EPA/600/R-08/096
June 2008
Mine Waste Technology Program
In Situ Source Control
Of Acid Generation Using
Sulfate-Reducing Bacteria
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 3, In Situ Source Control of Acid Generation Using Sulfate-Reducing Bacteria, funded by the U.S.
Environmental Protection Agency (EPA) and jointly administered by EPA and the U.S. Department of
Energy (DOE). This project addressed EPA's technical issue of Mobile Toxic Constituents - Water
through a field demonstration of a water treatment technology based on the use of sulfate-reducing
bacteria (SRB) at a remote inactive underground mine.
This project was undertaken to demonstrate the effectiveness of SRB technology to treat metal-laden
water flowing through and from an abandoned mine. The Lilly/Orphan Boy Mine, located in the Elliston
mining district of Montana near the capital city of Helena, was selected as the site for the field
demonstration. The Lilly/Orphan Boy Mine, active in the first part of the 20th century, was a relatively
small mine that produced lead ore, which was shipped to a smelter in Helena. After active mining ceased
in the 1950s, the mine workings subsequently flooded with groundwater and this eventually resulted in
acid rock drainage (ARD) discharging from the mine portal.
Under the MWTP, MSE Technology Applications, Inc. (MSE) demonstrated an innovative, in situ
biological technology to treat and control ARD emanating from the Lilly/Orphan Boy Mine. Cables were
installed to suspend platforms 30 feet below the static water level in the mineshaft that was open to the
surface. Organic matter, primarily cow manure and straw, was placed on the platforms in the shaft,
forcing the ARD coming from the mineshaft to pass through the organic matter before exiting the mine
through the portal. Dissolved metals were removed from the ARD entering the in situ bioreactor, and the
water subsequently flowed out of the mine through the downgradient portal. Because the SRB
technology also caused the shaft water pH to rise and the oxidation reduction potential to drop, the
amount of acid leaving the mine was substantially decreased. The bioreactor was activated in August
1994, and the water was analyzed for more than a decade (through July 2005). In general, the water has
seen a considerable reduction in dissolved metals concentrations, and the discharge pH has been increased
from a historic level of near 3 to a more neutral pH close to 6.
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 1
1.5 Site Selection 2
1.5.1 Location 2
1.5.2 Mine Site Geology and History 2
1.5.3 Mine Workings 2
1.6 Technology 3
1.7 Project Objectives 3
2. Technology Description 5
2.1 Metals Removal Mechanisms 5
2.1.1 Adsorption by Substrate 5
2.1.2 Biological Sulfate Reduction 5
2.1.3 Hydroxide Precipitation 6
2.2 Biological Sulfate Reduction 6
2.2.1 Microbial Description 7
2.2.2 Growth Parameters 7
2.2.3 Growth Requirements 7
2.2.4 Growth Inhibition Factors 9
3. Demonstration Description 11
3.1 Laboratory Testing 11
3.2 Substrate Adsorption Studies 11
3.3 Field Design and Construction 11
3.3.1 Monitoring Well Installation 12
4. Field Monitoring Results and Discussion 14
4.1 Dissolved Metals 14
4.1.1 Aluminum, Copper, and Cadmium 14
4.1.2 Zinc 15
4.1.3 Manganese 15
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Contents (Cont'd)
Page
4.1.4 Iron and Arsenic 15
4.2 Total Metals 16
4.3 Alkalinity 16
4.4 Physical Measurements 16
4.4.1 ORP 16
4.4.2 pH 17
4.4.3 Dissolved Oxygen 17
4.4.4 Temperature 17
4.4.5 Flow Rate 17
4.5 Other Chemical Measurements 17
4.5.1 Sulfate, Sulfide 17
4.5.2 Biochemical Oxygen Demand, Chemical Oxygen Demand 18
4.5.3 Nitrate, Ammonia 18
4.5.4 Volatile Fatty Acids 18
4.6 Molecular Microbiology 19
5. Economic Analysis 27
5.1 Evaluation Summary 27
6. Conclusions 28
7. Recommendations 30
8. References 31
Appendix A: Summary of Quality Assurance Activities A-l
Appendix B: Statistical Analysis B-l
Appendix C: Microbial Analysis Report C-l
Figures
3-1. Cross-section of underground mine subsurface SRB bioreactor 13
4-1. Aluminum concentrations 19
4-2. Copper concentrations 20
4-3. Cadmium concentrations 20
4-4. Zinc concentrations 21
4-5. Manganese concentrations 21
4-6. Iron concentrations 22
4-7. Arsenic concentrations 22
4-8. Sulfate concentrations 23
4-9. Sulfide concentrations 23
4-10. pH readings 24
4-11. ORP readings 24
VI
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Tables
Page
1-2. Demonstration Goals 4
4-1. Initial Sampling Dates 25
4-2. Monthly Sampling Events 25
4-3. Additional Sampling Events 26
4-4. Representative Lilly/Orphan Boy Water Chemistry 26
5-1. Costs for 3-gpm Systems 27
5-2. NPV of Costs for Lime Addition and SRB Technologies 27
vn
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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
DMW drift monitoring well
DNA deoxyribonucleic acid
DO dissolved oxygen
DOE U.S. Department of Energy
EPA U.S. Environmental Protection Agency
IAG Interagency Agreement Number
ID identification
MSE MSE Technology Applications, Inc.
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
SMW shaft monitoring well
SRB sulfate-reducing bacteria
VFA volatile fatty acid
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's (DOE) Environmental Management Consolidated Business Center. 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 In Situ Source Control Of Acid Generation Using Sulfate-Reducing Bacteria project was the result of
contributions by over 40 MSB employees. Of these, Ms. Suzzann Nordwick, Ms. Marietta Canty, Mr.
Tom Mclntyre, and Mr. Creighton Barry made significant contributions.
Special acknowledgment and thanks are extended to the members of the Newman family - owners of the
Lilly Lode and Orphan Boy mining claims during this demonstration project.
IX
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Executive Summary
The Mine Water Technology Program (MWTP), Activity III, Project 3, In Situ Source Control Of Acid
Generation Using Sulfate-Reducing Bacteria was funded by the U.S. Environmental Protection Agency
(EPA) and jointly administered by EPA and the U.S. Department of Energy (DOE). The project
addressed EPA's technical issue of Mobile Toxic Constituents - Water through a field demonstration of
an in situ sulfate-reducing bacteria (SRB)-based water treatment technology applicable to acid rock
drainage (ARD).
ARD is produced when metal sulfide minerals, particularly iron pyrite, come in contact with oxygen and
water. The resulting oxidation of the metal sulfide minerals dramatically increases their solubility in
water, causing the formation of an acidic metal-laden stream. Biological sulfate reduction with SRB can
be used to treat ARD. The bacteria convert the sulfate dissolved in the ARD to soluble sulfides, which
then react with the dissolved metal ions to rapidly precipitate stable metal sulfides.
The main purpose of conducting this field demonstration was to evaluate the use of SRB to mitigate the
effects of metal-contaminated ARD in situ. This field demonstration resulted in an effective, relatively
long-term test. The performance of the SRB treatment technology was demonstrated through the
collection and analysis of samples within the mine tunnel and at the mine portal. Dissolved metals
concentrations were the primary parameters monitored. However, periodically collected data also
included total metals, alkalinity, temperature, dissolved oxygen, pH, oxidation reduction potential, sulfate,
sulfide, biochemical oxygen demand, chemical oxygen demand, and volatile fatty acids. The effects of
the treatment were observed in nearly all of the analytical parameters measured.
The Lilly/Orphan Boy Mine, near Helena, Montana was selected as the site for the field demonstration.
The flooded subsurface mine workings were turned into an anaerobic biological reactor by suspending an
SRB-supporting organic substrate on a platform within the open mineshaft. While SRB technology is
commonplace now, its use to treat ARD was a novel concept in the early 1990s. Before the bioreactor
was activated in August 1994, concentrations of the major dissolved metals were typical of ARD water.
Analytical data taken over the course of the demonstration indicated that dissolved metals concentrations
had decreased considerably. In addition, the pH of the discharge was effectively increased from a pre-
demonstration value of about 3 to a more neutral value close to 6.
Data evaluation shows that overall metal removal was extremely high for aluminum, cadmium, copper,
and zinc, but lower for arsenic and iron. Data also indicates that higher metal removals were obtained
within the tunnel than at the portal. The pH of the mine water increased almost immediately after the
implementation of the technology. During spring runoffs, the pH was lower in the portal sample, but it
stayed near neutral in the tunnel. The spring runoff events influenced the water quality more noticeably at
the portal than in the tunnel due to oxygenated surface water runoff penetrating through the ground above
the portal and then solubilizing historic metal precipitates. Also, spring water quality was lower at the
portal due to a greater amount of ARD infiltration from fractures within the tunnel walls.
This demonstration was one of the pioneering efforts in SRB technology implementation at a mine site.
The field work proved the long-term effectiveness of using in situ SRB technology to treat acid rock
drainage at remote mine sites. Although new at the time, the technology was shown to significantly
improve water quality at an abandoned mine and was also more cost-effective than conventional
technologies.
ES-1
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1. Introduction
1.1 Project Overview
This document is the final report for Mine Waste
Technology Program (MWTP), Activity III,
Project 3, In Situ Source Control of Acid
Generation Using Sulfate-Reducing Bacteria. The
MWTP is a program funded by the U.S.
Environmental Protection Agency (EPA) and
jointly administered by EPA and the U.S.
Department of Energy 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 sulfate -
reducing bacteria (SRB) technology to treat acid
rock drainage (ARD) in situ at the Lilly/Orphan
Boy Mine located in the Elliston Mining district
near Helena, Montana.
This report presents field results gathered during
an 11-year field demonstration from August 1994
to July 2005. During this time, the ability of SRB
to treat and control metal-contaminated water was
evaluated. SRB technology relies on biologically
generated products to treat ARD inside the mine
before it discharges naturally from the mine portal
and into a surface stream.
The field portion of this project consisted of the
installation and operation of in situ bioreactors in
the mineshaft and in the portal level mine tunnel.
The field application consisted of placing a
biological reactor with organic substrate and SRB
inside the flooded mineshaft. In general, SRB
technology treats ARD by removing dissolved
metallic and anionic constituents from the water.
The SRB bioreactors biologically reduce sulfate to
sulfide and generate alkalinity that precipitates
metal sulfides and metal hydroxides, respectively,
from the ARD. Both of these reactions increase
the pH of the water, which decreases the potential
for additional acid.
1.2 Background
Prior to field testing, pilot-scale testing was
conducted in the laboratory in eight packed-bed
reactors that simulated conditions at the
Lilly/Orphan Boy Mine. The results of the
column tests are summarized in a previous
document (Canty, 1999). The tests were able to
demonstrate the effectiveness of SRB technology
to treat ARD, by increasing pH and removing
most metals. Design parameters were developed
from the column tests that were used in the
implementation of the field system.
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 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 purpose of the in situ source control
demonstration was to test and evaluate in situ
placement of SRB bioreactors and determine the
capabilities of this technique to remove dissolved
metals from the ARD emanating from the mine
workings. It was proposed that an in situ
configuration could allow for sustained SRB
growth by maintaining an organic support matrix
within the mine workings. Additionally, technical
information gained from this project would
provide technical and economic information on the
capabilities of this innovative application of SRB
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.
Biological sulfate reduction using SRB can be
used to treat ARD. The SRB reduce the sulfate
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dissolved in the ARD to form soluble sulfide and
the hydrogen sulfide generated by the biological
metabolism process react with most dissolved
metal ions to rapidly precipitate stable metal
sulfides. Besides lowering the concentrations of
sulfate and dissolved metals, the SRB process also
produces alkalinity in the form of bicarbonate
from the oxidation of the organic nutrients. This
in turn helps to buffer and decrease the acidity of
the ARD.
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 five
selection criteria: physical accessibility, legal
accessibility, physical shaft characterization,
hydrogeologic characterization, and shaft water
characterization (Mine Waste Report Activity I
Report, 1992). The site selected was the
Lilly/Orphan Boy Mine, a relatively small mine
with a sulfide-based geology that produced ARD
in flooded mine workings with the ARD flowing
from the mine portal.
1.5.1 Location
The Lilly/Orphan Boy Mine located in the Elliston
Mining District of Powell County, Montana, was
selected as the demonstration site. 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 Lilly/Orphan Boy Mine is situated on
patented claims in the Helena National Forest
about 11 miles south of Elliston.
1.5.2 Mine Site Geology and History
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.
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. Impurity metals included iron,
arsenic, and antimony. The general geology of the
Lilly Lode ore was sulfide mineralization with the
major minerals being pyrite, arsenopyrite, galena,
sphalerite, tetrahedrite, and chalcopyrite.
Production from the district was significant during
war years when notable amounts were supplied to
neighboring mills. The Grand Republic Mining
and Milling Company of Helena first staked the
Lilly lode claim in the late 1890s. They sunk a
250-foot shaft. In 1934, the Lindquest family
acquired the Lilly Lode, made the Lilly portal, and
drove in the Lilly tunnel. The Newman family
acquired the claim in 1941. The mine remained
active during World War II and even had a special
priority road built to transport ore. Records
indicated 1,000 tons of ore was shipped with most
coming from areas near the shaft.
Chemical properties of untreated Lilly/Orphan
Boy Mine water are shown in Table 1-1.
1.5.3 Mine Workings
Prior to the field implementation of the MWTP
Demonstration, the Lilly/Orphan Boy Mine
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consisted of a 250-foot shaft, four horizontal
workings, and some stoping (Figure 3-1). After
active operations ceased, the mineshaft naturally
flooded with water to the 75-foot level and
discharged about 3 gallons per minute (gpm) of
pH 3 ARD from the portal or adit associated with
the 75-foot level.
1.6 Technology
To treat the ARD discharging from the
Lilly/Orphan Boy Mine, an in situ SRB-based
system was constructed within the mine workings.
