EPA/600/R-02/053
                                       June 2002
 FINAL REPORT-SULFATE-REDUCING
       BACTERIA REACTIVE WALL
             DEMONSTRATION


MINE WASTE TECHNOLOGY PROGRAM
         ACTIVITY III, PROJECT 12
                IAGNO: DW89938870-01-1
                    Project Officer

                   Mr. Roger Wilmoth
               Office of Research and Development
            National Risk Management Research Laboratory
                  Cincinnati, Ohio 45268
               MSB Technology Applications, Inc.
            Mike Mansfield Advanced Technology Center
                  200 Technology Way
                    P.O. Box 4078
                  Butte, Montana 59702

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                                           Disclaimer

This report was prepared as an account of work sponsored by an agency of the United States Government.
Neither the United States Government nor any agency thereof, nor any of their employees, makes any
warranty, express or implied, or assumes any legal liability or responsibility for the accuracy,
completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that
its use would not infringe privately owned rights.  Reference herein to any specific commercial product,
process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or
imply its endorsement, recommendation, or favoring by the United States Government or any agency
thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the
United States Government or any agency thereof.

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                                            Foreword

The U.S. Environmental Protection Agency is charged by Congress with protecting the Nation's land, air,
and water resources.  Under a mandate of national environmental laws, the Agency strives to formulate and
implement actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, EPA's research program is providing data and
technical support for solving environmental problems today and building a science knowledge base
necessary to manage our ecological resources wisely, understand how pollutants affect our health, and
prevent or reduce environmental risks in the future.

The National Risk Management Research Laboratory is the Agency's center for investigation of
technological and management approaches for preventing and reducing risks from pollution that threatens
human health and the environment. The focus of the Laboratory's research program is on methods and
their cost effectiveness for prevention and control of pollution to air,  land, water, and subsurface resources;
protection of water quality in public water systems; remediation of contaminated sites, sediments, and
ground water; prevention and control of indoor air pollution; and restoration of ecosystems. The NRMRL
collaborates with both public and private-sector partners to foster technologies that reduce the cost of
compliance and to anticipate emerging problems. NRMRL's research provides solutions to environmental
problems by developing and promoting technologies that protect and improve the environment; advancing
scientific and engineering information to support regulatory and policy decisions; and providing the
technical support and information transfer to ensure implementation of environmental regulations and
strategies at the national, state, and community levels.

This publication has been produced as part of the Laboratory's strategic long-term research plan.  It is
published and made available by EPA's Office of Research and Development to assist the user community
and to link researchers with their clients.
                                                    E. Timothy Oppelt, Director
                                                    National Risk Management Research Laboratory

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                                      Executive Summary

This document is a final report on the performance of sulfate-reducing bacteria (SRB) bioreactors that
were constructed and operated for Mine Waste Technology Program (MWTP) Activity III, Project 12,
Sulfate-Reducing Bacteria Reactive Wall Demonstration.  The MWTP is funded by the U.S.
Environmental Protection Agency (EPA) and jointly administered by the EPA and the U.S. Department of
Energy (DOE) through an Interagency Agreement (IAG) and under DOE contract number DE-AC22-
96EW96405.

Efforts reported in this document focused on the demonstration of a passive technology that could be used
for remediation of thousands of abandoned mine sites existing in the Western United States that emanate
acid mine drainage (AMD). This passive remedial technology takes advantage of the ability of SRB to
increase pH and alkalinity of the water and to immobilize dissolved metals by precipitating them as metal
sulfides or hydroxides.

The SRB technology was demonstrated by constructing three bioreactors at an abandoned mine site
(Calliope Mine) in the vicinity of Butte, Montana. The bioreactors were fed by AMD emanating from a
large waste rock pile.  The quality of this AMD and its pH are related to the amount of atmospheric water
that infiltrates into the waste rock pile and leaches metals.  With the exception of the  first 8 months of
operation, atmospheric precipitation was well below normal. Consequently, the pH of the AMD increased,
and the load of metals in the AMD significantly decreased, bringing concentrations of iron, aluminum, and
manganese in the influent AMD below the target treatment levels for the project. The bioreactors operated
from December 1998 to July 2001 when they were then decommissioned.

Two bioreactors were placed below ground (Bioreactors II and III), and one was placed above ground
(Bioreactor IV). The aboveground bioreactor was built to  evaluate the effect of cold weather and freezing
on an SRB  system. In addition, Bioreactors II and IV were built with a pretreatment section to evaluate the
effect on the efficiency of the SRB of inducing an improved pH and oxidation-reduction potential (EH).

Each bioreactor was filled with a combination of organic matter, crushed limestone, and cobbles placed in
two or four discrete chambers. The first two chambers of Bioreactors II and IV constituted the
pretreatment section and included a chamber filled with organic matter and a chamber filled with crushed
limestone.  Following the pretreatment section, there was another chamber with organic matter and a
chamber filled with cobbles. A pretreatment section was not included in Bioreactor III in order to evaluate
its contribution to overall  bioreactor efficiency by comparison to Bioreactor II.

Bioreactors II and III, 71.5 feet and 61 feet in length respectively, were constructed below ground in  14-
foot-wide trapezoidal (4-foot-wide  bottom) trenches. Bioreactor IV, 72.5 feet in length, was constructed in
a 12-foot-wide metal half-culvert elevated above ground. The chambers filled with organic matter or
limestone were each 5 feet in length, whereas the chambers filled with cobbles were 50 feet in length.

The organic matter, an electron donor and carbon source for the SRB, was provided as an 80% to 20% by
volume mixture of cow manure and cut straw.  The cut straw was added to provide secondary porosity to
the mix and to prevent settling of the medium.  TerraCell™ material, commonly used in landscaping for
slope stabilization and made of high density polyethylene,  was used to form a cellular containment system
                                               in

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(CCS)1 to house the organic matter. The CCS prevented the organic matter from settling to the bottom of
the bioreactor, thus fostering the flow of AMD through the entire cross-sectional area without channeling.
Each layer (lift) of TerraCell™ was positioned at 60 degrees off the horizontal plane so that the cells of
each lift would be partially offset with respect to the cells of adjacent lifts.  Each lift was 6 inches thick (as
measured along the horizontal direction of flow) and contained 11-inch by 8.5-inch rhombohedral-shaped
cells.

The two belowground bioreactors (II and III) were designed to flow year-round.  The aboveground
bioreactor (IV) was designed to be shut down for winter to let it freeze while full of AMD.  The reactors
flowed at a rate of 1 gallon per minute for the majority of time. This flow rate corresponded to a calculated
5-1/2 day residence time for the AMD in Bioreactors II and IV and a 4-1/2 day residence time in
Bioreactor III. The residence time of the AMD in a single organic matter chamber was approximately 10
hours.

Bioreactor performance was monitored monthly by taking pH, EH, dissolved oxygen, and temperature
measurements and collecting samples of influent and effluent for chemical analysis. The analytes included
SRB population; alkalinity; and concentrations of sulfate, sulfide,  dissolved metals, aluminum, arsenic,
cadmium, copper, iron, manganese, and  zinc.

At the end of the project, the bioreactors were decommissioned, and the site was restored to nearly original
conditions.  The decommissioning activity also included an autopsy of the solid matrix material that was
not accessible during the operational time.  Autopsy sampling included collection of solid matrix samples
for chemical analyses to determine concentrations of total metals [aluminum (Al), arsenic (As), cadmium
(Cd), calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn], sulfate,
sulfide, nitrogen, phosphorous, and total organic carbon (TOC) in the chambers of organic matter and
limestone. Bacteriological analyses were also conducted to determine SRB population in the organic
substrate and in the limestone. Because  the cobbles did not have a visually discernible film of bacteria or
chemical precipitate, no solid matrix samples were collected  from these chambers.

Aqueous samples were also collected from the previously inaccessible bottom of the crushed limestone and
cobble chambers and analyzed for total and dissolved metals.

The autopsy on the bioreactors revealed  a convoluted biochemical environment that was probably caused
by the dramatic change in the AMD chemistry after the first 10 months of operation. The material
examined during the autopsy showed the mixed  results of processes that were occurring at low pH and a
reasonably high load of metals with the subsequent reactions that were characteristic for water of neutral
pH laden with much less of the dissolved metals.

Interpretation of monthly monitoring results combined with the autopsy findings allowed for the
formulation of a number of conclusions and recommendations, the most essential of which are listed below.
        'U.S. Patent No. 6,325,923
                                                IV

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The CCS worked very well in preventing settling of the organic matter and ensuring uniform flow of
AMD throughout the entire cross section of the organic carbon with no preferential flow paths
(channeling).

Configuring the bioreactors to accommodate flow in a horizontal plane (rather than in the vertical
direction) was successful. Problems that were experienced with reductions in flow rate turned out to
be associated with the AMD distribution system that was plugged by chemical precipitates. This
hindrance, however, is common to both configurations.

It takes some time for SRB population to be established in the bioreactors. Once established and
supplied with organic matter, they maintained a population of E+4 most probable
number (MPN)/milliliter or higher in the aqueous phase at temperatures ranging from 2 • C to
16-C.

The SRB average population of 2.06E+6 MPN/cubic centimeter in the solid matrix of organic matter
was two orders of magnitude greater than the SRB population present in the aqueous phase.

The AMD in the bioreactors was notably stratified with respect to oxidation-reduction potential that
was up to 400 millivolts lower at the bottom  of the bioreactors than at the top. Because maintaining
reduced conditions is required for SRB, the bioreactors should have been more carefully isolated
from atmospheric air. A plastic liner placed  on the top of bioreactors is preferred over the straw
bails used for this project.

Only Zn, Cu, and Cd were being removed as sulfides due to  SRB activities. Changes in
concentrations of other metals (Fe, Mn, Al, As), which do not necessarily precipitate as sulfide,
seemed to be affected by SRB only in an indirect manner by responding to increased pH caused by
SRB activity.

For the Calliope site climatic and hydrochemical conditions,  the thresholds for the removal of Zn,
Cd, and Cu were approximately 500 micrograms per liter ((ig/L), 5 (ig/L, and 80 (ig/L, respectively.
These thresholds were slightly lower for Bioreactors II and IV than for Bioreactor III, which did not
include a pretreatment cell. This indicates that the removal thresholds were dependent on the
configuration of the bioreactor but were  not affected by the shutdown and freezing of a bioreactor
during winter.

Most of the metal sulfides that were formed due to the SRB activity precipitated within the organic
matter. The same seems to be true for the rest of the metals that must have formed hydroxides and
carbonate compounds. The role of the cobble chamber was limited to a collection sump for a small
mass of precipitates that escaped the organic matter chambers.  This demonstrated that there was no
need for the large cobble chamber, which could have been substituted with a smaller "trap" sump.

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  •   The abundance of TOC present (20% by weight) in the organic matter chamber at the end of the
      project demonstrated that the bioreactors would have worked equally efficiently with a much smaller
      supply of organic carbon, provided the same residence time of AMD was maintained. Since the
      organic matter mass inhibits permeability, it is prudent to reduce the ratio of organic carbon to the
      permeability enhancing component (e.g., gravel, shell, etc.) and have more permeable medium.
      Since most of the material that caused plugging was found within and adjacent to the outlets of the
      AMD distribution system, there was a need to devise a system that would allow for occasional
      breakdown and removal of that material. Such a system might involve only a few outlets rather than
      the three dozen used in this design. It may include ports extended to the ground surface that would
      facilitate blowing in combustion engine exhaust to destroy plugging material that would then be
      removed by bailing.

Overall, the project documented that SRB technology, as applied in this demonstration, is effective in
removing Zn, Cu, and Cd by precipitating them as sulfides.  Removal mechanisms for Fe, Al, Mn, and As
were overshadowed by a dramatic change of the quality of the influent AMD. The results of the project
have also allowed the formulation of an important recommendation regarding the design and construction
of SRB bioreactors.
                                               VI

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                                         Contents

                                                                                     Page

Executive Summary	  iii
Tables	  ix
Figures  	   x
Acronyms and Abbreviations	  xi
Acknowledgment	  xii

1.  INTRODUCTION  	   1
   1.1   Problem Definition	   1
   1.2   Principles of the Sulfate-Reducing Bacteria Technology and its Application	   1

2.  SITE DESCRIPTION	   2
   2.1   Location	   2
   2.2   Acid Mine Drainage Source 	   2
   2.3   Bioreactor Layout and Configuration  	   2

3.  DESIGN AND CONSTRUCTION	   6
   3.1   Design Requirements  	   6
   3.2   Construction	   6
         3.2.1   Materials Used	   6
         3.2.2   Belowground Bioreactors	   7
         3.2.3   Aboveground Bioreactor	   7
         3.2.4   Sampling Locations and Ports  	   8

4.  OPERATION	  11
   4.1   Flow Rates	  11
   4.2   Acid Mine Drainage Levels	  11
   4.3   Sampling and Performance Monitoring	  11
   4.4   Decommissioning	  12
         4.4.1   Sampling	  12

5.  OPERATION PHASE RESULTS	  19
   5.1   Field Parameters	  19
         5.1.1   pH, EH, Dissolved Oxygen, and Temperature  	  19
         5.1.2   pH, EH, and Temperature Profiles	  20

   5.2   Sulfate-Reducing Bacteria Populations 	  21
   5.3   Sulfate and Sulfide	  21
   5.4   Alkalinity	  21
   5.5   Metals  	  22

6.  AUTOPSY RESULTS  	  41
   6.1   Aqueous Samples	  41
         6.1.1   Cobble Chambers	  41

                                           vii

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                                    Contents (Cont'd)
                                                                                       Page

          6.1.2   Limestone Chambers  	   42
          6.1.3   Sulfate and Sulfide Analyses  	   42

   6.2    Bioreactor Solid Matrix Samples  	   42
          6.2.1   Metals Concentrations in the Bioreactors  	   42
          6.2.2   Sulfate and Sulfide Analyses  	   44
          6.2.3   Total Organic Carbon  	   45
          6.2.4   Plugs in the Outlets of the AMD Distribution System Manifold	   45

   6.3    Flow Pattern	   45
   6.4    Sulfate-Reducing Bacteria Population on the Solid Matrix	   46
   6.5    Toxicity Characteristic Leaching Procedure  	   46

7.  QUALITY ASSURANCE/QUALITY CONTROL  	   58
   7.1    Background 	   58
   7.2    Project Reviews  	   58
          7.2.1   Internal Field Systems Review at the Demonstration Site  	   58
          7.2.2   External Technical Systems Audit  	   59

   7.3    Data Evaluation	   59
   7.4    Validation Procedures	   59
          7.4.1   Analytical Evaluation	   59

   7.5    Program Evaluation 	   60
          7.5.1   Field QC Samples  	   60
          7.5.2   Field Blanks	   60
          7.5.3   Field Duplicates	   60

   7.6    Summary	   60
8.  RECOMMENDED DESIGN IMPROVEMENTS	   65

9.  CONCLUSIONS	   66

10. REFERENCES	   69
                                            Vlll

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                                             Tables

2-1.   Acid Mine Drainage Analytical Data and Target Concentrations for Bioreactors	    3
4-la.  Flow and Water Level Measurements	   13
4-lb.  Water Level Profile in Bioreactor II	   15
4-2.   Monitored Parameters and Analytes	15
4-3.   Analysis for the Autopsy of the Bioreactors 	   16
5-1.   Field Parameters  	   23
5-2.   EH and pH Profiles for Bioreactors II and III	   26
5-3.   SRB Populations  	   27
5-4.   Sulfate and Sulfide Concentrations	   28
5-5.   Alkalinity	   29
5-6.   Metals Concentrations 	   30
6-1.   Analytical Results of Aqueous Samples Collected During the Autopsy	   47
      6-la.   Total and Dissolved Metals  	   47
      6-lb.   Iron Speciation	   47
      6-lc.   Sulfate and Sulfide	   47
6-2.   Comparison of the Dissolved Metals from the Autopsy and Monthly Sampling	   48
6-3.   Analytical Results of the Solid Matrix Samples Collected During the Autopsy  	   48
      6-3a.   Metal Concentrations	   48
      6-3b.   Nitrogen, Phosphorus, and TOC	   49
      6-3c.   Sulfide Acid-Base Accounting   	   49
      6-3d.   Analytical Results for Plugging Material in the Manifold of Bioreactor III  	   50
6-4.   Correlation Coefficients (•) for Total Metals Concentrations in the Solid Matrix of
      Bioreactors II, III, and IV	   50
7-1.   Data Quality Indicator Objectives	   61
7-2.   Summary of QC Checks for Critical Field pH Measurements and Dissolved Metals Analysis  ..   61
7-3   Summary of Qualified Data for MWTP Activity III, Project 12  	   62
                                               IX

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                                             Figures
                                                                                             Page

2-1.   Calliope site location map  	   4
2-2.   Layout of the bioreactors	   5
3-1.   Simplified cross-sections of Bioreactors II and IV	   8
3-2.   Cellular containment system for organic substrate	   9
3-3.   AMD distribution system manifold	   9
3-4.   End enclosure and intake sump for Bioreactors II and IV  	  10
3-5.   Sampling and monitoring point locations  	  10
4-1.   Bioreactor flow  	  17
4-2.   Water level in the bioreactors	  17
4-3.   Water level profile in Bioreactor II during early operation and the plugging episode of
      May 1999  	  18
5-1.   pHtrends	  32
5-2.   EH trends  	  33
5-3.   Dissolved oxygen trends	  33
5-4.   Temperature trends   	  34
5-5.   Bioreactor II pH and EH profiles for 12/6/99	  34
5-6.   Bioreactor III pH and EH profiles for 12/6/99	  35
5-7.   SRB populations  	  35
5-8.   Sulfate concentrations  	  36
5-9.   Soluble sulfide concentrations  	  36
5-10. Total alkalinity	  37
5-11. Zinc concentrations   	  37
5-12. Copper concentrations  	  38
5-13. Cadmium concentrations 	  38
5-14. Aluminum concentrations	  39
5-15. Arsenic concentrations	  39
5-16. Iron concentrations	  40
5-17. Manganese concentrations  	  40
6-la. Total vs. dissolved metals for the cobble chamber of Bioreactor II  	  51
6-lb. Suspended metals in the cobble chamber of Bioreactor II  	  51
6-2a. Total vs. dissolved metals for the cobble chamber of Bioreactor III	  52
6-2b. Suspended metals in the cobble chamber of Bioreactor III	  52
6-3a. Total vs. dissolved metals for the cobble chamber of Bioreactor IV	  53
6-3b. Suspended metals in the cobble chamber of Bioreactor IV	  53
6-4.   Iron speciation for autopsy aqueous samples	  54
6-5.   Total metals in the limestone and cobble chambers in Bioreactor II (aqueous samples)  	  54
6-6.   Total metals concentration in the solid matrix of Bioreactor II	  55
6-7.   Total metals concentration in the solid matrix of chamber 1 in Bioreactor III	  55
6-8.   Total metals concentration in the solid matrix of Bioreactor IV	  56
6-9.   Sulfate profiles in the solid matrix of the bioreactors	  56
6-10. Sulfide profiles in the solid matrix of the bioreactors	  57
6-11. Bioreactor III inlet-plug metals concentrations 	  57

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                                Acronyms and Abbreviations
ABA
Al
AMD
As
Ca
CaCO3
CCS
Cd
CO
Cu
DM
DO
DOE
EH
EPA
Fe
Fe2+
Fe3+
Fe(OH)3
FeS2
H2S
HCO3
HOPE
IAG
ICP
Mg
mil
Mn
MCL
MPN
MSE-TA
MWTP
Orl
Or2
ORP
pH
PVC
QA
QAPP
QC
SHE
SMCL
SRB
TCLP
TOC
TM
VFA
acid-base accounting (analytical method for sulfide)
aluminum
acid mine drainage
arsenic
calcium (also used as the designation for limestone chamber in figures)
calcium carbonate
cellular containment system
cadmium
cobble chamber (used only in tables or figures)
copper
dissolved metals
dissolved oxygen
U.S. Department of Energy
oxidation-reduction potential expressed with reference to standard hydrogen electrode
U.S. Environmental Protection Agency
iron
ferrous iron
ferric iron
ferrihydrite
iron disulfide (pyrite)
hydrogen sulfide
bicarbonate
high density polyethylene
Interagency Agreement
inductively coupled argon plasma
magnesium
1/1,000 of an inch
manganese
maximum contaminant level
most probable number
MSE Technology Applications, Inc.
Mine Waste Technology Program
organic matter chamber No.  1
organic matter chamber No. 2
oxidation-reduction potential measured using silver/silver chloride reference electrode
measure of hydrogen ion activity
polyvinyl chloride
quality assurance
quality assurance project plan
quality control
standard hydrogen electrode
suggested maximum contaminant level
sulfate-reducing bacteria
Toxicity Characteristic Leaching Procedure
total organic carbon
total metals
volatile fatty acids
                                              XI

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Zn               zinc
                                            xn

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                                    Acknowledgment

Work was conducted through the DOE National Energy Technology Laboratory at the Western
Environmental Technology Office under DOE Contract Number DE-AC22-96EW96405.
                                           Xlll

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                                       1.   Introduction
This document is a final report on the performance
of sulfate-reducing bacteria (SRB) bioreactors that
were constructed and operated for Mine Waste
Technology Program (MWTP) Activity III, Project
12, Sulfate-Reducing Bacteria Reactive Wall
Demonstration. The MWTP is funded by the U.S.
Environmental Protection Agency (EPA) and
jointly administered by the EPA and the U.S.
Department of Energy (DOE) through Interagency
Agreement (IAG) Number DW89938870-01-1 and
under DOE contract number DE-AC22-
96EW96405.

