CHARACTERIZATION, LEACHABlLlTY AND AClD MINE DRAINAGE

POTENTIAL OF GEOTHERMkL SOLID RESIDUES

Genandrialine Laquian Peralta

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy
Department of Chemical Engineering and Applied Chemistry
University of Toronto

Q Copyright by G. L. Peralta. 1997

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Geothermal Solid Residues
PhD 1997
Genandrialine Laquian Peralta
Department of Chernical Engineering & Applied Chemistry
University of Toronto

ABSTRACT

A solid waste is classified as hazardous if it contains sufficient leachable components to

contaminate the groundwater and the environment if disposed in a landfill. Solid residues
from three geothermal fields (Bulalo, Philippines, Cerro Prieto, Mexico, and Dixie Valley,
USA) containing S, Cu, Zn, and Pb at levels above earth's crustal abundance were studied
for their leachability. Several procedures were used to assess the potential mobility of the
elernents - protocol leaching tests, sequential chemical extraction, and acid mine drainage
potential test. In addition, whole rock analysis, optical and electron microscopy, X-ray
diffraction, radioactivity counting, and toxicity testing were also performed. The geothermal
residues are mostly silica (70%) with trace elements and have varying crystalline and
amorphous character. All the samples tested can be classified as nonhazardous since their
leachate quality is below the regulatory limits. Toxicity tests were negative and radioactivity
counts were within norm. Sequential extraction indicated the potential for metal release into
the environment but only under extreme conditions (pHs 2, 85-175 OC). Batch kinetic tests
identified that leaching of Pb in the presence of oxygen is diffusion-controlled with a rate
equation, r = 4.6 x 10" a, t ""+ 1.1 x 1O4 a, t''" mol.L'' .h-' . A batch reactor technique
(AMDP test) using the iron and suifur oxidizing bacteria, Thiobaci//usferrooxidans, was
developed for geothenal wastes to predict their acid mine drainage and bioleaching
potential. It was observecf that almost 100% of Cu and Zn in the Mexican scale and less
than 2% in the Philippine scale and sludge were released. Despite a significant Pb content,
only <6% leached from the Mexican scale. Geochemical thermodynamic modelling using
MINTEQAZ showed that much heavy metal content must be inaccessible to the leach
medium. The hazard and risk involved from geothermal residues were assessed to be very
low for the Mexican sludge and drilling mud and the American scale. However, a low but
manageable risk was attributed to the Philippine scale and sludge. The fine sized Mexican
scale was found to have medium risk that will require special handling prior to landfill
disposal.
 I grareruiiy acrtnowieage rne Toiiowing wno nave conrriDutea ro my r n u sruaies:
My supervisor Prof. Donald W. Kirk who was ever wise, helpful, patient, and friendly.
I was fortunate for having done my PhD under his supervision.
My Reading Cornmittee - Prof. Robert E. Jeivis, Prof. Vladirniros G. Papangelakis,
and Prof. Patricia L. Seyfried with the other examinen: Prof. D. Grant Allen, Prof. Grant
Ferris and Prof. Kostas Fytas (Universite' Laval, Quebec) for their comments and advice.
Professon, colleagues and friends who provided valuable contribution to my thesis
in t e n s of equipment, comments, and services: Prof. Greg Evans and Dr. Sandu Sonoc
for radioactivity counting, Prof. Patricia L. Seyfried and Ms. Sheree Yin for the toxicity
tests, Dr. John W. Graydon for the photomicrographs, XRAL Laboratory for elemental and
bulk analyses, Dr. Srebri Petrov for X-ray diffraction, Mr. Battista for transmission electron
microscopy, Mr. Fred Neub for assistance with light microscope and videotaping, Prof.
Grant Allen for the incubatorlshaker, Dr. Dmitri Rubisov for his suggestion on particle sire
analysis and leaching experiments, Dr. Karen Liu for support in optical microscopy with
image analysis, Mr. Durga Prasad for assistance with the autoclave and initial bacteria
culture, Mr. Jeff Bain and Prof. Charles Jia for assistance in geochemical modelling, Dr.
Martin H. Birley for introducing Endnote reference software, Dr. Taylor Eighmy for
introducing MINTEQA2, and Mr. Rene C. Peralta for computer support and maintenance.
O

