apparently due to
excess sulfide entering the field reactor. Since
the anaerobic reactors operated at lower-than-
design flow rates, excess sulfide flowed into
Reactor 4. The lower system flows resulted in
longer SRB bioreactor residence times and
caused greater sulfate reduction and excess
sulfide production. Excess sulfide was not
oxidized completely during the aeration step and
was detrimental to the aerobic reactor because 1)
it consumed oxygen needed for MOB growth; 2)
it was a reductant which reduced manganese as
it was oxidized; and 3) it created a reducing
environment in Reactor 4 which was unsuitable
for MOB growth but caused SRB proliferation.
Other reasons for poor Reactor 4 performance
were short-circuiting which resulted in a much
smaller effective reactor size than was designed
and too much depth for efficient oxygen
transfer.
6.2 Modifications to Original Reactor 4
The following modifications (in the order listed)
were made to improve aerobic reactor
performance. The effect of individual
modifications was not measured, but each
change appeared to enhance the overall
performance of Reactor 4.
1. A more vigorous passive aeration
system was installed prior to the reactor.
2. The reactor feed/discharge configuration
was modified to reduce short-circuiting
and use the full length of the reactor.
3. The reactor was treated with potassium
permanganate to decrease the SRB
population and to provide manganese
oxide seed material to enhance MOB
activity. The approximant dosages to
Reactor 4 was 0.2 g/L KMnO4.
4. Micro nutrients were added in an
attempt to stimulate MOB growth, the
dosages to Reactor 4 was 10 mg/1 of
carbon from the molasses, 1 mg/1 of
yeast extract, and 1.2 mg/1 of nitrogen as
ammonium.
In addition, attempts were made to develop an
indigenous MOB population in the laboratory
for inoculation in the field. However, the
establishment of a thriving MOB population in
the laboratory proved to be very difficult and
was abandoned.
6.3 Construction of New Reactor 4
Because the above modifications were
insufficient to produce the desired results and
because the project had a limited time frame, the
original reactor was abandoned. A new reactor
was built with a decreased reactor depth. The
new depth allowed for greater oxygen mass
transport which greatly improved aerobic
conditions. This change greatly enhanced the
overall performance of Reactor 4 and caused the
project to be a success as an integrated, passive
biological treatment system.
6.4 Aeration System
To increase aeration of the water prior to
entering Reactor 4, a longer piece of 8-inch
corrugated pipe was installed down the hill from
Reactor 3. Flow obstructions were added to the
pipe to create turbulence and improve aeration.
As a result of aeration improvements and
modifications to the reactor feed/discharge
configuration, Reactor 4 was able to more
efficiently oxidize sulfide and manganese and
achieve higher manganese removal.
6.5 Aeration Hold Tank
Another modification made during the project
that increased aeration with in turn improved
35
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MOB activity was the incorporation of a holding
tank immediately prior to Reactor 4.
6.5 Reactor 2
Since Reactor 1 efficiently removed iron, it was
determined that the second limestone bioreactor
(designed as the iron cleaning step) was not
essential and could be left out of future designs.
The limestone in Reactors 2 and 4 was a
dolomitic limestone rather than a high-calcium
limestone, which more readily produces
alkalinity. Dolomitic limestone was likely not
optimum for this application.
6.6 SRB Reactor Design
Another major finding resulting from this
project was the importance of enhancing
porosity since inlet plugging was caused by the
buildup of precipitates.
The ratio of settling material or manure to the
supporting matrix of walnut shells could be
modified to enhance porosity and hydraulic
conductivity. In another MWTP study, a
mixture of 80% walnut shells and 20% cow
manure worked very well to promote microbial
growth and prevent plugging.
6.7 Recommendations
The implementation of a passive integrated
ARD treatment system at the Surething Mine
proved to be a technologically challenging
demonstration. The following recommendations
were developed after completion of the data
analysis and review of the many lessons learned
from this field demonstration.
• There were many lessons learned in
bioreactor design over the course of this
demonstration. And the main
recommendation is to include reactor
maintenance in the design phase of future
projects.
Although the Surething Mine demonstration
lasted for over four years, that time was not
long enough to get long-term data on the
degradation of the walnut shells used in the
organic substrate. Future projects need to be
designed to determine the effective life for
substrate materials and to help optimize
reactor designs.
Microbiological community analysis needs
to be included in the initial project scope.
Additionally, microbiological community
analysis should play a larger role in future
MOB and SRB technology demonstrations
by collecting and analyzing samples over the
course of the demonstration. This would
allow comparison of results overtime.
These changes would help predict microbial
behavior in other MOB and/or SRB reactors
and allow for optimization of these
technologies.
Future projects that utilize MOB technology
in conjunction with SRB technology should
incorporate aggressive oxidation steps
between the two biological processes. This
would ensure that metals that are more
mobile in the reduced form (i.e. iron and
manganese) are oxidized and captured.
Future projects, that utilize passive
treatments in areas that experience large
seasonal flow variations, should work to
incorporate run-off surge storage capacity,
which would control the rate at which these
waters enter the system and allow optimal
retention times.
Collect soil samples downstream of the
discharge area and analyze increased plant
toxicity.
36
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7. References
Barghoorn, E. S. and R. L. Nichols, "Sulfate
Reducing Bacteria in Pyritic Sediments in
Antarctica," Science, 134, 90, 1961.