1.1  Problem Definition
Acid mine drainage (AMD) emanates from many
abandoned mines in the Western United States,
causing significant environmental problems by
contaminating surface waters and groundwater
with dissolved metals and raising their acidity.
Conventional active treatment of AMD is often not
feasible due to the remoteness of the site, the lack
of power, and limited site accessibility. Thus, for
such sites, there is a need for a passive  remedial
technology to immobilize metals and increase the
pH of the AMD.

Sulfate-reducing bacteria have the ability to
increase pH and alkalinity of the water and to
immobilize dissolved metals by precipitating them
as metal sulfides.  Some metals [e.g., aluminum
(Al)] are removed as hydroxides due to the increase
inpH.

1.2  Principles of the Sulfate-Reducing
Bacteria Technology and its Application
Acid mine drainage is a typical result of mining
sulfide-rich ore bodies. Acid mine water is formed
when sulfide-bearing minerals, particularly pyrite
[iron disulfide (FeS2)], are exposed to oxygen and
water as described by the following overall reaction
(1-1).
FeS2 + 15/4 O2 + 7/2 H2O — > Fe(OH)3 + 2SO42- + 4H+  (1-1)

This reaction results in increased acidity of the
water (lowered pH), increased metal mobility, and
the formation of dissolved sulfate.

When provided with an organic carbon source,
SRB are capable of reducing the sulfate to soluble
sulfide by using sulfate  as a terminal electron
acceptor; bicarbonate ions are also produced.  The
soluble sulfide reacts with some metals in the AMD
to form insoluble metal  sulfides (Reactions 1-2 and
1-3).  The bicarbonate ions increase the pH and
alkalinity of the water.

       SO42- + 2CH2O	> H2S + 2HCCV        (1-2)

   H2S + M2+	> MS + 2H+, where M = metal    (1 -3)

The SRB technology was demonstrated in the field
by engineering the SRB favorable conditions within
three bioreactors that were fed by AMD emanating
from an abandoned mine site (Calliope Mine) in the
vicinity of Butte, Montana. The bioreactors, on
which construction was  completed in November
1998, operated from December 1998 to July 2001
(32 months).  Performance of the  bioreactors was
monitored monthly by sampling and conducting
chemical and bacteriological analyses of the
influent and effluent of the bioreactors. The
bioreactors  were decommissioned in July 2001, and
samples of solid matrix from the bioreactors were
collected and analyzed for chemical, physical, and
bacteriological parameters. This report includes a
description of the site, reactor design and
construction,  details of the 32 months of
monitoring data, data acquired during the
decommissioning of the bioreactor, and data
interpretation.

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                                    2.   Site Description
2.1  Location
This project was conducted at the Calliope mine
(Calliope/Mary Emma Mining Claims) located
(Figure 2-1) in Silver Bow County, Montana, in
NW% of SE% and SW% of NE% both of SW% of
Section 10, T3N, R7W. The majority of the site
where the bioreactors were installed is located on
the Calliope mining claim, Mineral Survey
No. 2972. The Mary Emma mining claim, Mineral
Survey No. 5478,  which is adjacent to the north of
the Calliope, was also used for some installations.

2.2  Acid Mine Drainage Source
The abandoned Calliope mine site includes a
collapsed adit discharging water into a large waste
rock pile.  The exposed volume of the waste rock
pile is estimated to be 66,000 cubic yards;
however, the approximately 50-foot-tall pile that is
visible at the present time may not constitute the
entire volume of mine waste disposed at the mine.
The bottom part of the pile has probably been
covered with fill material that was placed there
during the construction of Interstate Highway  15
(1-15) and forms a distinct morphological shelf
extending westward from the lower pond and the
toe of the present waste rock pile.

The AMD discharging from the collapsed adit is of
relatively good quality with the pH ranging from
6.5 to 7.  This AMD flows over the top of the
waste rock and accumulates in a small,
approximately 50-foot-diameter flow-through pond
(Upper Pond). Overflowing the Upper Pond, the
AMD forms a surface drainage that, flowing on the
surface of the waste rock pile, reaches another
flow-through pond (the Lower Pond, which is 35
feet in diameter).  In the Lower Pond, the AMD
mixes with low pH subsurface seepage that enters
the pond  along its banks and through its bottom.
This subsurface seepage is fed by atmospheric
precipitation that infiltrates the waste rock pile and
reappears on the surface at the toe of the pile.  This
seep is enriched in metals with a pH
ranging from 2.6 to 3.6.  Under natural conditions,
the Lower Pond overflows and drains to an
adjacent gulch.  For this project, approximately
20% of the AMD that flows through the Lower
Pond was diverted for treatment in three engineered
bioreactors that were built at the site to
demonstrate the SRB technology.

The quality of the Lower Pond water and its pH are
related to the amount of atmospheric precipitation
that infiltrates into the waste rock pile and leaches
metals. With the exception of the first 8 months of
bioreactor operation,  atmospheric precipitation was
well below normal. Consequently, the quality of
the Lower Pond water significantly improved after
the first 10 months.  Table 2-1 includes analytical
information on the water quality of the influent to
the bioreactor system.

2.3  Bioreactor Layout and Configuration
All three bioreactors (denoted II, III, and IV) were
designed and constructed in parallel downstream
from the Lower Pond (Ref 1) (Figure 2-2).  This
allowed the AMD to be piped to and treated by the
bioreactors using gravity flow.  The bioreactors,
constructed in the fall of 1998, were designed to
evaluate the SRB technology applied in slightly
different environmental conditions. Bioreactors II
and III were placed below ground, and Bioreactor
IV was placed above  ground. The belowground
bioreactors were built to minimize temperature
changes and to prevent freezing. The aboveground
bioreactor was built to evaluate the effect of cold
weather and freezing  on the system. In addition,
Bioreactors II and IV were built with a
pretreatment section to evaluate the effect on the
efficiency of the SRB to improve pH and
oxidation-reduction potential (EH). Due to budget
constraints, Bioreactor I was not constructed.

Each bioreactor was filled with a combination of
organic matter, crushed limestone, and cobbles
placed in two or four discrete chambers
(Figure 2-2).  The first two chambers of

-------
Bioreactors II and IV constituted the pretreatment
section and included a chamber filled with organic
matter and a chamber filled with crushed limestone.
Following the pretreatment section was a primary
treatment section that included a chamber with
organic matter and a chamber filled with cobbles.
A pretreatment section was not included in
Bioreactor III in order to evaluate its contribution
to overall bioreactor efficiency by comparison with
Bioreactor II.

Each media component was expected to play an
important role in the treatment train:

   -  organic matter was the nutrient (the electron
      donor)  for the SRB;
   -  for the pretreatment section, a chamber with
      organic matter was included to lower the EH
      of AMD;
                                  -  crushed limestone provided buffering
                                     capacity to increase the alkalinity of AMD in
                                     the pretreatment section; and
                                  -  cobbles placed in the last chamber of each
                                     bioreactor were to provide a stable surface
                                     for bacterial attachment.

                               Bioreactors II and III, 71.5 feet and 61 feet in
                               length respectively, were constructed below ground
                               in 14-foot-wide trapezoidal (4-foot-wide bottom)
                               trenches. Bioreactor IV, 72.5 feet in length, was
                               constructed in a 12-foot-wide metal half-culvert
                               elevated above ground. The chambers filled with
                               organic matter or limestone were each 5 feet in
                               length, whereas the chambers filled with cobbles
                               were 50 feet in length.
     Table 2-1. Acid Mine Drainage Analytical Data and Target Concentrations for Bioreactors
        Analyte
                Concentration (u,g/L)
AMD (maximum)	AMD (minimum)
                                                          Target Effluent
     *Suggested maximum contaminant level (SMCL)
     ** Maximum contaminant level (MCL)
     * ** Secondary maximum contaminant level
     NA = not applicable
Comments
Aluminum
Cadmium
Copper
Iron
Manganese
Zinc
Arsenic
Sulfate
pH
14,100
41.9
3,050
8,670
3,770
11,100
10.9
229,000
7.52
11.0
3.1
2.8
8.0
690
990
1.1
69,800
3.29
1,000
5
100
1,000
2,000
4,000
NA
NA
6 to 8
50 to 200 ng/L*
5 ng/L**
1,300 ng/L**
300 |Ig/L***
50 |Ig/L***
5,000 |Ig/L***
50 ng/L**
250,000 |Ig/L***


-------
Figure 2-1.  Calliope site location map.

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               Organic
               Matter
   Crushed Limeston
       Organic
       Matter
     Cobbles
                                                                   Pretreatment
                                                                   Section
                                                                 Lower
                                                                 Pond,
                                                                 Source
                                                                 of AMD
   -Treated
    AMD
    Discharge
    Piping
                                             Above
                                             Ground
                                             Bioreactor IV
-Below
 Ground
 Bioreactors
 II & III
Figure 2-2. Layout of the bio reactors.

-------
                               3.  Design and Construction
3.1  Design Requirements
Several functional and operational constraints were
identified before the design of the bioreactors
began. The most important constraints are listed
below with their design solutions.

•  The entire system needed to be passive. This
   condition was satisfied allowing for gravity flow
   by incorporating the site topography and flow
   control instrumentation into the design.

•  Construction of the bioreactors had to allow for
   investigation of the impact of subfreezing
   temperature on SRB activity. This requirement
   was satisfied by designing an aboveground
   bioreactor with features similar to one of the
   belowground bioreactors.

•  Construction of the bioreactors had to allow for
   control of the water level to simulate seasonal
   droughts, if deemed appropriate. This
   requirement was achieved by constructing intake
   sumps where the hydraulic head could be
   controlled through a system of valves and
   overflow piping.

•  The chambers with organic matter had to be
   designed so they fostered permeation of the
   AMD through the entire cross-sectional area
   (without channeling) and prevented settling of
   the medium. A cellular containment system1
   (CCS) was built into the organic carbon
   chambers to satisfy these requirements.

3.2  Construction
Construction activities at the Calliope began in
August 1998 and were completed in October 1998.
Excavation and grading for construction of all
bioreactors and associated piping was completed
while maintaining the existing slope grade of 2.5%.
This grade was maintained to provide for natural
runoff in the SRB construction area and, more
importantly, to avoid the influx of surface runoff
        'U.S. Patent No. 6,325,923
into the belowground bioreactors.  Figure 3-1 is a
simplified longitudinal cross section through the
bioreactors.  Bioreactor III differs from the other
two bioreactors by having no pretreatment section
(i.e., it consists only of one organic matter chamber
and a chamber with cobbles).

3.2.1  Materials Used
All bioreactors were constructed with similar
materials. Whenever possible, off-the-shelf, acid-
resistant building materials were used.  These
materials and their use are described below.

•  Schedule 40 polyvinyl chloride  (PVC) piping,
   nontreated finished lumber, 40-mil woven
   geotextile, and 40-mil PVC liner.  The PVC
   liner, sandwiched between woven geotextile,
   was used for lining the bottom and the sides of
   each bioreactor.

•  A heavy gauge, multiple section, galvanized
   steel half culvert with fabricated steel end-walls
   was assembled to form an elevated trough to
   house the aboveground bioreactor.

•  Precast reinforced concrete was used for the
   inlet and outlet walls for the belowground
   bioreactors. Precast reinforced  concrete was
   also used for the  intake sumps, and the end
   enclosures required for construction of the
   belowground bioreactors.

•  TerraCell™ material, commonly used in
   landscaping for slope stabilization, made of high
   density polyethylene (HDPE), was used to form
   a CCS to house and support the organic matter.
   Each layer (lift) of TerraCell™  was  6 inches
   high and contained 11-inch by
   8.5-inch rhombohedral-shaped cells.

•  The organic matter was provided as an 80% to
   20% by volume mix of cow manure and cut
   straw. The cut straw was added to provide
   "secondary" porosity to the mix and prevent
   settling of the medium.

-------
•  The mixture of cow manure and straw was
   installed in the CCS, which consisted of 10 lifts
   of TerraCell™ material (Figure 3-2) and would
   limit settling (if it occurred) of the organic
   matter to each individual cell. The TerraCell™
   lifts were positioned at 60 degrees off the
   horizontal plane so the cells of each lift would
   be partially offset (only partially overlapping)
   with respect to the cells of adjacent lifts.  Such a
   configuration promoted migration of AMD
   along the organic matter chamber in a wavy-
   shaped flow line  and facilitated the packing of
   each individual cell with the organic matter.
   The TerraCell™  material (lifts) was individually
   fastened to a grid of 2-inch by
   4-inch lumber positioned at the top of the
   organic matter chamber. The grid was
   supported by 6-inch by 6-inch wood beams
   positioned across the reactor above  its top.

•  The limestone chambers of Bioreactors II and
   IV contained crushed limestone that was 3 to
   5 inches in size.  This crushed limestone was
   placed directly on the last CCS lift of the first
   organic matter chamber. The front face of the
   limestone chamber also sloped 60 degrees off
   the horizontal plane to provide a support surface
   for the second organic matter chamber.

•  Cobbles (mostly  granodiorite), which were 3 to
   5 inches in diameter, filled the remaining portion
   of the bioreactors.

•  Two lifts of straw bales, sized 16 inches by
   18 inches by 48 inches and placed on top of the
   belowground bioreactors, were used to create a
   32-inch-thick layer to provide thermal
   insulation.  Only one lift of straw bales was
   used for the aboveground bioreactor.

3.2.2  Belowground Bioreactors
The belowground bioreactors, II and III, were
constructed in lined  trapezoidal cross-section
trenches. The liner system, which was the same for
the below- and aboveground bioreactors, consisted
of a 40-mil PVC liner  sandwiched between two
layers of a 40-mil woven geotextile.  The latter was
used to provide additional abrasion resistance for
the PVC liner for all subsequent construction
activities.

Steel-reinforced cement end-walls, with the
appropriate piping penetrations, were precast at the
site and installed on top and behind the liner system
for the AMD inlet and outlet, respectively.  Unique
to the construction of the belowground reactors
was the embedding of a 2-inch inlet manifold for
the AMD distribution system (Figure 3-3) into the
60-degree precast inlet end-wall. This distribution
system allows the AMD to enter the  bioreactors
and flow evenly throughout the CCS.

A precast reinforced concrete intake  sump to
control water levels within the bioreactor was
installed directly upgradient of each bioreactor
(Figure 3-4).  Water levels within the bioreactor
were controlled via the intake sump.  A hose,
connected to an overflow drain line, was installed
in all intake sumps to control (raising or lowering)
the water level in each bioreactor.  Water was
piped from these intake sumps to the bioreactor
manifold distribution systems.

Precast reinforced concrete end enclosures (i.e.,
large manholes) were installed at the  end of the
belowground bioreactors to control flow rate and to
house equipment to monitor bioreactor
performance. Effluent from the bioreactors was
piped from the end enclosures and applied to the
land surface in the gulch (see Figure  3-5).

3.2.3  Aboveground Bioreactor
The aboveground bioreactor (IV) was constructed
using multiple sections of a heavy gauge,
galvanized steel culvert with fabricated steel end-
walls.  Individual sections were joined together
using carriage bolts. The aboveground bioreactor
was lined in a manner similar to the belowground
bioreactors.

Embedding the manifold in this reactor was not
possible because the end-wall was fabricated from
steel; therefore, the inlet manifold was placed on
top of the liner system and supported with wooden
blocking material placed on the 60-degree end-wall
under the liner system.  An end enclosure was not

-------
required for this reactor as it was constructed
above ground. Other features including flow
control and monitoring equipment and the intake
sump were like those used for the belowground
bioreactors.
3.2.4  Sampling Locations and Ports
The bioreactors were equipped with a number of
sampling ports to monitor performance of each
bioreactor. The ports were either installed at the
selected locations of the piping system that was
assembled to supply the bioreactors with AMD and
to discharge the treated effluent (see Figure 3-5) or
were constituted by piezometers that were installed
within the body of the bioreactors (Figure 3-1).
       Figure 3-1.  Simplified cross-sections of Bioreactors II and IV.

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                                  BOTTOM  OF BIOREACTORS
                                           S DE   VEW
Figure 3-2.  Cellular containment system for organic substrate.
                                                                               2" ELBOW OUTLET
                                                                                  ^CEMENT END
Figure 3-3.  AMD distribution system manifold.

-------
2" DISCHARGE PIPE
                                                      GLOBE VALVE^

                                                               pH PROBE-
                                                                 2" HOSE -
                                                                              -4" OVERFLOW  PVC PIPE FOR
                                                                               ALL TREAMENT TRENCHES.
  Figure 3-4.  End enclosure and intake sump for Bioreactors H and IV.
                             Sampling and Monitoring Points Locations
 Discharge from adit
                                                                   Monitored Parameters
                Upper Pond
1 /
CQ3 	 ^^ / Waste Rock Pile
^•f ~y, -^V^ Water percolatin
//\^f~~~"\ \ through Waste Rock
/ 1 ~-i>«- — N — "
NX ^^-- Lower Pond
1 \>Y^^
\ / i \ Th
V- 	 L }^ r
/ — MK/
Surface drainage ^^^
from Upper Pond Valve box ^^
to Lower Pond
J
^^ I
Overflow from Lower Pond /
to the gulch /
/ AMD
' supply line to 	 *-
each reactor
Overflow from the
supply system \ )
to the gulch ^4^-~^
Int
Fh B - Bacteria count
g" F- Flow rate
1 Selected metals L' ™aterlevel
g A- < Sulfate P_ ^ermperature of ^^
Pile Sulfide t_ Temperature at straw-
[CSSTft-yAdd.^, „. --".^ure
IVa- Sampling point ID
; supply lines
ie reactors
Limestone Aboveground bioreactor (IV)
|V 1 IVb IVc / IVd IVe
^-^
Intake Organ
sump carbo
— CA,T,F,L,P") 	
Ilia
Below-groun
Below-groun
"^^4
/ / ^
B t A,L,T
4
F,P 	
I t
n Cobbles End enclosure
\ \ i
H B t A,L,T
F,P 	
lib He 4 Hid He
d bioreactor w/o pre-treatment section (III)
d bioreactor with pre-treatment section (II)
lib lie \ lid lie
•v B t / A,L,T
•-, *
7'p —
\ / T
\ 1 End enclosure
Housing for
ambient
temperature
measurement
/y
Aa\
Discharge
line
&
Land application
of treated AMD
	 J(
  Figure 3-5.  Sampling and monitoring point locations.
                                                    10

-------
                                        4.  Operation
4.1  Flow Rates
The bioreactors operated from December 1998 to
July 2001 (32 months). The two belowground
bioreactors (II and III) were designed to flow year-
round. The aboveground bioreactor (IV) was
designed to be shut down for winter to let it freeze
while full of AMD.  The reactors flowed at a rate
of 1 gallon per minute (gpm) for the majority of
time (Figure 4-1 and Table 4-la). For 4 months in
the summer of 2000, the flow rate was doubled to
nearly 2 gpm. Although the flow for Bioreactor IV
was shut down for the winter, a center portion of
this bioreactor did not freeze due to a small (0.05-
gpm) leak through the liner that must have been
inadvertently perforated during construction.
Although the location of the leak was not defined,
the rate of the leak was determined by measuring
the water level changes with the valve on the
influent closed.

A flow rate of 1 gpm corresponds to a 5-1/2 day
calculated residence time for the AMD in
Bioreactors II and IV and a 4-1/2 day residence
time in Bioreactor III.  The residence time of the
AMD in a single organic matter chamber was
approximately 10 hours for the flow rate of 1 gpm.