The World Bank and the University of the Philippines (UP) for my scholarship
especially Dr. Francisco L. Viray and Dr. Reynaldo Vea of UP as well as Dr. Estrella F.
Alabastro, Ms. Lydia Tansinsin, and Ms. Teody Dayoan of DOSTESEP.
The geothermal community especially Dr. Marcelo Lippmann of Lawrence Berkeley
Laboratoiy, California for his advice, networking and assistance. Geothermal companies
who provided samples for this study: Philippine Geothermal Inc., Oxbow Geothermal Corp.
Nevada, USA, and Gerencia de Proyectos Geotermoelectricos, CFE of Mexico.
Colleagues and friends in the laboratory notably Dr. John Graydon, Mr. Cam Nhan,
and Mr. Chris Chan for being there to listen and iend assistance. The administrative staff
of our department and the International Student Centre especially Liz Paterson.
My dear friends whom I cannot al1 mention here but are listed in my Christmas card
directory. Some friends who through e-mail have sent technical and moral support
especially Ms. Jane Y. Gerardo, Dr. Efren F. Abaya, Dr. Martin H. Birley, Mr. Robert Bos,
Mr. Florencio Ballesteros, Mrs. Dionisia Ali, Dr. Keryn Lian, and Dr. Michael Gattrell.
Relatives particularly rny parents Antonio and Gloria Laquian and parents-in-law
Paterno and Remedios Peralta k r their prayers and full support.
My wonderful family - husband Gil Renato (Rene), my two sons, Kevin and Patrick -
for their invaluable support, patience, understanding, affection, massages, and share of
household chores. I dedicate this thesis to them.
Abstract ii
Acknowledgments iii
Table of Contents iv
List of Figures viii
List of Tables x
Nomenclature xi
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Objectives 2
1.3 Thesis Overview 2
CHAPTER 2 BACKGROUND AND LITERATURE REVIEW 3
2.1 Environmental Impacts of Geotherrnal Residues 3
2.1.1 Geothermal Energy and its Environmental Impacts 3
2.1.2 Treatment and Disposal Practices of Geothermal Residues 6
2.2 Guidelines for Waste Classification and Regulation 7
2.2.1 Average Crustal Abundance of Elements 7
2.2.2 .Leachate Quality Criteria 8
2.2.3 Permissible Heavy Metal Concentrations for Agricultural Use 9
2.2.4 Naturally Occurring Radioactive Materials IO
2.3 Techniques for Waste Characterization IO
2.3.1 Chemical Analysis IO
2.3.2 X-ray Diffraction 11
2.3.3 Radioactivity Counting 12
2.3.4 Optical Microscopy 12
2.3.5 Evaluation of Toxicity 12
2.3.6 Weathering Tendency 13
2.4 Leaching Protocol Tests 13
2.4.1 Principles of Leaching 13
2.4.2 Batch Versus Column Leaching 16
2.4.3 Agitated Extraction Procedures 16
 2.4.4 Sequential Chernical Extraction
2.5 Microbial Leaching
2.5.1 Acid Mine Drainage
2.5.1.1 Occurrence
2.5.1.2 Role of Microorganisms
2.5.2 Mechanism of Bacterial Leaching
2.5.2.1 Direct Method
2.5.2.2 Indirect Method
2.5.3 Factors Affecting Bacterial Leaching
2.5.3.1 Composition of Leaching Medium
2.5.3.2 Oxidation-Reduction Potential
2.5.3.3 Temperature
2.5.3.4 Hydrogen Ion Concentration
2.5.3.5 Agitation and Oxygen Transfer
2.5.3.6 Particle Size and Substrate Concentration
2.6 Prediction of Acid Mine Drainage Potential
2.6.7 Prediction Procedures
2.6.1.1 Static (Initial) Tests
2.6.1.2 Kinetic (Confirmation) Tests
2.6.2 Laboratory Scale Bioleaching Techniques
2.6.2.1 Stationary Flask Technique
2.6.2.2 Shake Flask Technique
2.7 Geochemical Equilibrium Modeling
2.7.1 Different Thermodynamic Models
2.7.2 Applications
CHAPTER 3 METHODS AND PROCEDURES
3.1 Waste Characterization
3.1.1 C hemical Analysis
3.1.2 Radioactivity Counting
3.1.3 X-Ray Diffraction
3.1.4 Optical Microscopy
3.2 Toxicity Testing
 3.3 Sequential Chemical Extraction
3.4 Accelerated Weathering Test
3.5 Protocol Leach Tests
3.5.1 Leachate Extraction Procedure (LEP)
3.5.2 Toxicity Characteristic Leaching Procedure (TCLP)
3.6 Extended Leach Tests
3.6.1 Oxic Conditions
3.6.2 Anoxic Conditions
3.7 Preliminary Acid Mine Drainage Potential Test
3.8 Acid Mine Drainage Confirmation Test
3.8.1 Bacteria Culture and Medium
3.8.2 Acclimation of Inoculum
3.8.3Acid Mine Drainage Potential Test
3.9 Batch Kinetic Experiments
3.9.1 Effects of Agitation, Temperature, and Sterilization
3.9.2 Monitoring and Sampling
3.10 MicrostructuralAnalysis
3.10.1 Light Microscopy with Image Analysis
3.10.2 Transmission Electron Microscopy
3.1 1 Geochemical Modeling
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Waste C haracterization
4.1.1 Chemical Analysis
4.1.2 Radioactivity
4.1.3 X-ray Diffraction
4.1.4 Optical Microscopy
4.1.5 Toxicity Testing
4.1.6 Weathering test
4.2 Protocol Leaching Tests
4.3 Extended Leach Tests
4.3.1 Oxic Conditions
4.3.2 Anoxic Conditions
 sequential Chemicai txtraction
Preliminary Acid Mine Drainage Potential Test
Confirmation of Acid Mine Drainage Potential
4.6.1 Bacterial Growth and Acclimation
4.6.2 Acid Mine Drainage Potential (AMDP) Test
Batch Kinetic Experiments
4.7.7 Effect of Sterilization
4.7.2 Effect of Agitation and Temperature
4.7.3 Effect of Bacteria
Reaction Rates and Mechanisms
4.8.1 Chemical Leaching Kinetics
4.8.2 Metal Solubilization Rate
Cornparison Between Acid Leaching and Bioleaching
4.9.1 Between TCLP and LEP
4.9.2 Between TCLP and AMDP
4.9.3 Evaluation of the AMDP Procedure
Geochemical Modeling
Sumrnary
Risk Assessrnent of Geothermal Residues
CHAPTER 5 CONCLUSIONS
CHAPTER 6 PROPOSED FUTURE STUDIES
REFERENCES
APPENDICES
A -Trial Experiments Prior to Procedure Development
B - Bacterial Density Estimation for Thiobacillus ferrooxidans
C - Calculation for the Preliminary Acid Mine Drainage Potential Results
D - About the Geochemical Model MINTEQAZ
E - Input Data Derivation for Geochemical Model
F - Sample Output of Geochemical Modelling
G - Uoff Acid Mine Drainage Potential Test
H - Results of Toxicity Testing
1 - Additional Dissolution Kinetics Data
Fig. 2.1 Schematic model of a hot-water geothermal system
Fig. 2.2 Schematic drawing showing the leaching of an ore particle
Fig. 4.1 Photomicrographs of Philippine scale, PSC (1OOx, 250x)
Fig. 4.2 Photomicrograph of Philippine sludge, PSL (50x)
Fig. 4.3 Photomicrograph of American scale, ASC (50x)
Fig. 4.4 Photomicrograph of Mexican drilling mud, MDM (50x)
Fig. 4.5 Photomicrograph of Mexican scale, MSC (1000~)
Fig. 4.6 Photomicrograph of Mexican sludge, MSL (1OOx, 50x)
Fig. 4.7 Comparison of extent of leaching between LEP and TCLP
Fig. 4.8 Comparison of extended TCLP leaching of Pb coarse and fine
Fig. 4.9 Dissolution behavior in the oxic extended TCLP
Fig. 4.1 O Dissolution behavior in the anoxic extended TCLP
Fig. 4.1 1 Sequential extraction results for PSC and PSL
Fig. 4.12 Sequential extraction results for ASC and MDM
Fig. 4.13 Sequential extraction results for MSC and MSL
Fig. 4.14a Photomicrograph of T. ferooxidans : high density (800x)
Fig. 4.14b Photomicrograph of T. ferooxidans : medium density (800x)
Fig. 4 . 1 4 ~ Photomicrograph of T. fenooxidans : low density (800x)
Fig. 4.14d Photomicrograph of T. ferooxidans with a cloud-like capsule
Fig. 4.15 TEM photomicrograph of various sizes of T. ferrooxidans (60K x)
Fig. 4.16 T E M photomicrograph of T. fenooxidans about to partition (99K x)
Fig. 4.17 pH-Eh change over tirne in AMD experiments
Fig. 4.18 Leaching in agitated AMD experiments : PSC, PSL and MSC
Fig. 4.19 Leaching in stationary AMD experiments : PSC, PSL and MSC
Fig. 4.20 Variations in pH over time for batch kinetic AMD experiments
Fig. 4.21 Variations in Eh over time for batch kinetic AMD experiments
Fig. 4.22 Effect of sterilization on metal bioleaching : PSC
Fig. 4.23 Effect of sterilization on metal bioleaching : PSL
Fig. 4.24 Effect of sterilization on metal bioleaching : MSC
Fig. 4.25 Effect of agitation and temperature on metal bioleaching: PSC
Fig. 4.26 EfFect of agitation and temperature on metal bioleaching : PSL
Fig. 4.27 Effect of agitation and temperature on metal bioleaching : MSC 97
Fig. 4.28 Effect of bacteria on metal bioleaching : PSC 98
Fig. 4.29 Effect of bacteria on metal bioleaching : PSL 99
Fig. 4.30 Effect of bacteria on metal bioleaching : MSC 1O0
Fig. 4.31 Dissolution kinetics of Pb in Mexican scale 102
Fig. 4.32 Overall metal bioleaching, percent over time for MSC 103
Fig. 4.33 Solubilization rate for Cu, Zn, and Pb in the Mexican scale 104
Table 2-1 Average abundance of the elements in crustal rocks
Table 2-2 Leachate quality criteria
Table 2-3 Limits of heavy metal concentration in sludges for agriculture
Table 2-4 Allowable values for metals in the European Union
Table 2-5 Summary of sorne static AMD test methods
Table 2-6 Summary of some kinetic AMD test methods
Table 3-1 Summary procedure for sequential chemical extraction
Table 3-2 Comparison of protocol leaching tests
Table 3-3 Culture medium for Thiobaci//usfenooxidans
Table 3-4 Input data for modeling protocol leach tests
Table 4-1 Chernical analysis of selected geothermal samples
Table 4-2 Crustal abundance ratio of selected geothermal residues
Table 4-3 Concentrations of radionuclides in the geothermal residues
Table 4-4 X-ray diffraction data of selected geothermal residues
Table 4-5 Preliminary acid mine drainage potential test results
Table 4-6 Maximum solubilization rate in batch process
Table 4-7 Comparison between BC Research Confirmation and AMDP Tests
Table 4-8 Sumrnary of results from geochemical modeling of the Mexican scale 109
Table 4-9 Hazard and risk rating for Mexican geothermal residues 114
Table 4-10 Hazard and risk rating for American and Philippine
geothermal residues 715
Seothemal samples :