The technology consisted of establishing a reactor
system 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 system
configuration provided good conditions for SRB
growth by supplying organic materials and other
nutrients. For more detailed information see
Technology Description (Section 2).
1.7 Project Objectives
The project objective was to develop technical
information on the ability of SRB, as a source
control treatment technology, to slow or stop the
process of acid generation and, thus, improve
water quality at a remote mine waste site. The
specific purpose of the field demonstration was to
show that SRB technology could treat an in situ
acidic aqueous waste by removing toxic dissolved
metallic and anionic constituents and neutralizing
the pH. The goal of the demonstration was to
achieve the effluent parameters summarized in
Table 1-2.
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.
The effluent parameters in Table 1-2 were derived
from a State of Montana discharge permit and
agreed to by EPA in the project quality assurance
project plan (QAPP).
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 SRB technology to
control acid generation and treat water, but also
focused on the feasibility and appropriateness of
using this technology at such a site under specific
conditions.
Successful achievement of the project goals was to
be quantified by measuring dissolved metals
concentrations, which would verify the ability of
SRB to treat metal contamination associated with
ARD. The drainage emanating from the
Lilly/Orphan Boy Mine was initially monitored for
reduction of dissolved sulfate, reduction of
dissolved heavy metals, and pH. A detailed
discussion of the sampling can be found in the
project-specific QAPP (MSB, 1994).
Table 1-1. Baseline Lilly/Orphan Boy Portal Water Chemistry
Pre-Treatment
Portal Water
(average from
September 1993 to
August 1994)
Fe
[milligrams
per liter
(mg/L)]
14.05
Zn
(mg/L)
19.4
Al
(mg/L)
7.36
Mn
(mg/L)
5.46
As
(mg/L)
0.08
Cd
(mg/L)
0.24
Cu
(mg/L)
0.33
scv2
(mg/L)
213
pH
3.44
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Table 1-2. Demonstration Goals
Parameter Goal
pH between 6 and 8
Sulfate reduction of 8%
Dissolved aluminum < 1.0 mg/L
Dissolved arsenic < 0.05 mg/L
Dissolved cadmium < 0.1 mg/L
Dissolved copper < 0.1 mg/L
Dissolved iron < 1.0 mg/L
Dissolved zinc < 4.0 mg/L
Dissolved manganese < 2.0 mg/L
Biochemical oxygen demand (BOD) < 4.0 mg/L
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2. Technology Description
The following section provides a detailed
description of the SRB technology available in the
literature that was used as the technical basis for
the design of the pilot- and field-scale tests. In
addition, a description is presented of the other
metal removal mechanisms anticipated to occur
within an organic-based system designed to
promote SRB activity.
2.1 Metals Removal Mechanisms
Although the purpose of the field testing was to
evaluate the use of SRB to mitigate metal-
contaminated wastewaters in situ, 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.
2.1.1 Adsorption by Substrate
The binding of metal ions by organic matter can
play an important role in removing these ions from
solution. Some adsorption most likely occurred at
the Lilly/Orphan Boy Mine. Three categories of
macromolecular, colloidal, or 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 + If" (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 the metal ion.
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).
2.1.2 Biological Sulfate Reduction
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. The formation of metal sulfides,
most of which are quite insoluble at a low EH and
a neutral pH, is very rapid. Therefore, biological
sulfate reduction likely occurred at the
Lilly/Orphan Boy Mine. Biological sulfate
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reduction is described in more detail in
Section 2.2.
2.1.3 Hydroxide Precipitation
Of the metals of interest in the Lilly/Orphan Boy
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
example, ferric hydroxide precipitation was
viewed as an unlikely occurrence, given the
reducing conditions present in the system, which
make sulfate reduction and the presence of ferric
ion mutually exclusive. Aluminum hydroxide is
the only stable hydroxide in this pH and EH range.
Therefore, aluminum removal by hydroxide
precipitation most likely occurred at the
Lilly/Orphan Boy Mine.
2.2 Biological Sulfate Reduction
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.
SRB decompose simple organic compounds using
sulfate as the terminal electron acceptor, thus
producing hydrogen sulfide. Additionally, other
bacteria are capable of reducing less oxidized
sulfur compounds (i.e., elemental sulfur and
thiosulfate) to produce hydrogen sulfide.
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.
SRB
SO42 +2CH2O -> H2S + 2HCO3 (4)
H2S -> 2H+ + 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. The Tennessee Valley
Authority has the most experience in constructing
wetlands for the treatment of ARD from coal
mines, which are typically aerobic systems
designed for iron removal (Brodie et al., 1988).
Historically, PMDTS were constructed as shallow
ponds resembling natural wetlands focusing on
plant uptake of metals as an important role in
metals removal of these systems (Brodie et al.,
1989). However, the most important method of
metals removal in PMDTS has become recognized
as biological sulfate reduction in the anaerobic
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zone of the system. In fact, plant uptake of metals
is no longer recognized as a necessary element of
a PMDTS. At the Big Five Tunnel PMDTS in
Idaho Springs, Colorado, no uptake of metals into
the plants could be demonstrated. Consequently,
the focus of PMDTS has moved toward metals
removal and generation of alkalinity by biological
sulfate reduction through optimization of an
anaerobic, reducing environment.
2.2.1 Microbial Description
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, and Desulfonema (Hunter, 1989).
The dominant species of SRB belong to the genera
Desulfotomaculum and Desulfovibrio (Cohen and
Staub, 1992).
2.2.2 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 °C 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.
2.2.3 Growth Requirements
Temperature, Reduction Potential
SRB are capable of tolerating a wide variety of
temperatures, salinities, and pressures and
demonstrate considerable adaptability to new
conditions of temperature (Postgate, 1984). The
major prerequisite for growth is an anaerobic
environment with EH near -100 mV (Postgate,
1984). Mesophilic SRB grow best at temperatures
between 30 °C to 42 °C, but tolerate temperature
swings between -5 °C to 50 °C (Postgate, 1984).
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A temperature of 37 °C was found to be optimum
using a bioreactor with wood chips as the organic
substrate (Turtle et al., 1969). On the contrary,
low temperatures have been reported to have a
considerable suppressing effect on biological
sulfate reduction (Davison et al., 1989 and
Kuyucak and St-Germain, 1993). At temperatures
below 10 °C, SRB performance may be lowered
60 to 80% (Kuyucak et al., 1991). Because an in
situ application of biological sulfate reduction
would involve a cool environment, SRB
effectiveness at a low temperature was of
particular relevance during the literature search.
Further literature review revealed opposite results
on the cold temperature capabilities of SRB. SRB
have demonstrated the ability to increase their
numbers in cold weather, thus compensating for
lower individual activity (Cohen and Staub, 1992).
Sulfate reduction was observed in an Antarctic
pool (Postgate, 1984). It has been suggested that
more SRB in nature function at below 4 °C
(psychrophiles) than above 5 °C, largely because
of their abundance in ocean sediments (Postgate
1984). Finally, Herlihy and Mills, (1985) reported
similar SRB activity rates in both winter and
summer. In general, literature information shows
that while SRB growth rates may be slower at low
temperatures, some growth does occur, and there
is ample evidence that a low temperature SRB
reactor would function to treat ARD.
pH
SRB tolerate pH values ranging from well below 5
to 9.5 (Postgate, 1984), but the specific bacterial
growth rate and the removal rate of metals have
been shown to be strongly influenced by pH. A
pH of 6 has been reported to be optimum for both
SRB growth rate and removal rate of metals
(Hunter, 1989). Desulfovibrio is reportedly
inhibited at pH less than 5 (Postgate, 1984).
However, it should be noted that these
microorganisms are capable of creating
microenvironments conducive to their growth
(Hunter, 1989). For example, in a constructed
wetland receiving ARD with pH < 3, the pH of the
pore water in the substrate ranged from 6 to 7
(Hedinetal., 1989).
Substrate
To determine an appropriate substrate to be used
during the pilot-scale testing, a literature review
was conducted. Several types of substrates, as
well as additives were identified.
SRB require a substrate composed of simple
organic compounds (Postgate, 1984). Davison et
al. (1989) reported the effects of substrates on
SRB activity. The substrates used in these
experiments included the following: spent
mushroom compost, peat, corn wastes, rice waste,
decomposed wood chips, and composted cow
manure. Decomposed wood chips and composted
cow manure gave the highest activity rates and
demonstrated the greatest buffering capacity. The
other substrates tested often yielded near zero SRB
activity. However, addition of pH raising
additives to the poor growth substrates resulted in
substantially increased activity. Cohen and Staub
(1992) reported that results from the Big Five
Tunnel PMDTS indicated that peat was an
ineffective substrate even when limestone was
added. Additionally, mushroom compost worked
well but only in conjunction with very low ARD
flow rates. Dvorak et al., (1991) reported that
sulfate reduction and metal retention increased in a
reactor with the addition of lactate. Postgate
(1984) reported that growth of a strain of SRB on
an unfamiliar carbon source might require a
metabolic adjustment that delays growth.
Another study was performed comparing three
cellulosic materials (straw, timothy hay, and
alfalfa hay) on their abilities to sustain microbial
treatment of ARD (Bechard et al., 1993). Of these
three cellulosic materials, alfalfa hay sustained
microbial treatment for the longest period of time.
However, the study determined that a more readily
available carbon source, such as sucrose, was
often needed to keep the cellulosic systems
operating. Cellulosic materials have been used in
conjunction with other substrate materials, such as
cow manure, to act as a long-term carbon source,
as well as a bulking agent (Cohen and Staub,
1992).
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In biochemical reactions under anaerobic
conditions, a consortium of "acid forming
bacteria" convert complex organic substrates into
aliphatic acids. Acetic, propionic, butyric acids,
the major aliphatic acids produced, are considered
simple organic compounds that can be used by
SRB. In other words, substrate effectiveness
appears to rely on the presence of other
heterotrophic bacteria to convert complex organic
compounds into the simple compounds required
by SRB. Cohen and Staub (1992) found that a
substrate composed of cow manure and
decomposed wood chips produced a high SRB
activity rate; the data suggested that a consortium
of heterotrophic bacteria existed in the substrate
that decomposed complex organics into simple
ones. In addition, cow manure was found to have
the buffering capacity and nutrient composition
necessary to enhance SRB activity. Cow manure
has been described as the ideal substrate for
biological sulfate reduction because of its
effectiveness and low cost (Cohen and Staub,
1992).
Other bacteria in anaerobic systems also use
simple organic compounds as a food source. For
example, "methane-forming" bacteria, or
methanogens, are capable of converting the
aliphatic acids into methane by cleavage of the
carboxylic acid group (COOH) and carbon dioxide
reduction (Sundstrom and Klei, 1979). However,
in an environment rich in sulfate, SRB effectively
compete with methanogenic populations for the
available aliphatic acids. While SRB can grow at
low concentrations of hydrogen, methanogens are
greatly hindered because their hydrogen uptake
systems cannot function at low hydrogen
concentrations. In addition, SRB have an
increased affinity for both acetic acid and
hydrogen in comparison to methanogenic
populations (Postgate, 1984). Also, a relationship
between SRB and methane-producing bacteria has
been noted. In natural environments, sediments in
which sulfate reduction is actively taking place
will often lie above sediments in which methane
production is occurring. It has been postulated
that SRB may have the capacity to use methane as
a substrate (Postgate, 1984).
Although past research has reported the treatment
capacity of SRB based on metal loading rates per
unit area of substrate (Kleinmann, 1990 and Cohen
and Staub, 1992), recent developments have
concluded that treatment capacity (flow rate) is
more accurately represented on the basis of
substrate volume (Euler, 1992).
Sulfate
In biological sulfate reduction, sulfate ions act as
an oxidizing agent for the dissimilation of organic
matter. SRB assimilate a small amount of reduced
sulfide ions, but essentially all sulfide is released
into the surrounding fluid. The process is
generally less effective at very low concentrations
of sulfate (Hedin et al, 1989) and (Kuyucak and
St-Germain, 1993). However, as long as sulfate
reduction remains the dominant electron acceptor
process occurring, methanogenesis would still
occur under anaerobic conditions.
Iron
Desulfovibrio shows an exceptionally high
requirement for iron. The iron is needed in cell
constituents, such as ferredoxin and cytochrome c
(Postgate, 1984). The dissolved iron reacts with
the H2S to form iron sulfide, therefore reducing the
dissolved iron concentration. Amino acids, which
may be a nutritional requirement for SRB growth,
are capable of chelating Fe2+ and thereby
inhibiting iron sulfide precipitation.
Consequently, specific amino acids may function
to make iron more readily available to SRB
(Dvorak et al., 1991).
2.2.4 Growth Inhibition Factors
While the presence of oxygen inhibits SRB
activity, SRB can survive long exposure to oxygen
and become active again when returned to an
anaerobic environment (Postgate, 1984). High
metal concentrations, particularly copper, may
also inhibit SRB growth. An SRB inhibitory
copper concentration of 5-50 mg/L of copper
sulfate was reported by Saleh et al. (1964).
However, Noboro and Yagisawa (1978) reported
rapid bacterial growth at a copper concentration as
high as 100 mg/L when a lactate substrate was
used.
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Lovley and Phillips (1994) reported inhibition of
SRB with Fe3+ in laboratory experiments. The
suggested cause for this inhibition was iron-
reducing bacteria, which have an energetic
advantage over the sulfate reducers. Few other
studies have indicated an inhibitory effect of iron.
In fact, several studies have reported significant
SRB growth with high concentrations of iron in
the wastewater of concern, for example, acid mine
water.
Two additional metal ions have been reported to
inhibit SRB growth. Postgate (1984) denoted the
selenate ion (a competitive antagonist of sulfate
reduction) and the molybdate ion (which depletes
the organism's ATP pool). However, both ionic
species are generally expected to be present in
only very small quantities in ARD.
Postgate (1984) reported a cyanide concentration
of 1 to 5 mol/mL as being a metabolic inhibitor.