Flow through Bioreactors III and IV was
maintained as desired for most of the
demonstration.  However, the flow rate through
Bioreactor II started to decrease in May 1999 and
ceased at the beginning of June 1999.  The flow
rate was restored in July 1999 after the upgradient
cell with organic matter was chemically treated and
blown out with  air using an air compressor to
remove biofouling and associated plugging.
Similar behavior in Bioreactor II was observed
again in May 2000.  In this case, the permeability
of the upgradient chamber was restored by
sparging it with combustion engine exhaust.
Cessation of flow  in Bioreactor II (indicated in
Figure 4-1) in March 2001 was actually caused by
sediment that accumulated within the inlet valve.
The flow was restored by cleaning the valve.
4.2  Acid Mine Drainage Levels
Acid mine drainage levels (or water levels, as it is
also referred to in this document) in the bioreactors
were controlled by  setting their levels in the intake
sumps.  In general, water level was maintained to
just below the top surface of the  5-foot-thick layer
of cobbles. A diagram of water level changes in
the bioreactors is presented in Figure 4-2, where
water level elevations are plotted with reference to
the bottom of each bioreactor at  its outlet.  For
Bioreactor II, which experienced two plugging
episodes, the water level dropped close to the outlet
level, as shown in Figure 4-3, for the episode on
June 1999.  In each case, most of the water level
drop took place between the inlet sump and the first
organic matter chamber, indicating plugging within
or immediately adjacent to the AMD distribution
system. Numerical values of flow and water level
measurement for the diagrams in Figures 4-2 and
4-3 are compiled in Table 4-la and 4-lb,
respectively.

4.3  Sampling and Performance
Monitoring
Performance of the bioreactors was monitored
monthly by taking measurements manually and
collecting samples of influent and effluent for
chemical analysis.  All aqueous samples were
collected and then analyzed by the HKM Analytical
Laboratory following the quality assurance project
plan (QAPP) (Ref  2). In general, samples were
submitted to the laboratory as raw water with the
exception of samples for dissolved metals.  All
samples were preserved as required by the QAPP.

In addition, an attempt was made to monitor the
pH, temperature, flow rate, and water level of the
influent and effluent using stationary transducers or
sensors and recording the measurements using data
loggers.  The receding interval was set for
  OO               O
30 minutes during the first 8 months of operation
and for every 4 hours thereafter.  However, the
reliability of the transducer-generated
measurements was  unacceptable  due to either
                                               11

-------
deterioration of the signal because of organic and/
or chemical coating or repetitive failures of the data
loggers. Therefore, the performance reported in
this document is based on the records derived
through monthly sampling events. The list of
measured or analyzed parameters is include in
Table 4-2, which also includes references to the
sampling locations and monitoring ports shown in
Figure 3-5.

4.4  Decommissioning
The site was decommissioned beginning in July
2001 in accordance with regulatory guidelines and
requirements imposed by the Montana Department
of Environmental Quality. The majority of
components of the system were either removed
from the site or abandoned in place (if they were
located in the subsurface), and the site was restored
to its predemonstration condition.  The only
infrastructure remaining  at the site are two
subsurface inlet sumps and the subsurface piping
system to feed the inlet sumps with AMD from the
Lower Pond. This infrastructure is currently used
for investigations conducted for MWTP Project 24,
Improved SRB.

The decommissioning process also included
autopsy sampling of the interior of each reactor to
evaluate how the SRB material was used and if
undesired preferred flow paths were developed.
The autopsy sampling was neither included in the
project work plan (Ref 3) nor in the project QAPP
(Ref 2); it was conducted as a value-added
investigation to the project to substantiate
recommendations for technology improvements and
enhance lessons learned conclusions.

Autopsy sampling focused on the collection of solid
matrix material that was  inaccessible during the
operational phase of the project. The majority of
this visual inspection and sample collection took
place within 30 feet of the respective AMD intake
to the reactors (i.e., in chambers containing organic
matter and limestone). Cobble chambers were
inspected adjacent to the respective organic matter
chambers.
Consistent with the above sampling needs, the
cobble chambers for Bioreactors II and III were
abandoned in place. Organic matter and limestone
was excavated, examined, and reburied in place.
All material of aboveground Bioreactor IV was
removed from the half-culvert and examined in the
same manner as the material from Bioreactor II.
After examination and sampling, the material from
Bioreactor IV (organic matter, limestone, cobbles)
was spread on or near the existing waste rock pile.
Other materials (e.g., lumber, TerraCell™,
aboveground portions of inlet and outlet enclosures,
PVC monitoring pipes, and metal culvert) were
salvaged or disposed in a local landfill.
Underground pipes that were left in place were
plugged. The straw bales covering the bioreactors
were used for mulch associated with the final
restoration of the site.

4.4.1   Sampling
Autopsy sampling included collecting solid matrix
samples for chemical analyses to determine
concentrations of total metals [aluminum (Al),
arsenic (As), cadmium (Cd), calcium (Ca), copper
(Cu), iron (Fe), magnesium (Mg), manganese
(Mn), zinc (Zn)], sulfate, sulfide, nitrogen,
phosphorous, and total organic carbon (TOC) in
the organic matter and limestone chambers.  The
EPA Toxicity Characteristic Leaching Procedure
(TCLP), Method 1311 (EPA SW-846) was used
for samples of the organic matter retrieved from the
upstream organic matter  chamber. Bacteriological
analyses were conducted to determine the SRB
population in the organic matter and in the
limestone. Because the cobbles did not have a
visually discernible film of bacteria or chemical
precipitate, no solid matrix samples were collected
from this chamber.

Aqueous samples were collected from the bottom
of the limestone and cobble chambers and analyzed
for the same total metals  as those listed for the
samples of solid matrix retrieved from the organic
matter chamber.  The aqueous samples from the
cobble chambers were also analyzed for dissolved
metals that included Al, As, Cd, Cu, Ca, Fe, Mg,
Mn, and Zn.  The analytical work performed for
the autopsy phase is summarized in Table 4-3.
                                               12

-------
Table 4-3 indicates that the analytical work
performed for Bioreactors II and IV was identical.
Each upstream organic matter chamber (Orl) of
Reactors II and IV had 5 samples of organic matter
recovered for chemical analyses from the 10 CCS
lifts that made up each chamber.  The samples
were collected from the center portion of the lifts,
starting with lift No. 9 and then every other lift.
Two additional organic matter samples (one from
lift No.  9 and one from lift No. 2) were recovered
from each Orl and analyzed for SRB population.
A single organic matter sample was collected from
Orl and analyzed by TCLP.  Thus, samples from
eight locations were collected from each upstream
organic matter chamber.

A single organic matter sample (lift No.5) was
recovered from each downstream  organic matter
chamber (Or2) and analyzed for the identical
constituents as the samples collected from the Orl
chamber, including the SRB population count but
excluding the TCLP.
                                      Each limestone chamber was sampled for a
                                      nonaqueous sample (a precipitate coating on the
                                      crushed limestone) and an aqueous sample
                                      (stagnant water from the base of the chamber).
                                      The suite of analyses for the nonaqueous sample
                                      was the same as for the samples collected from the
                                      Or2 chamber. An aqueous sample collected from
                                      each limestone chamber was analyzed for total
                                      metals, sulfide, and sulfate only.

                                      For the cobble chambers, stagnant water that
                                      accumulated near the base of the bioreactor was
                                      stirred, and one aqueous sample for each bioreactor
                                      was collected and analyzed for total and dissolved
                                      metals, sulfide, and sulfate.

                                      Analytical work performed for Bioreactor II was
                                      identical as that for the upstream chamber of
                                      organic matter (Orl) and the cobble chamber of
                                      Bioreactors II and IV.
Table 4-la. Flow and Water Level Measurements
    Date
   Bioreactor II
Flow   Water level (ft from
(gpm)  bottom of Bioreactor)
                                         Bioreactor III
                               Bioreactor IV
Flow
 Water level (ft from
bottom of Bioreactor)
Flow
Water level (ft from bottom
      of Bioreactor)
12/11/98
12/14/98
12/22/98
1/4/99
2/3/99
2/4/99
3/3/99
4/5/99
5/5/99
5/7/99
5/20/99
6/3/99
6/25/99
6/27/99
7/6/99
7/12/99
7/14/99
7/15/99
7/19/99
7/21/99
7/30/99
8/11/99
8/20/99
8/29/99

0.974
0.822
0.998
0.827

0.996
0.99
0.611


0



0.191





1.064


4.15




4.03



1.63
1.42

3.81
3.55
3.15

2.8
2.66
6.43
5.36
4.62
4.13
3.4
2.63

0.557
0.633
1.008
0.859

1.04
0.696
0.537


0.578



0.954





0.802


4.5




4.41



3.57
4.09

4.38
4.28
4.06

3.88
3.8
5.46
5.54
5.43
5.33
5.38
3.66

0.798
0
0
0

0
0
0


0



0.861





0.487


4.58




4.87



4.62
4.66

4.53
4.48


3.7



4.49
4.31
4.1
3.92
                                               13

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Table 4-la. Flow and Water Level Measurements
                 Bioreactor II                  Bioreactor III
Bioreactor IV
Date
9/1/99
9/8/99
9/21/99
9/24/99
9/27/99
10/7/99
10/26/99
11/9/99
12/8/99
1/6/00
1/10/00
2/3/00
2/8/00
3/6/00
3/16/00
4/5/00
5/8/00
5/25/00
5/28/00
6/7/00
6/14/00
6/16/00
6/18/00
6/30/00
7/5/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
3/27/01
4/4/01
4/9/01
5/9/01
6/6/01
7/9/01
Flow Water level (ft from „ Water level (ft from „ Water level (ft from bottom
(gpm) bottom of Bioreactor) bottom of Bioreactor) of Bioreactor)

1.002



0.947

0.862
0.868
0.877

0.976

0.789

1.009
0.863


1.071





1.868
1.324
1.969
1.247
1.428
1.029
0.891
0.815
0.883
0
0
0.983
0.854
0.812
0.919
5.41
5.43
4.89
4.92
5.34

5.06
4.97
4.99

4.6

4.03
2.66
6
5.67
3.98
2.49
4.28
3.29
2.89
1.93
0.47
4.95
4.63
4.46
4.12
2.57
4.38
4.62
5.21
5.19
4.3
0.84



4.81
4.61
4.24

1.063



0.962

1.004
0.921
0.763

0.91

0.809

0.602
1.019


1.89





1.793
1.104
2.116
1.823
1.105
0.715
0.897
0.916
0.754


0.886
0.752
0.706
0.786
4.08
2.92
4.51
4.47


3.96
3.81
3.74

3.66

3.59
3.27

3.58
5.57


4.05





4.3
4.21
4.58
4.55
4.59
4.53
4.63
4.58
4.35



4.06
3.9
3.75

1.012



1.273

0.537
0
0

0

0

0
0


0





1.964
1.693
2.528
2.048
1.278
0
0
0
0
0
0
0
0
0.917
0.988
4.06
4.69
4.2
4.13


4.37
4.48
4.12

4.45

4.49
4.61

4.43
4.68


4.61
4.51
4.35



4.32
4.18
3.28
3.71
4.44
3.96
4.26
3.83
4.28



4.41
4.54
4.49
                                                    14

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             Table 4-lb. Water Level Profile in Bioreactor II
                    Point
                                            Water level - feet above bottom of Bioreactor
                12/11/1998
                                                                                 05/20/1999
influent sump
Hfd
Hgd
II hd
II bd
Hcd
Hid
II jd
II kd
II Id
II dd
5.01
4.94
4.66
4.66
4.66
4.66
4.65
4.66
4.64
4.65
4.65
4.96
2.93
2.62
2.01
1.92
1.93
1.92
1.93
1.92
1.92
1.91
Table 4-2. Monitored Parameters and Analytes
       Measurement
Planed Sample Frequency1
                                                           Sample Location2
                                                                                 Reported frequency and/or comments
Dissolved metals (Al, As, Cd,
Cu, Fe, Zn, Mn)
Alkalinity, SO4, EH, DO,
Soluble sulfide (HS)'
Volatile fatty acids (VFA)
PH
SRB count
Water temperature
Flow rate
Water level
Air temperature at straw/rock
interface
days 10, 20, 30, every
month thereafter
days 10, 20, 30, every
month thereafter
days 10, 30, every 3rd
month thereafter
every half-hour
days 10, 30; every 3rd
month thereafter
every half-hour
every half-hour
every half-hour
days 10, 20, 30; every
month thereafter
Ilia, lid, Hid, IVd
Ilia, lid, Hid, IVd
Ilia, lid, Hid, IVd
Ilia, He, Hie, I'VE
lib, Illb, IVb
Ilia, lid, Hid, IVd
He, Hie, I'VE
lid, Hid, IVd
He, IIIc, IVc
As planned
As planned
Increased to every month
Monthly; probe got coated
Increased to every month
monthly; data logger malfunctioned
monthly; data logger malfunctioned
monthly; data logger malfunctioned
Incomplete data; probes corroded
   Ambient air temperature
     every half-hour
                                                          Ambient temperature
                                                               housing
1 Samples for Bioreactor IV were not taken during the months in which freezing occurs
2 See Figure 3-5 for locations
'Addressed in Section 8, Quality Assurance/Quality Control
No records; instrument broke3
                                                         15

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Table 4-3.  Analysis for the Autopsy of the Bioreactors
Total No.
of
analyses
3
24
3
24
24
24
19
19
19
10
169
Number of
Locations
Analyzed
parameter
TCLP1
TM2
DM3
Fe Spc7
Sulfide8
Sulfate9
N13
pl4
TOC15
SRB

Sample
Orl4
I10
5io

5io
5io
5io
5io
5io
5io
210

8
IV (Aboveground)
Chamber
Ca5 Or24 CO6

211,12 jlO

211,12 jlO
211,12 jlO
211,12 jlO
I12 I"
I12 I"
I12 I"
I12 I"

3 2

1"
1"
1"
1"
1"





1
Total
1
9
1
9
9
9
7
7
7
4
63
14
Reactors
III
Chamber
Total
Orl CO
I10 1
510 1" 6
1" 1
510 1" 6
510 1" 6
510 1" 6
5io 5
5io 5
5io 5
210 2
43
8 1 9
II
Chamber
Orl Ca Or2
I10
= 10 911'12 1 10

= 10 911'12 1 10
cio 211'12 1 10
cio 211'12 1 10
J10 jl2 jlO
clO 112 110
clO 112 110
210 1 12 1 10

832
Total
CO
1
1" 9
1" 1
1" 9
1" 9
1" 9
7
7
7
4
63
1 14
1) Toxicity Characteristic Leaching Procedure, EPA Method 1311 (samples of organic matter)
2) Total metals, EPA Method 6010A/Method 3005
3) Dissolved metals, EPA Method 6010A/Method 3005
4) Orl4 and Or24 the upstream and downstream organic matter chambers, respectively (see Figure 2-3)
5) Limestone chambers (see Figure 2-3)
6) Cobble chambers (see Figure 2-3)
7) Fe Spc (Iron Speciation) (Standard Method 3500-Fe);
8) Sulfide, EPA Method 376.1
9) Sulfate, EPA Method  375.2
10) Organic matter (nonaqueous)
ll)Aqueous sample (211'12 -limestone/cobble chambers - one of two samples is aqueous)
12) Nonaqueous sample (film, slime, or precipitate coating),
     (211-12- limestone/cobble chambers-one of two samples is nonaqueous)
13) Total nitrogen
14) Phosphorous
15) Total organic  carbon
                                                             16

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                                        Bioreactors Flow
  11/1/98   1/30/99   4/30/99  7/29/99  10/27/99  1/25/00  4/24/00   7/23/00   10/21/00   1/19/01   4/19/01   7/18/01
                                                 Date

Figure 4-1. Bioreactor  flow.
                                  Water Level in Bioreactors
Bioreactor II plugging       Bioreactor II plugging        I
                                                                    March 2001
                                                                     Bioreactor II inlet valve plugging
  11/1/98   1/30/99  4/30/99   7/29/99   10/27/99   1/25/00   4/24/00   7/23/00  10/21/00  1/19/01   4/19/01   7/18/01
                                                 Date

Figure 4-2. Water level in the bioreactors.
                                                  17

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                     Water Level Profile in Bioreactor II
           During Early Operation and Plugging Episode of May 1999
                                 II bd    II cd    II id

                                  Piezometer series
                                                                         II dd
Figure 4-3. Water level profile in Bioreactor II during early operation and the plugging
episode of May 1999.
                                      18

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                               5.  Operation Phase Results
Results reported in this section include
measurements and analyses performed on the
aqueous samples.  They include parameters
measured in the field, bacteriological analyses of
the SRB population, and chemical analyses for
sulfate, sulfide, alkalinity, and selected dissolved
metals.

5.1  Field Parameters
Field measurements are compiled in Tables 5-1 and
5-2 and are presented in a graphical form in the
figures that are individually referred to in this
section of the report. Other field parameters (i.e.,
the flow rate and water levels in the bioreactors)
are presented in Section 4 together with other
information regarding operation of the bioreactors.

5.1.1  pH, EH, Dissolved Oxygen, and
Temperature
Diagrams 5-1, 5-2, 5-3, and 5-4 present pH, EH,
dissolved oxygen (DO), and temperature,
respectively, for the  influent and effluent from each
bioreactor during the 32-month operation period.
These results are also tabulated in Table 5-1.

As mentioned in Section 2.2, the quality of water in
the Lower Pond improved significantly after the
first  10 months of operation.  This is also
evidenced in the pH trends diagram (Figure 5-1)
where pH of the intake water (Lower Pond AMD)
increased from a minimum value of 3.29 in May
1999 to 7.14 in January 2000 and then stayed at
this or a slightly lower level for the duration of the
project, with the exception of the spring months.

The initial increase in effluent pH (including June
1999) can largely be attributed to alkalinity present
within the organic matter rather than to the
presence of limestone. This is indicated by an
insignificant pH difference in the effluent from
Bioreactors II and III, the latter having no
limestone pretreatment chamber.  As the SRB
became established and the effluent pH from each
bioreactor dropped to 8,  7.5, and 7 for Bioreactors
IV, II and III  respectively, the pH differential
between the influent and effluents could be
attributed to SRB activity.  The slightly lower pH
of the effluent from Bioreactors II and IV during
this period may have been due to the limestone
chambers in these bioreactors.

Values of pH of the effluent from Bioreactor III fell
below the pH values of its influent during the
period from August 2000 to April 2001. However,
the subsequent decrease of the influent pH in May
and June 2001 to a value of 5 had no effect on the
effluent pH, indicating that the bioreactor was still
capable of improving the quality of the AMD.
This decrease in the influent pH did not impact the
pH of the other two reactors.

Oxidation reduction potential (ORP) measurements
in the field were taken using an ORP electrode with
a silver/silver-chloride reference electrode. Since
the EH is  defined (Ref 4) as a voltage reading with
a reference to the standard hydrogen electrode
(SHE), these field values were converted to the EH
values by adding 200 millivolts (mV) and corrected
for temperature to report it for 25 • C.  The
corrected EH values are presented in Figure 5-2.

The EH diagram shows that with the exception of a
few time periods, the most noticeable being from
late summer 1999 through winter 1999/2000 for
Bioreactors II and IV and late  summer through fall
of 1999 for Bioreactor III, the EH values were
positive,  indicating an apparent oxidizing
environment. There was, however, a significant
difference in EH of the influent and the effluent
AMD; the former being in the  300-mV to 400-mV
range for most of the operating time. This
difference indicates that the bioreactors
significantly lowered the EH. Moreover, the
bioreactors  fostered conditions favorable for the
SRB population growth, as indicated further in this
document.  Therefore, it is postulated that the EH
measurements of aqueous samples collected at the
outlets of the bioreactors (location A in Figure 3-5)
may not reflect the EH present  in the "pockets" of
organic matter where the SRB lived. Additionally,
                                               19

-------
the EH and pH profiling of the bioreactor (see
Section 5.1.2) showed that the EH measured at the
outlet of the bioreactors was higher than that
measured for aqueous samples collected from the
organic matter chambers where most of the sulfate
reduction took place.

The DO diagram (Figure 5-3) indicates that the
oxygen level ranging from 4 milligrams per liter
(mg/L) to 14 mg/L in the influent decreased in the
bioreactors to less than 1 mg/L for the majority of
the operation time. However, the 14 mg/L peak in
the influent DO concentration in December 2000
was also reflected as a maximum DO concentration
in Bioreactor III, which had no limestone chamber
and only one organic matter chamber. Moreover,
both peak values correlate well with very high EH
values recorded for Bioreactor III at the same time
(Figure 5-2).

The temperature of the influent and effluent are
presented in Figure 5-4.  The temperatures reflect
seasonal variation and change from 0.5 • C to
15.5 -C for the  influent and 1 -Cto 16 'Cforthe
effluent from both belowground bioreactors.
Temperature of the effluent from these two
bioreactors was very similar throughout the
operating period with the exception of the first
4 months when  the effluent from Bioreactor II was
up to 2 • C higher. This difference is attributed to
the fermentation processes of the organic matter
that took place at the beginning of the operation
and were more intensive in Bioreactor II, which
contained two organic matter chambers.