PSC - Philippine scale

PSL - Philippine sludge
ASC - American scale
MRM - Mexican drilling mud
MSC - Mexican scale
MSL - Mexican sludge
Terms and Procedures :

ABA - acid base accounting

AC - acid consumption
AMD - acid mine drainage
AMDP - acid mine drainage potential
APP - acid production potential
ARD - acid rock drainage
BCRIT - British Columbia Research initial test
BCRCT - British Columbia Research confirmation test
DMSO - dimethylsulfoxide
DSTP - direct sediment testing procedure
ICP - inductively coupled plasma mass spectrometry
LEP - leachate extraction procedure
LOI - loss on ignition
NORM - naturally occuring radioactive materials
ORP - oxidation - reduction potential
SCE - sequential chernical extraction
TCLP - toxicity characteristic leaching procedure
TEM - transmission electron microscopy
XRF - X-ray fluorescence
XRD - X-ray diffraction

Institutions :

AECB - Atomic Energy Control Board

ATCC - American Type Culture Collection
APHA - American Public Health Association
BNL - Brookhaven National Laboratory
CANMET - Canada Centre for Mineral and Energy Technology
EU - European Union (formerly European Community)
ICRP - International Commission on Radiological Protection
UNSCEAR - United Nations Scientific Cornmittee on the Effects of lonizing Radiation
Uoff - University of Toronto
USEPA - United States Environmental Protection Agency
WHO - World Health Organization
W C - Wastewater Technology Centre
 -
Hac acetic acid
Ac - acetate ion
Co- original concentration (mol. L1)
k, - rate constant for coane fraction (mo1.L-'. h -%)
4 - rate constant for fine fraction (rno1.L-'. h -')
a, - coarse fraction '

a, - fine fraction (1 -a,)

t - leaching time, h
Bq - Becquerel
Ci - curie
pCi - picocurie
pSv - microsievert
pm - micron
ppm - part per million

Particle size :

-125 p m = less than 125 pm

-4mm = less than 4 mm
-9.5 + 6 mm = between 6 - 9.5 mm

Conversion:

1 Bq = 27 pCi
1 pCi = 1 x 10''2Ci
1 pm = IO4m
1 ppm = 1 mg/L (in dilute solution)
CHAPTER 1 INTRODUCTION

1.5 Background
Development of appropriate waste management methods requires fundamental
understanding of the physicochemical properties and leaching behavior of the waste
material. Multidisciplinary techniques from chernical engineering, metallurgy,
hydrometallurgy, geology, microbiology, process mineralogy, biohydrometallurgy and
environmental engineering can be utilized to develop an acceptable waste characterization
program. Time and money can be saved in waste management if there was a thorough
knowledge of the characteristics of the waste, the long-term leaching behavior and an
assessment of the risk involved. The same principles are applicable to geothermal wastes.
Geothermal energy has received increasing attention as an attractive alternative to
fossil-fueled energy sources since it is economical and creates less environmental
pollution. It is widely used in almost 25 countries. The exploration and utilization of
geothermal resources generate solid residues such as scale, sludge, and drilling mud. In
this study, geothermal residues were studied in liquid-dominated geothermal systems in
the Philippines (scale and sludge) and Mexico (scale, sludge, and drilling mud). Scale only
was obtained from the US as sludge was not generated in that vapor-dominated
geothermal system. Since these solids contain Fe, Cu, Zn, and Pb at levels above normal
soils, they have been labelled as hazardous and require special treatment and disposal.
There is very little information on geothemal residues with respect to their characteristics,
leaching behavior, and environmental hazard. This study will contribute largely to the
understanding of their true nature in order to be able to recommend appropriate measures
for their management.
In disposing geothermal residues in a landfill, the most serious threat to the
environment is leaching of the toxic components (such as heavy metals) to groundwater.
There is concern that the leachate will contaminate the aquifer which will eventually affect
the human population through ingestion if such an aquifer was used for drinking water or
for irrigation of agricultural lands. Further investigation of conditions and mechanisms
under which metals might be eventually released would clarify any potential environmental
contamination.
1.2 Objectives
The main objective of this thesis was to understand the leaching behavior of
geothemal residues in a landfill environment. It will consider microbial action in evaluating
the environrnental impact of disposing these wastes on land. Specifically, this work will
predict and estimate the possible environmental effects of geothermal wastes in a landfill
through the study of their (a) physico-chemical characteristics, (b) leachability, and ( c) acid
mine drainage potential.