Cohen, R. R. H. and M. W. Staub, Technical
Manual for the Design and Operation of a Passive
Mine Drainage Treatment System, prepared for
the U.S. Bureau of Reclamation by the Colorado
School of Mines, Golden, Colorado, 1992.
Doshi, Sheela M., Bioremediation of Acid Mine
Drainage Using Sulfate-Reducing Bacteria,
National Network of Environmental Management
Studies Fellow University of Indiana, for the U.S.
Environmental Protection Agency Office of Solid
Waste and Emergency Response Office of
Superfund Remediation and Technology
Innovation Washington, D.C., August 2006.
Dugan, P. R., C. I. Randies, J. H. Turtle, B.
McCoy, and C. MacMillan, The Microbial Flora
of Acid Mine Water and Its Relationship to
Formation and Removal of Acid, State of Ohio
Water Resources Center, Ohio State University,
1968.
Dvorak, D. H., R. S. Hedin, H. M. Edenborn, and
S. L. Gustafson, "Treatment of Metal-
Contaminated Water Using Bacterial Sulfate
Reduction: Results from Pilot-Scale Reactors",
U.S. Bureau of Mines Pittsburgh Research Center,
International Conference on Abatement of Acidic
Drainage, Montreal, September 1991.
Figueroa L, Seyler J, Wildeman T (2004)
Characterization of organic substrates used for
anaerobic bioremediation of mining impacted
waters. Proceedings, International Mine Water
Association Conference, Jarvis, A., ed, September
20-25, 2004, Newcastle, England pg. 43-52
Gusek JJ (2002) Sulfate-reducing bacteria design
and operating issues: is this the passive treatment
technology for your mine drainage? Proc, 2003
National Assoc of Abandoned Mine Land
Programs Annual Conf, Park City, UT, USA,
Technical paper session 15 part 3, p 1-14
Hunter, R. M., Biocatalyzed Partial
Demineralization of Acidic Metal Sulfate
Solutions, Doctoral Thesis, Montana State
University, Bozeman, Montana, 1989.
Johnson, Karen L. and Paul L. Younger, Rapid
Manganese Removal from Mine Waters Using an
Aerated Packed-Bed Bioreactor, J Environ Qual
34:987-993,2005.
Lee, J. M., Biochemical Engineering, Prentice-
Hall, Inc., Englewood Cliffs, New Jersey, 1992.
Marble, Justin C., Timothy L. Corley, Martha H.
Conklin, and Christopher C. Fuller,
"Environmental Factors Affecting Oxidation of
Manganese in Final Creek, Arizona" USGS
Geological Survey Water - Resources
Investigations Report 99-4018A Volume 1 of 3.
U.S. Geological Survey Toxic Substances
Hydrology Program - Proceedings of the
Technical Meeting, Charleston, South Carolina,
March 8-12, 1999.
McClernan, H.G., Metallic mineral deposits of
Powell County, Montana, Montana Bureau of
Mines and Geology: Bulletin 98, 69 p. 1976.
McGregor R, Blowes D, Ludwig R, Pringle E,
Pomeroy M (1999) Remediation of heavy metal
plume using a reactive wall. Proc, 5th International
In Situ and On-Site Bioremediation Symp, San
Diego, CA, USA, Battelle Press, vol 5(4) p 19-24
Middleton, A. C. and A. W. Lawrence, "Kinetics
of Microbial Sulfate Reduction'" Journal of the
Water Pollution Control Federation, July 1977.
37
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MSB Technology Applications, Inc., Quality
Assurance Project Plan (QAPP) for an Integrated,
Passive Biological Treatment System, Revision 2.
Mine Waste Technology Program, Activity III,
Project 16. April 2001.
Nordwick SM, Marek Zaluski, Brian Park, and
Diana Bless, Advances in Development of
Bioreactors Applicable to the Treatment of ARD,
7th ICARD, St. Louis MO., March 26-30, 2006.
Nordwick, Suzzann, Marek Zaluski, Brian Park,
John Trudnowski, Bioreactor Research for the
Treatment of Acid Mine Drainage, Eighth
International In Situ and On-Site Bioremediation
Symposium, Baltimore, Maryland, June 6-9, 2005.
Nordwick SM, Bless DR (2002). Integrated,
passive biological treatment process for acid mine
drainage, abstract and poster. U.S. EPA
Conference on Hardrock Mining: Issues Shaping
the Industry, Denver, Colorado, May 2002
Postgate, J. R., The Sulfate-Reducing Bacteria,
2nd Edition, Cambridge University Press,
Cambridge, 1984.
Rankin, M.W. (Mining Geologist), Newman Bro's
Properties, Elliston Mining District, Powell
County, Montana, July 1950.
Robbins, Eleanora I., Timothy L. Corley, and
Martha H. Conklin (1999). Manganese Removal
by the Epilithic Microbial Consortium at Final
Creek near Globe, Arizona. USGS Water-
Resources Investigations Report 99-40ISA
Volume 1 of 3, Volume 1 - Contamination From
Hard-Rock Mining, USGS Toxic Substances
Hydrology Program - Proceedings of the
Technical Meeting, Charleston, South Carolina,
March 8-12, 1999.