The above-freezing temperatures inside the
aboveground bioreactor (IV) during winter were
caused by a small (approximately 0.05-gpm) leak
from the bioreactor that prevented the bioreactor
from freezing solid, as anticipated by the design.

The 2 • C higher temperature of the effluent from
Bioreactor IV during summer is attributed to its
exposure to higher ambient air temperature because
of its aboveground location.
5.1.2  pH, EH, and Temperature Profiles
Although profiling the bioreactors for pH and EH
values was not in the original sampling plan, it was
conducted to assist in data interpretation. The pH
and EH measurements of the AMD at all deep and
shallow piezometers for each bioreactor were taken
four times per month beginning in November 1999.
The results obtained were similar for all
measurement events (Table 5-2).  Figures 5-5 and
5-6 present the results of measurements taken in
December 1999 for Bioreactors II and III,
respectively. Measurements taken in Bioreactor IV
are not plotted because they depict nonflowing
conditions (the reactor was shut down for winter).

The pH profiles document very little change along
the flow in both bioreactors with the exception of
the first organic matter chamber where pH values
dropped by approximately 0.2 units upon the
influent entry.  This slight decrease in the pH
values was probably caused by the release of
protons when hydrogen sulfide (H2S) reacted with
dissolved metals and precipitated them as metal
sulfides as shown in Reaction 1-3.

The pH increased approximately 0.5 units in the
limestone chamber in Bioreactor II and in the
cobble chamber in Bioreactor III. Worth noting is
a small difference in the pH measurements taken in
the shallow and deep piezometers in the cobble
section. The slightly lower values of pH at shallow
depth correlates well with the higher EH
measurements taken at the same locations.

The EH values were more  differentiated than the pH
readings for both bioreactors.  The AMD flow in
the bioreactors were notably stratified with respect
to Eg, which in Bioreactor II was up to 400 mV
higher in the shallow piezometers than in the  deep
piezometers. This difference, thus also
stratification, diminished downgradient and close to
the outlet of the bioreactors due to mixing of the
water flowing through the cobble section. This
mixing process was increased by the placement of
the outlet pipe located 6 inches above the bottom of
the bioreactors.
                                               20

-------
The water mixing process seems to also be
responsible for higher values of EH at the
monitoring location at the outlet of the bioreactors
in comparison to measurements taken at the bottom
of the first organic matter chambers. This
difference was approximately 50 mV and 150 mV
for Bioreactors II and III, respectively.

5.2  Sulfate-Reducing Bacteria Populations
The first 8 months of operation can be described as
a period in which the microbial populations were
established within the bioreactors.  It should be
noted that the bioreactors were  started in the winter
when temperatures were not ideal for microbial
growth. As the bioreactor temperatures (Figure 5-
4) began to increase in April and May 1999, an
increase in SRB populations (Figure 5-7 and Table
5-3) was also seen. During the  second winter of
operation, the well-established SRB population was
not affected by the low temperatures.

The correlation of the SRB population with EH can
be seen in Figure  5-2. The EH decreased to -80 mV
for Reactor III in  September 1999 and
-200 mV for Reactor IV in October 1999. During
the same time, the SRB populations grew to a level
of 2E+5 most probable number (MPN) for Reactor
III.  There was a small decrease in the SRB
population during the winter of 1999-2000, with a
subsequent increase in the spring of 2000. This
increasing trend ended at the same time as the flow
rates were doubled, indicating that SRB might have
been flushed out at that flow velocity. The
subsequent decrease in the SRB population through
the winter of 2000/2001  is considered a delayed
effect of doubling the flow rate  in the summer of
2000. When the temperature increased in the
spring of 2001, the SRB population returned to
above the E+4 MPN level.

Based on the metals-removal results explained
further in this document (Section 5.5), the
population of SRB at the E+4 MPN level worked
well for the geochemical and climatic conditions
present at the Calliope site.  It is worth noting,
however, that this SRB population should not be
considered the optimum and/or  recommended
population for a SRB bioreactor in general. This is
because the activity of the SRB is probably more
important than the population size.  In other words,
a small population of SRB that are very active may
be more efficient than a large population of less
active cells.  Reliable methods for the direct
measurement of SRB activity are not currently
available for routine sample analysis. Methods
have been developed based on the uptake of
radioactively labeled sulfate; however, these
require specialized equipment and have an inherent
safety hazard that makes routine use difficult to
justify.  Such methods were not included in the
monitoring program for the Calliope site.

5.3  Sulfate and Sulfide
Sulfate and sulfide concentrations in the influent
and effluents of the bioreactors (Figure 5-8 and
5-9, respectively, and Table 5-4) do not give
conclusive results regarding sulfate reduction rates.
These diagrams are included in this report mainly
for documentary purposes. The main reason for
the inconclusiveness of the sulfate records could be
a high concentration of sulfates in the organic
matter built in the reactors, as indicated by often
higher concentrations of sulfate in the effluent than
in the influent.  The use of analytical results for
sulfide is limited due to the analytical procedure
selected for the project. As this procedure did not
require filtering of the sampled material, some of
the sulfide detected might have come from
suspended metal sulfides. This is especially true
for sulfide analyses of the influent AMD that show
up to 4 mg/L of sulfide, which should not exist in
dissolved form under EH and pH conditions of
influent and in the presence of dissolved Cu.
Moreover, there was a noticeable H2S odor coming
from the influent AMD.

5.4  Alkalinity
Analytical data for alkalinity, expressed as
milligrams of calcium carbonate (CaCO3), are
presented in Figure 5-10 and Table 5-5. Although
this diagram shows the total alkalinity, it is actually
a bicarbonate (HCO3~) alkalinity for most of the
operating time.  Only during the first 3 months of
operation  (2 months for Bioreactor II) did
                                               21

-------
carbonate alkalinity (CO32~) contribute to the total
alkalinity, with the hydroxide alkalinity never
detected.

The alkalinity of a typical AMD is zero, as
indicated on Figure 5-10, where, until October
1999, alkalinity of the influent AMD at the intake
location was zero. As the AMD at the site
improved (due to the change of climatic conditions)
and its pH increased to 6.5, the alkalinity of the
influent AMD increased to the range of 20 mg/L to
30 mg/L, with the exception of March 2001  when
it peaked to 189 mg/L.

The alkalinity of the treated AMD is a product of
sulfate reduction by SRB that use organic carbon
as the electron donor, as described by Reaction 1-2.
Therefore, the alkalinity buildup in the effluent
from all bioreactors at the Calliope site is a good
indication of biochemical reactions taking place in
the bioreactors.

5.5  Metals
The primary objective of this project was to assess
various configurations of the bioreactors and
monitor their ability to produce a high-quality
effluent (Ref 3). It was the goal of the field
demonstration to achieve the effluent
characteristics given in Table 2-1.

These target concentrations were set arbitrarily at
the beginning of the project when the quality of
AMD in the Lower Pond was at its worst or close
to it. The quality of the AMD improved with time
due to climatic conditions, and some of the metals
(Al, Zn, and Mn) became irrelevant because their
concentrations in the influent were already close to
or even below the target concentrations.

Analytical results for concentrations of seven
dissolved metals (Zn, Cu, Cd, Al, As, Fe, and Mn)
in the influent and effluent of the bioreactors are
compiled in Table 5-6. These monthly results are
also presented in a graphical form in the figures
that are individually referred to in this section.

Changes in Zn concentrations are presented in
Figure 5-11.  During the first 7 months of the
demonstration, Zn concentrations were rising as the
sorptive capacity of the organic matter was being
filled.  During this period, the percent of Zn
removal was as high as 99%.  Once the sorptive
capacity was filled, Zn concentration stabilized at a
threshold of approximately 500 (ig/L for Reactors
II and IV and 800 (ig/L for Reactor III. The
slightly lower percent of removal in Reactor III is
attributed to a smaller total supply of organic
carbon, as this reactor has only one chamber with
organic matter.

Concentrations of Cu are presented in Figure 5-12.
Much of the Cu removal observed during the first 7
months of operation can be attributed to
adsorption. Once sorption sites fill and the SRB
populations become established, Cu was removed
through SRB activity to a threshold level of
50 (ig/L on average for Bioreactors II and IV and,
again, a slightly higher threshold level of
approximately 80 (ig/L for Bioreactor III.

Cadmium concentrations are presented in Figure 5-
13 (in linear scale).  Similarly to Zn and Cu, the
removal of Cd observed during the first 7 months
of operation can be attributed to adsorption. Once
sorption sites fill and the SRB populations become
established, Cd was removed through SRB activity
to a threshold level ranging between 4 (ig/L to 5
(ig/L, until March 2001 when the Cd concentration
in the influent dropped to approximately 3 (ig/L for
a 2-month period. Cadmium concentration in the
effluent in the same time period decreased to less
than 1  (ig/L.

Figure  5-14, which presents the concentration of
Al, also shows a reduction of concentration in the
effluents, but only for the first 10 months of
operation. During this time, Al was removed due
to adsorption on the organic matter, similarly to
Zn, Cu, and Cd. After September 1999 when the
Al concentration in the influent decreased to
100 (ig/L or below level, there is no indication of
further removal of Al.

Figure  5-15, which presents As concentrations,
indicates that As content did not decrease in the
bioreactor effluents but rather the effluents were
                                                22

-------
enriched with As for most of the operating time.
The reason behind such behavior is two-fold.  First,
it was found by Canty (Ref 5) that the manure
obtained from a similar source but used for another
demonstration site, contained elevated (in
comparison to the AMD) levels of As. Thus, it is
prudent to assume that  some As was flushed from
the organic matter chambers down to the sampling
points.  Second, as Robins and Huang (Ref. 6)
explain, under oxidized conditions, as present for
the source of AMD, Fe3+ (ferric iron) precipitates
as ferrihydrite Fe(OH)3, which effectively adsorbs
As. However, under reducing conditions, such as
in the bioreactors, ferrous iron (Fe2+) becomes the
predominant iron species. Because Fe2+ is much
more soluble than Fe3+, it is released into solution
along with the previously adsorbed As.  Because
the analytical work for  the operation phase of the
project did not include  speciation of Fe, it is
impossible to determine which of the above-
described mechanisms was predominantly
responsible for the high concentrations of As in the
effluent.

Iron concentrations, presented in Figure 5-16,  show
that with exception of the initial 8 months, Fe
concentration in the influent was significantly lower
than that in the effluents.  During the initial
8-month period, Fe seemed to be removed by the
bioreactors due to initially high sorptive capacity of
the organic matter.
Higher dissolved Fe concentration in the effluent
than in the influent (beginning in July 1999) can be
explained by a very possible, but never measured,
difference in dissolved versus total Fe
concentrations in the influent. As stated earlier in
this document, under oxidizing conditions and
nearly neutral pH, such as those present in the
Lower Pond, the prevailing form of iron (Fe3+)
precipitates as Fe(OH)3. Because the AMD that
was piped from the Lower Pond was not filtered,
it certainly brought suspended Fe(OH)3 to the
bioreactors.  It is possible that under reduced
conditions in the bioreactors, Fe3+ in Fe(OH)3 was
reduced to Fe2+ and released to the aqueous phase
as dissolved Fe, increasing its concentration in the
effluent.

The Mn diagram (Figure 5-17) indicates that the
bioreactors were not efficient in lowering
concentrations of Mn for most of the
demonstration.  In fact, reduction of Mn
concentration took place only at the beginning of
the project and close to its end (for Bioreactor II).
At the beginning of the demonstration, Mn was
sorbed (like  other metals) to the organic matter. At
another date in late winter through early spring
2001, Mn was efficiently removed in Bioreactor
III. In this case, the efficient removal of Mn
coincides with an unexplained increase in DO
concentration (Figure 5-3) that was subsequent to
the also unexplained peak of EH in the winter of
2000/2001.
Table 5-1.  Field Parameters
     Location
                    Date
                                  pH
                                             £„ (mV)
                                                           DO (mg/L)
                                                                             Temperature (* O
Influent 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
3.87
6.01
4.51
5.70
5.62
3.29
3.64
4.19
5.14
6.08
6.68
6.58
6.87
7.14
6.81
6.61
700
426
571
466
480
726
611.4
315.4

403
309
357
294.4
247.5
302
334
6.8
8.85
10.19
11.19
8.29
6.06
3.93
6.39
probe maliiinction
8.61
8.94
9.76
9.94
10.5
8.81
8.89
1.6
1.1
0.9
0.6
0.5
4.6
9.9
15.3
14.4
9.7
7.3
4.3
1.3
0.7
1.2
4.7
                                                23

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Table 5-1. Field Parameters
Location Date
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
DH
6.07
6.83
5.80
6.24
6.48
6.83
6.92
7.08
7.40
6.94
£„ CmV)
368
363
409
366
342
310
267
267
422
probe
DO Cma/L)
8.42
8.59
7.66
7.53
7.43
7.89
8.89
9.03
13.84
11.49
Temperature (• O
5.2
7.5
15.4
13.3
15.5
10.5
6.6
4.3
1.6
1.2
malfunction
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
Bioreactor II 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
7.52
7.10
7.33
6.48
4.85
5.01
9.95
9.51
8.75
8.42
8.37
8.29
7.87
7.61
7.68
7.09
7.14
7.20
7.69
7.29
7.46
7.37
6.98
6.79
7.25
7.10
6.76
7.31
6.73
7.06
7.17
6.98
230.9
365
251
32
394
401
76
252
342
378
353
87
62.8
106.5

-76
-125
-36
-62
-35
29
42
-68
-74
78
16
151
-14
-34
133
330
probe
10.49
9.67
9.93
probe malfunction
7.1
0.51
0.63
1.09
0.93
0.11
0.04
0.95
0.46
probe malfunction
0.2
0.24
0.53
0.43
0.39
0.48
0.30
0.51
0.21
0.29
0.27
0.31
0.61
0.27
0.46
2.58
0.82
1.5
2.7
3.8
8.3
8.5
16.5
4.9
5.9
4.1
2.4
2.1
6.1
8.9
13.2
15.4
14.1
9.2
6.4
4.4
2.4
1.9
2.9
2.9
5.3
10.6
13.5
15.8
12.9
12.0
6.3
3.7
2.9
malfunction
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
Bioreactor III 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
1 1/9/99
12/8/99
1/6/00
7.21
7.74
6.83
6.61
6.76
6.76
9.12
9.32
9.34
8.43
7.78
7.56
7.62
7.54
7.03
7.27
7.02
6.75
7.21
6.98
296
-83
289
94
98
15
219
279
403
444
328
273
187.2
72.9

-82
-81
37
59
68
0.56
0.59
0.48
probe malfunction
0.47
0.05
0.18
0.44
0.76
0.17
0.04
0.08
0.15
probe malfunction
0.21
0.24
0.27
1.18
1.43
2.2
2.7
2.9
4.8
9.1
12.4
4.2
3.4
2.2
1.7
1.3
5.4
9.6
13.8
15.0
14.8
10.9
6.3
3.2
2.2
24

-------
Table 5-1. Field Parameters
Location Date
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01

2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
BioreactorlV 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01

2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
DH
6.92
7.00
6.82
6.84
6.63
6.39
6.35
6.45
6.45
6.77
6.73
6.65

6.79
6.64
6.82
6.7
6.8
6.26
9.98
9.87
9.68
9.19
8.37
8.63
8.27
7.59
8.12
7.80
7.90
7.91
8.25
7.96
8.17
7.88
7.92
8.06
7.49
7.23
6.99
7.81
7.54
7.57
7.84
7.70

8.47
7.68
7.75
7.79
7.18
6.97
£„ CmV)
259
145
76
-49
41
114
196
164
241
187
569
probe
maliunction
379.4
113
334
-48
161
34
69
339
337
346
135
17
53.6
77.3
DO Cma/L)
0.66
2.23
0.34
0.12
0.27
0.91
0.67
2.53
1.79
0.91
5.69
5.28

3.91
3.80
3.22
probe maliunction
0.43
0.15
0.40
0.24
0.46
0.06
0.28
0.09
0.15
probe maliunction
-82
-100
-191
-60
-31
95
53
-87
-33
37
37
30
-53
-31
98
289
probe
maliunction
242
-16
254
-87
39
-40
0.2
0.25
0.28
0.3
0.31
0.77
0.28
0.37
0.08
0.27
0.3
0.30
0.45
0.31
0.24
0.60
1.06

0.73
0.50
0.48
probe maliunction
0.42
Temperature (• O
1.6
3.6
4.1
6.7
11.8
14.1
15.7
12.9
10.0
5.8
2.8
2.5

2.1
2.0
3.1
4.4
6.8
13.7
1.0
0.5
0.3
0.1
0.1
2.0
10.3
16.2
17.0
13.8
7.6
5.6
2.1
0.7
0.0
1.6
3.1
5.0
14.1
14.5
17.3
12.4
7.5
5.5
0.8
1.2

0.5
0.4
0.8
2.6
8.2
18.1
25

-------
Table 5-2. EH and pH Profiles for Bioreactors
Location Piezometer
Influent
Bioreactor II II fd
II gd
II hd
II bd
II cd
Hid
II jd
II kd
II Id
II fs
II gs
II hs
libs
lies
His
II js
II ks
Ills
Bioreactor III III fd
Illbd
Illgd
Hied
Illhd
HIM
Illjd
Illfs
Hlbs
Hlgs
IIIcs
HIlis
mis
IIIjs
418.20
131.9
-100.7
-63.3
19.5
-10.3
-56.2
-56.5
-61.8
-57.8
328.5
312.1
380.4
33.9
-35.2
-76.5
-80.5
-82.3
-76.8
-13.5
41.5
81.2
108.2
87.6
50.4
34.9
116.7
78.3
62.9
55.3
41.8
23.9
II and III
EH
12/6/99 1/11/00
438.8
53.2
-135
-101.2
-91.6
-111.7
-110.5
-108
-99.9
-87.4
331.7
284.3
114.7
105.3
49.7
33.5
-26.8
-77.9
-95.4
-46.5
-153.2
-110.8
-72.8
-55.5
-36.9
16
97.3
47.1
56.2
65.7
50
53.5
301.9
174
-38
-11
-21
-15
-18
-13
-1
12
150
70
84
52
32
32
1
-14
88
-23
-16
1
22
47
115
127
95
97
110
96
126
2/9/00
302
243
83
22
-25
0
-10
-13
4
29
dry
214
192
106
81
24
11
23
42
150
60
71
98
114
141
259
dry
202
111
229
231
219
245
12/6/99
6.79
6.2
7.01
7.19
7.46
7.55
7.55
7.55
7.6
7.53
7.01
7.31
7.59
7.3
7.08
7.13
7.13
7.25
7.25
6.36
7.61
7.58
7.47
7.41
7.15
6.99
7
7
6.99
7.04
6.65
6.83
PH
1/11/00
6.78
5.76
7.03
7.06
7.38
7.4
7.45
7.45
7.48
7.45
dry
6.76
7.43
7.19
7.14
7.23
7.19
7.19
7.19
6.19
7.27
7.3
7.15
7.05
6.93
6.73
6.85
6.82
6.73
6.69
6.62
6.56
2/9/00
6.67
5.79
7.1
7.36
7.59
7.43
7.6
7.7
7.65
7.78
dry
7.29
7.13
7.27
7.34
7.72
7.74
7.6
6.37
7.19
7.12
6.74
6.81
6.71
6.55
6.52
6.32
6.14
6.44
6.51
6.59
26

-------
Table 5-3. SRB Populations
                Date
Bioreactor II
SRB populations (SRB/mL)
      Bioreactor HI
Bioreactor IV
12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/12/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/16/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
1.40E+03
2.00E+02
9.20E+00
1.80E+00
1.10E+00
1.70E+01
1.70E+02
2.80E+03
2.80E+04
1.40E+06
4.50E+05
4.50E+04
1.70E+04
1.20E+04
1.10E+04
2.40E+04
1.70E+06
2.00E+03
1.70E+04
1.40E+05
1.10E+05
9.20E+04
4.00E+04
2.80E+02
2.10E+03
4.50E+03
7.80E+03
7.80E+03
2.40E+04
1.40E+05
9.30E+04
1.70E+04
1.10E+04
1.70E+01
1.70E+01
l.OOE-01
1.80E+00
8.10E+00
2.40E+02
7.80E+03
2.00E+03
2.40E+03
2.80E+04
2.00E+05
4.50E+04
4.50E+03
1.10E+04
1.10E+04
1.70E+04
1.10E+05
1.10E+05
1.10E+05
2.00E+04
4.50E+04
1.10E+04
1.40E+04
2.00E+03
1.40E+03
2.40E+03
4.50E+03
2.00E+03
2.10E+03
4.50E+03
1.40E+04
2.80E+04
4.50E+04
7.80E+01
1.40E+02
l.OOE-01
1.80E+00
8.10E+00
2.40E+02
2.00E+03
1.10E+04
1.40E+04
1.40E+04
2.40E+04
4.50E+04
1.40E+04
2.00E+03
2.40E+03
1.10E+04
1.40E+04
7.80E+04
7.80E+04
4.50E+04
6.80E+04
2.00E+04
2.00E+03
2.40E+02
2.40E+03
1.40E+04
1.40E+04
2.10E+04
2.40E+04
7.80E+04
2.00E+04
2.10E+04
4.50E+04
                                                   27