1.3 Thesis Overview

The thesis is composed of six chapters and an appendix. The first chapter is the
introduction which is essentially a thematic foreword about the research along with the
objectives and expected output. The second chapter is a brief background of the study as
well as a review of related literature specifically about geotherrnal energy and its residues,
environrnental impacts, acid and microbial leaching, protocol tests. regulatory limits, acid
mine drainage and geochemical modelling. The third chapter describes of the experimental
work, computer modelling, materials used, methods, measurements, and analytical
techniques. In Chapter 4, under results and discussion, experimental and computational
results are given along with a discussion of their implications in relation to the objectives.
The conclusions arising from the study are listed in Chapter 5 while the proposed future
studies are in Chapter 6. At the end of the six chapters are nine appendices supporting the
main body of the thesis.
CHAPTER 2 BACKGROUND AND LITERATURE REVIEW

2.1 Environmental Impacts of Geothermal Residues

The following section is an introduction to geothermal energy and some of its
importantenvironmental concerns. Past and present waste management practices are also
summarized.

2.1.7 Geothennal Energy and its Environmental Impacts

Geothermal energy for power generation has received increasing attention as an
attractive alternate energy source both due to its environmental and economic advantages.
Where it is abundant and economical to exploit, geothenal power has been used for a
number of years to commercially generate electricity. Geothermal power dates back to
1913 in ltaly and has since spread over the Pacific Rim [Il.It is now an important source
of power in more than 25 countries worldwide with potential in 40 countries [ l , 21.
The environmental impact of any electric power production system is reflected in the
number and complexity of the steps in the fuel and production cycle. Since geothermal
power plants use naturally occurring stearn, there is no need for the complex stearn-
generating systems or extensive mining, processing, storage, or transportation facilities
that are required for other thermal power plants.
The creation of geothermal resources begins with a source of heat - hot or molten
rock, lying close to the earth's surface as shown in Figure 2.1 [3]. The high temperature
rock zone is overlain by a permeable rock formation containing water from precipitation
which rises upward as it is heated by the rock below. Generally the flashed steam process
is used for electricity generation from hot-water systems. In this process, as the hot water
under a very high pressure, is pumped out of the reservoir by wells and as it nears the
surface and the pressure decreases, about 20% of the fluid boils and "flashes" into steam.
Separators are used to separate the steam from the water and the former is directed to
turbines for power generation. Two types of geothermal system exist : liquid-dominated
(hot water) and vapor-dominated (steam) with the former more common worldwide and
having greater environmental concerns. The exploration and utilization of geothermal
resources generate residues such as scale, sludge, and drilling mud. Scale is deposited
in steam gathering systems, wellbores, separaton, and turbine blades and is manually
removed during preventive maintenance shutdown. The water leaving the separators is
available for further processing depending on its mineral content. It is woled and allowed
to partially evaporate in a cooling or thermal pond. In this cooling pond at atmospheric
pressure and lower temperature, silica precipitates and settles at the bottom of the pond
to form a residual sludge. The supernatant liquid is either reinjected or discharged to a
body of water. Drilling mud is a by-product of drilling operations during the exploration and
development of the geotherrnal well field.

Figure 2.1 Schernatic model of a hot-water geothermal system, adapted from Muffler
and White (1978).
 There is a widespread belief that geothemal resources represent a relatively "clean"
non-polluting energy source, hence the increased public interest in geotherrnal
developrnent. While it is true that geotherrnal resources offer significant environmental
advantages over fossil and nuclear energy, there are also adverse local impacts on land,
water, and air as summarized by Brown and several geothermal practitioners and El-
Hinnawi [4,5]. It was reported that CO,, H,S, As, B,and Hg are species of most concern
in geothermal power plants in New Zealand (61and in the Philippines [7]. Bowen and
Axtrnann had made two of the early studies on environrnental impact of a geothermal
power plant which recognized emissions to air (CO, and H, S) and water (thermal
discharges) as the main problems [8,9]. In the US, Hahn had identified exposure to heat
and noise, H,S, NH,, hazardous chemicals and wastes as the major occupational health
hazards associated with geotherrnal energy development from drilling to power production
[IO). Relatively unknown prior to development and an aftermath of power generation is the
formation of solid residues (especially scale and sludge) from several processes. These
residues have not been widely studied but were reported to contain elevated amounts of
trace elernents notably, arsenic, copper, zinc and lead which may be the result of the
wastes being enhanced with metals from the rock formation or salts from the reservoir
121. For a 433 MW geothermal power plant at lrnperial Valley, California, an
fluids [Il,
estirnated 50,000 tonslyear of production wastes were generated [12].
There is a scarcity of reports on the characteristics of scale and sludge precipitates
in geothermal brines. There is hardly any literature about drilling mud. Dissolved silica is
a major constituent in these geothermal effluents which are currently managed by means
of holding ponds before the aqueous component is discharged to natural waterways or
reinjected to geothermal wells [13]. Significant research effort on the chemistry of scales
has been carried out and reported [14-171. Wong and Shugarman studied a silica-rich
sludge containing high levels of lead, copper and zinc and reported ways of reducing their
concentrations to acceptable levels [15]. Hickman reported the presence of arsenic, sulfur,
and lead accumulating as cooiing tower basin sludge [16] which is quite different from the
cooling pond sludge. The sludges produced by geothermal operations have been called
by different narnes : geothermal residues, residual sludge, silica slurry, sump sludge,
holding pond sludge and cooling tower sludge. The fint five terms refer to the same
sludge, mainly silica containing various inorganic elements, which precipitates in the
holding pond while the geothermal brine is cooling. The cooling tower sludge originates
in the cooling tower basin.
Several researchers have studied the characteristics of scale and sludge. Webster
and Kukacka found large amounts of arsenic, lead, cadmium and çhromium in geothemal
residues in the US [18]. Karabelas et al determined the characteristics of scales from a
geothermal power plant in Milos, Greece and found them enriched with silicates as well as
heavy metal sulfides of lead, zinc, copper and iron [19]. Gallup and Reiff characterized
geothermal scale deposits from Salton Sea, California and Tiwi, Philippines, using
Mossbauer spectroscopy and X-ray diffraction [20]. It was found that the bulk of the mineral
phases present were microcrystalline, poorly crystalline or glassy. He concluded that the
iron silicate deposited in scale was derived pnrnarily from geothermal brine with co-
deposition of poorly-crystalline steel corrosion products. Premuzic et al have studied
sludges for almost eight years but mostly for treatrnent purposes with little effort on
characterization [ I l , 21-25]. Most of these studies only examined the physicochernical
characteristics without investigating the long term stability or mobility of the heavy metals
under changing environmental conditions.