Skousen J, Sexstone A, Ziemkiewicz P (2000)
Acid mine drainage treatment and control. In:
Barnhisel R, Daniels W, Darmody R (eds),
Reclamation of Drastically Disturbed Lands,
American Soc of Agronomy, Madison, WI, USA,
p 131-168
Stumm, W. and J. J. Morgan, Aquatic Chemistry,
An Introduction Emphasizing Chemical Equilibria
in Natural Waters, Second Edition, John Wiley
and Sons, New York, New York, 1981.
Tsukamoto TK, Miller GC (2002) Sustainable
bioreactors for treatment of acid mine drainage at
the Leviathan Mine. Presented at Conf on
Hardrock Mining 2002, Denver, CO, USA, new
media CD platform session 10, p 3
Vail, W.J., and R.K. Riley. (1997). The abatement
of acid mine pollution using the Pyrolusite
Process. In: Proceedings, Nineteenth Annual
Conference, National Association of Abandoned
Mine Lands Program. August 17-20,1997, Davis,
WV.
Welch, E. B., Ecological Effects of Waste Water,
Cambridge University Press, New York, New
York, 1980.
Wildeman, T., J. Dietz, J. Gusek, and S. Morea, S.,
Handbook for Constructed Wetlands Receiving
Acid Mine Drainage, Risk Reduction Engineering
Laboratory, Office of Research and Development,
1993, p. 3-1 to 3-20.
Wildeman T, Updegraff D (1998) Passive
bioremediation of metals and inorganic
contaminants. In: Macalady DL (Ed), Perspectives
in Environmental Chemistry, Oxford University
Press, New York, USA, p 473-495
Young, J. W. "The Bacterial Reduction of
Sulfates," Can. J. Res. (B), 14, 49-54, 1936.
Zhang, Jinghao, Leonard W. Lion, Yarrow M.
Nelson, Michael L. Shuler and William C.
Ghiorse, Kinetics of Mn(II) oxidation by
Leptothrix discophora SSI, Geochimica et
Cosmochimica Acta 66(5) (2002) 773-781
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39
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Appendix A
Summary of Quality Assurance Activities
40
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FIELD AND LABORATORY DATA VALIDATION
Mine Waste Technology Program
Activity III, Project 16—Integrated, Passive Biological Treatment at the Surething Mine
BACKGROUND
In July 2001, sampling officially began for Mine Waste Technology Program (MWTP) Activity III,
Project 16—Integrated, Passive Biological Treatment at the Surething Mine. The objective of the project
was to investigate the effectiveness of using a biological system to treat the acid rock drainage emanating
from the adit of the Surething Mine. All of the field and laboratory data for sampling events has been
evaluated to determine the usability of the data.
In order to determine the effectiveness of the biological process being demonstrated, several sampling
points were designated, and a variety of analyses were assigned to each point. Several analyses were
performed on the collected samples either in the field at the Surething Mine near Elliston, Montana or at
the HKM Laboratory (now the MSB Laboratory) in Butte, Montana.
The analyses to be performed were specified in the QAPP and each analysis was classified as critical or
noncritical. A critical analysis is an analysis that must be performed in order to achieve project
objectives. A noncritical analysis is an analysis that is performed to provide additional information about
the process being tested. Critical analyses in the final version of the QAPP for this project are listed
below:
• Flow rate at influent and effluent sampling locations and
• Dissolved Metals (Al, As, Cd, Cu, Fe, Mn, Pb, Zn) at influent and effluent sampling locations.
Noncritical analyses for this project are listed below:
• pH;
• Temperature;
• Oxidation Reduction Potential (ORP)
• Dissolved Oxygen (DO)
• Sulfate;
• Sulfide;
• Nitrate-Nitrite as Nitrogen;
• Total Ammonia as Nitrogen;
• Alkalinity;
• Sulfate-reducing Bacteria Counts;
• Biochemical Oxygen Demand (BOD);
• Dissolved Metals at intermediate sampling points; and
• Total Metals (Al, As, Cd, Cu, Fe, Mn, Pb, Zn).
The QC objectives for each critical analysis are outlined in the QAPP and were compatible with project
objectives and the methods of determination being used. Control limits for each of these objectives are
established for each critical analysis. The QC objectives for this project are outlined in Table A-l below.
41
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Table A-l. Precision, accuracy, MDL, and completeness objectives for critical analyses.
Parameter
Flow rate
Al
As
Cd
Fe
Mn
Pb
Zn
Unit
gpm
jlg/L
MDL1
0.5
300
10
2
1600
300
4
300
Precision2
N/A
20% RPD
Accuracy3
N/A
75-125%
recovery
Completeness4
95%
95%
1 Method detection limits were based on 10% of target action levels and rounded to the nearest ppb, tens
of ppb, or hundreds of ppb as appropriate.
2 Relative percent difference of analytical sample duplicates.
3 Percent recovery of matrix spike, unless otherwise indicated.
4 Based on number of valid measurements, compared to the total number of samples.
For noncritical analyses, QC objectives are determined by using standard guidelines that exist, or by
applying reasonable control limits in order to determine the usability of the data that was generated in the
field or in the laboratory for the noncritical analyses.
PROJECT ASSESSMENTS
Two assessments were performed during this project:
• Technical Systems Review by MSB in October 2001 and
• Technical Systems Review by EPA in September 2002.