-------
Table 5-4. Sulfatt
Date
12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/12/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/8/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
s and Sulflde
Influent
102
115
113
104
143
135
229
200
122
118
109
99
93
88
81
84
83.2
103
88.3
91.6
92.5
78.5
81.4
82.9
69.8
73.3
79.7
72.4
60.6
1,400
81.6
118
101
Concentrations
Sulfate (mg/L)
II III
250
80
145
111
122
193
281
304
136
92
113
73
87
83
82
82
73.8
100
81.2
82.4
72.7
95.4
88.5
59.2
73.4
65.9
68.1
55.4
8 1
196
131
128
88.7
Values enveloped by lines are considered outliers
172
111
123
115
152
178
223
197
126
80
76
88
100
89
85
87
81.4
95.6
69
84.8
93.5
78.5
83.8
81.5
86.2
79.7
74.9
56.4
59.6
73.7
113
113
102
and were
rv
148
114
143
137
186
187
300
457
326
74
92
106
99
98
125
152
114
69.2
127
91.9
64.7
80.3
93.1
86
84.7
163
208
263
251
204
241
121
79.7
not grafted
Influent
3.1
2.8
3.7
0.0
0.0
0.0
0.0
0.0
0.0
2.7
1.2
0.0
1.0
1.1
3.1
1.6
1.37
1.5
1.7
2.6
5.1
4.3
3
3.1
3.6
3
3
4.5
5
2.6
2.8
2.2
2.8

Sulflde
II
1.2
2.2
6.4
1.2
1.3
0.0
0.0
15.0
0.0
2.8
3.2
0.0
9.6
1.4
4.8
2
1.13
1.5
1.3
2.3
4.8
4.9
3.2
5.2
3.4
3.2
2.6
3
3.7
2.2
1.7
1.3
3.9

(mg/L)
III
2.3
2.9
2.4
1.6
2.4
0.0
0.0
2.2
0.0
4.1
3.0
0.0
0.6
3.2
3.9
1.2
1.53
2
3.7
2.9
4.7
7.2
2.3
5.6
2.3
3.3
2
4
4.5
1.7
1.9
1.6
4.1

rv
2.1
3.3
1.9
1.2
0.0
0.0
1.2
1.3
0.0
2.0
1.3
1.2
0.9
0.8
4.2
1.3
1.45
1.2
0.85
2.8
4.7
6.0
3
3.2
2.6
3.2
2.9
4.6
5
2.5
2.6
2
2.4

28

-------
Table 5-5. Alkalinity
            Date
                                     Intake
Total Alkalinity (mg/L as CaCO3)
  II                   III
                                                                                                          IV
12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/12/99
9/8/99
10/7/99
11/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/8/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
14.4
12.4
24.8
19.6
16.2
0.0
14.0
11.0
18.0
19.4
18.2
22.2
20.8
28.0
21
25.4
189
25.2
16
0
0
215.0
111.0
105.0
95.0
95.8
102.0
126.0
474.0
128.0
247.0
238.0
124.0
54.0
53.6
70.4
79.0
90.4
100.0
87.2
74.2
152.0
54.6
45.8
122.0
62.2
63.8
54.0
70.2
95.8
90.4
70.0
83.4
83.8
277.0
192.0
93.8
95.8
94.2
86.0
85.4
99.0
78.2
359.0
248.0
75.8
26.4
35.0
37.0
41.0
35.2
47.0
126.0
38.4
27.2
29.8
23.6
28.0
39.6
26.2
29.2
26.8
24.2
40.2
46.6
75.0
33
294.0
238.0
223.0
203.0
275.0
251.0
241.0
262.0
214.0
290.0
446.0
87.8
90.4
68.2
125.0
129.0
146.0
220.0
426.0
77.2
124.0
81.8
59.6
56.4
65.2
102.0
120
154
25.8
286
347
202
97
                                                           29

-------
Table 5-6. Metals Concentration
Location Date Al
Influent 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
1 1/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
Bioreactorll 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
1 1/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2400.0
1670.0
2080.0
788.0
1160.0
3750.0
14100.0
8770
1580
779
140
26.6
35
93.1
18
11
44.5
134
32
154
35.3
17.3
36.1
18.5
18.9
18.9
73.4
45.5
18
31
40.5
1650
587
39.5
24.6
22.8
30.8
11.2
10.7
13.8
111
49.3
97
128
35.0
15.8
21.3
30.6
16.5
24.6
130
56.5
47.6
55.2
17.3
17.3
98.2
49.6
31.8
18.9
Metals concentration (ng/L)
As Cd Cu
2.8
2.5
4.4
5.2
3.6
3.9
1.8
3.1
1.3
2.5
6.5
6.2
6.7
5.5
10.9
5.1
6.1
3.5
7.6
2.1
1.5
1.5
2.5
1.9
2.1
3.3
5.7
4.7
3.4
2
1.6
1.1
1.1
15.6
10.0
7.4
5.1
3.4
4.3
3.0
26.5
5.4
14.9
7.7
5.0
5.6
5
5.4
2.6
1.6
15.6
10
2.8
6.8
3.6
2.3
3.6
3.5
4.7
6.8
15.2
18.1
14.9
16.0
12.1
20.1
41.9
31
16.5
17.9
16.4
12.7
7.3
9.3
10.9
7
8.2
7.3
8.4
8.6
6.3
5.7
3.7
5.7
5.1
4.3
4.3
4.3
2.8
3.1
4.8
8.8
12.1
3.9
3.9
3.9
4.8
4.8
4.8
4.8
2.5
2.5
3.9
4.6
4.9
4.7
5.5
4.7
3.5
3.5
3.5
3.5
3.2
3.2
3.4
3.4
3.9
4.3
4.3
4.3
884.0
737.0
689.0
477.0
518.0
1020.0
3050.0
2090
451
366
200
68.9
78.8
82.8
7.2
53
114
274
145
310
174
81.4
92.7
71.4
44.2
34.1
28.1
2.8
26
30
162
635
459
29.4
11.5
3.3
4.8
4.3
47.5
7.8
75.2
25.6
96.6
95.9
36.8
19.4
23.2
15.8
12.8
21.4
72.2
49.7
21
81.8
17.7
5.9
89.1
57
68.8
31.5
Fe
524.0
377.0
603.0
357.0
265.0
713.0
7220.0
2960
111
281
372
50.6
154
15.7
115
35.9
20.1
124
27.2
73.1
20.5
31.9
15.5
15.5
20.1
19.5
19.5
19.5
8
13
25.6
70.2
195
140.0
77.6
46.9
93.7
35.5
54.2
97.5
1300
686
868
983
545
284
395
352
314
305
1810
1230
111
2110
261
197
926
578
533
234
Mn
1620.0
2020.0
1700.0
1830.0
1620.0
2120.0
3770.0
2860
1600
1950
1950
1580
1320
1170
1420
1130
1220
1360
1270
1230
1170
1070
1090
1140
1110
1000
882
787
690
840
947
1460
1190
35.6
31.1
39.5
117.0
115.0
357.0
551.0
999
964
1040
1340
885
774
765
785
913
982
1000
1140
1000
1440
984
836
1050
564
500
296
Zn
3740.0
4420.0
3610.0
3570.0
3190.0
4870.0
11100.0
7890
3370
3790
3520
2700
2130
2140
2080
1890
2050
2460
2020
2230
1840
1500
1580
1570
1420
1240
1270
1090
990
1000
1570
3180
2720
75.6
18.6
22.3
22.3
43.9
128.0
249.0
388
717
704
1230
1180
750
111
453
176
210
751
467
331
409
372
189
707
517
404
419
30

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Table 5-6. Metals Concentration
Location Date Al
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
Bioreactor III 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
1 1/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
Bioreactor IV 12/14/98
12/22/98
1/4/99
2/3/99
3/3/99
4/5/99
5/5/99
6/3/99
7/12/99
8/11/99
9/8/99
10/7/99
1 1/9/99
12/8/99
1/6/00
2/3/00
3/6/00
4/5/00
5/8/00
6/7/00
7/6/00
46.5
19
140
130
47.8
118
43.8
40.0
22.8
43.4
16.9
10.7
45.3
23.7
59.6
335
141
52.1
10.1
26.4
19.9
9.4
31.2
68.5
240
50.9
25.3
24.6
49.6
48
83.3
18.9
18.9
18.9
22
85
143
125
80.3
54.2
27.0
29.8
45.2
35.4
18.8
29.5
9.7
57.2
37.7
49.7
14.5
25.3
10.1
13
18.6
21.8
32.9
87.3
43.4
23.4
Metals concentration (ng/L)
As Cd Cu
3.3
2.2
4.7
11.4
5.5
4
17.1
11.3
5.0
6.2
4.4
4.6
3.5
3.1
2.9
8.9
12.7
3.5
4.2
3.2
3.5
2.4
1.6
3.6
11.7
3
2.2
1.5
2.8
2.3
3.3
1.9
6.1
3
3.1
2
4.5
4.3
2.7
26.3
21.6
29.1
19.7
19.6
14.6
17.1
26.3
3.4
13.4
28.7
2.3
3.4
2.6
7.8
8.8
10.1
8.8
16.3
9.6
1.6
4.3
0.1
0.3
4.8
4.8
5
3.9
3.9
3.9
4.8
4.8
4.8
4.8
2.5
3.1
7
3.9
3.9
4.7
4.7
4.8
3.5
3.5
3.5
4.7
4.4
3.2
3.4
4.4
3.5
5.6
4.3
4.3
4.3
1
2
9.1
4.8
4.8
3.9
3.9
3.9
4.8
4.8
4.8
4.8
2.5
2.5
3.9
3.9
3.9
4.7
4.7
4.7
3.5
3.5
3.5
3.7
3.2
3.2
23.7
8
100
142
50.6
110
65.5
20.3
8.6
18.1
17.5
6.4
43.4
36.4
28.3
140
69.2
22.5
16.1
14
19.5
18.3
51.8
65.6
114
49.3
96.8
45.8
68.8
69.5
128
24.3
23
20.4
16
65
155
142
122
103.0
79.4
33.0
39.5
44.8
32.3
36.3
36.2
8.0
28.1
32.6
6.4
23.5
17.2
16.9
39.7
47.1
34.6
44.1
47
20.1
Fe
460
110
600
454
638
707
229.0
92.4
57.8
69.8
43.9
49.8
149.0
202
337
1380
875
661
302
163
91.2
95.8
323
1240
3910
1070
115
124
116
115
161
43.9
34.3
25.2
31
150
282
494
638
417.0
155.0
126.0
95.7
230.0
180.0
166.0
301
410
530
782
142
304
141
188
206
325
434
1900
761
448
Mn
871
600
2400
721
849
996
105.0
54.5
33.1
124.0
436.0
768.0
2100.0
1940
1510
3100
1360
1260
1230
1150
1170
1200
1270
1500
1450
1130
1120
886
631
631
493
310
129
73.5
76
280
212
3040
2470
19.2
20.4
19.3
27.5
168.0
215.0
454.0
983
1310
1070
1480
751
883
811
934
1060
1070
1150
2190
1000
1120
Zn
648
48
300
1170
518
899
91.0
34.8
35.8
35.9
60.9
241.0
459.0
1190
1030
1100
847
753
364
659
934
707
682
893
1070
723
956
784
1040
1330
1590
836
871
937
790
850
1720
1590
1010
118.0
53.3
40.6
119.0
72.8
51.3
194.0
421
306
672
595
278
491
544
438
466
471
410
442
463
348
31

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Table 5-6. Metals Concentration
    Location
                       Date
                                        Al
                                                            Metals concentration (ng
                                                     As
                                                                   Cd
                                                                                Cu
                                                                                               Fe
                                                                                                           Mn
                                                                                                                          Zn
8/10/00
9/7/00
9/25/00
10/31/00
12/4/00
1/8/01
2/12/01
3/14/01
4/9/01
5/9/01
6/6/01
7/9/01
17.3
17.3
17.3
45.4
18.9
51.1
18.9
85
22
55.8
81.7
42.3
1.5
1.8
1.5
2.5
4.1
7.2
5.5
6
8
9.6
4.1
1.1
3.4
3.4
3.4
4.3
4.3
4.3
4.3
0.4
0.7
4.8
4.8
4.8
10.2
9.8
6.3
52.9
16.9
13.6
18.5
530
35
33.9
132
18.2
87.7
67.1
124
506
1090
1590
937
1100
2900
3540
1940
572
837
629
794
942
1320
1810
2000
2300
2300
2610
1400
1270
269
222
145
383
170
203
315
220
250
211
505
292
                                                            pH Trends
                11/1/98    1/30/99    4/30/99   7/29/99    10/27/99   1/25/00    4/24/00   7/23/00   10/21/00   1/19/01   4/19/01    7/18/01
                                                               Date
          Figure 5-1.  pH trends.
                                                                32

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                                          EH Trends
                                              Date
Figure 5-2. EH trends.
                                    Dissolved Oxygen Trends
                                             Date
Figure 5-3. Dissolved oxygen trends.
                                              33

-------
                                           Temperature Trends
      11/1/98   1/30/99   4/30/99   7/29/99   10/27/99  1/25/00   4/24/00   7/23/00   10/21/00   1/19/01   4/19/01   7/18/01
                                                     Date
Figure 5-4.  Temperature trends.
                            Bioreactor II pH and EH Profiles for 12/6/99
                              limestone
                   organic carboin   ,  ,
                    0      :   chamber
                     chamber "
organic carl
  chamber I
 cobble
chamber
                                                                             •deep Eh
                                                                             •shallow Eh
                                                                             •deep pH
                                                                             1 shallow pH
                               IIG       II H       II B       IIC       III       II J
                              Piezometer nest (each piezometer is approximately 10 ft apart)
Figure 5-5.  Bioreactor II pH and EH profiles for 12/6/99.
                                                     34

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                               Bioreactor III pH and EH Profiles for 12/6/99
                        IIIF         IIIB        IIIG         IIIC        IIIH         III I

                              Piezometer nest (each piezometer is approximately 10 ft apart)
Figure 5-6.  Bioreactor III pH and EH profiles for 12/6/99.
                                             SRB Populations
        11/1/98  1/30/99  4/30/99  7/29/99  10/27/99  1/25/00   4/24/00  7/23/00  10/21/00  1/19/01   4/19/01   7/18/01

                                                    Date
 >	

Figure 5-7.  SRB populations.
                                                    35

-------
                                             Sulfate Concentrations
         11/1/98   1/30/99    4/30/99    7/29/99   10/27/99   1/25/00    4/24/00   7/23/00    10/21/00   1/19/01   4/19/01    7/18/01



                                                         Date
Figure 5-8.  Sulfate concentrations.
                                         Soluble Sulfide Concentrations
       11/1/98   1/30/99    4/30/99   7/29/99   10/27/99   1/25/00   4/24/00   7/23/00   10/21/00   1/19/01   4/19/01   7/18/01



                                                        Date
Figure 5-9.  Soluble sulfide concentrations.
                                                        36

-------
                                                       Total Alkalinity
          11/1/98    1/30/99   4/30/99    7/29/99    10/27/99    1/25/00    4/24/00    7/23/00    10/21/00   1/19/01    4/19/01    7/18/01
                                                                 Date
Figure 5-10.  Total alkalinity.
                                                    Zn Concentrations
                                                           -Intake
                                                            'II
                                                           -III
                                                            IV
                                                            II autopsy sample (total)
                                                            III autopsy sample (total)
                                                            IV autopsy sample (total)
                                                            II autopsy sample (dissolved)
                                                            III autopsy sample (dissolved)
                                                            IV autopsy sample (dissolved)
          11/1/98   1/30/99   4/30/99   7/29/99   10/27/99   1/25/00    4/24/00   7/23/00   10/21/00   1/19/01    4/19/01   7/18/01
                                                                Date
Figure 5-11.  Zinc concentrations.
                                                                37

-------
                                                   Cu Concentrations
  If1 l.E
-Intake
 'II
-III
 IV
 II autopsy sample (total)
 III autopsy sample (total)
 IV autopsy sample (total)
 II autopsy sample (dissolved)
 III autopsy sample (dissolved)
 IV autopsy sample (dissolved)
                                                             Date
Figure 5-12.  Copper concentrations.
                                                   Cd Concentrations
                                                                                    •Intake
                                                                                    'II
                                                                                    •III
                                                                                    IV
                                                                                    II autopsy sample (total)
                                                                                    III autopsy sample (total)
                                                                                    IV autopsy sample (total)
                                                                                    II autopsy sample (dissolved)
                                                                                    III autopsy sample (dissolved)
                                                                                    IV autoosv samole (dissolved!
       11/1/98  1/30/99  4/30/99  7/29/99  10/27/99  1/25/00  4/24/00  7/23/00  10/21/00  1/19/01  4/19/01   7/18/01
                                                             Date                                                          i
Figure 5-13.  Cadmium concentrations.
                                                             38

-------
                                                     Al Concentrations
                                                                            Intake
                                                                            II
                                                                            III
                                                                            IV
                                                                            II autopsy sample (total)
                                                                            III autopsy sample (total)
                                                                            IV autopsy sample (total)
                                                                            II autopsy sample (dissolved)
                                                                            III autopsy sample (dissolved)
                                                                            IV autopsy sample (dissolved)
                                                                Date
Figure 5-14.  Aluminum concentrations.
  I
                                                     As Concentrations
-Intake
 'II
•III
 •IV
 II autopsy sample (total)
 III autopsy sample (total)
 IV autopsy sample (total)
 II autopsy sample (dissolved)
 III autopsy sample (dissolved)
 IV autopsy sample (dissolved)
        11/1/98   1/30/99    4/30/99   7/29/99    10/27/99   1/25/00   4/24/00    7/23/00   10/21/00   1/19/01   4/19/01    7/18/01
                                                              Date
 >	
Figure 5-15.  Arsenic concentrations.
                                                               39

-------
                                                     Fe Concentrations
  I
-Intake
 'II
-III
 IV
 II autopsy sample (total)
 III autopsy sample (total)
 IV autopsy sample (total)
 II autopsy sample (dissolved)
 III autopsy sample (dissolved)
 IV autopsy sample (dissolved)
                                                                                                      \
          11/1/98    1/30/99   4/30/99    7/29/99   10/27/99   1/25/00    4/24/00   7/23/00   10/21/00   1/19/01    4/19/01    7/18/01
                                                                 Date

Figure 5-16.  Iron concentrations.
                                                     Mn Concentrations
  I
  •5  l.E+02
                                                           II autopsy sample (total)
                                                           III autopsy sample (total)
                                                           IV autopsy sample (total)
                                                           II autopsy sample (dissolved)
                                                           III autopsy sample (dissolved)
                                                           IV autopsy sample (dissolved)
                                                                Date
Figure 5-17.  Manganese concentrations.
                                                                40

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                                     6.   Autopsy Results
Reporting on autopsy sampling in this section is
descriptive in its character; it documents the results
obtained and includes basic qualitative
interpretation.  It is expected that a portion of the
autopsy data will be used in a quantitative manner
to improve and validate a computer program for
bioreactor design begun in one of the tasks of
another MWTP project (i.e., Project 24, Improved
SRB).

6.1  Aqueous Samples
Although the autopsy sampling focused on
collecting the solid matrix material that was
inaccessible  during the operational phase of the
project, it also  included aqueous samples collected
from the bottom of the bioreactors within chambers
filled with limestone cobbles. To minimize the
impact of atmospheric oxygen on the aqueous
samples, the samples were collected immediately
after the limestone cobbles were removed to
provide access to the treated AMD that remained at
the bottom of the bioreactors.  Aqueous samples
collected at the bottom of the cobble chamber were
analyzed for total and dissolved metals, iron
speciation, sulfate, and sulfide. Samples collected
at the bottom of the limestone chambers were
analyzed for the same analytes with the exception
of dissolved metals. Analytical results are
compiled in  Table 6-1 (a, b, c). The concentrations
of selected analytes are  also presented in graphical
form in the figures that are individually referred to
in this section.