2.1.2 Treafrnent and Disposal Practices of Geothermal Residues

Most geothermal residues have posed disposal problerns to geothermal operators
since some have been reported to contain toxic elements at elevated concentrations above
normal soils [12, 16, 20, 281. Landfilling is widely practiced as a disposal option since it is
inexpensive and perceived to have low environrnental impact.
Most effluents of liquid-dominated geothermal systems are generally elevated in
dissolved salts, boron, ammonia, arsenic, and heavy metals 1291. Such effluents are
currently managed by means of holding ponds which discharge to natural waterways or are
reinjected back into suitable geothermal wells [13]. While reinjection is generally favored,
it is very expensive (30% of total capital cost of steam-water gathering system) and can
affect the energy potential of the geothermal resources through lowering of reservoir
temperature [5] and plugging of the formation adjacent to the reinjection wells [8, 301.
The popular disposal option practiced by rnany geothermal operators is landfilling
onsite. Technologies such as solidification1stabilization [18], bioleaching of heavy metals
126, 271, and entombmenvlandfilling 1311 have been investigated to address the disposal
problem of geothermal residues. Premuzic at al used biotechnology for thermophilic metal
leaching using mixed cultures of Thiobacillus femooxidans and Thiobacillus thiooxidans
at 55 OC using an agitated bioreactor [25]. Field operators in the Philippines are currently
studying alternative methods of handling and permanently isolating the wastes [32].In
1985 in the USA, Dobryn reported that an estimated one million dollars per year was being
spent at a typical 50 MW geothermal power plant for disposal of 175 kglday of solid
residues [33]. He also found the cost of the biological waste treatment plant (0.17-
0.23centslkwh) was the sarne as the cost of hauling the solid waste to a hazardous
disposal site ($1,022,000 per year or 0.23 centslkwh). Either cost accounts for about 5%
of the cost of generating electricity from geothermal power [33]. In addition, as the cost of
disposat increases, the long term liabilities also increase at even greater proportion with
lirnited space available for landfilling.
The disposal cost for regulated wastes was five times the cost of non-regulated
wastes and had doubled during the period 1985 to 1991 [25]. Brookhaven National
Laboratoiy (BNL) paid US$ 550 per metric ton for disposal of geothermal sludge in 1991
while the corresponding non-regulated waste cost only US$ 110 per metric ton. Haulage
or shipping cost for sludge having chromium, lead and radium was US$ 1400 per m3while
treated sludge (without toxic components) was only US$270 per m3,providing a five-fold
saving. It was reported that BNL obtained a 60070% savings in disposal cost using the
biochemical waste detoxification technology but which nevertheless require intensive
271.
power supply and capital investment [26,

2.2 Guidelines for Waste Classification and Regulation

Several known regulatory limits and criteria are presented in this section. These will
be used later in the Results and Discussion for comparison purposes. Normal values for
certain elements and radioactive materials in soi1 are also given.

2.2.7 Average Cnistal Abundance of Eiements

The earth's crust consists almost entirely of oxygen compounds, especially silicates
of aluminum, calcium, magnesium, sodium, potassium and iron [34].In Table 2-1 are
presented the data on the average abundance of the elements in the earth's crust that are
of importance in environmental analyses.
Table 2-1 Average Abundance of the Elements in Crustal Rocks

Major Crustal Minor Crustal

Elements Average, % Elernents Average, ppm

2.2.2 Leachate Quality Cntena

The leachate quality criteria in Table 2-2 were derived by rnultiplying 100 times the
WHO Drinking Water Standards [35,361. This is to account for dilution and attenuation
effects in the groundwater if the waste is disposed in a landfill and the elements leach out.
Several leaching protocol tests which will be discussed in Section 2.4 use these criteria to
evaluate whether a certain waste is hazardous or not. If the concentrations in the extracted
leachate are below these lirnits, then the waste is classified as nonhazardous and can be
disposed as a non-regulated waste. Severai countries worldwide including the US, Canada,
Philippines and Mexico use these criteria for regulatory purposes.
Table 2-2 Leachate Quality Criteria, mglL

Element Concentration Element Concentration

2.2.3 Permissible Heavy Meta1 Concentrations for Agricultural Use

Sewage sludges may be disposed on agricultural lands provided that they meet the
heavy metal concentrations shown in Table 2-3 which was reported by Weber et al and
cited by Tyagi [37]. Only the values for Canada and the median for several European
Union (EU) countries are listed here although limits are also given for Co, Mn, Mo, and Ni.

Table 2-3 Maximum Permissible Heavy Metal Concentration in Sludges Considered to

be Acceptable for Agricultural Lands, mglkg dry weight

Element Canada EU Median 1 Elernent Canada EU Median

As 75 10
Cc! 20 7
Cr - 1000
Cu - 1100

Moreover, in Table 2-4 is presented a summary of the norms providing guidelines

to the European Union of concentration limits for soils and sludges as reported by Davis
and cited by Tyagi [37]. Davis also observed that more than half of the sludges disposed
did not conforrn to these guidelines. However, only metals in the dissoived form are
available for plant uptake. Therefore, in addition to knowing the elemental composition of
sludges, speciation through X-ray diffraction or sequential extraction is important in
determining potential harm to the environment.
Table 2 4 Lirnit Values for Metals in the European Union

2.2.4 Naturally Occumng Radioactive Materials

In the Salton Sea, California, Gallup and Featherstone studied the control of
naturally occurring radioactive materials (NORM) that precipitate from geothermal brines
[38]. Their treatment efficiency was based on a reduction of radium concentrations in
geothermal scales and sludges below 0.2 Bqlg (5 pCilg), an anticipated NORM regulation
for solid wastes. Ra-226 and Ra-228 were present in concentrations ranging from 9 -15
Bqlg (250-400 pCi1g). Conversion units used are: 1 becquerel (Bq) is equivalent to 27
picocuries (pCi) while one picocurie is equivalent to 1 x 1012 Ci. The same limit of 5 pCi$
was used by Prernuzic et al in their work which removed NORM from geothermal sludges
using bioleaching technology (26,271.
In Canada, the Atomic Energy Control Board has proposed radioisotope release
concentrations (C-123) but these have not been approved up to this writing [39].Maximum
permissible release concentrations for Ra-226 to the atmosphere, to sewer, and to landfill
or incinerator are 0.007 Bqlm3, 10 BqlL, and 0.3 Bqlkg, respectively. They are based on
the annual dose criterion of a maximum of 50 pSvIyr which is only a srnall fraction of both
the average annual dose received by members of the general public in Canada from
natural background radiation and the regulatory dose limit of 5 mSvIyr for the public [39].