10
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3. Demonstration Description
Demonstration of SRB technology consisted of the
following major phases with several sub-phases as
described below.
• Phase I - Laboratory testing
• Phase la - Bench-scale substrate adsorption
study
• Phase II - Field demonstration at a remote
mine site
- Design
- Implementation
- Monitoring
3.1 Laboratory Testing
Technical parameters required for the field
application were developed from laboratory
testing conducted by MSB between January 15
and March 25, 1994 in Butte, Montana.
Sediments collected at the Lilly portal were used
as the source for the laboratory SRB populations.
Water pumped from the Lilly mine shaft was
collected in large plastic containers and
transported to Butte to be used as the ARD feed
for the laboratory test. Following the laboratory
testing, the field demonstration commenced in
August 1994 and continued through July 2005.
The laboratory tests were designed to support the
field demonstration by identifying functional
parameters using ambient conditions comparable
to the Lilly shaft water. Eight 4-foot vertical
Plexiglas organic substrate packed-bed SRB
reactors were operated at 8 °C and were fed Lilly
mine shaft ARD continuously in an upflow
configuration at a reactor hydraulic retention time
of 120 hours over 60 days. During this time,
numerous physical and chemical parameters were
monitored.
The experimental design consisted of a 3x2 full
factorial design, allowing for the comparison of
two different bacterial preparation methods (no
preparation and prepared) and three organic
substrate-layering methods (no gravel, mixed, and
layered). In the prepared tests, SRB were first
grown for two weeks at 20 °C in a sodium sulfate
solution.
Laboratory column testing and subsequent
laboratory analysis showed that metal removal
occurred due to both adsorption and sulfide
precipitation, however, the amount of metal
removal by either mechanism was not quantified.
The total dissolved metal removal efficiencies
reached 99% for zinc, 99% for aluminum, 96% for
manganese, 98% for cadmium, and 96% for
copper. Iron and arsenic removal was not as
effective but was slightly more effective in the
reactors with prepared bacteria. This was
attributed to high levels of iron and arsenic
contamination in the organic substrate.
3.2 Substrate Adsorption Studies
Throughout the project, many questions were
raised regarding the mechanism for metals
removal. Although the majority of metals removal
is credited to SRB through sulfide precipitation,
some metals removal can be attributed to
adsorption by the organic substrate. To help
quantify this removal mechanism, exploratory
laboratory column testing was conducted with the
objective of determining the extent to which
metals are removed via adsorption. For this
testing, the total metals removal of sterilized
organic substrate was compared to that of
unsterilized organic substrate. Results indicated
that the sterilized substrate was still able to remove
aluminum and manganese, so it is likely that these
two metals had some removal as a result of initial
adsorption onto the substrate.
3.3 Field Design and Construction
The SRB field demonstration was designed to use
the flooded subsurface mine workings of the
Lilly/Orphan Boy Mine as an "in situ biological
reactor" (Figure 3-1). Two platforms were
suspended by cables in both sides of the two-
compartment shaft 30 feet below the static water
level and were secured at the surface. An organic
substrate consisting of approximately 70% cow
11
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manure, 20% decomposed wood chips, and 10%
alfalfa straw was placed in the shaft and supported
by the platforms. The percentages are
approximant, as the substrate was prepared by
mixing about four parts cow manure, one part
wood chips, and one part straw in a concrete mixer
while in the field. Some substrate was placed on
the platform in the shaft and the remainder was
placed in the horizontal adit by drilling holes from
the surface and pumping material into the tunnel.
In addition, two injection wells were drilled into
the main portal tunnel of the mine (Lilly Tunnel)
so that substrate could also be placed into this
underground space. Therefore, the ARD flowed
upward through the substrate in the shaft (artesian
water flow has been observed at the mine) and
horizontally through the substrate in the tunnel.
The biological reaction took place in the substrate
regions, and the treated water subsequently flowed
out of the mine through the portal. Because the
technology caused the shaft water pH to rise and
the EH to fall, the amount of acid generation within
the mine was decreased. Monitoring of the field
demonstration began once the substrate was placed
in late August 1994 and continued for almost 11
years.
3.3.1 Monitoring Well Installation
Original plans called for sampling water
emanating from the mine portal and comparing
these samples to historical influent values to
determine bioreactor effectiveness. However, as
the demonstration progressed, the tunnel was
suspected of re-contaminating the water after it
passed through the bioreactor. Therefore,
monitoring wells were installed to obtain samples
from within the mine tunnel to realistically
evaluate the bioreactor. One monitoring well was
drilled into the tunnel downgradient of the
injection well and the injection well was also fitted
to be a monitoring well. These monitoring wells
allowed for the collection of samples before the
water traveled the full length of the tunnel to the
portal.
Extensive background testing was performed prior
to initiating the field demonstration. This data was
used to assess the effectiveness of the treatment
technology by comparing effluent parameters to
historical influent values. As the demonstration
progressed, however, it was decided that this
background data was outdated, and a better
method would be necessary to monitor influent
and effluent at the same time. Therefore, two
angled groundwater monitoring wells were
installed in September 2003 to monitor the ARD
at a point prior to entering the biomass in the shaft.
These wells monitor confined and unconfmed
groundwater in the main shaft and a drift.
Prior to well installation, the mine workings were
evaluated based on available maps. However,
different subsurface maps placed the adit level at
different depths. For example, Aikin (1950)
shows a cross-sectional view of the Lilly workings
that placed the main underground workings at
102 feet below the collar of the shaft. By contrast,
Rankin (1950) showed the same workings 74 feet
below the collar.
Elevation data acquired at the site indicated that
the elevation difference between the collar of the
shaft (6,810.6 feet) and the elevation just above
the collapsed adit of the main underground
workings (6,746.8) was 63.8 feet. The elevation
point above the collapsed adit was at least 7 or
8 feet above the adit. This means that the
approximate elevation between the shaft collar and
the workings near the adit was 71 feet below the
collar of the shaft, similar to Rankin's figure of
74 feet. Therefore, it was determined that the best
estimate of vertical drill depth would be based on
Rankin's 1950 map of the workings.
Two deviated monitoring wells were installed in
September 2003. The shaft monitoring well
(SMW) was completed in groundwater associated
with the shaft and the drift monitoring well
(BMW) was completed in groundwater associated
with the main underground drift immediately east
of the shaft. The SMW intercepted the shaft at a
depth of 123 feet and the BMW intercepts the drift
just east of the Lilly shaft 15 feet back.
12
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HEADFRAME SUPPORT
CABLES SECURED AT
SURFACE •
PORTAL
SAMPLES
TUNNEL
SAMPLES
CAVED
Figure 3-1. Cross-section of underground mine subsurface SRB bio reactor.
13
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4. Field Monitoring Results and Discussion
The first technology evaluation-sampling event
was conducted September 6, 1994 after the shaft
bioreactor was installed in August 1994. The final
sampling was conducted on July 14, 2005. For the
first few months of the demonstration, twice-
monthly sampling events were conducted.
Monthly sampling was then conducted for five and
a half years. After the decision was made to
extend the project, sampling events were reduced
to bimonthly and then to three times per year.
Additionally, as the project proceeded, the number
of analyses performed on each sample was
minimized to reduce project costs. A total of 92
sampling events were conducted. Twice monthly
sampling dates are listed in Table 4-1. Monthly
sampling events are listed in Table 4-2, and
additional sampling events are listed in Table 4-3.
The analytical results of samples taken from
within the horizontal mine tunnel and at the portal
of the mine are presented within the following
sections. Data is presented in graphs and includes
dissolved metals, pH, and EH. A summary of the
quality assurance (QA) activities from the project
specific QAPP are contained in Appendix A. All
data, with the exception of two temperature
measurements from 1994, were determined to be
usable.
The organic substrate was placed in the mineshaft
between August 27 and August 31, 1994. Data
prior to this date represents the water chemistry
before treatment. The data shows that, for nearly
all parameters, tunnel removal efficiencies were
better than those for the portal. This is addressed
in the discussion of the individual parameter, and
has generally been attributed to untreated materials
entering the flow between the tunnel sampling
point and the adit. The statistical analysis of the
metals analysis data is contained in Appendix B
along with the QA data summary table.
4.1 Dissolved Metals
As with most acidic mine effluents, the water
emanating from the Lilly/Orphan Boy 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.
Dissolved metals samples were collected from
both the tunnel and the portal during the field
demonstration and were analyzed for aluminum,
arsenic, cadmium, copper, iron, manganese, and
zinc. These metals were chosen based on their
pre-demonstration concentration in the
Lilly/Orphan Boy Mine ARD.
Chemical properties of untreated Lilly/Orphan
Boy Mine water are shown in Table 4-4 along
with typical post-treatment water chemistries for
the tunnel water and mine portal effluent.
4.1.1 Aluminum, Copper, and Cadmium
Concentrations of dissolved aluminum, cadmium,
and copper were significantly reduced to below
detection limits shortly after the addition of the
organic substrate. This would be expected since
the solubility of these metals is very dependent on
pH. Similar dramatic results were observed in
both the tunnel and at the portal for most of the
year. However, the portal concentration of all
three metals increased for the period of high flow
during the spring runoff months of May and June.
By contrast, samples from within the mine tunnel
showed very little seasonal variation.
As shown in Figure 4-1, the concentration of
aluminum in the portal showed regular seasonal
variation for the first 6 years of operation.
Although data is limited, it appears that the spring
runoff values have been going up in recent years.
The general trend of the graph is upward, which
may indicate that the effectiveness of the SRB
reactor is diminishing. After almost 11 years of
operation, the nutrient sources are becoming
depleted and the ability of SRJ3 to produce sulfide
and precipitate metals has decreased. However,
since the water was not sampled in winter months
for the last years of the demonstration, it is not
known if this trend is real. Additional data would
be needed to evaluate further. Figures 4-2 and 4-3
14
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for copper and cadmium in the portal show a
similar upward trend.
4.1.2 Zinc
Dissolved zinc was not removed as effectively as
aluminum, copper, and cadmium. Data shown in
Figure 4-4 indicates that zinc is removed to below
detection limits in the tunnel, but rebounds prior to
the portal. In fact, zinc measured at the portal
never reached the target level of 4.0 mg/L. As
with other metals, portal zinc concentrations
increased considerably during spring runoff. The
higher removal of zinc observed in the tunnel of
the mine is attributed to the more reduced
conditions in the tunnel as compared to the portal.
As with other metals, Figure 4-4 shows an upward
trend that may be indicative of the bioreactor's
depletion of essential nutrients.
4.1.3 Manganese
Dissolved manganese was not removed as
effectively as aluminum, copper, and cadmium.
As shown in Figure 4-5, concentrations of
manganese in the tunnel were reduced below the
target value of 2 mg/L several times, primarily
during the first 2 years of operation.
Concentrations of manganese at the portal
remained at or above pre-demonstration levels
showing high correlation to spring runoff times.
This same phenomenon was observed during this
project's laboratory pilot-scale testing and was
attributed to the fact that manganese does not form
a solid that can precipitate at the pH and EH
conditions of this system. Effective manganese
removal requires oxidizing conditions and a higher
pH.
4.1.4 Iron and Arsenic
Dissolved iron data was inconsistent throughout
the demonstration and never met the target
concentration of 1.0 mg/L (Figure 4-6). During
the first few years, dissolved iron was removed
more effectively within the tunnel than at the
portal. Immediately after the addition of the
organic substrate, the dissolved iron concentration
actually increased in portal samples. This trend
was reversed during the high flow rate that
occurred during spring runoff. An increase in
dissolved arsenic was observed shortly after the
implementation of the technology at the mine as
well. Although only a slight increase in arsenic
concentration was observed in the tunnel, a large
increase was observed at the portal (from near the
instrument detection limit of 0.0336 mg/L to a
high of 14.7 mg/L). An understanding of iron and
arsenic chemistry helps explain these phenomena.
Under oxidizing conditions, ferric iron precipitates
as Fe(OH)3 (ferrihydrite) and very effectively
adsorbs arsenic. However, under reducing
conditions, ferrous iron becomes the predominant
iron species, which is much more soluble than
ferric iron. When the highly soluble ferrous iron
was released into solution, arsenic, which had
been previously absorbed on to the iron, was
released also. This took place during the
demonstration when the underground workings of
the mine were transformed from an oxidizing
environment to a reducing one.
The concentration of arsenic in the mine portal
water varied considerably during the
demonstration. As shown by Figure 4-7, arsenic
in the portal followed the same trend as the iron
levels. As with iron concentrations, arsenic
concentrations also increased during each spring
run off. The system consistently demonstrated
that it could recover. Most notable was the 2004
event in which arsenic spiked to 38 parts per
million. These increases in iron and arsenic
concentrations during spring runoff indicate a
reversal of the conditions imposed by the SRB
technology and similar to the historical conditions
of the mine. As the flow rate increased in the
mine system and the retention time decreased, the
SRB were likely unable to produce enough soluble
sulfide to precipitate the metals, maintain a low
reduction potential, and neutralize the water
through bicarbonate production. Consequently,
the pH fell and the reduction potential increased
while the system became more oxygenated.
Ferrous iron is oxidized to ferric iron under these
conditions, precipitates, and absorbs the arsenic.
Conversely, as the system recovered from high
spring runoff, the reduction potential appeared to
15
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decrease and the pH and iron concentration
appeared to increase.
4.2 Total Metals
In addition to the dissolved metals analyses,
samples were collected for total metals analysis on
17 occasions during the first year of the field
demonstration. These were obtained from both the
tunnel and the portal and analyzed for aluminum,
arsenic, cadmium, copper, iron, manganese, and
zinc. In comparison with dissolved metals
concentrations, total metals results tend to be more
variable due to the presence of relatively large
particles of organic or other substances containing
high concentrations of a particular metal.