6.1.1  Cobble Chambers
Figures 6-la and b, 6-2a and b, and 6-3a and b
include diagrams of dissolved versus total and
suspended metals for Bioreactors II, III, and IV
respectively. Values used for these diagrams are
tabulated in  Table 6-la.  Figures denoted with the
letter "a" show total versus dissolved metals, and
figures denoted with the letter "b" present
suspended metals calculated as the difference
between the  total and dissolved metals.
The autopsy dissolved metal concentrations
compare well to the corresponding metals
concentrations of the final sampling event as
documented in Table 6-2. The differences may be
attributed to a 1-week gap between the sampling
events and the different sampling locations. In
general, the dissolved metal concentrations were
higher than during the final monthly sampling event
with the biggest difference for As, Al, and Fe.  The
autopsy dissolved and total metal concentrations
are also marked in  Figures 5-11 through 5-17.

Although not detected by a meter, the smell of H2S
was present during sampling of the "soupy" grey
water that accumulated at the bottom of the
bioreactors.  This smell was indicative of reducing
conditions and sulfate reduction at these locations.
Such judgement was supported by results of
speciation analyses for dissolved  Fe (Figure 6-4
and Table 6-lb) in  the aqueous samples collected
from the cobble and limestone chambers. Ferric
iron (Fe3+) for those samples was found only in the
sample taken from  the limestone chamber of
Bioreactor II and the limestone and cobble
chambers of Bioreactor IV.  The maximum
concentration of Fe3+ for these locations was
0.17* g/L or 9% of Fe2+ in the limestone chamber
of Bioreactor II.  However, the analyses for sulfide
and sulfate in the dissolved phase (Table 6-lc)
show only 3.9 mg/L, 2.2. mg/L, and  4.2 mg/L
concentrations of sulfide for Bioreactors II, III, and
IV respectively.  This indicates that only a small
portion of dissolved metals in reduced form were
balanced in the solution by sulfides.  These were
probably Zn, Cu, Cd, and a  small portion of Fe.

The rest of the metals dissolved in solution,
including high concentrations of Ca (Table 6-la),
are electronically balanced by a high concentration
of sulfate (Table 6-lc), hydroxide, and
bicarbonate.  Concentrations of the latter two were
not analyzed for the autopsy samples.
                                                41

-------
Series "b" diagrams that show a high load of
suspended metal compounds in a "soupy" solution
attested to the successful operation of the
bioreactors.  Provided no suspended metals drained
from the bioreactors through their outlets, the mass
of metal compounds found in the lower portion of
the bioreactors together with metal compounds that
accumulated within the organic matter and on the
limestone should be related to the mass of metals
removed from the influent AMD.

The analytical data available do not allow for
determination of chemical compounds that
contained these metals.  However, considering the
observed (during the autopsy on the bioreactors)
orange-color precipitate present on the PVC liner
and the HDPE of the CCS, it is possible that solids
in the "soupy" water also included significant
amounts of ferric hydroxide. It is also
hypothesized that Al present as a suspended solid
was in the form of hydroxides.

6.1.2  Limestone Chambers
Analytical results for total metals for aqueous
samples collected at the bottom of the limestone
chambers of Bioreactors II and IV resemble those
obtained for aqueous samples of the cobble
sections, as documented in Figure 6-5 for
Bioreactor II. Thus, despite the lack of analytical
data for dissolved metals concentration for this
section of the bioreactors, it is assumed that the
concentrations of suspended metal compounds in
the limestone section were  similar to those for the
cobble section.

6.1.3  Sulfate and Sulfide Analyses
Table 6-lc includes analytical data for sulfate and
sulfide in the aqueous sample collected during the
autopsy of the bioreactors. The table shows that
sulfate concentrations were as high as 352 mg/L in
Bioreactor III.  The highest concentration of
sulfide, 4.2 mg/L, was found in Bioreactor IV.
6.2  Bioreactor Solid Matrix Samples
Analytical data for solid matrix samples collected
during the autopsy of the bioreactors are included
in Table 6-3 (a, b, c, d), and they are also presented
in graphical form in the figures that are
individually referred to in this section.  Also
presented in this  section are analytical data of
material that was found plugging the manifold for
the AMD distribution system.

6.2.1  Metals Concentrations in the
Bioreactors
Figures 6-6, 6-7, and 6-8 present the distribution of
metals in the solid matrix of the first three
chambers of Bioreactors II, III, and IV,
respectively. These values are also compiled in
Table 6-3a. Concentration of metals within the
organic matter (denoted in the figures as organic
matter chambers 1  and 2) was determined with
respect to the total dry weight of the sample
collected. For the limestone chamber, the
precipitate present on the limestone was scraped,
and metal concentrations were determined with
respect to the dry weight, not including the weight
of the limestone. As  stated in Section 4.4.1, the
cobbles that comprised the last chamber of each
reactor did not have a visually discernible film of
chemical precipitate, thus the metal concentrations
in the solid matrix of these chambers were
considered null.

Concentrations of metals that accumulated within
the solid matrix of the bioreactors were measured
in thousands of milligrams per kilogram with the
exception of those for Cd and As. These high
metals concentrations demonstrated that a large
load of metals  was retained within the organic
carbon material,  thus indicating the bioreactors
were efficient in  removing metals from the influent
AMD. Unfortunately, the analyses performed
using the inductively coupled argon plasma (ICP)
method do not allow for distinguishing of metals
that were adsorbed by organic matter from  those
that precipitated  as chemical compounds due to
SRB activities.
                                                42

-------
Evidence of the biological removal of metals by the
SRB comes from other analytical results conducted
for the project.  First, the high concentrations of
sulfide in the organic matter, as reported in Section
6.2.2, need to be stoichiometrically balanced.
Metals that form amorphous metal sulfides, a
chemical compound that can only be biogenic,
probably provided this balance. Secondly, as
stated in Section 5.5, Mn that is not biologically
removable at the low pH level was efficiently
removed only at the beginning of the operation
when it was sorbed to the organic  matter. After the
sorption capacity of the organic matter was
exhausted, Mn stayed in the aqueous phase while
other metals (Zn, Cu, and Cd) were still being
removed. Removal of these metals, with no
organic-matter adsorption capacity present, can
only be explained by formation of biogenic metal
sulfides.

Because of a small population of samples collected,
the diagrams in Figure 6-6 through 6-8 may be
affected by spatial variability of metal
concentrations.  Nevertheless, the  diagrams indicate
some general features and trends that might not be
explainable and/or clearly conclusive and are worth
noting as general observations.

•  Aluminum concentrations decrease along the
   flow paths within the first organic matter (Orl)
   and limestone chambers, then slightly increased
   in the second organic matter chamber (Or2)  for
   Bioreactors II and IV.

•  In general, Fe demonstrated a gentle trend of
   decreasing concentrations, though not without
   exceptions (mostly in Bioreactor IV).  High
   concentrations of Fe in the first lift for all
   bioreactors agree with visual observations
   during the autopsy process when the orange-
   color precipitate was observed  on the HDPE
   walls of the CCS and within the organic matter
   itself. This precipitate, iron hydroxide, was
   most abundant within the first CCS lift.
•  Zinc seemed to be evenly distributed within Orl
   of Bioreactor II, but its concentration peaked in
   the centers of Orl of Bioreactors III and IV.

•  There is a distinct trend of decreasing
   concentration for Cu along the flow direction for
   all three bioreactors.

•  There seems to be an increasing trend for Mn
   concentration along the flow path, with the
   exception of a noticeable decrease in Mn
   concentration in the limestone chamber of
   Bioreactor IV.

•  Cadmium concentrations behaved erratically  for
   Bioreactors III and IV but had a decreasing
   trend for Bioreactor II. In all three bioreactors,
   Cd concentrations varied the most of all the
   metals presented.

•  There was a decreasing trend for As
   concentrations within Bioreactors II and III and
   a similar but less obvious trend for Bioreactor
   IV.  All bioreactors demonstrate much higher
   concentration of As in the most upgradient CCS
   lift (No. 1) than in the rest of the solid matrix
   sampled.  For Bioreactor II, these much-
   elevated As concentrations extend to the second
   sampling point, lift No.3.

•  The elevated concentrations of As at the very
   front portion of the bioreactors correlate very
   well with high concentrations of Fe at the same
   location.

Statistical correlation coefficients calculated for all
possible pairs of the aforementioned metals are
presented in Table 6-4. Outlined with heavy lines
are three correlation coefficients that because of
their high values need to be considered meaningful
despite possible spatial variability of the metal
concentrations that might have been missed by a
small population of samples. These correlations
coefficients  are for Al and Cu, As and Fe, and Cd
andZn.
                                                43

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High correlation of Al and Cu may be explained by
known observations that they both precipitate
quickly when aqueous conditions change to a less
acidic and more reducing environment.  In such a
case, Al precipitates as hydroxides, and Cu
precipitates as sulfide.

A strong dependancy of As and Fe has already
been explained in Section 5.5 by referring to a
statement by Robins and Huang (Ref 6) that As
effectively adsorbs to Fe(OH)3. As mentioned
earlier in this section, observations during the
autopsy indicated an abundance of Fe(OH)3
precipitant in the bioreactors. The high correlation
coefficient between As and Fe in the bioreactors
support findings of Robins and Huang.

High correlation coefficient for concentrations of
Cd and Zn were expected due to the similarity in
chemical behavior of these two elements. As it
appears, a two orders of magnitude difference in
their concentrations in the aqueous solutions
(Figures 5-11 and 5-13) corresponds to their
precipitates in the bioreactors where they also
differ by two  orders of magnitude.

6.2.2  Sulfate and Sulfide Analyses
Figures 6-9 and 6-10 present diagrams for sulfate
and sulfide profiles in the solid matrix of the
bioreactors, respectively.  These diagrams were
plotted based on values included in Table 6-3c that
were obtained from acid base accounting (ABA)
analyses. The sulfide diagram
(Figure 6-10) was assembled by adding values for
insoluble sulfide to values of pyritic sulfides (both
present in Table 6-3c) and converting the sum from
percent to mg/kg.

Figure 6-9 shows  different changes of sulfate
concentrations within the first organic matter
chamber of each bioreactor.  However, considering
the high value for lift No. 7 in Bioreactor IV as  an
outlier or a sulfate "nugget," there is some
resemblance of concentration of sulfates in
Bioreactors II and IV. In both bioreactors,  the
concentration of sulfates decrease toward the
limestone chambers and then rise to a similar level
in the second organic matter chamber.  Sulfate
concentrations in Bioreactor III, which does not
include a limestone chamber, shows a general
rising trend throughout the organic matter chamber.
These features of the sulfate profiles show the
advantage of placing a limestone chamber in the
bioreactors.

Sulfide concentrations (Figure 6-10) within the first
organic matter chamber are generally similar,
except for CCS lift No. 9 in Bioreactor II. In
general, sulfide concentrations in the organic matter
chambers are above the 4,000 mg/kg level and are
twice as high as concentrations of sulfates.  The
decrease of sulfide concentration in lift No. 9 of
Bioreactor II to 3,300 mg/kg remains a conundrum.
Its explanation can be sought in the fact that the
relatively small number of samples collected (one
every second lift) increases the chance for some
samples not being representative of the actual
hydrochemical conditions prevailing in the sampled
medium.

Numerical values for sulfide concentrations in the
solid matrix of organic matter are more than three
orders of magnitude greater than those for the
aqueous phase in the limestone and cobbles
chambers (Table 6-lc). The actual difference was
even greater because of the conversion of mg/kg for
the solid matrix to mg/L for the aqueous phase.
This is because 1 cubic decimeter of solid matrix,
which equals 1 liter in volume, weighs more than 1
kilogram.

Sulfide load in organic matter, together with metal
concentrations as addressed in Section 6.2.1, is
indicative of metal sulfides precipitating in the
organic matter chamber due to SRB activities.
This postulate together with the observation of no
precipitated metal in the cobble chambers of the
bioreactors, indicates again that the role of the
cobble section was limited to a sump for a small
mass of precipitates that escaped from the organic
matter chambers.
                                                44

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6.2.3  Total Organic Carbon
Numerical data for TOC concentration in the
bioreactors solid matrix are included in Table 6-3b.
As expected, they indicate that the lowest TOC
concentrations were associated with the limestone
chambers: 8.7 % and 1.2% by weight for
Bioreactors II and IV respectively.  The TOC
concentrations within the organic matter chambers
ranged from 17% to 24% with the majority of
values above 20%. These values of TOC
concentrations document that there was plenty of
organic carbon remaining in the organic matter
chambers after 32 months of bioreactor operation.
Although, TOC concentrations for the initial fresh
organic matter were not measured, the high TOC
values measured during the autopsy may indicate
that the depletion of organic carbon would not be a
factor for the efficient removal of metals even if
these bioreactors were operating for several more
years.

6.2.4  Plugs in the Outlets of the AMD
Distribution System Manifold
Many inlets in all three bioreactors were plugged
with chemical precipitates located within the last
1 inch of the L-shaped inlet. Because the outlets
(also called bioreactor inlets) were 2 inches in
diameter, so were the plugs. The plugs had the
appearance and consistency of gel.  The upper half
of a plug, the one facing the incoming AMD, was
black.  The lower half, which was in contact with
the organic matter, was light brown. Analytical
data for a plug collected from one of the outlets in
Bioreactor III are tabulated in Table 6-3d and
presented in graphical form in Figure 6-11.

As implied by a sharp boundary between black and
brown portions of the plug, the concentrations of
metals in each portion were different.  The main
differences were the concentrations  of Al, Fe, Ca,
and Mg (the last two are not shown in the figures).
The Al concentration in the black portion of the
plug was much higher (approximately 540%
higher) than the Al concentration in the brown
portion.  Conversely, the concentrations of Fe, Ca,
and Mg were lower in the black portion (i.e.,
approximately 45%, 26%, and  15%, respectively,
of those in the brown portion).

With the exception of Fe, Al, and As, the metal
concentrations in the plug were in the same range
as those determined for samples collected from the
CCS lift. Iron concentration in the brown portion
of the plug was nine-fold of that found in the
adjacent CCS lift and constituted 12.7% of the
weight of the plug. Since it is expected that the
majority of the Fe present in that place in the
system was associated with Fe(OH)3, this
compound made up 24% of weight for the lower
portion of the plug. An elevated (five times higher
than in adjacent CCS lift) concentration of As was
certainly the result of its adsorption to Fe(OH)3, as
explained earlier in this report.

Aluminum concentration in the upper portion of the
plug was more than six-fold of that determined for
the adjacent CCS lift. Assuming its presence as a
hydroxide, this compound made up  16% of weight
for the upper portion of the plug.

The high percentage of Fe and Al hydroxides in the
plug indicates a high probability that the bioreactor
inlets were plugging mainly due to chemical
reactions; the sediment carried by the unfiltered
AMD was a secondary reason for plugging.

6.3  Flow Pattern
Visual observations made during the autopsy of the
bioreactors indicated no preferential flow paths
developed during the 32-month operation period.
This means that the AMD flowed and was treated
throughout the entire cross section of the organic
matter.  Most of the individual cells of the CCS
were found full of organic matter. Probably less
than 5% of cells were voided of the  organic matter
in their top portions, with voids never taller than 2
inches.  There was no discernible pattern of these
voids that seemed to be uniformly distributed
throughout each individual CCS lift.
                                               45

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6.4  Sulfate-Reducing Bacteria Population
on the Solid Matrix
Although the SRB population in the aqueous phase
of the bioreactors was measured during the
operating period, no measurements of SRB
population attached to the solid matrix of the
bioreactors were made.  To measure the final
population of SRB within the solid matrix, samples
of both the organic matter and limestone were
collected during the autopsy and submitted to a
bacteriological laboratory for an SRB count. The
results returned were expressed in MPN of SRB
per gram (MPN/g) of wet weight of the solid
matrix for organic matter and in MPN  of SRB per
sample for the limestone.

Therefore, the results needed to be converted and
expressed as a function of the volume of the
matrix. This conversion was needed to compare
the SRB population in the aqueous and solid
phases using the same dimensions (i.e., MPN per
volume).

To make the aforementioned conversion for the
organic matter, an attempt was made to determine
its particle density.  This presumably simple task
was not successful because standard methods used
for determination of particle density of solids did
not seem to be applicable for organic matter.
Therefore, the SRB population per unit volume
was determined as a function of particle density.
To accomplish this, a geometric mean of MPN/g
for SRB populations in eight samples collected
from all three bioreactors was calculated.  This
value, 1.83E+06 MPN/g, and the average
volumetric moisture content of 58.5% were used to
determine SRB populations as a function of
particle density. Thus, the values  for SRB
populations were expressed as MPN per unit
volume of wet organic matter, and they ranged
from 1.45E+06 MPN per cubic centimeter
(MPN/cc) to 2.63E+6 MPN/cc for the  particle
density ranging from 0.5 grams per cubic
centimeter (g/cc) to 2.05 g/cc, respectively.
Assuming particle density of 1.3 g/cc, the MPN for
SRB population in 1 cc of wet organic matter was
2.06E+6 MPN/cc. This population of SRB
attached to the solid matrix of the organic matter is
two orders of magnitude greater than the value of
2.81E+4 MPN/milliliter (mL) calculated as a
geometric mean for three aqueous samples
collected during the last sampling event  on July 9,
2001.

To determine SRB population per unit volume of
the limestone chamber, an average surface area for
the limestone was calculated. This was  necessary
because the SRB populations on the limestone were
determined by the bacteriological laboratory by
scraping the biofilm from the sampled limestone
and then expressing the population of SRB as
MPN per sample.  The calculations performed
resulted in a determination of SRB population at
8.2E+4 MPN/cc.  This SRB population is of the
same order of magnitude as for the aqueous
samples. The unit of cubic centimeter for the
limestone population estimate is used in this case
for comparison purposes only. Actually, a more
representative unit of volume would have been
cubic meter since the limestone ranged in size from
5 centimeters (cm) to 15 cm in diameter.

6.5  Toxicity Characteristic Leaching
Procedure
The results of TCLP testing demonstrated that the
solid matrix of the bioreactors did not exhibit
characteristics of toxicity as it was documented by
concentrations  of Cd and As, the only metals listed
in 40 Code of Regulations that might be of concern
for the project. Cadmium and As concentrations in
leachate from samples  of organic carbon matter
were 7.6 and 136 times lower, respectively, than
the regulatory levels that would classify this matrix
as hazardous waste.
                                              46

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

Table 6-la. Total and Dissolved Metals

Location Al
Bioreactor II cobble . ,, OA
. . UoU
chamber
Bioreactor III
cobble chamber
Bioreactor IV . „
cobble chamber

Location Al
Bioreactor II ^1QQ
limestone chamber
Bioreactor II cobble ,_. „„
chamber
Bioreactor III *,A~,nn
,,. , , 34300
cobble chamber
Bioreactor IV 7^00
limestone chamber
Bioreactor IV 9240
cobble chamber
Table 6-lb. Iron speciation

As Cd
29.3 4.8

16.8 4.8
8.5 4.8

As Cd
251 86.1
53.1 5.3

53.1 5.9
Dissolved metals (• g/L)
Ca Cu Fe Mg
13500 86.8 2910 29400

87000 220 2830 20000
124000 38.7 981 33300
Total metals samples (• g/L)
Ca Cu Fe Mg
1040000 2450 43000 55100
152000 951 52200 45100

109000 592 42300 36200
30 11.4 539000 506 13200 42800
20 4.8

122000 159 12700 35200


Mn Zn
1760 506

2720 1030
1980 329

Mn Zn
7270 20400
3000 2150

5280 3770
3160 6390
2170 856

Iron Species (mg/L) (nonfiltered samples)
Location
Bioreactor II limestone chamber
Bioreactor II cobble chamber
Bioreactor III cobble chamber
Bioreactor IV limestone chamber
Bioreactor IV cobble chamber
Table 6-lc. Sulfate and Sulfide
Fell
1.9
3.6
2
0.93
1

Fe III Fe Total
0.17 2.1
<0.05 3.5
<0.05 2.1
0.057 0.99
0.076 1.1

% Fe Recovery
100
100
100
100
100

Sulfate and Sulfide concentrations (mg/L) (nonfiltered samples)
Location
Bioreactor II limestone chamber
Bioreactor II cobble chamber
Bioreactor III cobble chamber
Bioreactor IV limestone chamber
Bioreactor IV cobble chamber






Sulfate
3091
2401
352
225
1591
Sulfide
3.2
3.9
2.2
3
4.2
'Corrected values obtained using inductive coupled plasma (ICP) method
                                                                47