2.3 Techniques for Waste Characterization

2.3.7 Chernical Analysis
It is vital to know the chemical composition of a waste sample at the outset since it
will be an important basis for subse~uentwaste characterization steps. Chernical analysis
is perfonned as whole rock analysis after total digestion with aqua regia and hydrofluoric
acid. This is a method commonly employed for determining the chemical composition of
geochemical samples. X-ray fluorescence (XRF) spectrometry is used for analyzing the
major species and inductively coupled plasma emission (ICP) spectrometry for the trace
elements.

2.3.2X-ray Diffraction
X-ray diffraction (XRD) is a unique technique that identifies the compounds of
crystalline materials [40]. X-rays that impinge upon atomic layers of a material cause the
atoms to vibrate and emit energy of the same wavelength as that of the incident X-ray. The
diffraction pattern contains information about the crystallinity, phase composition,
orientation and lattice stresses of the samples. The peak positions and intensity in the
diffraction patterns of crystalline materials provide information about crystal structure and
lattice parameters. A typical diffraction pattern for an amorphous material is a broad
spectrum with no prominent sharp peaks relating to long range periodicity.
In a diffractometer, the signal intensities recorded by the X-ray detector as it slowly
traverses around the circle, can be plotted on a chart. The values obtained from this chart,
with its abscissa showing 20 values and its ordinate showing signal intensities, can be used
to identify minerals by comparing signal intensities and the appropriate d-spacing values
against standard values contained in powder diffraction data files [41]. The &values can
be calculated using the Bragg equation
nh = 2 d sin8
where n is a small integer (usually 1)
A is the wavelength of the incident beam
d is the distance between adjacent atomic planes, and
0 is the angle between the incident beam and the reflecting crystal plane.

XRD has a number of limitations [42]. With multi-phase samples there is a

significant possibility of line overlap and the three strongest lines may not be due to the
same substance. It is not possible to identify noncrystalline or amorphous substances
since these do not register normal diffraction patterns. Components in a mixture occurring
below 1 to 2% by weight are not detected since these are insuffident quantities of the
materials to give rneasurable diffraction lines.

2.3.3 Radioactivity Counting

Gamma radiation is detected and measured using various methods [40]. A common
equipment is a gamma spectrometer equipped with a high purity germanium detector. The
sample is contained in a small via1 and placed in a cylindrical hole with Pb shield. The
principle is similar to pulse-height analysers which consist of one or more pulse-height
selectors that are configured to provide energy spectra. A single channel analyser typically
has a voltage range of perhaps 10V or more with a window of 0.1 to 0.5 V. Multichannel
analysers typically contain several hundred separate channels, each of which acts as a
single channel that is set for a different voltage window. The signal from each channel is
then fed to a separate counting circuit, thus permitting simultaneous counting and
recording of an entire spectrum.

2.3.4 Optical Microscopy

For light microscopy, powdered samples are mounted in epoxy resin on glass and
polished into thin sections of 1 cm diarneter to reveal the interna1structure and morphology
of the particles. Inthe reflected light microscope, the light from a high intensity source
enters the instrument through a side tube, and is then reflected perpendicuiarly by a
system of rnirrors on to a polished specimen held on the microscope stage 1411. A
photornicrographic equipment is mounted on the microscope ta be able to take
photographs of the specimen under investigation. A video camera feeding to a computer
may be attached to the microscope to transfon a light microscope to an image analysis
system. The images can be manipulated and enhanced by viewing the computer screen
instead of the eyepiece. It is also possible to Save images in cornputer format and to do
size measurement, particle counting, and video recording.

2.3.5 Evaluafion of Toxicity

Two microbial colorimetric bioassays: SOS-Chromotest and Toxi-Chromotest have
been developed recently to detect genotoxic and toxic activities of chemicals,
phanaceuticals, and food stuffs. The tests have also been applied to environmental
samples such as water, sewage and sediments [43]. The Toxi-Chromotest toxicity
bioassay is based on the ability of toxicants to inhibit the de novo synthesis of an inducible
enzyme, B-galactosidase. The arnount of de novo synthesized enzyme is determined by
a colorimetric reaction.
The SOS-Chromotest is a qualitative method of detecting the presence of
genotoxicants [44, 451. A genotoxicant or genotoxin is any DNA-damaging agent, e.g.,
mutagen which attacks the genome (DNA) part of the ceIl. A genetically engineered strain
of E. coli PQ37 developed at the lnstituit Pasteur, France produces 8-galactosidase in
response to genotoxins. Genotoxins affect living cells by altering or creating lesions in the
DNA structure causing mutations through faulty base pairing during the excision and repair
pathway. The effect of toxins on living cells is more rapid and simply causes cell death.
On the other hand, the Toxi-Chromotest involves a different strain of lyophilized E. coli K12
OR85 bacteria (EBPI Canada) produced by arbitrary bombardment with UV light.

2.3.6 Weathering Tendency

Accelerated weathering experiments can provide further insights into long-term
behaviour of waste samples in a landfill environment. Chemical weathering is related to
such factors as climate, topography, parent material, and time with temperature and
moisture flux as the major environmental variables affecting weathering rates [46,47]. This
can be simulated in the laboratory by agitating the samples continuously for months in a
gyratory incubator-shaker at elevated temperature thereby promoting physical and
chemical changes which usually are very slow processes. Weathering caused by min,
wind and Sun can, over time, release the heavy metals that are found in rock or soi1
samples.