TECHNICAL SYSTEMS REVIEW BY MSE
A technical systems review was conducted on October 11, 2001 by MSE. The TSR was based on the
project-specific QAPP and included: personnel, facilities, and equipment; documentation; calibration of
equipment; and sampling procedures. There were no findings identified, but the following
recommendations were made: as-built the system because there were changes from the design of the
system presented in the QAPP; update the QAPP to reflect actual procedures for field measurements; and
sample caps were not wrapped in Parafilm® as outlined in the sampling procedure in the QAPP.
Technical Systems Review by EPA
A TSR was conducted by EPA September 10-12, 2002. The scope of the audit included sampling and
analytical laboratory activities. One finding related to deviations from the QAPP was noted because
deviations were significant compared to the QAPP on file at EPA. Observations were noted in the
following areas: flow rate measurement should be better defined so project personnel know when to use
the flume and when to use the manual bucket check; the impact of bypass line into Compartment 2 should
be determined; changes in design should be documented in an amended QAPP; use of the flow cell for
pH, ORP, DO should be documented in an amended QAPP; sampling times were not being properly
recorded, so it was recommended that field personnel begin to documents sampling times; the QAPP
described SW-846 methods, but the laboratory was using CLP methods, so it was recommended that this
42
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be documented in the updated QAPP document; review of cadmium analysis to determine if ICP-AES is
providing an appropriate detection limit; laboratory QC for GFAA should be corrected in the QAPP; and
change in method used for sulfide analysis should be documented in the QAPP. An amended QAPP was
submitted to EPA, and all corrective actions were implemented.
VALIDATION PROCEDURES
Data that was generated for all critical and noncritical analyses was validated. The purpose of data
validation is to determine the usability of all data that was generated during a project. Data evaluation
was 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 (RPDs) 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;
• that the data in the report submitted by the laboratory to project personnel can be verified from
the raw data generated by the laboratory.
Measurements that fall outside of the control limits specified in the QAPP, or for other reasons are judged
to be outlier are flagged appropriately to indicate that the data is judged to be estimated or unusable. An
analytical evaluation was performed to determine the usability data that was generated by the HKM
Laboratory for the project. Laboratory data validation was performed using USEPA Contract Laboratory
Program National Functional Guidelines for Inorganics Data Review as a guide.
On several occasions, accuracy could not often be quantified because the matrix spike added to the
samples was not high enough for several metals. This occurred for sampling events when the influent
sample was selected by the laboratory analyst for QC analyses (preparation blank, matrix spike, and
duplicate). In these cases, the serial dilution analyses were reviewed to ensure that matrix interferences
were not present. The serial dilution recoveries were consistently within control limits.
ANALYTICAL EVALUATIONS
Several analytical evaluations of field and laboratory data were performed over the life of this multi-year
project.
Field Logbook Evaluation
An examination of the field logbook for this project found that all sampling and calibration information
was present. Sampling personnel also documented any additional information about unique conditions
that could impact the project data.
Field data validation was performed to determine the usability of the data that was generated during field
activities. The usability was determined by verifying that correct calibration procedures of field
instruments were followed. The following analyses were performed in the field:
43
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• pH;
• temperature;
• oxidation/reduction potential;
• Specific conductance (SC)
• dissolved oxygen; and
• flow rate.
For each sampling event, calibration of the meters used was performed correctly and associated QC
checks were performed. All field data is considered usable.
PROGRAM EVALUATION
The program evaluation focused on the following areas:
• Chain of Custody (COC) Procedures;
• Sampling and Data Completeness;
• Field Blanks; and
• Field Duplicates.
Chain of Custody Procedures
All information provided in the Chain of Custody (COC) Forms for this project was complete and
accurate.
Sampling and Data Completeness
All samples that were supposed to be collected, were collected when possible. The chief reason
preventing sample collection was limited access to the mine during the winter. The project was extended
to include more sampling events than originally planned, so the impact of missed sampling events was
minimal on the amount of project data available for evaluation.
Field QC Samples
Field blanks and duplicates were collected at each sampling event for dissolved metals. The point of
collection rotated between sampling locations. All field QC samples were within control limits; however,
for the 9/2/04 sampling event, the field blank and the field duplicate were mixed up. The field blank
results were reported for the field duplicate sample ID and vice versa. This problem was corrected in the
project database.
ANALYTICAL RESULTS
Similar to the field blank/field duplicate being confused with one another, for the 11/7/01 event, the
influent and effluent samples were mixed up. This error was also corrected in the project database.
The other major analytical challenge was spike recoveries for arsenic and lead. The arsenic spike
recoveries had a positive bias on two occasions and a negative bias on one occasion, while the lead spike
recoveries that were outside control limits indicated a negative bias.
44
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Table A-2. Summary Qualified Data for MWTP Activity III, Project 16.