Concentrations of total copper and aluminum
showed similar trends in that lower, stable values
were observed in the tunnel samples compared to
higher, more sporadic concentrations in the portal
water. Total zinc and cadmium concentrations in
the portal samples were higher than those in the
tunnel samples. In addition, both total zinc and
cadmium concentrations rose in the portal samples
during spring runoff, while this trend was not
observed within the mine tunnel.
Total manganese concentrations were lower in the
tunnel water samples than the portal samples
during the first part of the year, then reversed and
became higher than the portal samples during the
latter part of the year. Similar to the dissolved
manganese results, this observation is attributed to
the lack of formation of manganese compounds at
the pH and EH of the system.
Total arsenic and iron results were very similar to
those for dissolved arsenic and iron. Total arsenic
and iron concentrations were both higher in the
portal water samples compared to the tunnel water.
This phenomenon was previously explained by
arsenic and iron chemistry in Section 4.1.
4.3 Alkalinity
Alkalinity typically increases as a result of SRB
activity. Therefore, alkalinity, as calcium
carbonate (CaCO3), was analyzed in samples taken
at the mine portal for the first 3 years of the
demonstration. The total alkalinity of the portal
water prior to the addition of the organic substrate
was less than 10 mg/L as CaCO3. After the
addition of the organic substrate, alkalinity
concentrations increased, although results were
variable. The highest alkalinity concentration (64
mg/L) was measured one month after the
implementation of the technology. Overall,
effluent alkalinity was not a concern during the
demonstration, as other portal water samples did
not show alkalinity above the detection limit of 10
mg/L.
4.4 Physical Measurements
Oxidation reduction potential (ORP), pH,
dissolved oxygen (DO), flow rate, and temperature
were measured on 15 occasions during the first
year of the field demonstration.
4.4.1 ORP
ORP is a helpful measurement for assessing SRB
growth potential because the organisms require a
reducing environment for optimal growth. SRB
can also help produce a reducing environment if
one does not already exist. ORP is measured in
mV, with zero being neither oxidizing nor
reducing. Positive values indicate an oxidizing
environment while negative numbers specify a
reducing environment. Prior to the addition of the
organic substrate, the reduction potential in the
mine water was about +400 mV. The addition of
organic matter caused the EH to drop sharply in
both the tunnel and the portal water samples
within the first few weeks to around -50 and
+50 mV, respectively. Over the course of the
demonstration, it was found that the reduction
potential was generally lower in the tunnel than at
the mine portal. This has been attributed to the
likely possibility that the water eventually exiting
the portal becomes contaminated with more
oxidized surface water sources. On one occasion
the reduction potential within the tunnel rose
above that measured at the mine portal. The
reduction potential measured within the portal
mine water rose sharply during spring runoff due
to the addition of fresh oxidized water.
16
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4.4.2 pH
pH is a relatively simple measurement of acidity
or alkalinity that can indicate major changes in the
condition of the mine water. As stated previously,
SRB typically prefer neutral pH, but they can
function at lower values and are able to raise the
pH of their surroundings by consuming hydronium
ions and producing bicarbonate. Within a few
weeks of substrate addition to the underground
mine workings, the pH increased from about 3 to
near neutral within the tunnel and to about 6 at the
mine portal. As the demonstration progressed, pH
measured at the portal remained neutral for the
most part but dropped back to 3 during spring
runoff events. Within the tunnel of the mine,
however, the pH remained circumneutral at all
times, including during spring runoff. The
stability of the pH within the tunnel indicates that
the technology would be able to provide a stable
treatment environment.
4.4.3 Dissolved Oxygen
It was necessary to reduce the level of DO in the
mine water since oxygen is detrimental to SRB
growth. Heterotrophic bacteria (aerobes and
facultative anaerobes) use oxygen as a terminal
electron acceptor as they use carbon as an energy
source for growth. After available oxygen is
consumed, anaerobic organisms, such as SRB can
proliferate. Historical DO levels within the portal
water were measured at about 6 mg/L prior to the
implementation of the technology. Within a few
months of substrate addition, DO levels dropped
and remained less than 2 mg/L, the ideal condition
for SRB growth.
4.4.4 Temperature
Water temperatures recorded in the tunnel and at
the portal ranged from 3.5 °C to 12.2 °C and
3.8 °C to 7.5 °C, respectively. Temperatures
recorded from tunnel water samples were higher
on average than portal samples by 1.3 °C. This
difference in temperature may be attributed to the
tunnel water mixing with other sources before
discharge at the portal. The SRB that thrive in this
environment are classified as psychrophilic since
they are able to grow at temperatures less than
15 °C. (Morita and Moyer, 2007).
4.4.5 Flow Rate
Due to poor access and the dilapidated condition
of the mine portal, the flow rate of ARD was not
easily measured. During the first year of the
demonstration, field instrumentation was used in
an attempt to measure flow. However, this later
proved difficult to continue because much of the
flow went underground immediately before the
portal. During the first year the portal flow rate
remained fairly constant at less than 2 gpm.
However, spring runoff caused the flow rate to
climb to ahigh of 7.6 gpm during May 1995.
4.5 Other Chemical Measurements
In addition to metals measurements, the following
parameters were measured: sulfate, sulfide, BOD,
chemical oxygen demand (COD), nitrate,
ammonia, and volatile fatty acids (VFA).
4.5.1 Sulfate, Sulfide
As stated previously, SRB reduce sulfate to sulfide
during the course of their growth process. These
parameters were measured to indirectly verify the
existence and proliferation of SRB in the mine
water. Sulfate and sulfide samples were measured
routinely for the first 6 years of the field
demonstration, and then occasionally after that. In
general, concentrations of sulfate measured in the
tunnel were lower than those at the mine portal.
Because organic substrate was located in the shaft
and in the tunnel of the mine, these are the regions
in which sulfate reduction would occur. Prior to
the addition of organic substrate, baseline sulfate
concentration in the underground mine workings
was analyzed at 274 mg/L. Over the course of the
demonstration, sulfate concentrations mostly
stayed below this level (Figure 4-8).
Prior to the addition of organic substrate,
concentrations of soluble sulfide in the mine water
were below the instrument detection limit. After
the placement of the cow manure substrate,
variable and sporadic concentrations of sulfide
were measured in both the tunnel and the portal at
the same sampling events in which sulfide was
analyzed (Figure 4-9). There was some variability
of the concentrations that may have been caused
by the highly reactive nature of sulfide and the
17
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analytical difficultly in quantifying this parameter.
Sulfide generation is a good indicator of SRB
activity because sulfate is not reduced to sulfide
simply from a decrease in reduction potential;
microbial action is required.
4.5.2 Biochemical Oxygen Demand,
Chemical Oxygen Demand
In environmental chemistry, the COD test is
commonly used to indirectly measure the amount
of organic compounds in water. BOD is a
chemical procedure for determining how fast
biological organisms use up oxygen in a body of
water. It is used in water quality management and
assessment, ecology, and environmental science.
BOD is not an accurate quantitative test, although
it could be considered as an indication of the
quality of a water source.
Addition of organic and biological activity can
substantially impact oxygen demand. For the first
3 years, BOD and COD samples were collected at
the portal to determine the oxygen demand
imposed on the mine water and the receiving
stream (Telegraph Creek) as a result of the organic
substrate. BOD and COD levels rose within a few
weeks of substrate addition from background
levels of 4 and 83 mg/L reaching highs of 18.5 and
246 mg/L, respectively. Both parameters dropped
dramatically within a few months of organic
placement to approximately 4 and 40 mg/L,
respectively and analysis was discontinued.
BOD concentrations were elevated for six months
after technology implementation and then returned
to background levels.
4.5.3 Nitrate, Ammonia
Nitrate and ammonia are present in natural organic
matter (such as cow manure) and they can be
produced as a result of biological activity. Nitrate
and ammonia samples were analyzed for the first
3 years to determine discharge levels to Telegraph
Creek, which might be subject to state regulatory
discharge requirements.
Nitrate levels were measured for the first 4 years
of the project. They generally remained low
throughout this part of the demonstration. The
typical range was 0.05 to 3 mg/L. Ammonia
levels rose sharply within the first few weeks after
the addition of the organic substrate reaching a
high of 11.8 mg/L and gradually declined over the
remainder of the 4-year monitoring period,
reaching a low of 0.3 mg/L. These results only
include the nitrogen component of the nitrate and
ammonia for ease of comparison, in keeping with
EPA conventions.
Ammonia concentrations were elevated for a
longer period, but returned to background
concentrations after one year. BOD
concentrations dropped more quickly than the
ammonia concentrations because the BOD
measurement was carbonaceous BOD and not
nitrogenous BOD. Oxidation of carbonaceous
organics occurs more quickly than nitrogenous
organics because of a longer lag phase in the
growth of denitrifying bacteria (Barghoorn and
Nichols, 1961). After the organic substrate was
added to the mine, the BOD was depleted as the
abundant heterotrophic microorganisms present in
the substrate utilized the carbonaceous organic
material. The longer period of time required for
the decrease of ammonia was attributed to the
limited amount of oxygen available in the system
for nitrification of ammonia to nitrite/nitrate.
Nitrite/nitrate subsequently underwent anaerobic
denitrification.
4.5.4 Volatile Fatty Acids
As discussed previously, VFA are produced by
heterotrophic microorganisms by the breakdown
of more complex organic substances. According
to Kleikemper et al. (2002), SRB are known to use
VFAs as a food source, although not all genera are
capable of utilizing acetate. It appears that acetate
levels rose during the first few months of the
demonstration, then enough acetate-utilizing SRB
became established, and began to feed on the
acetate.
The following VFAs were measured during the
first year of the field demonstration: acetate,
propionate, iso-butyrate, normal-butyrate, and
formate. VFAs were monitored to help determine
18
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which simple organic compounds the SRB were
utilizing from the cow manure substrate. Higher
concentrations of VFAs were observed in the mine
tunnel than at the portal. Acetate concentrations
rose sharply in the mine tunnel after the addition
of the organic substrate. The concentration of
acetate peaked after three months and then began
falling. Propionate, iso-butyrate, normal-butyrate,
and formate concentrations were variable.
4.6 Molecular Microbiology
At the conclusion of the project, a sample was
collected from the shaft bioreactor and examined
at Montana State University's Center for Biofilm
Engineering using molecular community
analytical techniques. Deoxyribonucleic acid
(DNA) was extracted from the sample - both from
the liquid and from the soil. Method details are
given in Appendix C. This included the
extraction, purification, and amplification by
polymerase chain reaction (PCR) of DNA,
assessment of community profiles, and sequencing
of amplified DNA to identify species.
Molecular analysis results were significant in that
they confirmed the presence of SRBs and
indicated a diverse bacterial community. A total
of 56 clones had analyzable sequences that were
further analyzed using Basic Local Alignment
Search Tool (BLAST). Returned sequenced
results included two SRB (Chloroflexi bacterium
and Flexibacter) and a sulfur reducing bacteria
(Thermococcales archaeon). Other identified
sequences included three sulfur oxidizers, three
compost isolates, four Proteobacteria, and one
iron/manganese-reducing anaerobe.
Aluminum Concentrations - vs Time
1-94 Aug-95 Aug-96 Aug-97 Aug-98 Aug-99 Aug-00 Aug-01 Aug-02 Aug-03 Aug-04 Aug-05
Sample Date
Figure 4-1. Aluminum concentrations.
19
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Copper Concentrations - vs Time
1190
-10 !-=^- ^JJ8$B^roKS»Qooc6isX*3Kca»$g3oa^^ pro
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
Sample Date
Figure 4-2. Copper concentrations.
Cadmium Concentrations - vs Time
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
ample Date
Figure 4-3. Cadmium concentrations.
20
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Zinc Concentrations - vs Time
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
Sample Date
Figure 4-4. Zinc concentrations.
Manganese Concentrations - vs Tim
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
Sample Date
Figure 4-5. Manganese concentrations.
21
-------
Iron Concentrations - vs Time
Target Cone. (1.0) Baseline Cone. (13.8)
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
Sample Date
Figure 4-6. Iron concentrations.
Arsenic Concentrations - vs Time
40 n
May-93 May-94 May-95 May-96 May-97 May-98 May-99 May-00 May-01 May-02 May-03 May-04 May-05
Sample Date
Figure 4-7. Arsenic concentrations.
22
-------
Sulfate Concentrations - vs Time
Tunnel
• Portal
Target (196) Baseline (213) — x- Shaft
700 -i
0
Aug-94 Aug-95 Aug-96 Aug-97 Aug-98 Aug-99 Aug-00 Aug-01 Aug-02 Aug-03 Aug-04 Aug-05
Sample Date
Figure 4-8. Sulfate concentrations.
Sulfide Concentrations - vs Time
Porta —*— Shaft
94 Aug-95 Aug-96 Aug-97 Aug-98 Aug-99 Aug-00 Aug-01 Aug-02 Aug-03 Aug-04 Aug-05
Sample Date
Figure 4-9. Sulfide concentrations.
23
-------
pH - vs Time
Aug-94 Aug-95 Aug-96 Aug-97 Aug-98 Aug-99 Aug-00 Aug-01 Aug-02 Aug-03 Aug-04 Aug-05
Sample Date
Figure 4-10. pH readings.
ORP - vs Time
•Tunnel
Portal Baseline (242) —K—Shaft --a- Drift
600
-100
-200
Aug-94 Aug-95 Aug-96 Aug-97 Aug-98 Aug-99 Aug-00 Aug-01 Aug-02 Aug-03 Aug-04 Aug-05
Sample Date
Figure 4-11. ORP readings.