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Table 6-2.  Comparison of the Dissolved Metals from the Autopsy and Monthly Sampling
Sample
Bioreactor , ,.
location
Date
Zn Cu
Dissolved metal concentration (ng/L)
Mn Cd As Al
All1 07/9/01 899 110 996
Bottom2 07/17/01 506 87 1,760
AIII1 07/9/01 1,010 122 2,470
III
Bottom2 07/17/01 1,030 220 2,720
AIV1 07/9/01 292 18 1,270
IV
Bottom2 07/16/01 329 39 1,980
1 See Figure 3-5 for location
2 Bottom of the cobble chamber adjacent to the organic matter chamber
Tables 6-3. Analytical Results of the Solid Matrix Samples Collected During
Table 6-3a. Metal Concentrations
Location
Bioreactor II manure
chamber 1 lift 1
Bioreactor II manure
chamber 1 lift 3
Bioreactor II manure
chamber 1 lift 5
Bioreactor II manure
chamber 1 lift 7
Bioreactor II manure
chamber 1 lift 9
Bioreactor II limestone
chamber
Bioreactor II manure
chamber 2 lift 5
Bioreactor III intake
plug - brown
Bioreactor III intake
plug - black
Bioreactor III manure
chamber 1 lift 1
Bioreactor III manure
chamber 1 lift 3
Bioreactor III manure
chamber 1 lift 5
Bioreactor III manure
chamber 1 lift 7
Bioreactor III manure
chamber 1 lift 9
Bioreactor IV manure
chamber 1 lift 1
Bioreactor IV manure
chamber 1 lift 3
Bioreactor IV manure
chamber 1 lift 5
Bioreactor IV manure
chamber 1 lift 7
Bioreactor IV manure
chamber 1 lift 9
Bioreactor IV limestone
chamber
Al
7830
13700
10900
6880
4700
2650
3730
10300
55800
8600
4020
6060
5500
4550
15100
13000
10900
5680
3890
2400
5
5
6
5
5
5
the Autopsj
Metal concentrations of the solid matrix samples (ing/Kg)
As Cd Ca Cu Fe
42.1
31.4
23.6
22.8
15.4
8.3
10.1
400
485
76.1
24
19.1
17.8
15.8
109
18.2
14.9
22
14.4
4.2
23.4
31.9
21.6
28.6
12.8
3.8
2.4
25.7
31.2
24.3
10.1
35.5
5
18.7
10.5
10.8
59.3
19.4
0.92
0.91
8490
10400
6350
12300
38800
208000
11500
5700
1480
9560
13100
10500
15400
12600
2740
6130
9460
16600
19100
274000
1470
2970
2260
975
438
163
147
7340
8560
1480
241
596
579
203
2400
2340
1670
562
107
58.9
11400
9960
6910
9040
7010
4150
7280
127000
57800
14000
7710
8280
8970
8370
22900
7390
6580
7830
9210
4050
4
29
3
17
1
9
Mg
5170
9890
6990
7640
6480
5110
5920
3980
613
6180
6670
7130
6680
5210
4750
6130
7600
5290
4930
4800
118
1,380
80
353
42
162
Mn
931
902
387
1200
884
740
866
25200
35100
637
940
783
912
1180
263
402
668
655
800
185
Fe
707
2,910
638
2,838
572
981
Zn
4450
3790
3470
4270
2720
1000
3010
2530
2100
3630
3820
5790
2900
4880
1660
1600
7690
6400
1610
566
                                                 48

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Table 6-3a. Metal Concentrations
      Location
                        Al
                                  Metal concentrations of the solid matrix samples (ing/Kg)
                                   As         Cd        Ca         Cu         Fe
                                                                                         Mg
                                                                                                  Mn
                                                                                                             Zn
Bioreactor IV manure
chamber 2 lift 5
4700
            19.9
                      0.92
                                18500
                                            178
                                                      10300
                                                                 7110
                                                                           1500
                                                                                     583
Table 6-3b. Nitrogen,  Phosphorus, and TOC

                         Analytical chemistry for solid matrix samples (mg/Kg)
              Location                   Total Nitrogen	Phosphorous
                                                           TOC (%)
Bioreactor II manure chamber 1 lift 1
Bioreactor II manure chamber 1 lift 3
Bioreactor II manure chamber 1 lift 5
Bioreactor II manure chamber 1 lift 7
Bioreactor II manure chamber 1 lift 9
Bioreactor II limestone chamber
Bioreactor II manure chamber 2 lift 5
Bioreactor III manure chamber 1 lift 1
Bioreactor III manure chamber 1 lift 3
Bioreactor III manure chamber 1 lift 5
Bioreactor III manure chamber 1 lift 7
Bioreactor III manure chamber 1 lift 9
Bioreactor IV manure chamber 1 lift 1
Bioreactor IV manure chamber 1 lift 3
Bioreactor IV manure chamber 1 lift 5
Bioreactor IV manure chamber 1 lift 7
Bioreactor IV manure chamber 1 lift 9
Bioreactor IV limestone chamber
Bioreactor IV manure chamber 2 lift 5
10251
14623
12259
12141
12983
3056
13086
10917
15533
10406
11074
6689
8308
13528
11063
10501
9920
3963
10682
58.2
86.1
53
62.1
124
144
65.9
74.4
104
44.1
119
72.4
60.4
90.4
78.9
66.1
68.4
22
168
20.0
20.0
22.3
23.4
17.2
8.7
23.3
22.6
21.6
20.7
21.2
21.4
24
22.1
23.5
21.6
23.9
1.2
16.9
Table 6-3c.  Sulflde Acid-Base Accounting
                                         Sulfide Analysis by Acid-Base accounting
Location
Bioreactor II manure chamber
lliftl
Bioreactor II manure chamber
1 lift 3
Bioreactor II manure chamber
1 lift 5
Bioreactor II manure chamber
1 lift 7
Bioreactor II manure chamber
1 lift 9
Bioreactor II limestone
chamber
Bioreactor II manure chamber
2 lift 5
Bioreactor III manure
chamber 1 lift 1
Bioreactor III manure
chamber 1 lift 3
Bioreactor III manure
chamber 1 lift 5
Bioreactor III manure
chamber 1 lift 7
Bioreactor III manure
chamber 1 lift 9
Total
% Sulfur
0.79
0.70
0.63
0.69
0.46
0.15

0.87

0.76
0.82
1.12
1.02
1.10
J.J.UI »» tHC»
Extractable S
% Sulfate
0.20
0.19
0.03
0.18
0.07
0.07

0.25

0.23
0.28
0.44
0.33
0.52
HQ Extractable S
% Insoluble Sulfide
0.04
0.03
0.14
0.05
<0.01
<0.01

0.06

0.04
0.08
0.13
0.11
<0.01
HNO3 Extractable S
% Pyritic Sulflde
0.48
0.44
0.41
0.41
0.33
0.17

0.50

0.37
0.32
0.41
0.41
0.56
Residual S
% Organic
0.08
0.05
0.06
0.05
0.07
0.02

0.06

0.12
0.14
0.14
0.17
0.06
                                                        49

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Table 6-3c.  Sulflde Acid-Base Accounting
                                        Sulflde Analysis by Acid-Base accounting
Location
Bioreactor IV manure
chamber 1 lift 1
Bioreactor IV manure
chamber 1 lift 3
Bioreactor IV manure
chamber 1 lift 5
Bioreactor IV manure
chamber 1 lift 7
Bioreactor IV manure
chamber 1 lift 9
Bioreactor IV limestone
chamber
Bioreactor IV manure
chamber 2 lift 5
Total
% Sulfur
0.68
0.72
0.84
1.19
0.90
<0.01

0.69
AAJJl »» illCl
Extractable S
% Sulfate
0.21
0.23
0.27
0.81
0.29
<0.01

0.29
HQ Extractable S
% Insoluble Sulflde
0.06
0.02
0.03
<0.01
0.06
<0.01

<0.01
HNO, Extractable S
% Pyritic Sulflde
0.32
0.42
0.46
0.47
0.49
<0.01

0.32
Residual S
% Organic
0.10
0.05
0.08
0.14
0.07
<0.01

0.08
Table 6-3d.  Analytical Results for Plugging Material in the Manifold of Bioreactor HI

                                       Total metals in plugging material (ing/Kg)
       Location
                         Al
                                   As
                                             Cd
                                                        Ca
                                                                  Cu
                                                                             Fe
                                                                                       Mg
                                                                                                 Mn
                                                                                                           Zn
Reactor III intake plug -
brown
Reactor III intake plug -
black
10300
55800
400
485
25.7
31.2
5700
1480
7340
8560
127000
57800
3980
613
25200
35100
2530
2100
 Table 6-4. Correlation Coefficients (•) for Total Metals Concentrations in the Solid Matrix of Bio reactors H, HI, and
 TV
Al
Al 1
As
Cd
Cu
Fe
Mn
Zn
As
0.602
1





Cd
0.475
0.095
1




Cu
0.9703
0.5278
0.476
1



Fe
0.5622
0.9434
0.0175
0.4464
1


Mn
-0.376
-0.281
0.0003
-0.39
-0.096
1

Zn
0.1745
-0.0387
0.8375
0.1559
-0.0747
0.1254
1
                                                       50

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                               Total vs Dissolved Metals
                        for the Cobble Chamber of Bio reactor II
                        Fe
                                                             Cu
                                                        Metals concentration in ug/L
                                                                # Mn
                                                                  D Total metals

                                                                  D Dissolved metals
                                                     Cd
Figure 6-la. Total vs. dissolved metals for the cobble chamber of Bioreactor II.
                                  Suspended Metals
                        in the Cobble Chamber of Bioreactor II
                                                       Metals concentration in ug/L
                                                             Cu
                                                               # Mn
                                                              D Total minus Dissolved
Figure 6-lb. Suspended metals in the cobble chamber of Bioreactor IL
                                             51

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                                   Total vs Dissolved Metals
                           for the Cobble Chamber of Bio reactor III
                                                        Metals concentration are in ug/L
                                                            Cu
                                                                Mn
                                                                   D Total metals
                                                                   D Dissolved metals
Figure 6-2a. Total vs. dissolved metals for the cobble chamber of Bioreactor IIL
                                      Suspended Metals
                            in the Cobble Chamber of Bioreactor III
                                                       Metals concentration in ug/L
                         Fe
                                                             -? Mn
                                                              D Total minus Dissolved
                                                    Cd
Figure 6-2b.  Suspended metals in the cobble chamber of Bioreactor IIL
                                              52

-------
                                   Total vs Dissolved Metals
                           for the Cobble Chamber of Bioreactor IV
                                                      Metals concentration are ug/L
                                                                  D Total metals
                                                                  D Dissolved metals
                                                     Cd
Figure 6-3a. Total vs. dissolved metals for the cobble chamber of Bioreactor IV.
                                      Suspended Metals
                           in the Cobble Chamber for Bioreactor IV
                                                         Metals concentration in ug/L
                                                               s Mn
                                                               D Total minus Dissolved
Figure 6-3b. Suspended metals in the cobble chamber of Bioreactor IV.
                                             53

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                          Iron Speciation for Autopsy Aqueous Samples
                    Bioreactor II    Bioreactor II cobble Bioreactor III cobble   Bioreactor IV   Bioreactor IV cobble
                  limestone chamber     chamber        chamber      limestone chamber      chamber
                                                Location
Figure 6-4.  Iron speciation for autopsy aqueous samples.
                          Total Metals in the Limestone and Cobble Chambers
                                   in Bioreactor II (Aqueous Samples)
Figure 6-5.  Total metals in the limestone and cobble chambers in Bioreactor II (aqueous samples).
                                                54

-------
              Total Metals Concentration in Solid Matrix of Bioreactor II
                                                                Limestone    Chamber 2
                                       Sample location
Figure 6-6. Total metals concentration in the solid matrix of Bioreactor IL
                      Total Metals Concentration in Solid Matrix
                            of Chamber 1 in Bioreactor III
                                           1II/5/1

                                       Sample location
Figure 6-7. Total metals concentration in the solid matrix of Chamber 1 in Bioreactor III.
                                            55

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             Total Metals Concentration in Solid Matrix of Bioreactor IV
                                                                  Limestone    Chamber 2
                                        Sample location
Figure 6-8. Total metals concentration in the solid matrix of Bioreactor IV.
                       Sulfate Profiles in Solid Matrix of Bioreactors
  .2 5000
„. 1st organic chamber
Flow 	 ^. &




^~ •!! solid matrix
^^^^III solid matrix
• • >IV solid matrix
• II aqueous matrix (mg/L)
• III aqueous matrix (mg/L)
A IV aqueous matrix (mg/L)
/
i
\
i
\
f
\
g
i
i
i S
t ^r
/ /^^/ l

^^\" '"'
Ss x^x^
^^' "~H
limestone
chamber







~^A''
2nd i cobble
organic ! chamber
chamber

i
	 i 	
|
i
f \
V I
/
\ i
                                          7th lift
                                           Location
                                                    9th lift
Figure 6-9. Sulfate profiles in the solid matrix of the bioreactors.
                                              56

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                           Sulflde Profiles in Solid Matrix of Bioreactors
  -a
Flow _
"X
^M
^





1st organic chamber
x/"s-r-
«• „. _S / ^
^/'""
+
^~ "II solid matrix
^^^^III solid matrix
~ • 'IV solid matrix
• II aqueous matrix (mg/L)
III aqueous matrix (mg/L)
A IV aqueous matrix (mg/L)

^***7*
	 * ' »
X '
X t
\ 1
\ •
. 1
\ •
\
'



limestone
chamber


/
/
V /
1 /
1 J
g
• ,
* 1
« f
« 1
2nd
organic
ch^nber
/
/
f
g
i
r
g
g
g


cobble
chamber







                                  5th lift
                                             7th lift       9th lift
                                              Location
Figure 6-10.  Sulfide profiles in the solid matrix of the bioreactors.
       I
                            Bioreactor III Inlet-Plug Metals Concentration
                                    D Reactor III intake plug -
                                     lower (brown) portion
                                    • Reactor III intake plug -
                                     upper (black) portion
Figure 6-11. Bioreactor III inlet-plug metals concentrations.
                                                  57

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                        7.   Quality Assurance/Quality Control
7.1  Background
Following is a summary of the Quality Assurance
(QA) activities associated with the project.
Samples were collected according to the schedule
outlined in the approved project-specific QAPP
document. Performance data were collected
monthly for 32 months. All field and laboratory
data available had been evaluated to determine the
usability of the data. Dissolved metals (Al, Cd,
Cu, Fe, Mn, and Zn) analysis and field pH
measurements were classified as critical analyses
for this project.  A critical analysis is an analysis
that must be performed in order to determine if
project objectives were achieved. Data from
noncritical analyses were also evaluated.

7.2  Project Reviews
During the project, the following evaluations were
performed:

-  internal field systems review at the
   demonstration site; and
-  external Technical Systems Audit (TSA).

7.2.1  Internal Field Systems Review at the
Demonstration Site
A field systems review was performed on October
10, 1999, at the Calliope Mine.  The field  systems
review included a review of the following items:

-  personnel, facilities, and equipment;
-  documentation (chain-of-custody, logbooks);
-  calibration of equipment; and
-  sampling procedures.

No concerns were identified during the audit.

7.2.1.1  Personnel, Facilities, and Equipment
Personnel present during the audit included John
Trudnowski, MSB Project Engineer; Rod  Schwab,
MSB Sampler; and Ken Reick, MSB Project QA
Officer. The Project Engineer and Sampler were
knowledgeable about the demonstration and their
duties and responsibilities at the demonstration site.

7.2.1.2  Documentation
Chain-of-custodies (COC) were reviewed at the
demonstration site, and all COC procedures were
being followed. The project logbooks were also
reviewed.  The sampling logbook was very
thorough and included spaces where specific
information was required.  Sampling personnel
were familiar with the logbook format and COC
procedures.  The sampling logbook did not conform
to the Standard Operating Procedures (SOP)
because the pages of the logbook were not
numbered consecutively as stated in the SOP.

7.2.1.3  Calibration of Equipment
Field equipment was used to measure pH, dissolved
oxygen, temperature, and EH potential. This
information was recorded in the project logbooks.
All meters were properly calibrated prior to
performing measurements.  Standard operating
procedures were available at the demonstration site
and explained how to calibrate/operate the meters.
Sampling personnel were familiar with the SOPs
and requirements for routine calibration of the
various meters.

Measurement of pH in the water samples is a
critical measurement and, therefore, has had quality
control (QC) objectives already assigned.  The
quantitative objectives are accuracy, precision, and
completeness.  The absolute difference between the
measurement and the buffer pH is reported as
accuracy.  Precision is based on consecutive
determinations of accuracy. During sampling,
accuracy was determined by measuring the
appropriate buffer; however, it was performed only
once (this QC check was well within the required
limits). Because consecutive determinations were
not performed, precision could not be determined.
For future sampling events, the samplers  were
                                              58

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required to determine the accuracy and precision of
the pH measurements.

As a corrective action for the audit observations, an
amended QAPP was prepared.  The need for
accuracy and precision measurements associated
with critical field measurements were reiterated in
annual MSB internal sampling refresher training.

7.2.1.4  Sampling Procedures
A review of sampling activities was also performed
during the systems review.  All sample collection
procedures and equipment decontamination
procedures were followed by sampling personnel.

7.2.2  External Technical Systems Audit
In addition to the internal field systems review by
MSB, an external TSA of both the project and the
HKM Laboratory was performed by Joe Evans of
Science Applications International Corporation
(subcontractor to EPA) during the week of
September 25, 2000.  There were no findings
resulting from  the audit; however, there were three
observations. All three observations related to
making minor  changes to the QAPP to more
accurately reflect field procedures. An amended
project-specific QAPP was prepared as a result of
the audit.

7.3  Data Evaluation
The data quality indicator objectives for field pH
measurements  and dissolved metals analysis were
outlined in the  QAPP and were compatible with
project objectives and the methods of determination
being used. The data quality indicator objectives
were method detection limits (MDLs) for accuracy,
precision, and completeness. Control limits for
each of these objectives are summarized in
Table 7-1.

In addition to the data quality indicators listed in
Table 7-1, HKM also analyzed internal QC checks,
including calibration, calibration verification
checks, calibration blanks, method blanks, and
laboratory control samples. These QC checks have
also been evaluated for the purpose of this data
review.

7.4  Validation Procedures
Data that were generated throughout the project
were validated. The purpose of data validation is
to determine the usability of data generated during
a project. Data validation consists of two separate
evaluations: 1) an analytical evaluation and 2) a
program evaluation.

7.4.1  Analytical Evaluation
An analytical evaluation is performed to determine
the following:

-  that all analyses were performed within
   specified holding times;
-  that calibration procedures were followed
   correctly by field and laboratory personnel;
-  that laboratory analytical blanks contain no
   significant contamination;
-  that all necessary independent check standards
   were prepared and analyzed at the proper
   frequency and that all remained within control
   limits;
-  that duplicate sample analysis was performed at
   the proper frequency and that all Relative
   Percent Differences (RPD) were within
   specified control limits;
-  that matrix spike sample analysis was
   performed at the proper frequency; and
-  that all spike recoveries (%R) were within
   specified control limits.

Measurements that fall outside of the control limits
specified in the QAPP, or for other reasons were
judged to be outlier, were flagged appropriately to
indicate that the data were judged to be estimated
or unusable.

An analytical evaluation was performed to
determine the usability of data that were generated
by the HKM Laboratory for the project.
Laboratory data validation was performed using
                                               59

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Reference 7 as a guide. The QC criteria outlined in
the QAPP were also used to identify outlier data
and to determine the usability of the data for each
analysis. A summary of QC check results for the
critical dissolved metals and pH analyses are
presented in Table 7-2. All data requiring flags are
summarized in Table 7-3. In addition to the
analytical evaluation, a program evaluation was
performed.

7.5  Program Evaluation
Program evaluations include an examination of
data generated during the project to determine:

-  that all samples, including field QC samples,
   were collected, sent to the appropriate
   laboratory for analysis, and were analyzed and
   reported by the laboratory for the appropriate
   analyses;
-  that all field blanks contained no significant
   contamination; and
-  that all field duplicate samples demonstrated
   precision of field as well as laboratory
   procedures by remaining within control limits
   established for RPD.

Program data that were inconsistent or incomplete
and did not meet the QC objectives outlined in the
QAPP were viewed as program outliers and were
flagged appropriately to indicate the usability of the
data.

7.5.1  Field QC Samples
In addition to internal laboratory checks, field QC
samples were collected to determine overall
program performance.

7.5.2  Field Blanks
None of the field blanks collected for the project
showed significant contamination for dissolved
metals analysis, with two exceptions.  The field
blank collected on June 3, 1999, showed significant
contamination (greater than the Contract Required
Detection Limit under the Contract Laboratory
Program). Iron samples with concentrations less
than 10 times the contamination concentration in
the field blank were flagged "U."  A "U" flag
indicates the data are undetected below the
associated value. Another field blank collected on
August 11, 1999, showed significant contamination
for manganese analysis; however, all of the sample
concentrations were greater than 10 times the
contamination, so no samples required a flag.
Early in the project, several field blanks showed
significant contamination for cadmium  and zinc.
The problem was investigated and traced to the
holding tank being used to store deionized (DI)
water. A clean tank solved the problem.  Samples
were not flagged because the contamination source
was not linked to contamination problems resulting
from sampling procedures.