Date1
4/19/00—
prior to
system
start-up
7/24/01
9/14/01
10/11/01
11/7/01
7/11/02
9/11/02
11/12/02
Sample ID
Surething
Irrfl
Effl
Influent
Effluent
PT1
PT2
PT3
Influent
Effluent
SP1
SP2
SP3
SP4
Influent
Effluent
INF
EFF
SP1
SP2
SP3
SP4
INF
EFF
SP1
SP2
SP3
SP4
INF
EFF
SP1
SP2
SP3
SP4
Analysis
Dissolved Cd
Total Fe
Total Zn
Dissolved As
Dissolved As
Dissolved Pb
Dissolved
Metals
Sulfate
Sulfide
Alkalinity Forms
Total Metals
Dissolved
Metals
Dissolved As
Dissolved Pb
QC
Criteria
Matrix Spike
Matrix Spike
Analytical
Duplicate
Matrix Spike
Influent and
Effluent mixed up
Samples were
received by
laboratory in a
cooler without ice
or custody seals.
Matrix Spike
Matrix Spike
Control
Limit
75-125% recovery
75-125% recovery
<20%RPD
75-125% recovery
N/A
Proper procedures
should have been
followed, ideal
temperature is
<4°C.
75-125% recovery
75-125% recovery
Result
193.9%
137.3%
132.6%
24%
69.6%
67.3%
N/A
Samples were
improperly
packaged and
sample
temperatures had
reached 16°C.
130.9%
63.4%
Flag2
J
J
J
J
N/A
J
J
J
Comment
Flag this sample "J", as estimated.
Flag these samples "J", as estimated.
Flag these samples "J", as estimated.
Flag these samples "J", as estimated.
Ensure that the data for influent and
effluent are reported correctly in project
database.
Flag all samples for this event "J", as
estimated.
Flag associated samples for this event
"J", as estimated.
Flag associated samples for this event
"J", as estimated.
45
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Date1
10/8/03
11/13/03
6/7/04
9/2/04
6/13/05
Sample ID
INF
EFF
SP1
SP2
SP3
SP4
INF
SP1
SP2
SP3
INF
SP1
SP2
SP3
Field Blank
and Field
Duplicate
INF
EFF
SP1
SP2
SP3
SP4
Analysis
Total As
Diss Pb
Total Pb
All Dissolved
Metals
Total As
Dissolved As
QC
Criteria
Matrix Spike
Matrix Spike
Matrix Spike
Field Blank and
Field Duplicate
Matrix Spike
Control
Limit
75-125% recovery
75-125% recovery
75-125% recovery
Samples should be
labeled correctly
75-125% recovery
Result
142.8%
66.8%
61.2%
Samples were
mixed up
54.1%
60.7%
Flag2
J
J
J
N/A
J
Comment
Flag associated samples for this event
"J", as estimated.
Flag associated samples for this event
"J", as estimated.
Flag associated samples for this event
"J", as estimated.
No action required.
Flag associated samples for this event
"J", as estimated.
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.
46
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CONCLUSION
Activity III, Project 16—Integrated Passive Biological Treatment project provided much needed long-
term data for passive treatment of ARD. While this report identified minor data quality issues, the data
generated from the project is considered good quality data. One lesson learned for this project that can be
utilized on future projects is that the QAPP should be amended when changes on the project occur.
A-47
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Appendix B
Statistical Analysis
-------
Introduction
This report describes the statistical data analysis of the metals concentration data for the influent and
effluent water from the Sure Thing Mine treatment process. The Sure Thing Mine water treatment process
was designed, constructed and operated by MSB under the Mine Waste Technology Program (MWTP),
Activity III, Project 16 funding. The statistical analysis was completed according to the specifications set
forth in the MWTP, Activity III, Project 16 quality assurance project plan (QAPP) (MSB, April 2001).
Project Objective
The project objective as stated in the QAPP was to achieve a 75% reduction in the dissolved metals in the
effluent water for As, Al, Cd, Cu, Fe, Mn, Pb, and Zn when compared to the influent water. The percent
removal of dissolved metals was calculated according to the following:
(influentConc. InletFlowRate-EffluentConc. OutletFlowRate]
%removal = -^ r '- 100%
(InfluentConc. InletFlowRate)
The percent removal data was to be graphed for each metal for the duration of the project and the mean
percent removal calculated for the entire project and seasonal periods if fluctuations in percent reduction
are noted.
Data Analysis Procedure
The data used for the analysis was provided by the project and included inlet and outlet flow data and
dissolved metals concentration data sampled monthly on a semi-regular basis starting in July 2001 and
continuing through October 2005 for a total of 61 sampling events. Sampling events were occasionally
missed due to weather related site access issues. The data provided for this analysis had been reviewed
and validated by the MSB quality control officer according to the specifications in the QAPP (MSB, April
2001).
Inlet and Outlet-Flow Rate Data
The inlet flow-rate data were 95% complete; inlet flow-rate data were not available for 3 of the 61
sampling events. The outlet flow-rate data were 56% complete; outlet flow-rate data were missing for 27
of the 61 sampling events. Outlet flow-rate data were often not available due to system plugging or
freezing. Outlet flow-rate was estimated for 26 of the 27 sampling events without outlet flow-rate data
based on the assumption that, for a closed system, the outlet flow should approximately equal the inlet
flow. The ratio of measured outlet flow to inlet flow was calculated for all sampling events in which both
flow-rate measurements were available. The mean value of the outlet to inlet flow-rate ratios is 0.825
(standard deviation equal to 0.277) and the median value is 0.904. The ratios were skewed towards higher
values. Therefore, the median values were used to estimate outlet flow-rate for sampling events missing
outlet flow-rate data. The median ratio was also used to estimate two inlet flow-rate values from
-------
measured outlet flows. One sampling event did not have either inlet or outlet flow data available; and
therefore, no estimate of the flow-rates for that sampling event was possible. The flow-rate data and
associated evaluation are included as Attachment 1.