24
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Table 4-1. Initial Sampling Dates
7-Sep-93
20-May93
1-Oct 93
18-May-94
l-Jun-94
14-Jun-94
28-Jun-94
21-M-94
8-Aug-94
19-Aug-94
6-Sep-94
20-Sep-94
4-Oct-94
19-Oct-94
l-Nov-94
15-Nov-94
Table 4-2. Monthly Sampling Events
1994
13-Dec-94
1995
26-Jan-95
16-Feb-95
14-Mar-95
18-Apr-95
18-May-95
22-Jun-95
ll-Jul-95
8-Aug-95
15-Sep-95
12-Oct-95
16-Nov-95
20-Dec-95
1996
23-Jan-96
21-Feb-96
26-Mar-96
9-Apr-96
23-May-96
18-Jun-96
18-Jul-96
22-Aug-96
19-Sep-96
23-Oct-96
2-Dec-96
1997
21-Jan-97
18-Feb-97
5-Mar-97
15-Apr-97
28-May-97
30-Jun-97
31-M-97
27-Aug-97
30-Sep-97
16-Oct-97
ll-Nov-97
4-Dec-97
1998
27-Jan-98
18-Feb-98
24-Mar-98
23-Apr-98
20-May-98
18-Jun-98
29-M-98
25-Aug-98
28-Sep-98
21-Oct-98
24-Nov-98
30-Dec-98
1999
14-Jan-99
18-Feb-99
31-Mar-99
29-Apr-99
19-May-99
17-Jun-99
21-M-99
30-Aug-99
29-Sep-99
20-Oct-99
3-Nov-99
28-Dec-99
2000
20-Jan-OO
9-Feb-OO
3-Mar-OO
20-Apr-OO
25-May-OO
15-Jun-OO
13-Jul-OO
25
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Table 4-3. Additional Sampling Events
2000 2001
14-Sep-OO 31-Jan-Ol
7-Nov-OO 28-Mar-Ol
29-May-Ol
2-Aug-Ol
3-Oct-Ol
5-Dec-Ol
Table 4-4. Representative Lilly/Orphan Boy
Fe Zn
(mg/L) (mg/L)
Baseline (Average
1993 to August 13'8 19'4
1994)
2002
31-Jan-02
15-May-02
27-Aug-02
2003
4-Jun-03
21-Aug-03
16-Sep-03
2004 2005
27-May-04 26-May-05
21-M-04 14-Jul-05
12-0ct-04
Water Chemistry
Al
(mg/L)
7.36
Mn As
(mg/L) (mg/L)
5.46 0.08
Cd Cu SO42 R
(mg/L) (mg/L) (mg/L) P
0.24 0.33 213 3.4
Tunnel - May 2002 9.7 O.01 O.02 1.51 0.04 O.005 O.002 21.0 6.6
Portal - May 2002 28.4 12.5 0.51 5.44 3.66 0.064 0.041 223 5.2
26
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5. Economic Analysis
5.1 Evaluation Summary
As part of the project, an economic analysis was
conducted by MSB in 1998. The full report was
issued separately as document MWTP-128. The
following information has been summarized from
that report.
A cost comparison was conducted on the
innovative SRB technology installed at the
Lilly/Orphan Boy Mine and a baseline lime-
addition technology. It was assumed that ARD
would be treated for an indefinite amount of time
and, therefore, the life-cycle cost analysis for each
technology was completed using a 30-year period.
SRB technology had higher capital and startup
expenses but had lower net present value (NPV)
than lime treatment. These up-front costs were
diminished over the 30-year period used in
calculating the NPV. In comparison, the higher
operating expenses of lime addition resulted in a
higher overall cost than the passive SRB
technology. However, for operating periods of
less than 10 years, lime treatment approached the
total costs of SRB. Cost analysis was calculated
for two flow rates: 3 gpm and 100 gpm. Cost
results are presented in Table 5-1 and NPV results
are shown in Table 5-2.
Table 5-1. Costs for 3-gpm Systems
Small Scale System: 3 gpm
Capital/Startup Costs
Description
Materials & Supplies
Equipment
Installation
Preliminary Laboratory Analysis
Ponds
TOTAL
Description
Labor
Laboratory & Field Testing
Materials & Supplies
Maintenance/Miscellaneous
Sludge Removal
Consumables
TOTAL
Lime Addition
t
3>
$ 12,623
$ 15,378
t
3>
$ 12,687
S 40,688
Annual Operating & Maintenance Costs
Lime Addition
$ 31,200
$ 22,500
$
$ 7,500
$ 4,500
$ 917
S 66,617
$
$
$
$
$
S
$
$
$
$
$
$
S
Sulfate Reducing Bacteria
8,848
5,805
90,002
28,258
-
132,913
Sulfate Reducing Bacteria
11,760
35,275
551
2,185
-
-
49,771
Table 5-2. NPV of Costs for Lime Addition and SRB Technologies
NPV of Costs for 30- Year Period
Technology 3 gpm
Lime Addition $1,221,128
SRB $1,014,845
100 gpm
$1,826,382
$1,527,523
27
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6. Conclusions
The following conclusions were drawn based on
the data presented.
• Metals data showed that overall metal removal
was higher for aluminum, cadmium, copper,
and zinc, than for arsenic, manganese, and
iron.
• The data also indicated that higher metal
removals were obtained within the tunnel than
at the portal. This difference can be explained
by either (1) historic precipitates within the
tunnel acted as a new source of metals and re-
contaminated the water or (2) additional
sources of ARD entered the flow as it passed
through the tunnel.
• The pH of the mine water increased almost
immediately after implementation of the
technology, which was attributed to the
buffering capacity of the organic substrate.
• During spring runoff periods, pH and water
quality were lower in the portal than in the
tunnel, where pH remained near neutral. This
was likely due to oxygenated surface water
runoff penetrating through the ground above
the portal, flowing into the tunnel, and then
solubilizing historic metal precipitates or
becoming re-contaminated as it passed
through the tunnel where other ARD
infiltration was present.
• The pH of the mine water increased almost
immediately after technology implementation.
This initial increase in pH was attributed to the
buffering capacity of the organic substrate.
During the first spring runoff, the pH dropped
in samples collected at the mine portal, but the
pH remained near neutral in the tunnel. The
pH decrease at the portal was attributed to
spring runoff influencing the water quality at
the portal. Higher spring flow rates allowed
larger volumes of water to be re-contaminated
by historic precipitates within the tunnel or
contaminated from additional fractures within
the tunnel. Also, as the portal is closer to the
surface, it is possible that more highly
oxygenated surface water entered the portal
outflow stream and solubilized new metal
contaminants as it passed through the ground
and tunnel.
During all spring runoff time frames, metal
concentrations generally rose in samples
collected at the portal. However, metal
concentrations remained steady in samples
collected from within the tunnel during the
same periods. Again, larger flows of
oxygenated surface water percolating into the
tunnel and flowing into the portal area likely
caused the dissolution of historic precipitates
and increased the flow of additional ARD
sources.
At least some of the reduction of sulfate and
metals within the underground mine system
was caused by the action of SRB. This was
evident by measured decreases of sulfate,
detection of soluble sulfide within the mine
water, and other changes that are typically
associated with the action of SRB.
An increase in iron and arsenic concentrations
was observed in the portal discharge water
shortly after implementing the technology and
regularly throughout the demonstration. This
is best explained by the reduction of insoluble
ferric iron to the more soluble ferrous form.
Historic ferrihydrite within the system was
reduced to ferrous iron when the technology
induced a reducing environment within the
mine water. Arsenic adsorbed to ferrihydrite
was also released when the iron was
mobilized, increasing its concentration in the
effluent.
BOD concentrations were elevated for six
months after technology implementation and
then returned to background levels. Ammonia
concentrations were elevated for a longer
period but returned to background
concentrations after one year. This was
attributed to the limited amount of oxygen
available in the system for nitrification of
ammonia to nitrite/nitrate. Nitrite/nitrate
28
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subsequently underwent anaerobic off conditions and was inconsistent in
denitrification. achieving effluent design goals.
Although of sufficient capacity to provide • The upward trend in dissolved metals
significant water treatment much of the year, concentration indicates that the in situ
the system was undersized for high flow run- bioreactor is nearing the end of its useful life.
29
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7. Recommendations
The following recommendations were developed
after completion of the data analysis from this
field demonstration.
• Collect samples from Telegraph Creek
upstream and downstream of the portal area
and analyze for metals. This data will show
the influence of the portal discharge on the
receiving stream. In addition, the results will
indicate any metal removal occurring in the
natural wetlands located just downgradient of
the portal. If the wetlands are effectively
removing the iron and arsenic remaining in the
portal discharge after treatment by the SRB
technology, then an oxidizing, polishing step
may not be needed.
• Continue to monitor the metals removal
efficiency from the mine and within the tunnel
every few years to assess the long-term
impacts of the organic addition. Sampling
should be done during both low and high flow
rates every 2 to 3 years. Longevity of SRB-
treatment technology is not known because in
situ treatments are a relatively recent
development. The Lilly/Orphan Boy Mine
Demonstration may be the longest running in
situ hard rock mine treatment in the nation
using SRB technology. Longer-term data
would help future projects determine the
effective life for substrate and help optimize
designs.
• Collect samples from within the organic
substrate for molecular microbiological
analysis several times in the future. The
results could be compared with previous
results so that microbial community changes
overtime could be documented. These
changes would help predict microbial behavior
in other SRB projects and allow optimization
of this technology.
• Future projects that utilize SRB technology
should incorporate an oxidation step
downstream of the reduction process. 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.
• In this study, only the effluent analysis was
known. It is recommended that future in situ
field technology demonstrations commit the
necessary funding to allow for measurement
of mine water flows through the treatment
system and design, as best as possible, a
method to collect feed samples throughout the
demonstration. These are needed in order to
fully evaluate the treatment efficiency of any
system.
The implementation of an SRB technology at the
Lilly/Orphan Boy Mine provided an innovative
solution.
30
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8. References
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Montana. A thesis submitted to the Department of
Geology in partial Fulfillment of the Requirements
for the Degree of Bachelor of Science in
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Barghoorn, E. S. and R. L. Nichols, "Sulfate
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Bechard, G., S. Rajan, W. D. Gould,
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Brodie, G. A., D. A. Hammer, and D. A.
Tomljanovich, "Treatment of Acid Drainage with
a Constructed Wetland at the Tennessee Valley
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Chelsea, Michigan, pp. 201-209, 1989.
Brodie, G.A., D. A. Hammer, and D. A.
Tomljanovich, "Constructed Wetlands for Acid
Mine Drainage Control in the Tennessee Valley,"
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Canty, M., "Overview of the Sulfate-Reducing
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Cohen, R. R. H. and M. W. Staub, Technical
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U.S. Bureau of Reclamation by the Colorado
School of Mines, Golden, Colorado, 1992.
Davison, W., C. S. Reynolds, and E. Tipping,
"Reclamation of Acid Waters Using Sewage
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274, 1989.
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.
Euler, J. H., Determination of the Effects of
Varying the Surface Area to Volume Ratio
Configuration ofBioreactors Treating Acid Mine
Drainage, Master's thesis, Colorado School of
Mines, Golden, Colorado, 1992.
Hedin, R. S., R. Hammack and D. Hyman, "The
Potential Importance of Sulfate Reduction
Processes in Wetlands Constructed to Treat Mine
Drainage" in Constructed Wetlands for
Wastewater Treatment, Lewis Publishers, Chelsea,
Michigan, 1989.
Herlihy, A.T., and Mills, A.L., Sulfate reduction in
freshwater sediments receiving acid mine
drainage. Appl. Environ. Microbiol., v. 49 pp.
179-186, 1985.
Hunter, R. M., BiocatalyzedPartial
Demineralization of Acidic Metal Sulfate
Solutions, Doctoral Thesis, Montana State
University, Bozeman, Montana, 1989.
31
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Kleikemper, J., M.H. Schroth, W.V. Sigler, M.
Schmucki,.S.M.Bernasconi, and J. Zeyer, Activity
and Diversity of Sulfate-Reducing Bacteria in a
Petroleum Hydrocarbon-Contaminated Aquifer,
Applied And Environmental Microbiology, Apr.
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Kleinmann, R. L. P., "Acid Mine Drainage in the
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Carbondale, IL, July, 18-19, pp. 1-1 - 1-8, 1990.
Kuyucak, N. and P. St-Germain, In Situ Bacterial
Treatment of AMD in Open Pits, Phase II -
Detailed Laboratory Evaluation of Nutrients,
Start-up Conditions, and Scale-up Parameters,
Noranda Technology Centre Research Report,
Pointe-Claire, Quebec, 1993.
Kuyucak, N., D. Lyew, P. St-Germain and K.G.
Wheeland, "In Situ Bacterial Treatment of AMD
in Open Pits," Proceedings of 2nd International
Conference on the Abatement of Acidic Drainage,
Sept. 91, Montreal, Vol. 1, pp. 336-353, 1991.
Lee, J. M., Biochemical Engineering, Prentice-
Hall, Inc., Englewood Cliffs, New Jersey, 1992.
Lovley, D.R. and E.J.P. Phillips, Novel Processes
for Anaerobic Sulfate Production from Elemental
Sulfur by Sulfate-Reducing Bacteria, Appl
Environ Microbiol., 60(7): 2394-2399, July 1994.
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of Microbial Sulfate Reduction'" Journal of the
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Identification and Technology Prioritization
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Water and Acid Generation, Montana College of
Mineral Science and Technology, Butte, Montana,
1992.
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Mine Waste Technology Program, MWTP-128,
December 1998.
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Situ Source Control of Acid Generation Using
Sulfate-Reducing Bacteria, Mine Waste
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Applications of Bacterial Leaching, pp. 321-344,
New York: Academic Press (1978).
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Properties, Elliston Mining District, Powell
County, Montana, July 1950.
Saleh, A.M., R. Macpherson, and J.D.A. Miller,
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32
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33
-------
Appendix A
Summary of Quality Assurance Activities
-------
Summary of Quality Assurance Activities
Mine Waste Technology Program
Activity III, Project 3, Phase 2
(Field Testing of Sulfate Reducing Bacteria
at the Lilly Orphan Boy Mine)
BACKGROUND
On September 6, 1994, sampling officially began for Mine Waste Technology Program (MWTP) Activity
III, Project 3, Phase 2 — Field-Testing of Sulfate Reducing Bacteria (SRB) at the Lilly/Orphan Boy
Mine. The objective of the project was to investigate the effectiveness of using SRBs to treat the acid
mine drainage at a remote mine site and obtain a high quality effluent.