2.4 Leaching Protocol Tests

2.4.7 Principles of Leaching
Leaching for environmental purposes has been derived from hydrornetallurgy
principles in which metals are recovered from low-grade and submarginal ores. Through
leaching of the ore, precious metals are solubilized and subsequently recovered by
processing the aqueous stream. In the environmental field, leaching tests have been used
to determined whether the elements of environmental concern, now called contarninants,
will solubilize and pollute the environment, e.g. surface water or groundwater.
Leaching, in a hydrometallurgical sense, is a typical heterogenous process, in which
a solid phase (ore particle), a liquid phase (leach solutions), and a gaseous phase (0, and
sometimes CO,) are al1 involved. It is depicted schematically by the ore leaching process
in Figure 2.2. According to this rnodel, the oxidant diffuses into the ore particle and reacts
with the mineral grains. As the leaching front moves into the particle, some mineral grains
present in veinlets or as discreet disseminated particles, may only be partially leached and
the inner core of the particle may remain unleached (top figure) [146]. As time increases,
the reaction zone moves further inward and a diffusion mechanism becomes the rate-
controlling or slowest step as shown in the lower figure which was adapted from Rossi [48].
For environmental purposes, leaching tests involve contacting the waste material
with a liquid to detemine which components dissolve. Prior to contact with the waste, the
liquid is called the leachant; after contact, it becomes the leachate. Various types of leach
tests have been developed that differ in parameters such as leachant composition, method
of contact, liquid-to-solid ratio, agitation, contact time, and temperature, in order to
investigate the chemical and mass-transport phenornena involved in leaching. Leaching
tests are undertaken with several objectives but the most important ones are identification
of leachable constituents, classification of hazardous wastes, and risk assessment of land
disposal. There are more than 26 protocols, either as standards or accepted research
tools that are available [49-511 but only three will be used in this study.
It is recognized that laboratory leaching procedures which attempt to simulate field
conditions are never adequate to predict long-term leachability [49]. This is because
leaching typically occurs very slowly and the test would only represent a time period
equivalent to the test duration. Thus short tests are actually accelerated procedures that
cause significant matrix alteration, erosion and eliminate mitigating effects such as redox
potential which is controlled by biological activity. These accelerated tests produce results
that would never be observed under field conditions. Protocol leaching tests, which
because of their nature must use accelerated conditions are often an exaggeration of the
real environment with a very large safety margin. Nevertheless, they are quite useful for
regulatory purposes in spite of their shortcomings because they provide quick information
about the wastes to policy and decision-makers.
 Leached rim

Reaction zone

structure

Figure 2.2 Schernatic drawing of leaching of an ore particle containing

a disseminated metal sulfide and the reaction zone (top) with
a plot of concentration versus time showing a diffusion-controlled
leaching mechanism (bottom), adapted from Rossi [48].
2.4.2 Batch Versus Co/umn Leaching
Both column and batch leaching methods can generate useful leachates to evaluate
potentially hazardous wastes. However. the relative accuracy of predicting levels leached
from landfilled wastes remains uncertain. Reproducibility in experimental data is a factor
of two better for batch methods than column rnethods. The disctepancy was attributed to
channelling in the column during leaching [52]. The batch extraction method is simple and
more reproducible with controlled agitation while the column method is more realistic in
simulating leaching processes under field conditions.
According to Jackson [52],the batch method is more aggressive than the colurnn
method in extracting elernents of concern presumably because of the better contact of the
waste with the leachant during rotary agitation. Batch tests can be set up and used
routinely by laboratory personnel more easily than the column method. Better
reproducibility associated with the batch method facilitates more satisfactory interlaboratory
cornparisons required in regulatory extraction procedures.

2.4.3 Agitafed Extraction Procedures

Nurnerous protocol leaching tests are available but the rnost widely used are
Toxicity Characteristic Leaching Procedure (TCLP) in the US [36]and the Leachate
Extraction Procedure (LEP) in Ontario, Canada [35].These procedures assume that the
wastes are destined for a sanitary landfill which is dominated by municipal solid waste.
Both methods use a 20:l liquid to solid ratio, the rotary extraction mechanisrn and acetic
acid extracting solution. The main differences are that extraction for the Canadian LEP is
six hours longer with intermediate pH adjustments and at slower rotation speed (10 rpm
against the 30 rprn of TCLP). These procedures are described more in detail under
experimental work. These tests are single measurements which assume that equilibrium
was attained in the laboratory within one day. Batch kinetic tests could provide kinetic
information over a period longer than one day but are not currently used as protocol tests.

2.4.4 Sequential Chernical Extraction

Sequential chernical extraction (SCE) is not a protocol procedure but is a more
aggressive test for leachability and provides speciation information. It involves several
extractions using successively stronger reagents and temperatures. each intended to
remove one phase from the sample. The basis of the sequential extraction procedure is
the existence of specific fractions in a solid material which can be selectively extracted by
the corresponding reagents [53].Most SCE procedures were adapted for environmental
samples such as contaminated soils, sediments, dust, and fly ash 153-611. In this study,
the procedure used is a version of the most widely used scheme of Tessier et al [55] which
was based on the work by Gupta and Chen [54].
Sequential extraction has been popular, though tirne consuming to perform, since
it can provide detailed information on the origin, mode of occurrence, biological and
physicochemical availability, mobilization, and transport of trace metals [54, 55. 62, 631.
The Tessier procedure involves five extraction stages, designated A to E, used to leach
metals associated with different chernical phases in the residues [55]. The various fractions
have been classified as Fraction A represents the extraction of elements that are soluble
in or are in an exchangeable form with water; Fraction B represents the extraction of
elements from carbonate minera1 phases susceptible to mild acid conditions; Fraction C
represents the extraction of elements bound with iron and rnanganese oxides; Fraction D
represents the elements bound with sulfides which may be decomposed under oxidizing
conditions; and lastly, Fraction E represents the elements that are associated with
unreactive minerais, mainly silicates, which can incorporate metals within their crystal
structures. This lasi fraction may contain metals that are not expected to be released in
solution to the environment under normal conditions, even over a long time.

2.5 Microbial Leaching

Geothermal solid residues have been found to have similar components (silica and
921 and therefore experience from the mining industry
metal sulfides) to mine tailings [28,
may be applicable to the geothermal industry. The following section will deal with the
occurrence of acid mine drainage and the various mechanisms of bacterial leaching.

2.5.1 Acid Mine Drainage

2.5.i .1 Occurrence
Acid mine drainage (AMD) is a problem commonly found in coal and copper mines
whereby sulfide materials rejected during the mining of coal and metal mines and
deposited in mine tailings or heaps are oxidized to sulfates. This in tum releases runoff with
high acidity and heavy rnetals causing pollution to the environment over a long period of
time [64-661. Acidic drainage also causes severe corrosion problems to mining and
ancillary equipment [67].In western USA, the Forest Service estirnated that between
20,000 and 50,000 sites (including abandoned and operating mines) are currently
generating acid on forest lands and that drainage from these mines is affecting between
8,000 and 16,000 km of streams [68].The annual volume of acid-generating waste rock
or tailings produced by the Canadian mineral industry is estimated at 140,000 dry
tonneslyear [69].The sulfide oxidation is microbially enhanced by the presence of iron
and sulfur oxidizing bacteria such as Thiobacillus ferrooxidans, that c m survive at low pH
(< 3.5) and high temperature (up to 60 OC). Since geothermal residues possess
characteristics resembling mine or rock tailings and have also been traditionally disposed
in open dumps, the investigation of possible AMD potential was most prudent.