Dissolved Metals Data
The dissolved metals data for the influent water included data from 34 sampling events (inlet and outlet
flow rates, as discussed above were often measured on multiple times in a single month while samples for
laboratory analysis were only acquired once in each month that a sampling event occurred.) The effluent
samples included data from 25 sampling events. All data were used regardless of the qualifier; for values
reported below the detection limit, the detection limit was used as a conservative upper bound. Sufficient
data (both dissolved metals and flow-rate) were available to calculate 24 percent-removal values for each
dissolved metal (70% of total number of available sampling events). For each dissolved metal, the data
used for the calculations, summary statistics (including a histogram plot), and a t-test comparing the data
to the 75% criteria are included as attachment 2. The results are summarized in Table B-l.
Table B-l. Summary of statistical values calculated for each dissolved metal % removal.
Dissolved
Metal
As
Al
Cd
Cu
Fe
Mn
Pb
Zn
Mean %
Removal
91%
100%
99%
100%
99%
63%
96%
99%
Standard
Deviation
9%
0%
1%
1%
2%
35%
1%
1%
Median
93%
100%
99%
100%
100%
74%
99%
100%
Minimum
Value
67%
99%
96%
96%
89%
0%
77%
96%
Maximum
Value
100%
100%
100%
100%
100%
100%
100%
100%
Lower
Confidence
Level of
Mean
(LCL)
87%
100%
98%
100%
98%
48%
93%
99%
t-
statistic
9.239
424.5
80.12
136.7
53.03
-1.671
15.95
125.8
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A comparison of means (t-test) was done with the null hypothesis being that "the mean percent removal
was less than or equal to 75%." And, the alternative hypothesis being that "the mean percent removal was
greater than 75%." The lower confidence level (LCL) was used to evaluate the hypothesis. If the LCL
was above 75%, then the null hypothesis was rejected and it was concluded that the mean percent removal
was greater than 75%.
All but one of the listed dissolved metals met the 75% mean removal criteria. The exception was
manganese. For the other metals, there was generally around 99% to 100% removed with very small
confidence intervals about the means. For manganese, the t-statistic was much closer to the rejection
region (t < -1.717: p = 0.05 and 23 degrees of freedom); however, manganese did pass the t-test. The
manganese data probably reflects changes to the system that were made to try and increase the manganese
removal as suggested by the scatter plot of the data.
Bootstrap Analysis
A bootstrap analysis of the percent-reduction data for each dissolved metal was completed using S-
Plus™. A mean, standard deviation, and confidence interval were calculated using bootstrapping for each
dissolved metal. The results from bootstrapping were compared to the results obtained using standard
statistical calculations (i.e., no resampling of population) and are summarized in Attachment 3. Generally,
no significant differences were observed between the results, suggesting that bootstrapping is unnecessary
with this data and that the sample populations were sufficient to describe the mean dissolved metals
removal.
References
MSB Technology Applications, Inc., April 2001. Quality assurance project plan for an integrated, passive
biological treatment system, Mine Waste Technology Program, Activity III, Project 16.
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Attachment 1 - Flow Data
12
10
o
§ 6
Histogram of Outlet to inlet Flow Rate Ratios
0.2308 0.3910 0.5513 0.7116 0.8719 1.0321 1.1924 More
Ratios
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Attachment 2 - Dissolved Metals Data
Scatter Plot of Arsenic Removal
1T)0/
mn%
to
^ «n% -
c
o
'.u Rn%
3
T3
(1)
iv 4n%
£
70% -
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• *
0 5 10 15 20 25 30
Sample Number
18
16
14
S12
o 10
a)
8
6
4
2
0
Histogram of Arsenic Removal
70% 80% 90% 100% More
% Removal
-------
190%
mn% -
ft no/.
c
o
•f fio% -
3
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£
90% -
n%
c
Scatter Plot of Aluminum Removal
) 5 10 15 20 25 3
Sample Number
0
Histogram of Aluminum Removal
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
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70%
80% 90%
% Removal
100%
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190%
100%
ft no/.
c
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•f R0% -
3
•O
0)
(V 40%
£
90% -
n%
c
Scatter Plot of Cadmium Removal
) 5 10 15 20 25 3
Sample Number
0
Histogram of Cadmium Removal
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
c
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70%
80% 90%
% Removal
100%
More
-------
190%
mn% -
ft no/.