It should be noted that some of the site characterization samples discussed in this report were taken prior
to September 6, 1994, and other samples were taken during sampling events that are not outlined in the
project specific quality assurance project plan (QAPP); however, all of the field and laboratory data for
sampling events from 8/1994 to 9/2003 has been evaluated to determine the usability of the data.
In order to determine the effectiveness of the SRB process being demonstrated, several sampling points
were designated, and a variety of analyses were assigned to each point. Just as sampling activities were
initiated, however, several of the sampling points were no longer viable due to pipes breaking because of
freezing and duplication of results from several different sampling locations. Sampling continued at the
three remaining viable points (PT3, PT6, and MW). Several analyses were performed on the collected
samples either in the field at the Lilly/Orphan Boy Mine, the MSB Laboratory, HKM Laboratory, or at
the State Laboratory, which performed biochemical oxygen demand (BOD) analysis until the MSB
Laboratory acquired the capabilities to perform the analysis. In September 2003, two monitoring wells
were installed to monitor samples of the water in the shaft and water in the drift. The influent sampling
location was destroyed when the technology was deployed.
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 for this project were:
- pH;
- temperature;
- flow rate;
- sulfate;
- total suspended solids (TSS);
- nitrate-nitrite as nitrogen;
- total ammonia as nitrogen;
- solid sulfide;
- BOD;
- soluble sulfide; and
- dissolved metals (Al, As, Cd, Cu, Fe, Mn, Zn).
A-l
-------
Noncritical analyses for this project were:
- EH;
- dissolved oxygen (DO);
- alkalinity;
- hydrogenase activity;
- chemical oxygen demand (COD);
- total nitrogen;
- total phosphorous;
- total organic carbon (TOC); and
- total recoverable metals (Al, As, Cd, Cu, Fe, Mn, Zn, Pb and Hg).
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. The QC objectives are method detection limits
(MDLs), accuracy, precision, and completeness. Control limits for each of these objectives are
established for each critical analysis. 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.
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 validation
consists of two separate evaluations: an analytical evaluation and a program evaluation. An analytical
evaluation is performed to determine that:
• all analyses were performed within specified holding times;
• calibration procedures were followed correctly by field and laboratory personnel;
• laboratory analytical blanks contain no significant contamination;
• all necessary independent check standards were prepared and analyzed at the proper frequency
and all remained within control limits;
• duplicate sample analysis was performed at the proper frequency and all relative percent
differences (RPDs) were within specified control limits;
• matrix spike sample analysis was performed at the proper frequency and all spike recoveries
(%R) were within specified control limits; and
• 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. All
QC outliers for the sampling events covered by this report are summarized in Table A-l. In addition to
the analytical evaluation, a program evaluation was performed.
Program evaluations include an examination of data generated during the project to determine that:
• all information contained in chain-of-custodies (COCs) is consistent with the sample information
in field logs, laboratory raw data, and laboratory reports;
A-2
-------
• all samples, including field QC samples were collected, sent to the appropriate laboratory for
analysis, and were analyzed and reported by the laboratory for the appropriate analyses;
• all field blanks contain no significant contamination; and
• all field duplicate samples demonstrate precision of field as well as laboratory procedures by
remaining within control limits established for RPDs.
Program data that is inconsistent or incomplete and does not meet the QC objectives outlined in the
QAPP are viewed as program outliers and are flagged appropriately to indicate the usability of the data.
Both the analytical and program evaluations consisted of evaluating the data generated in the field as well
as in the laboratory.
ANALYTICAL EVALUATION
Several analytical evaluations of field and laboratory data were performed over the life of this multi-year
project.
Field Logbook Evaluation
Field data validation included an examination of the field logbook that was created for this project. The
field logbook typically contains all of the information that is available regarding:
- sampling information/conditions; and
- sample treatment/preservation.
Sampling Information/Conditions
Sampling conditions and information such as weather conditions, date of sampling, and time of sampling
should be specified in the field logbook for each sampling event. Information should also be provided to
specifically identify why a sample could not be collected. An examination of the logbook for this project
found that on several occasions information was lacking in some of these areas, particularly with respect
to weather information and the time of day the samples were collected. Sampling personnel should also
document any additional information about unique conditions that could impact the project data.
Information should be complete for each sampling event even if some information must be repeated from
previous sampling events. Providing excessive information is better than providing too little information.
Sample Preservation/Treatment
All of the preservatives required for each analysis are clearly listed in the field logbook; therefore, it was
assumed that all of the samples were properly treated/preserved prior to delivery to the appropriate
laboratory.
Field Data Validation
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. In addition, the QC parameters of precision and accuracy calculated in the
field were compared to those specified in the QAPP. Any data that falls outside of the control limits must
be considered outlier and flagged appropriately. The analyses performed in the field were:
A-3
-------
pH (critical);
temperature (critical);
flow rate (critical);
EH (noncritical); and
DO (noncritical).
pH
The pH meter was to be calibrated using two known buffer solutions that would bracket the measured pH.
To determine the accuracy of the pH meter, a third known buffer in the calibration range was to be
measured twice. Accuracy was defined as the absolute difference between the accepted value of the third
known buffer solution and the measured value of the third known buffer solution. Precision was defined
in the QAPP as the absolute difference between the two measured values of the third known buffer
solution. The QC control limits established for pH measurements for both precision and accuracy were
O.lpHunit.
For each sampling event, calibration of the pH meter was performed correctly. Although sampling
personnel either did not calculate the QC control limits or calculated them incorrectly for pH analysis,
enough information was available to determine that the pH measurements were within control limits, with
one exception. For the first sampling event, no duplicate measurement was taken making it impossible to
determine the precision of the measurements; therefore, pH data from this event should be flagged "J" as
estimated.
Temperature
The pH meter was also used to determine temperature using the thermistor contained in the pH probe.
The thermistor was calibrated against a thermocouple, which was standardized by a National Bureau of
Standards (NBS) thermometer. Because of the cost of replacement and the increased likelihood of
damage to the NBS thermometer in the field, calibration was performed in MSB's uptown office, prior to
departing for the Lilly/Orphan Boy Mine. The thermocouple was standardized against the NBS
thermometer at room temperature or roughly 20 °C. The typical temperature measurements in the field
varied greatly, but were generally much lower than 20 °C. A suggestion was made to sampling personnel
that the thermocouple be standardized against the NBS thermometer at a temperature closer to the
temperature that will be measured in the field.
For all sampling events, however, calibration procedures were in compliance with the QAPP, and enough
information was provided to determine that all temperature data was within the control limits of 1.0 °C
for precision and accuracy as specified in the QAPP, with two exceptions. For the first sampling event
(Day 1—09/06/94) no temperature calibration information was provided; therefore, the temperature data
generated for the first sampling event should be flagged "R" as unusable. For the second sampling event
(Day 8—09/13/94), the precision of the measurements could not be calculated because no duplicate
measurement was recorded; therefore, the temperature data generated on 09/13/94 should be flagged "J"
as estimated.
Flow Rate
Flow rate was measured by diverting the flow from a weir to a 1-liter or 500-mL graduated cylinder and
noting the amount of time it took to reach a certain volume. Flow rates were then calculated by dividing
A-4
-------
the volume collected by the amount of time required to reach that volume. The measurement was
duplicated to determine the precision of the measurement. The QC control limit established for precision
was a RPD between duplicate measurements.
All flow rate data is considered useable with the exception of the first sampling event (Day 1—09/06/94).
A duplicate measurement of flow rate was not taken; therefore there is no information about the precision
or accuracy of the measurements. Flow rate data from 09/06/94 should be flagged "J" or estimated.
Several flow rate measurements throughout the project could not be taken due to the freezing of the water
at the mine during the winter months.
EH
Because EH was not identified as a critical parameter in the QAPP, there were no specific QC objectives
assigned to this analysis; however, the data generated was still examined to determine if the instrument
was properly calibrated. The calibration information for EH was documented in the field logbook for each
sampling event, with the exception of the first sampling event (Day 1-09/06/94). Because no calibration
information was provided, the EH data should be flagged "J" or estimated for Day 1.
Dissolved Oxygen
Because DO was not identified as a critical parameter in the QAPP, there were no specific QC objectives
assigned to this analysis; however, the DO data that was generated was still examined to determine if the
instrument had been properly calibrated. The calibration information was lacking for the majority of the
sampling events for DO analysis. Generally, the DO meter is calibrated with sodium sulfite solution,
which has a DO of 0%, and then calibrated in air to achieve a DO of 100%; adjustments are then made to
account for barometric pressure and salinity. All of this information should have been provided in the
logbook. During the Day 52 sampling event, the DO measurement was not performed because of a dead
battery; however, the battery probably should have been replaced by this time because on Day 37 and Day
45, sampling personnel noted that the DO meter had a low battery. Because of the lack of calibration
information provided, all DO data should be flagged "J" or estimated.
Problems encountered with field measurements were caught early in the project. New data sheets were
generated to facilitate collection of all data as well as documentation of calibration. Field data from
1995-2003 had very few problems.
Laboratory Data Validation
Laboratory data validation was performed to determine the usability of the data that was generated at the
laboratories analyzing samples for the project. The bulk of the analyses were performed at MSB
Laboratory. BOD analysis was performed at the State Laboratory in Helena during the first 8 sampling
events until the MSB Laboratory was capable of performing the analysis.
The analyses performed in the laboratory were:
- sulfate (critical);
- TSS (critical);
- nitrate-nitrite as nitrogen (critical);
- total ammonia as nitrogen (critical);
A-5
-------
- solid sulfide (critical);
- BOD (critical);
- soluble sulfide (critical);
- alkalinity (noncritical);
- hydrogenase activity (noncritical);
- COD (noncritical);
- total nitrogen (noncritical);
- total phosphorous (noncritical);
- TOC (noncritical); and
- dissolved metals (critical) and total recoverable metals (noncritical).
Laboratory data validation was performed using Laboratory Data Validation: Functional Guidelines for
Evaluating Inorganics Analyses (USEPA, 1988) as a guide where applicable to each individual analysis.
For critical analyses, the QC criteria outlined in the QAPP was also used to identify outlier data and to
determine the usability of the data for each analysis. When data validation was initiated, the MSB
Laboratory was not sending sufficient information to perform a complete and thorough data validation on
the non-metal, wet chemistry analyses. The QA/QC summaries that were submitted with the reports were
lacking information about calibration blanks, and the raw data was not submitted, making sample result
verification impossible. An informational request was made to the laboratory, and laboratory personnel
quickly responded by submitting all of the requested information. Once the information was received,
data validation of all wet chemistry analyses was completed. Flagged data is summarized in Table A-l.
Metals Analysis
Dissolved metals analysis was classified as critical in the QAPP, while total metals analysis was classified
as noncritical; however, all metals analyses were evaluated using the QC criteria specified in the QAPP
for dissolved metals and the Functional Guidelines for Evaluating Inorganics Analyses (USEPA, 1988).
All metals results are considered usable; however, some samples had to be flagged "J" as estimated for
certain analytes. Refer to Table A-l for a summary of metals data requiring qualification. The amount of
qualified data has decreased as the HKM Laboratory became more proficient with the matrix of samples
from this project, which was complicated by the addition of organic matter. A sample preparation
modification to add hydrogen peroxide to the samples removed the previous interferences caused by the
organic matter. For years 1999 through 2003, no metals data, with the exception of a field duplicate in
August 2003, required qualification. It should also be noted that, as the project progressed, less samples
were collected.
Once the analytical portion of the evaluation was completed, the program evaluation was initiated.
PROGRAM EVALUATION
The program evaluation focused on:
• COC procedures;
• sampling and data completeness;
• field blanks; and
• field duplicates.
A-6
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Chain-of-Custody Procedures
All information provided in the COC forms for this project was complete and accurate with one notable
exception. On the COC from the 09/20/94 sampling event, the field QC sample numbers are transposed.
The field blank whose sample ID was 9 was listed with the laboratory ID of W183; however, the sample
bottle was labeled W184. As a result, the data for the field blank was reported under the field ID of the
field duplicate and vice versa. The samples were checked at the laboratory, and this scenario was
verified; therefore, all data was considered usable. A minor finding was that for two sampling events,
10/04/94 and 11/15/94, laboratory personnel signed the COCs as received by the laboratory in the wrong
location.
Sampling and Data Completeness
All samples that were scheduled to be collected, were collected when possible. Flow rate measurements
could not be taken on several occasions due to pipes freezing at the mine during winter months. A DO
measurement was not taken on 10/25/94 because the meter had a dead battery. A solid sulfide sample
was not collected during the 11/15/94 sampling event as scheduled due to breaking cables that ran from
the surface to the sampling containers immersed in the substrate in the shaft. All samples collected were
analyzed and reported for the appropriate parameters in the field or at the laboratory.
Field QC Samples
When data validation was initiated, it became apparent that the number of field QC samples being
collected was not sufficient when compared with the field QC sampling scheme outlined in the QAPP.
Corrective action was implemented so that additional field QC samples would be taken during the
remaining sampling events and the number of field blanks and duplicates would be in compliance with
the QAPP. Field blanks and duplicates requiring qualification are summarized in Table A-l with other
flagged data.
Project Reviews
Several project reviews were performed as noted below.
• EPA Field Technical Systems Review—November/December 1994;
• MSB Technical Systems Review at Lilly/Orphan Boy Mine—November 1995;
• MSB Technical Systems Review at Lilly/Orphan Boy Mine—August 2001; and
• MSB Technical Systems Review at Lilly/Orphan Boy Mine—August 2002.