7.5.3  Field Duplicates
All field duplicates collected were within control
limits for all analyses, with the four exceptions. A
field duplicate collected on January 8, 2001, was
out of control for aluminum analysis. While EPA
does not specify control limits for field duplicates,
the data reviewer is allowed discretion when
evaluating field duplicates.  For this project,
precision control limits of • 35% RPD were used
for field duplicates.  As a result, the samples from
the January 8, 2001, event were flagged "J." A "J"
flag indicates that the associated value is estimated.
Field duplicates samples collected on April 5,
1999, and February 12, 2001,  were out of control
for copper analyses, resulting in samples from
these events being flagged "J"  for copper. A field
blank collected on February 3, 1999, was out of
control for iron analysis, and associated samples
were flagged "J."

7.6   Summary
All data from the HKM Laboratory were validated
according to EPA guidelines and the project-
specific QAPP. Some of the data were flagged for
various reasons and are summarized in Table 7-3.
                                                60

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The importance of calibration of field meters
should be reiterated to sampling personnel because
the lack of calibration resulted in critical pH data
from two sampling events being discarded.  On a
positive note, the data were very organized,  which
made the data evaluation process much easier.
                                     MWTP, Activity III, Project 12 presented unique
                                     challenges for the sampling and analytical team.
                                     While several of the data points were qualified for
                                     various reasons, this multi-year project produced
                                     high quality data.
Table 7-1. Data Quality Indicator Objectives
   Parameter
Matrix
Unit
MDLa
Precision1"
Accuracyc0
Completeness11
       pH         Aqueous        SU          N/A            ±0.1e              ±0.1f             95%
 Dissolved Metals   Aqueous        jjg/L           5             -20%            75-125%           95%
 aMDLs were based on what is achievable by the methods and what is necessary to achieve project objectives and account for
 anticipated dilutions to eliminate matrix interferences. MDLs were adjusted as necessary when dilutions of concentrated
 samples are required.
 bRelative percent difference of analytical sample duplicates.
 'Percent recovery of matrix spike, unless otherwise indicated.
 dBased on number of valid measurements compared to the total number of samples.
 Trecision of pH measurements was based on consecutive determinations of accuracy.
 'Accuracy of pH measurements was based on the absolute difference between accepted value of the buffer and the measured
 value of the buffer.

Table 7-2. Summary of QC Checks for Critical Field pH Measurements and Dissolved Metals Analysis
 Analysis
 Field pH Measurements
             Mean Absolute Difference (Precision)
             0.02 SU
                                   Range of Absolute Differences (Precision)
                                   0-0.09 SU
 Analysis
 Dissolved Al
 Dissolved As
 Dissolved Cd
 Dissolved Cu
 Dissolved Fe
 Dissolved Mn
 Dissolved Zn
 Analysis
 Field pH Measurements
            Mean RPD for Sample Duplicates
            9.9%
            7.4%
            2.8%
            8.4%
            4.6%
            2.6%
            8.2%
            Mean Absolute Difference (Accuracy)
            0.02 SU
                                   Range of RPDs for Sample Duplicates
                                   0-28.3%
                                   1.3-18.8%
                                   0.9-8%
                                   0-50.6%
                                   0-12.6%
                                   0-8.4%
                                   0-70.3%
                                   Range of Absolute Differences (Accuracy)
                                   0-0.09 SU
 Analysis
 Dissolved Al
 Dissolved As
 Dissolved Cd
 Dissolved Cu
 Dissolved Fe
 Dissolved Mn
 Dissolved Zn
            Mean Matrix Spike Recovery
            101%
            95.5%
            99.6%
            100.4%
            101.1%
            102.2%
            98.2%
                                   Range of Matrix Spike Recoveries
                                   82-131%
                                   58.9-114%
                                   88-110%
                                   84-115%
                                   82.2-134%
                                   79.3%-127%
                                   69.6-114.8%
                                                    61

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Table 7-3.
Date1
12/23/98





01/04/99

02/03/99



Summary of Qualified Data for MWTP Activity IE, Project 12
Sample Analysis QC
ID Criteria
BDRB Dissolved Analytical
D- Cu Duplicate
Blank
IAD
IILD
IIIA
IVKD
IVKDD
IVKDS
HID Alkalinity Initial
IIIJD (forms) Calibration
IVKD Verification
IIIA

ID Dissolved Field
IIIA Fe Duplicate
IIIAFC
IIIJD
IILD
IVKD
Control Result Flag2 Comment
Limit
• 20% RPD 50.6 J Samples were flagged "J"
due to an out-of-control
field duplicate.





90- 110% recovery 63% R Initial calibration
recovery verification had <75%
recovery; therefore, the
data should be removed
from consideration.
• 35% RPD 47.4% RPD J Flag samples "J" for out-
of-control field duplicate.



04/05/99
Field pH
Holding Time    Analyze
                immediately
                                                                        Hours
                                                                        elapsed
04/05/99    ID
            IIIA
            IIIAFC
            IIIJD
            IILD
            IVKD

06/03/99    ID
            IIIA
            IIIAFC
            IIIJD
            IILD
            IVKD
Dissolved
Cu
Field
Duplicate
• 35%RPD
                     165% RPD
                       Because pH readings were
                       not performed
                       immediately in the field
                       but instead analyzed in the
                       laboratory following
                       transport to Butte, the pH
                       readings from this
                       sampling event should be
                       considered estimated and
                       flagged with a "J."

                       Flag samples  "J" for out-
                       of-control field duplicate.
Dissolved
Fe
Field Blank
 100 ppb
128 ppb
                                                    U
Flag samples with
concentrations less than 10
times the contamination
concentration  "U" due to
out-of-control field blank.
                                                           62

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Date1
7/12/99



08/11/99




08/11/99
04/05/00




06/07/00





12/04/00


Sample
ID
BDRB
FB
ID
IIIA
IIIAFC
IIIJD
IILD
IVKD
BDRB
FB
ID
IIIA
IIIAFC
IIIJD
IILD
IVKD

BDRB
FB
ID
IIIA
IIIAFC
IIIJD
IILD
IVKD
BDRB
FB
ID
IIIA
IIIAFC
IIIJD
IILD
IVKD
Intake
IIBD
IIIBD
IVB
IILD
IIIJD
IVKD
Analysis QC
Criteria
Dissolved Matrix Spike
Zn
Analytical
Duplicate



Dissolved Matrix Spike
Zn




Field pH Calibration
Dissolved Matrix Spike
As




Dissolved Serial Dilution
Zn




Field pH Calibration
Field ORP

Control Result
Limit
75- 125% recovery 69.6%
• 20% RPD 70.3%



75- 125% recovery 72.8




Required each Not
sampling event performed
75- 125% recovery 58.9




• 10% difference 15.7%





Required each Not
sampling event performed
Required each Not
sampling event performed

Flag2 Comment
J Zinc results should be
flagged "J" as estimated
for out-of-control spike
and duplicate.



J Zinc results should be
flagged "J" as estimated
for out-of-control matrix
spike.



R Flag samples "R" as
unusable due to lack of
calibration documentation.
J Arsenic results should be
flagged "J" as estimated
for out-of-control matrix
spike.



J Zinc results should be
flagged "J" as estimated
due to out-of-control serial
dilution analysis.



R Flag samples "R" as
unusable due to lack of
calibration documentation.


63

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Date1 Sample Analysis QC
ID Criteria
1/8/01 Intake Dissolved Field
IIBD Al Duplicate
IIIBD
IVB
IILD
IIIJD
IVKD
2/12/01 Intake Dissolved Field
IIBD Cu Duplicate
IIIBD
IVB
IILD
IIIJD
IVKD
3/14/01 IIBD Sulfate Outlier



4/09/01 Intake Sulfate Outlier



7/20/01 RCIIC Dissolved Matrix Spike
OB Al
RCIIIC
OB Dissolved
RCIVC Fe
OB
Dissolved
Mn
Control Result Flag2 Comment
Limit
• 35% RPD 40. 1% RPD J Flag samples " J" for out-
of-control field duplicate.




• 35% RPD 40. 1% RPD J Flag samples " J" for out-
of-control field duplicate.




Rosner's Test for l,400mg/L R Sample is outlier
Outliers is an outlier according to Rosner' s test.
at a 5% Data were transformed to
significance achieve normal
level distribution prior to
performing outlier test.
The data should be
removed from further
consideration.
Rosner's Test for 8 mg/L is an R Sample is outlier
Outliers outlier at a according to Rosner' s test.
5% Data were transformed to
significance achieve normal
level distribution prior to
performing outlier test.
The data should be
removed from further
consideration.
75-125% recovery 130.7% J Flag samples "J" for out-
of-control matrix spike.
134.5%


127.5%

1 Date that the samples were collected.
2 Data Qualifier Definitions:

 U-The material was analyzed for but was not detected above the level of the associated value (quantitation or detection limit).
 J-The sample results are estimated.
 R-The sample results are unusable.
 UJ-The material was analyzed for but was not detected, and the associated value is estimated
                                                            64

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                     8.   Recommended Design Improvements
To minimize AMD stratification with respect to
ORP and thus increasing bioreactors efficiency,
the bioreactors need to be covered with a plastic
liner that would minimize oxygen intrusion
either directly from the atmosphere or through
atmospheric precipitation.  The layer of two lifts
of straw bails used at the demonstration site was
not sufficient.

Because the biochemical reactions take place
within the organic matter where they also
precipitate, there is no need for a large cobble
chamber.

If a preventive "trap sump" to collect a small
mass of precipitates that might escape from the
organic matter chamber needs to be  included in
the design, it should have its outlet placed high
above its bottom. A trap sump could be
designed as a flow-through container or a small
lined retention pond filled with cobbles
supporting a plastic liner covering the pond.
Other designs could also be considered,
provided they would minimize the possibility of
agitation of the collected effluent of the
bioreactor by atmospheric conditions (wind and
precipitation) or human and animal encounters.

The abundance of TOC present in the organic
matter chamber at the end of the project
demonstrates that the bioreactors would have
worked equally efficiently with a much smaller
supply of organic carbon, provided the same
residence time of AMD was maintained. Since
most of the organic matter mass inhibits
permeability, it is prudent to reduce the ratio of
organic matter and the permeability enhancing
component (e.g., gravel, shell, etc.) and have a
more permeable medium.

Although not explicitly indicated by the
demonstration project results, the straw added to
the organic matter does not seem to be an
appropriate material to increase the permeability
of the medium. More rigid material like walnut
shells or even gravel, added in high proportion
to the organic matter, is needed to effectively
increase permeability.

Since most of the plugging material that
restricted the flow was found within and
adjacent to the outlets of the AMD distribution
system, there is a need to devise a system that
would allow for occasional breakdown and
removal of the plugging material. Such a
system may need to involve only a few outlets
rather than the three dozen used in this
demonstration. It might include ports extended
to the ground surface that would facilitate
blowing in combustion engine exhaust to
destroy plugging material that would then be
removed by bailing.

If any instrumentation for automatic
measurements of the AMD quality and  quantity
is used, it is recommended that a filter be
installed upstream of the sensor.
                                            65

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                                    9.   Conclusions
A CCS built in the bioreactors worked very well
in preventing settling of organic matter and
ensuring uniform flow of AMD throughout the
entire cross section of the organic matter with
no preferential flow paths (channeling).

Most of the individual cells of the CCS were
found full of organic matter, with probably less
than 5% of the cells void of the organic matter
in their top 2-inch portions. This indicates that
the organic matter could have been packed less
tightly and still conform to the design
parameters that allowed approximately 3 inches
of settling.

Configuring the bioreactors to accommodate
flow in a horizontal plane (rather than in the
vertical direction) was successful. Problems
that were experienced with reduction in the flow
rate turned out to be associated with the AMD
distribution system that plugged with chemical
            J         r   OO
precipitates.  However, this hindrance is
common to both configurations.

It takes some time for the SRB to be established
in a new bioreactor.  Once established and
supplied with organic carbon, they maintained a
population of E+4 MPN/mL or higher in an
aqueous phase at temperatures  ranging from 2
•Cto 16-C.

Winter freezing of a well-established SRB
population has little or no effect on their activity
for the remainder of the year.

The SRB average population of 2.06E+6
MPN/cc attached to solid matrix of organic
matter was two orders of magnitude greater than
the value of 2.81 E+4 MPN/mL calculated as an
average SRB population in the  aqueous samples
collected during the last sampling event on
July 9, 2001.
The SRB population attached to the limestone
was of the same order of magnitude (E+4) as for
the aqueous samples.

High sulfide load in the organic matter, together
with high concentrations of metals, is indicative
of metal sulfides precipitating in the organic
matter chamber due to SRB activities.

A drop in the SRB population in July of 2000,
which paralleled the 100% increase of the flow
rates, might indicate flushing out of the bacteria
at that flow velocity.

Although it appeared a limestone chamber
slightly increased effluent pH, its role was not
dominant for the overall performance of the
bioreactors.

The EH values measured in the most
downgradient piezometer show that with the
exception of a few periods, the EH values were
positive. However, data acquired during the EH
profiling of the bioreactor document that the EH
values in the deep portion of the organic matter
were approximately 50 mV and 150 mV lower
for Bioreactors II and III respectively. This is
an important observation because it confirms
that reduced conditions, which are required for
the SRB activity, were present within the
portions of the bioreactors where most of the
biochemical reactions took place and where
most metals precipitated.

The bioreactors were notably stratified with
respect to EH. In Bioreactor II, it was up to
400 mV lower at the bottom of the bioreactor
than close to the surface. This difference, thus
also stratification, diminished downgradient and
close to the outlet from the bioreactors due to
mixing of water flowing through the cobble
section.  It is postulated that the EH
                                             66

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stratification was caused by an inadequate
isolation of atmospheric air at the top of the
bioreactors.

The alkalinity buildup in the effluents is a good
indication of biochemical reactions (i.e., sulfate
reduction) taking place in the bioreactors.

Data acquired from the project indicate that only
Zn, Cu, and Cd were being removed as sulfides
due to SRB activities.  This statement is based
on the stoichiometric balance that includes
analytical data for metals and sulfide and is also
supported by Mn data that indicate that the
adsorptive capacity of the organic matter was
exhausted after 8 months of operation.

Changes in concentrations of other metals,
which precipitated not necessarily as sulfides,
seem to be affected by the SRB only in an
indirect manner, by responding to changes of
chemical conditions caused by the SRB.

Zinc removal thresholds of 500 (ig/L for
Bioreactors II and IV and 800 (ig/L for
Bioreactor III seem to be independent of influent
concentrations.

Copper removal thresholds of 50 (ig/L for
Bioreactors II and IV and 80 (ig/L for
Bioreactor III also seem to be independent of
influent concentrations.

Cadmium removal thresholds of 5 (ig/L that
prevailed for most of the operating time
decreased when the Cd concentration of the
influent dropped to 1 • g/L.

Different Zn and Cu removal thresholds for
Bioreactor III than the respective removal
thresholds for Bioreactors II and IV indicate
that the thresholds depend on the configuration
of the bioreactors but are not affected by the
closing up and freezing of a bioreactor during
winter.
A slightly lower metal removal efficiency in
Bioreactor III that contained only one chamber
with organic matter may indicate that the
residence time of 10 hours within the organic
carbon matter is close to minimal. This
residence time may vary for different climates.

Bioreactor III, with only one organic matter
chamber and no limestone chamber, was
noticeably less efficient in creating a reducing
environment and also less efficient in removing
dissolved metals.  It is precarious to
discriminate whether Bioreactor III was less
efficient due to the absence of a limestone
chamber or a second organic matter chamber.

Evidence of metal sulfides precipitating in the
organic matter chambers, together with the
observation of no precipitate in the cobble
chambers, indicates that the cobble chamber
was not essential for biochemical reaction.

For this demonstration, the role of the cobble
chamber was limited to a collection sump for a
small mass of precipitates that escaped from the
organic matter chambers.

The autopsy on the bioreactors revealed a
convoluted biochemical environment that was
probably caused by a dramatic change  in the
AMD chemistry after the first 10 months of
operation. The environment examined during
the autopsy included mixed results of processes
that were occurring first at a low pH and a
reasonably high load of metals with the
subsequent reactions that were characteristic for
water with a neutral pH laden  with much less
dissolved metals.

Aqueous samples collected during the autopsy
indicated that only a small portion of the
dissolved metals in reduced form were  balanced
in the solution by sulfides. These were probably
Zn, Cu, Cd, and a small portion of Fe.  Other
metals dissolved in solution, including high
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concentrations of Ca, were electronically balanced
by high concentration of sulfate, bicarbonate, and
assumed-present hydroxide.

•  It is  hypothesized, based on the analytical data
   and visual observation during the autopsy on the
   bioreactors, that water accumulated at the
   bottom of the bioreactors contained large
   amounts of suspended ferric and Al hydroxides.

•  The high concentration of metals that
   accumulated with the solid matrix of the
   bioreactors demonstrated that a large load of
   metals was retained within the organic matter
   material, thus indicating the bioreactors were
   efficient in removing metals from the influent
   AMD.

•  For the autopsy data, there was a trend of
   decreasing concentrations for some metals as
   they were retained along the flow path within
   the first organic matter chamber.  These metals
   include Al, Fe, Cu, and As. The reason behind
   such a behavior remains a conundrum.

•  A high correlation coefficient (• = 0.9434) for
   concentration trends for As and Fe in the  solid
   matrix of the bioreactors seem to support the
   inference that the higher As concentration in the
   effluents rather than influent was controlled by
   sorption processes of As to Fe(OH)3.

•  In general, sulfide concentrations in the organic
   matter chambers were above the 4,000 mg/kg
   level and were twice as high as concentrations
   of sulfates. This large sulfide load together with
   high metal concentrations is indicative of metal
   sulfides precipitating in the organic matter
   chamber due to  SRB activities.
Although TOC concentrations for the initial
fresh organic matter were not measured, the
high TOC values measured during the autopsy
strongly indicate that the depletion of organic
carbon would not be a factor for the efficient
removal of metals even if these bioreactors were
operating for several more years.

Plugging of the bioreactors took place within or
immediately adjacent to the AMD distribution
system. This statement is supported by the
water level decrease between the inlet sump and
first organic matter chamber. Also supporting
this conclusion were the observations  during the
autopsy that more chemical precipitate was
present in the front portion of the bioreactors
than downstream.

The high percentage of Fe and Al hydroxides in
the plug removed from the AMD inlet to
Bioreactor III indicates a high probability that
bioreactor inlets were plugging mainly due to
chemical reactions; the sediment carried by the
AMD was a secondary reason for plugging.

The reliability of transducer-/sensor-generated
measurements was poor especially for the pH
sensors that were coated with precipitate within
several weeks. Part of the problem was a very
slow flow that did not allow for dynamic
cleaning of the sensor.  The low flow  rate also
inhibited measurements because of the necessity
of using a small diameter vortex that was prone
to plugging.

If sulfide concentrations in aqueous samples
from the influent and effluent are to be
indicative of the bioreactor performance, they
need to be filtered prior to laboratory  analysis.
In addition, the initial and final concentrations
of sulfate and sulfide in the organic matter need
to be known.
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                                      10.  References
1.   Zaluski, M., M. Foote, K. Manchester, M.
    Canty, M. Willis, J. Consort, J. Trudnowski,
    M. Johnson, and M.A. Harrington-Baker,
    "Design and Construction of Bioreactors with
    Sulfate-Reducing Bacteria for Acid Mine
    Drainage Control," Proceedings of The Fifth
    International In Situ and On-Site
    Bioremediation Symposium, Battelle Press,
    San Diego, CA, April 19-22, 1999.

2.   MSB Technology Applications, Inc., Quality
    Assurance Project Plan for Sulfate-Reducing
    Bacteria Reactive Wall Demonstration
    Project, Mine Waste Technology Program;
    Activity III, Project 12, Phase 2,  1998.

3.   MSB Technology Applications, Inc., Work
    Plan-Sulfate'-Reducing Bacteria Reductive
    Wall Demonstration, Mine Waste
    Technology Program; Activity III, Project 12,
    Phase 2, 1998.
4.   Greenberg, A.F., L.S. Clesceri, A.D. Eaton,
    "Standard Methods for Examination of Water
    and Wastewater," American Public Health
    Association, Section 2580 B, 1992.

5.   Canty, M., "Overview of the Sulfate-Reducing
    Bacteria Demonstration Project
    under the Mine Waste Technology Program,"
    Mining Engineering, June 1999.

6.   Robins, RG. and J.C.Y. Huang, "The
    Adsorption of Arsenate Ion by Ferric
    Hydroxide," Proceedings of the Arsenic
    Metallurgy Symposium, TMS/AIME Annual
    Conference, Phoenix, AZ, January 1988.

7.   EPA, USEPA Contract Laboratory Program
    National Functional Guidelines for
    Inorganics Data Review, 1994.
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