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3
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£
90% -
n%
c
Scatter Plot of Copper Removal
) 5 10 15 20 25 3
Sample Number
0
Histogram of Copper Removal
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
c
O
n
70%
80% 90%
% Removal
100%
More
-------
190%
mn% -
ft no/.
c
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•f fio% -
3
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£
90% -
n%
c
Scatter Plot of Iron Removal
•
) 5 10 15 20 25 3
Sample Number
0
Histogram of Iron Removal
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
c
O
n
70%
80% 90%
% Removal
100%
More
-------
Scatter Plot of Manganese Removal
190%
mn% -
sn%
c
o
•f fio% -
3
•D
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0 5 10 15 20 25 30
Sample Number
-I 9 __
I ^
i n
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1
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a- °
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Histogram of Manganese Removal
70%
rn
n
r
80% 90%
% Removal
100%
More
-------
190%
100%
sn%
c
o
•f fio% -
3
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£
on% _
n%
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Scatter Plot of Lead Removal
•
^
) 5 10 15 20 25 3
Sample Number
0
Histogram of Lead Removal
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
c
O
n
70%
,— I
80% 90%
% Removal
100%
More
-------
190%
mn%
ft no/.
c
o
•f fio% -
3
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£
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n%
c
Scatter Plot of Zinc % Removal
) 5 10 15 20 25 3
Sample Number
0
Histogram of Zinc %
0 xic
C 13
(1)
o> 1 n
JI ID
LJ_
c
O
Removal
n
70%
80% 90%
% Removal
100%
More
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Attachment 3 - Bootstrap Results
Mean
Std Dev.
SE Mean
LCL Mean
UCL Mean
As
Parametric
Assumption
0.913
0.087
0.018
0.877
0.950
Bootstrap
0.913
0.084
0.018
0.865
0.940
Al
Parametric
Assumption
0.998
0.003
0.001
0.997
0.999
Bootstrap
0.998
0.003
0.001
0.996
0.999
Fe
Parametric
Assumption
0.993
0.022
0.005
0.984
1.003
Bootstrap
0.993
0.019
0.005
0.974
0.998
Mn
Parametric
Assumption
0.601
0.370
0.076
0.444
0.757
Bootstrap
0.601
0.361
0.076
0.445
0.745
Mean
Std Dev.
SE Mean
LCL Mean
UCL Mean
Cd
Parametric
Assumption
0.987
0.014
0.003
0.981
0.993
Bootstrap
0.987
0.014
0.003
0.981
0.992
Cu
Parametric
Assumption
0.996
0.009
0.002
0.993
1.000
Bootstrap
0.997
0.007
0.002
0.989
0.998
Zn
Parametric
Assumption
0.995
0.010
0.002
0.991
0.999
Bootstrap
0.995
0.009
0.002
0.990
0.998
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Appendix C
Microbial Analysis Report
C-l
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Center for Biofilm Engineering
Montana State University
Final Report
June, 2007
CHARACTERIZATION OF THE MICROBIAL COMMUNITY WITHIN
INTEGRATED BIOLOGICAL REACTORS AT THE SURETHING MINE
Submitted to: MSB Technology Applications, Inc.
Contact: Suzzann Nordwick
Address: PO Box 4078
Butte, MT 59701
Phone: (406) 494-0896
Email: suzzann.nordwick@mse-ta.com
Submitted by: Center for Biofilm Engineering
Contact:
Address:
Phone:
Email:
Elinor Pulcini, PhD
Montana State University-Bozeman
366 EPS Building
P.O. Box 173980
Bozeman, MT 59717-3980
(406) 994-1814 Fax: (406) 994-6098
elinor_p@erc.montana.edu
c-i
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Introduction
Mine Waste Technology Program (MWTP) Activity III, Project 16 "Integrated Passive Biological
Treatment Process Demonstration" involves the use of a passive biological reactor to treat acid mine
drainage (AMD). Phase Two of this EPA project was constructed on site at the Sure Thing Mine in
Southwest Montana. The goal of this project was to characterize the microbial community inhabiting the
organic substrate from an in-situ bioreactor at Sure Thing Mine in Southwest Montana with respect to the
presence of sulfate-reducing bacteria (SRB) and manganese oxidizing bacteria (MOB). Previous work
done by the Medical Biofilm Laboratory (MBL) at the Center for Biofilm Engineering, Montana State
University for MSE on the identification of species in samples from the Lilly/ Orphan Boy Mine using
molecular community analytical methods indicated the presence of SRBs. The Lilly/Orphan Boy sample
was a single point/time sample. Samples from the Sure Thing Mine have been taken from various points
within the bioreactor setup.
Methods
DNA was extracted from samples using the Ultra Clean Soil DNA kit (MoBio Laboratories Inc.) in which
the cells were lysed in a buffer and the DNA is extracted using the Savant 101 bead beater (Fast Prep).
The DNA was purified according to kit protocols and subsequently used as a template for polymerase
chain reaction (PCR).
PCR was performed on extracted DNA using primers specific for SRB and MOB as well as the
Eubacterial primer 357F and 518R (Integrated DNA Technologies) (Table C-l). The SRB primers are
specific for a sulfate reductase that is fairly well conserved among the various SRB species. The MOB
primers are specific for a Mn (II) oxidase found in Bacillus spores.
Primer reactions and DNA amplification were performed using a PTC-100 Programmable Thermal
Controller (MJ Research) using the following parameters: 94°C for 2 minutes, 15 cycles of 94°C for 45
seconds, 55°C for 45 seconds, 72°C for 45 seconds with a final extension step of 72°C for 7 minutes.
Verification of the presence of DNA was assessed in 1.5% agarose gels stained with ethidium bromide.
Table C-l. Primer sequences to be used for this project. (M = A or C, Y = C or T, R =G or A, K = G or
T, S = G or C, W = A or T, I = Inosine).