EPA Field Technical Systems Review
From November 29, 1994 to December 1, 1994, EPA conducted a technical systems review (TSR) of
field activities for several MWTP projects including Activity III, Project 3. Six concerns listed below
were identified during the TSR.
• Field personnel did not calculate QC results;
• The QAPP might need updating;
• There were no BOD QC results;
A-7
-------
• The mine drainage was analyzed for lead and mercury, but those parameters were not listed in the
QAPP;
• The arsenic objective as stated in the QAPP might not be achievable; and
• Corrective actions needed to be documented.
All of the concerns identified were addressed after the TSR.
MSE Technical Systems Review at Lilly/Orphan Boy Mine - November 1995
An audit of field procedures was performed at the Lilly Orphan Boy Mine in November of 1995. The
purpose of the audit was to ensure all corrective actions from the previous year's external audit had been
performed and that sampling procedures outlined in the project specific QAPP were being implemented.
There were no findings identified during the audit.
MSE Technical Systems Review at Lilly/Orphan Boy Mine - August 2001
An audit of field procedures was performed at the Lilly Orphan Boy Mine on August 2, 2001. The audit
focused on procedures outlined in the project-specific QAPP. Six findings were identified:
• The QAPP was deficient because it did not adequately describe sampling locations.
• Sample container was not cleaned in between sample locations. Also, a sample container cap was
dropped on the ground and used in this condition rather than replacing or cleaning the cap.
• The meter used to obtain temperature data was not calibrated as outlined in the QAPP.
• Minimum entries required by the MSE SOP for logbooks were not made.
• The flow rate measurement used different equipment than was outlined in the QAPP.
• Groundwater samples were obtained using procedures that may have contaminated the samples
(e.g., the retrieving string and bailer were placed on the ground).
MSE Technical Systems Review at Lilly/Orphan Boy Mine - August 2002
An audit of the sampling events at the Lilly Orphan Boy mine was conducted on 8/27/02. The purpose of
the audit was to ensure that specified corrective actions resulting from audit findings from a previous
audit conducted in August 2001, had been implemented. The audit focus was on the following areas.
• Adequacy of operator training;
• Presence of adequate sample containers;
• Zero head space in sample containers;
• Flow measurement accuracy; and
• Logbook maintenance.
Sampling personnel were John Trudnowski and Travis Hendrickson. Sampling began at 9:30 am.
Samples were obtained at three locations: PT6, MW, and PT3. The sampling went smoothly.
Decontamination procedures were observed and the sampling personnel appeared to be well trained and
experienced. Zero headspace was maintained in the samples. The pH of the samples was taken in order
to ensure the adequacy of the preservative. Calibration of field equipment fell within specified limits.
Flow measurements at PT3 were not taken due to deterioration of the weir. Sampling was concluded at
1:00 pm.
A-8
-------
The only finding was that the logbook and entry data specified in SOP G4 of the QAPP was not followed.
Sampling personnel pointed out that to take the master log in the field was not practical, particularly
during the winter when snowmobiles must be used get to the site. Therefore, a smaller ring-bound
logbook was used. The data was transferred to the master log when sampling personnel returned from the
field.
It was recommended that the QAPP be amended by removing SOP G4 and inserting the current logbook
practices in Section 4, Site Selection and Sampling Procedures.
A-9
-------
Table A-l. Summary Qualified Data for MWTP Activity III, Project 3, Phase 2.
Date1
All events
from 8/94
through
12/94
08/19/94
08/19/94
08/30/94
09/06/94
09/06/94
09/06/94
09/06/94
09/06/94
09/13/94
09/13/94
09/20/94
09/13/94
09/20/94
09/20/94
Sample ID or
Batch #2
PT3
PT3
C1567
PT3
PT3
PT3,6
Ptl,3& 6
PT3,6
C1585
PT3,6
C1625
C1625
C1625
C1625
C1670
Analysis
DO
NO3/NO2
TR Mets-Pb
NO3/NO2
Flow Rate
EH
pH
Temp
Diss-As
Temp
TR-As
Fe
Hg
TR-A1
TR Mets (Al,
As, Cu, Fe, Pb,
Hg)
QC Criteria
Calibration
Accuracy
Accuracy
Accuracy
Precision
Calibration
Precision
Calibration
Accuracy
Precision
Precision
Accuracy
Field
Duplicate
Control
Limit
Sodium Sulfite,
Air
75-125 %R
75-125 %R
75-125 %R
<5%RPD
Zoebell's solution
<0.1pHunit
NBS&
Thermocouple
75-125 %R
<1.0 °C
<20%RPD
75-125 %R
<20% RPD
Result
No calibration
documented
54%
132.2
48%
No duplicate
measurement taken
No calibration
documented
No duplicate
measurement taken
No calibration
documented
72.1
No duplicate
measurement taken
37.6%
25.7%
32.7%
39.2
Significantly higher
(3 1.3% to 93. 3%)
Flag3
J
UJ
J
UJ
J
J
J
R
J
J
J
J
J
Comment
Flag all DO measurements
"J".
Flag the associated sample
"UJ".
Flag all detectable samples
"J".
Flag the associated sample
"UJ"
Flag the associated sample
"J".
Flag the associated sample
"J".
Flag all associated samples
"J".
Flag all associated samples
"R".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag the associated samples
"J".
-------
Date1
10/04/94
10/11/94
10/18/94
10/25/94
11/01/94
11/08/94
11/08/94
11/22/94
12/20/94
2/7/95
8/8/95
12/20/95
3/26/96
3/26/96
4/9/96
5/23/96
Sample ID or
Batch #2
PT3
PT3
PT3
C1678
PT3
PT3
C1740
C1886
C2127
C2245
C2251
C2252
C2301
Analysis
NO3/NO2
NO3/NO2
TR-Cu
NO3/NO2
NO3/NO2
TR-Fe
Mn
TR-Cd
TR-A1
Diss-CD
TR-A1
As
Diss-Cd
TR-A1
As
Cu
Pb
Zn
QC Criteria
Accuracy
Accuracy
Precision
Accuracy
Accuracy
Serial
Dilution
Serial
Dilution
Matrix Spike
Precision
Field
Duplicate
Precision
Field
Duplicate
Control
Limit
75-125 %R
75-125 %R
<20% RPD
75-125 %R
75-125 %R
10% difference
10% difference
75-125% recovery
10% difference
>10% difference
5.53 ppb
41.8%
37.4%
5. 18 ppb
71.3%
60.3%
63.4%
81.4%
58.2%
Flag3
UJ
UJ
J
J
UJ
J
J
J
J
J
J
J
Comment
Flag all associated samples
"UJ"
Flag the associated samples
"UJ".
Flag all associated samples
"J".
Flag the associated sample
"J".
Flag the associated sample
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
-------
to
Date1
6/18/96
6/18/96
6/18/96
7/18/96
8/22/96
9/19/96
9/19/96
10/23/96
10/23/96
Sample ID or
Batch #2
C2358
C2358
C2358
C2385
C2465
PT3
PT6
MW
PT3
PT6
MW
PT3
PT6
MW
PT3
PT6
MW
Analysis
TR-Zn
TR-A1
Cu
TR-Hg
Diss-Zn
TR-A1
As
Cd
Cu
Pb
Zn
Sulfide
Diss-Al
Pb
TR-A1
Zn
Sulfate
QC Criteria
Field Blank
Field
Duplicate
Field Blank
Field Blank
Field
Duplicate
Matrix Spike
Field
Duplicate
Field
Duplicate
Field
Duplicate
Control
Limit
-------
Date1
1/21/97
2/18/97
2/18/97
3/5/97
5/29/97
5/29/97
7/31/97
8/1/97
Sample ID or
Batch #2
C2662
C2681
PT3
PT3
PT6
MW
C2787
PT3
PT6
MW
C2900
PT3
Analysis
TR-A1
As
Cd
Cu
Fe
Pb
Hg
Zn
TR-A1
As
Cu
Zn
BOD
Sulfide
TR-A1
Pb
Sulfide
TR-A1
As
Cu
Pb
Zn
BOD
QC Criteria
Field
Duplicate
Field
Duplicate
Test
Duration
CCV
Field
Duplicate
Field
Duplicate
Field
Duplicate
Blank
Control
Limit
<35%RPD
<35%RPD
5 days
90- 110% recovery
<35%RPD
<35%RPD
<35%RPD
Undetectable BOD
Result
95.5
71.1
67.4
74.7
29.8
68.0
89.7
64.1
57.5
40.3
43.0
28.3
6 days
117.6
113.1
117.9
41.8
54.4
77.7%
70.4
38.4
67.1
83.2
51.4
Result showed
BOD
Flag3
J
J
J
J
J
J
J
J
Comment
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
-------
Date1
10/16/97
10/16/97
12/4/97
12/4/97
5/20/98
5/20/98
6/19/98
8/26/98
8/21/03
5/24/04
Sample ID or
Batch #2
PT3
PT6
MW
PT6
PT3
PT6
MW
PT3
PT6
MW
PT3
PT6
MW
PT3
MW
PT6
PT3
PT6
MW
PT3
MW
PT6
PT6
Analysis
Sulfide
Diss-Zn
Diss-Cu
Fe
Mn
Diss-Hg
Sulfide
Diss-Al
Cu
Zn
Diss-Zn
Sulfide
Diss As
Diss/Tot Ca,
Mg, and S
QC Criteria
Calibration
Verification
Field Blank
Matrix Spike
Field Blank
Duplicate
Matrix Spike
Field Blank
Duplicate
Field
Duplicate
Analytical
Anomaly
Control
Limit
90-1 10% recovery
<20 ppb
75-125% recovery
Tot
Flag3
J
J
J
U
J
J
U
J
J
J
Comment
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all samples less than 2.2
ppb "U".
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all samples less than 363
ppb "U".
Flag all associated samples
"J".
Flag all samples "J" for
arsenic analysis
Flag all associated samples
"J".
-------
Date1
5/24/04
7/21/04
10/12/04
Sample ID or
Batch #2
PT6
PT3
MW
PT6
Shaft MW
Analysis
Nitrate/
Nitrite
Ammonia
Diss/Tot As
andS
QC Criteria
Negative
Matrix
Interference
Matrix Spike
Analytical
Anomaly
Control
Limit
No interference
75-125% recovery
DissTot
Flag3
J
J
J
Comment
Flag all associated samples
"J".
Flag all associated samples
"J".
Flag all associated samples
"J".
1 Date that the samples were collected.
2 MSB Laboratory Batch numbers are listed for the metals analyses early in the project, while Sample IDs are listed for all other analyses.
3 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.
-------
SUMMARY
While the majority of the findings of the analytical and program evaluations were minor and were
addressed, several lessons can be learned so that mistakes will not be repeated during future projects. The
following recommendations are suggested in order to improve future project and MWTP QA/QC.
Laboratory QA/QC:
• QA/QC summaries and raw data were submitted by MSB Laboratory; however, when another
laboratory will be performing project analyses, a QA/QC summary and raw data must be
requested, particularly if the analysis is considered critical.
Field QA/QC:
• Creating a standardized format for sampling logbooks may help sampling personnel better
understand exactly what information they are required to provide to remain in compliance with
the QAPP. For example, designating areas for sampling conditions, QC calculations, and
calibration information would assist sampling personnel in providing all of the required
information. Each logbook should be customized for a particular project by including the
appropriate sampling frequency, analyses, and QC requirements outlined in the project specific
QAPP.
• Assigning a unique field ID to each sample collected during the project would also facilitate in
distinguishing one sample taken at PT3 from a sample taken a week later at PT3 for reporting and
validation purposes. Perhaps the date sampled could be included in the field ID, such as, PT3-
10/04/94.
Project QA/QC:
• The objective for arsenic in the effluent was stated as <0.03 parts per million (ppm) in the QAPP.
The objective cannot be achieved when the instrument detection limit is 0.0336 ppm. Typically,
the detection limit should be at least a factor of 5 below the objective to ensure that conclusions
made about achieving the objective can be drawn with confidence. Had As been analyzed by
Furnace AA (IDL=0.001), this problem could have been avoided.
There was a great volume of data generated for this multi-year project, and while some of the data was
considered estimated for various reasons, the fact that the majority of the data was usable, with the
exception of one temperature measurement, underlines the fact that quality data has been generated for
MWTP projects. Project 3 provided a unique opportunity to apply the MWTP quality system to a long-
term project. Because the project evolved over time, the QA requirements also evolved. By the end of
the project, several QA/QC challenges were addressed.
A-16
-------
Appendix B
Statistical Analysis
-------
STATISTICAL SUPPORT FOR RESEARCH ACTIVITIES
GENERAL INFORMATION
Q A ID No.: N/A
EPA Technical Lead Person (TLP):
Title:
Support Provided by:
Contract No. 68-C-03-032
Project QA Category:
Diana Bless
N/A
Data Analysis Guidance for MWTP, Activity III,
Project 3: In Situ Source Control of Acid Generation
Using SRB
Neptune & Co.
Date Submitted:
09/28/04
REVIEW SUMMARY
Review Distribution Date
NRMRL-STD QA Manager
Telephone No.
01/04/05
Lauren Drees
569-7087
Endorsement Status
No. of Findings
No. of Observations
N/A
N/A
N/A
The data provided to EPA were analyzed by a statistician with respect to the project objectives. Since the
associated QAPP was not specific regarding data analysis procedures, one recommended approach is
provided in the attachment.
If you have any questions or need additional information, please contact the STD QA Manager.
cc: Helen Joyce
Suzzann Nordwick
Michelle Lee
B-l
-------
Data Handling
All data were used as reported regardless of the qualifier. Each metal had a total of 89 observations from
both locations (Point 3 and 6) with the exception of As which had only 88 observations from Point 6. All
data were used in the analyses even though the data from the last two sampling events for As at Point 3
and the first sampling event for Al at Point 6 appear to be outlying values. Since these three
concentrations fall outside the main body of the data (see Figures B-l and B-3), it is recommended they
be checked for assignable causes.
Data Analysis
Table B-l presents descriptive statistics for the data provided. Figures B-l through B-7 display
concentrations for each of the seven metals over time.
The results for Al, Cd, Cu, and Zn followed similar patterns. At Point 3, the pattern was cyclical with the
highest concentrations occurring in the summer months and the lowest in the winter months (see Figures
B-l, B-2, B-5 and B-7). At Point 6, the concentrations were independent of the change in season and
remained relatively constant throughout the duration of the investigation. The percentage of the 89
observations that were below the target effluent levels varied for the four metals. At Point 3, 0% of Zn,
44% of Al, 51% of Cd, and 68% of Cu observations were below their target effluent levels. At Point 6,
98% of Al, 99% of Cu, and 100% of both Cd and Zn observations were below their target effluent levels.
With the exception of the last two sampling events in 2004, As displayed a cyclical pattern at Point 3 that
was opposite from the pattern displayed by Al, Cd, Cu and Zn. The highest concentrations of As
occurred in the winter months and the lowest in the summer months (see Figure B-3). At Point 6, the
concentrations were independent of the change in season and remained relatively constant throughout the
investigation. At Point 3, 27% of the observations were below the target effluent level; this increased to
43% at Point 6.
Fe and Mn (see Figures B-4 and B-6) are similar in that their concentrations at Point 6 were not constant
like the other five metals but fluctuated throughout the investigation. There is no apparent seasonal trend
at either Point 3 or 6. None of the Fe concentrations at either location were below the target effluent
level. None of the Mn concentrations were below the target effluent level at Point 3, whereas 16% were
below the target level at Point 6.
B-2
-------
Table B-l. Descriptive Statistics (ppm) by Location
Metal
Al
Cd
As
Fe
Cu
Mn
Zn
Point
3
6
3
6
3
6
3
6
3
6
3
6
3
6
Target
1.0
1.0
0.1
0.1
0.05
0.05
1.0
1.0
0.1
0.1
2.0
2.0
4.0
4.0
Q*
12
81
6
77
24
41
0
0
38
81
0
0
0
0
UQ
77
8
83
12
65
47
89
89
51
8
89
89
89
89
Minimum
0.07
0.00
3.7
3.2
0.02
0.01
2.0
3.7
1.5
1.1
4.45
0.89
5.57
0.00
Median
1.18
0.02
96.7
4.3
1.69
0.06
21.3
21.6
33.3
2.6
5.63
3.36
15.40
0.02
Mean
2.68
0.28
106
5.1
3.48
0.26
20.7
24.2
105.7
18.9
5.71
3.15
14.86
0.09
Maximum
12.70
18.00
301
58.3
38.4
6.76
43.4
54.9
696.0
1140.0
8.14
5.67
28.80
2.98
Std. Dev.
2.90
1.91
74.7
5.8
5.90
0.76
12.6
13.0
147.4
120.8
0.61
1.03
5.01
0.35
= Number of qualified observations; UQ = the number of unqualified observations.
-------
Figure B-l. Al Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 1.0 ppm).
Al: Points
D)
C
O
c
o
O
1995
1 ' ' l1 "
1996
1997
1998
1999 2000
Date
2001
2002
2003
2004
CO
D)
C
O
c
0>
o
o
o .
Al: Points
1111 I" "
1995
1 ' ' I1 "
1996
1997
111 I1 ''
1998
1 ' 'I ' "
1999
2000
n\^
2001
^n •
2002
2003
2004
Date
Stars are qualified data.
-------
Figure B-2. Cd Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 0.1 ppm).
O
CO
Cd: Points
D)
C.
Q
o
o
•a
O
p
o
1995
1996
1997
1998
1999 2000
Date
2001
2002
2003
2004
o
o
o
CM
Cd: Points
C.
CD
O
O
p
o
1995
1996
1997
1998
1999 2000
Date
2001
2002
2003
2004
Stars are qualified data.
-------
Figure B-3. As Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent < 0.05 ppm).
Cd
D)
0)
o
c
o
O
!> s
c
g
1° o
r •
=**
•-•—»•
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Date
Stars are qualified data.
-------
Figure B-4. Fe Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 1.0 ppm).
Fe: Points
o>
E
o
O
o
o .
1995 1996 1997
1998 1999 2000 2001
Date
2002
2003
2004
Fe: Points
o>
E
ro
o
O
o
o .
1995
1 '' r "
1996
1997
1998
1999 2000
Date
2001
2002
I n
2003
2004
Stars are qualified data.
-------
Figure B-5. Cu Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 0.1 ppm).
Cu: Points
D)
E
O
^
o
CD
O
't
o
CN
O
q
o
1995
1996
1997
1998
I ........... I
1999 2000
Date
2001
2002
2003
2004
Cu: Points
O
O
CO
o
CD
O
CN
O
q
o
1995 1996 1997
1998 1999 2000
Date
2001
2002
2003
2004
Stars are qualified data.
-------
Figure B-6. Mn Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 2.0 ppm).
Cd
c
Q
o
o
c
0)
o
O
E CD -
Mn: Points
1995 1996 1997 1998 1999 2000 2001
Date
Mn: Points
2002
2003
2004
1995
1996
1997
1998
1999 2000
Date
2001
2002
2003
2004
Stars are qualified data.
-------
Figure B-7. Zn Concentrations Over Time for Points 3 and 6 (target maximum dissolved effluent 4.0 ppm).
Cd
o
c
0>
o
c
o
O
N
D)
c
o
c
0>
g
o
o
N
1O
_ CN
c
O
o .
Zn: Points
""I I I I I I
1995 1996 1997 1998 1999 2000
Date
Zn: Points
2001
2002
2003
2004
1995
1996
1997
1998
1999 2000
Date
2001
2002
2003
2004
Stars are qualified data.
-------
Table B-2. QA Data Summary of Dissolved Metals An alysis
Metal
Al
Cd
As
Fe
Cu
Mn
Zn
Point
Portal
Tunnel
Portal
Tunnel
Portal
Tunnel
Portal
Tunnel
Portal
Tunnel
Portal
Tunnel
Portal
Tunnel
Target
1.00
1.00
0.10
0.10
0.05
0.05
1.00
1.00
0.10
0.10
2.00
2.00
4.00
4.00
Q*
12
84
12
84
25
41
0
0
38
84
0
0
0
3
UQ
80
8
86
12
67
51
92
92
54
8
92
92
92
89
Minimum
0.07
0.00
3.70
3.20
0.02
0.00
1.97
3.71
1.50
1.10
4.45
0.89
5.57
0.00
Median
1.46
0.02
101.50
4.40
1.10
0.06
21.50
21.15
36.20
2.45
5.63
3.35
15.60
0.02
Mean
2.82
0.27
109.69
5.21
3.38
0.27
20.73
23.72
112.93
18.36
5.70
3.13
15.01
0.10
Maximum
12.70
18.00
301.00
58.30
38.40
6.76
43.40
54.90
686.00
1140.00
8.14
5.67
28.80
2.98
Std. Dev.
3.02
1.88
76.85
5.74
5.83
0.75
12.53
13.16
156.37
118.80
0.61
1.02
5.04
0.34
% Under Target
42%
98%
0%
0%
27%
42%
0%
0%
0%
0%
0%
16%
0%
100%
*Q = Number of qualified observations; UQ = number of unqualified observations.
B-ll
-------
Appendix C
Microbial Analysis Report
-------
Methods
DNA was extracted from the sample (both from the liquid and from the soil), from a positive control (P.
aeruginosd) and a negative control (sterile water) using the Bio-101 DNA Fast Prep Kit (QBioGene)
using a Savant 101 bead beater (Fast Prep).
Since different primers may preferentially prime different species, two Eubacterial primers were used:
1070F (5' ATG GCT GTC GTC AGC T 3') and 1392R (5' ACG GGC GGR GRG TAG 3') and using
518R (5'GTA TTA CCG CGG CTG CTG G 3') and 357F (5" CTA CGG GAG GCA GCA G 3')
(Integrated DNA Technologies). Primer reactions and DNA amplification will be 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.
PCR products were cloned using TOPO TA Cloning Kit (Invitrogen). Manufacturer's protocols were
followed using 4 ul of PCR product in the initial reaction. 40ul of the transformation mix was plated on
Luria Broth agar plates supplemented with kanamycin (LB+kan) and IPTG (isopropyl-beta-D-
thiogalactopyranoside). IPTG induces activity of beta-galactosidase, an enzyme that promotes lactose
utilization, by binding and inhibiting the lac represser and is used to induce /acZgene expression in
cloning experiments which is seen as blue versus white colonies on agar plates. Since it would be
unlikely that a contaminant would have both antibiotic resistance genes, white colonies were transferred
from the LB+kan plates to Luria Broth supplemented with ampicillin (LB+amp). Those tubes that became
turbid were used for the plasmid prep using the Wizard Plus SV Miniprep DNA Purification System
(Promega) following manufacturer protocols.
Template DNA was prepared for sequencing using the QIAprep Spin Kit (Qiagen) following
manufacturer's protocols. Samples were labeled, frozen and shipped overnight to Retrogen Inc,
(http://www.retrogen.com/) for sequencing using the Ml3 primer (5-CAC GAC GTT GTA AAA CGA C-
3'). This allows for better amplification of the PCR product for sequencing since it primes approximately
30 to 40 bases inside the template DNA, thereby removing poorly amplified DNA at the beginning of the
sequence. Sequencing data were received from Retrogen Inc. via email and were analyzed with BLAST
sequence searches utilizing (www.ncbi.nlm.nih.gov) to identify bacterial species.
Results
A total of 64 clones were sent to Retrogen Inc.for sequencing. Sixty of the 64 clones returned analyzable
sequences; four of the sequence reactions gave multiple sequences and were not analyzed using BLAST.
Possible reasons given by Retrogen Inc. for multiple sequences are heterogeneous DNA templates,
multiple priming sites for the primer, GC compression and repeated sequences in the DNA template.
Species were determined based on the following parameters:
• The sequences had to contain the TOPO vectors (the DNA sequences adjacent to the PCR
product insert).
• The deposited sequences had to have a minimum 90% match to the DNA sequences that were
analyzed through BLAST.
• Sequences that matched with less than 300 base pairs were eliminated.
C-l
-------
• Sequences that matched greater than 400 base pairs were eliminated.
All sequences analyzed with BLAST returned as soil microorganisms, there were no clinical isolates.
Sequence results:
Sulfate-reducing bacteria
Uncultured Chloroflexi bacterium isolate WB-7 16S ribosomal RNA
295/328 (89%) (Note: This one does not fit the exclusion criteria)
Uncultured Flexibacter sp. partial 16S rRNA gene, clone 150
348/351 (99%)
Sulfur reducer
Thermococcales archaeon T30a-17 partial 16S rRNA gene, clone T30a-17
310/310(100%)
Thiosulfate oxidation
Uncultured Bacteroidetes bacterium partial 16S rRNA gene, clone
299/311 (96%)
Uncultured Bacteroidetes bacterium clone BPC3_E09 16S ribosomal RNA gene, partial sequence
340/341 (99%)
Uncultured Bacteroidetes bacterium partial 16S rRNA gene, clone JG35+U2A-AG10
295/307 (96%)
Compost Isolates
Anoxybacillus toebii NS1-1 16S ribosomal RNA gene, partial sequence
327/343 (95%)
Planifilum fulgidum gene for 16S rRNA, partial sequence, strain:CO 170
348/352 (98%)
Uncultured bacterium pPD12 16S ribosomal RNA gene, partial sequence
321/339 (94%)
Proteobacteria
Uncultured beta proteobacterium clone DS087 16S ribosomal RNA gene, partial sequence
340/340 (100%)
Uncultured gamma proteobacterium clone C-CS3 16S ribosomal RNA
336/343 (97%)
C-2
-------
Uncultured gamma proteobacterium clone C-CS3 16S ribosomal RNA
350/354 (98%)
Uncultured beta proteobacterium partial 16S rRNA gene, clone NE62
348/351 (99%)
Other
Thermoactinomyces sp. LA5 16S ribosomal RNA gene, partial sequence
337/341 (98%)
Uncultured Acidobacteria bacterium clone BSR3LG05 16S ribosomal RNA gene
331/334(99%)
Uncultured bacterium NoosaAW69 16S ribosomal RNA gene, partial sequence
317/330(96%)
Uncultured bacterium clone S-Jos_62 16S ribosomal RNA gene, partial sequence
345/352 (98%)
Methylotenera mobila strain JLW8 16S ribosomal RNA gene, partial sequence
350/351 (99%)
Uncultured soil bacterium clone PAH-Bio-17 16S ribosomal RNA gene, partial sequence
302/303 (99%)
Uncultured bacterium clone CD207F01 16S ribosomal RNA gene, partial sequence
306/340 (90%)
Uncultured bacterium clone DUNssu095 (+1B) (OTU#107) 16S ribosomal RNA gene, partial sequence
350/351 (99%)
Uncultured bacterium NoosaAW69 16S ribosomal RNA gene, partial sequence
324/337 (96%)
Legionella donaldsonii gene for ribosomal RNA, small subunit
331/342(96%)
Uncultured bacterium clone KD4-59 16S ribosomal RNA gene, partial sequence
341/343 (99%)
Uncultured bacterium clone DUNssu095 (+1B) (OTU#107) 16S ribosomal RNA gene, partial sequence
349/351 (99%)
Pseudomonas spp from soil/root environments
Pseudomonas sp. NRS243 partial 16S rRNA gene, isolate NRS243
314/341 (92%)
C-3
-------
Microbiology of a wetland ecosystem constructed to remediate mine drainage from a heavy metal
mine
Frateuria sp. WJ64 16S ribosomal RNA gene, partial sequence
342/342 (100%)
Fe(III) and Mn(IV)-reducing anaerobe
Bacillus infernus TH-22 16S small subunit rRNA gene, partial sequence
325/339 (95%)
C-4
------- |