Type
Eubacteria
SRB
MOB
Designation
518R
357F
DSR-AB1F
DSR-AB4R
mnxGIF
mnxGIR
Primer Sequence (5' to 3')
GTA TTA CCG CGG CTG CTG G
CCT ACG GGA GGC AGC AG
ACS CAC TGG AAG CAC G
GTG TAG CAG TTA CCG CA
ACG CAT GTC TTT CAC TAT CAT GTT CAT
AAA TAA GTG GTC ATG GAA GAA CCA TGC
C-2
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Results and Discussion
One of the objectives of this project was to verify of the SRB species present in the Most Probable
Number (MPN) as compared to the SRB species present in the Sure Thing samples in order to assess the
effectiveness of MPN methods used. In addition, it was anticipated that analysis of these samples could
pinpoint the presence and activity of MOB and SRB at specific stages within the bioreactor in order to
assess the effectiveness of remediation technology for future projects. Unfortunately, the samples taken
from the Sure Thing mine were old. The recovery of DNA from these samples that truly represented the
microbial community profiles at the time of sampling may have been complicated by the age of the
samples.
Manganese Oxidizing Bacteria (MOB)
DNA was extracted from the samples and PCR was performed using the MOB primers mnxGI Forward
and mnxGI Reverse (Figures A and B). Ferrooxidans 23270 (an environmental isolate) was used as a
potential positive control. Unfortunately, it does not contain gene for manganese oxidation and was not
amplified using the MOB primers.
Bands were detected in the following samples which indicate the possible presence of manganese
oxidizers:
• Reactor 1, Hole 1,#1
• Reactor 1, Hole 1, #2
• Reactor 1, Hole 2, #4
• Reactor 1, Hole 3, #1
• Reactor 1, Hole 3, #3
• Reactor 1, Hole 3, #4
The faintness of the bands on these gels may either indicate that the numbers of bacteria analyzed by PCR
are very low in the sample or that the bacterial community structure degraded with time.
C-3
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1 234 567 8 9 10 11 12
Figure A. Results of PCR using MOB primers.
1) 100 bp Ladder
2) Reactor 1, Hole 3, #1
3) Reactor 1, Hole 3, #2
4) Reactor 1, Hole 3, #3
5) Reactor 1, Hole 3, #4
6) Reactor 3, Hole 1,#1
7) Reactor 3, Hole 1,#2
8) Reactor 3, Hole 1,#3
9) Reactor 3, Hole 1,#4
10) Reactor 3, Hole 2, #1
ll)Ferrooxidans 23270
12)100 bp Ladder
C-4
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2 34-567 8 9 10 11 12
Figure B. Results of PCR using MOB primers.
1) 100 bp Ladder
2) Ferrooxidans 23270
3) Ferrooxidans 29047
4) Reactor 1, Hole 1,#1
5) Reactor 1, Hole 1, #2
6) Reactor 1, Hole 1, #3
7) Reactor 1, Hole 1, #4
8) Reactor 1, Hole 2, #1
9) Reactor 1, Hole 2, #2
10) Reactor 1, Hole 2, #4
11) Positive control from Reactor 1, Hole 1, #1
12)100 bp Ladder
C-5
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Sulfate Reducing Bacteria (SRB)
DNA was extracted from the samples and PCR was performed using the SRB primers DSR-AB1 Forward
and DSR-AB4Reverse (Figures C).
Bands were detected in only two samples, Reactor 1, Hole 3, #3 and Reactor 1, Hole 3, #4.
Again, the faintness of these may probably indicates that the bacterial community structure degraded with
time.
1
3 4
7 8
Figure C. Representative agarose gel of PCR results using SRB primers.
1
2
o
J
4
5
6
7
8
9
Reactor 1 ,
Reactor 1 ,
Reactor 1 ,
Reactor 1 ,
Reactor 3,
Reactor 3,
Reactor 3,
Reactor 3,
Reactor 3,
Hole 3, #1
Hole 3, #2
Hole 3, #3
Hole 3, #4
Holel,#l
Hole 1, #2
Hole 1, #3
Hole 1, #4
Hole 2, #1
Eubacterial Primers
For community analysis, PCR was performed using Eubacterial primers 35f F and 518R using stock
Prevotella DNA as a positive control and sterile water as a negative control. Amplified DNA was
subsequently separated by Denaturing Gradient Gel Electrophoresis (DGGE) using a 40% to 60%
denaturing gradient in 8% to 12% polyacrylamide gels following recommended manufacturer protocols
(BioRad). Only 3 samples of the 25 samples that were extracted and PCR amplified produced positive
PCR results. DGGE was run on those 3 samples (Fig D). While separate bands in a DGGE gel may not
always be a positive indicator of a distinct species, the increase in band numbers does indicate a
potentially diverse community structure.
C-6
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Figure D. DGGE results using Eubacterial primers 357 F and 518 R.
A Reactor 1, Hole 1,#1
B Reactor 1, Hole 1, #2
C Reactor 1, Hole 2, #2
Results can be interpreted only as indications of what occurred in the in-situ bioreactor at the Sure Thing
Mine. Both sulfate reducing bacteria and manganese oxidizing bacteria appear to have been present in
Reactor 1. Unfortunately, the lack of positive indicators (PCR bands) in reactors 2 and 3 are not
necessarily definitive proof that there were no SRBs or MOBs present in those reactors due to the
degradation of the samples.
C-7
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