2.5.1.2 Role of Microorganisms

The reaction rate causing AMD is greatly accelerated by the presence of T.
ferrooxidans to as much as I O 6 - fold [70, 711. These bacteria promote indirect oxidation
of pyrite and other sulfides through the catalysis of the oxidation of ferrous ion to ferric ion
which is an effective oxidant. However, they may also catalyze direct oxidation of pyrite by
oxygen. These organisms act only as redox catalysts; they do not oxidize substrates or
reduce oxygen but mediate the reaction or electron transfer. In doing so, they obtain a
source of energy from these energy-yielding redox reactions for their metabolic needs.
Thiobacillus ferrooxidans tends to live in environments such as hot springs, volcanic
fissures, and sulfide deposits as well as oil brines, coal water, mine wastes, peat soil,
concrete and building stone [48, 721.
Thiobacillus fenooxidans is a chemoautotroph in the genus of Thiobacilli and can
derive metabolic energy from oxidation of iron and sulfur compounds [73-771.It requires
oxygen and carbon dioxide for its metabolism. The bacteria can draw energy from the
oxidation of ferrous ions to ferric ions as the sole energy source. The bacteria have been
observed to survive under anaerobic conditions by oxidizing sulfur with ferric ion serving
as the oxidant. This was reported by Sugio et al 1791,
Corbett et al and Goodman et al as
cited by Tyagi [37]and most recently by Pronk [78]. Thiobacillus ferrooxidans is
morphologically similar to Thiobacillus thiooxidans, a sulfur oxidizing bacterium, and both
have been found in heap and dump leaching of minerals where acid mine drainage is
suspected.
T. femoxidans is highly polymorphic. The shape of its cells Vary frorn large rods with
rounded ends, from 2 pm in length and 0.5 pm in diameter, to spheres, ovoids and rods
from 0.5 to 0.7 pm in length and 0.3 to 0.4 pm in diameter. White they c m occur singly or
in pairs, chains consisting of six to seven cells have also been observed. Two modes of
cell division have been recognized : constriction, the most common multiplication mode,
and partition.
It was reported that the combination of T. femoxidans and T. Thiooxidans is more
effective in bioleaching [22, 26, 371.The most robust leaching microorganisms are the
thermophilic (60°C) acidophilic species of the genus Sulfolobus. Of these, Thiobacillus
femoxidans is the easiest bacteria to cuiture in the laboratory since it requires less energy
at room temperature (25-35 OC).

2.5.2 Mechanism of Bacterial Leaching

Due to biochemical reactions, largely insoluble metal sulfides can be degraded to
soluble metal sulfates by direct and indirect methods of bacterial metabolism [37,70, 801.

2.5.2.1 Direct Method

In the direct mechanism, the metal sulfide is oxidized to metal sulfate :
bacteria
Mes + 2 0, -+ MeSO,

where Me is a bivalent metal. The heavy metal sulfides such as NiS, ZnS, COS, PbS, and
CuS are generally insoluble in aqueous acid leach media, while their sulfates have
solubility with the exception for lead sulfate which is sparingly soluble (K, of 1.6 x 109.
This is illustrated by pyrite oxidation which involves rapid oxidation of ferrous sulfate with
the rnediation of Thiobacillus ferooxidans. Equation 2 is the overall reaction for Equations
2a and 2b.

bacteria
4 FeSO, + 0, + 2 H,SO, -+ 2 Fe,(SO,), + 2 H,O
2.5.2.2 Indirect Method
In the indirect mode of bacterial action, the metal sulfide is oxidized by a ferric ion
without the direct participation of bacteria as shown below in Equation 3. This reaction
takes place geochemically under conditions of weathering and leaching. Ferrous ions can
be reoxidized as in Equation 2 and again ferric ions can act as the oxidizing agent. In
indirect leaching processes, Thiobacillus femoxidans catalyses ferrous ion oxidation which
takes place very slowly under normal conditions (Equation 5).

The elemental sulfur that has been set free in Equation 3 will be oxidized to sulfuric acid
mediated by bacteria in the following mode:
bacteria
S* + 1.50, + H20 + H$O,

In the same manner, the ferrous ion is reoxidized, mediated by the microorganisms to ferric
ion:

bacteria
2Fe2+ + 0.50, + 2 H + 2Fe3++ H20

and the iron redox cycle is repeated. The production of sulfuric acid will decrease the pH,
which will enhance further the solubilization of metals. It is also possible to form a yellow
insoluble precipitate called jarosite, KFe,(SO,),(OH), which can hamper transport
phenornena by coating the minerai. In bacterial leaching systems, it is desirable to prevent
jarosite generation because of the formation of diffusion barriers on mineral surfaces and
the scavenging of metal ions from the leach solution [81].
In the direct mode of bacterial oxidation, as shown in Equation 1, bacteria rnust
remain close tu the surface to be adsorbed onto the solid substrate where dissociation
takes place according to the solubility product of the metal sulfide :
The released sulfide moiety of the metal sulfide is then immediately captured by the
enzyrnic systern of bacteria and is oxidized to sulfate :

As the sulfide ion is oxidized to sulfate, further dissolution of MS as shown in Equation 6

will occur, shifting the reaction to the right. The prerequisite for bacterial oxidation of solid
sulfides according to this explanation is the availability of the substrate in the soluble form.
In contrast to the direct leaching of suifide minerals, the agent responsible for leaching
(H,SO,) is produced by bacterial oxidation.
Reactions for other sulfide minerals shown below were presented by Dutrizac and
MacDonald [26]:

ChalcopvHte
CuFeS, + 4 Fe '+

CuFeS, + 4 Fe3++ 2H20 + 30, -+ Cu 2 + +5 F e 2 + + 2H2S04 (9)

bacteria
2 CuFeS, + 8.50, + H,S04 -+ 2 CuSO, + Fe,(SO,), + HO
, (10)

bacteria
ZnS + 20, -+ ZnSO,

PbS + Fe,(SO,), -+ PbSO, + 2FeS0, + S0

bacteria
PbS+20, -+ PbSO,

2.5.3 Factors Affecthg Bacterial Leaching

The effkiency of rnicrobial leaching processes depends mainly on the bacterial
activity and on the chemical and mineralogical composition of the sample (80, 821.The
bacterial activity is influenced by several environmental factors, such as composition of
the leaching medium, Eh, pH, temperature, particle size, and oxygen availability. A
maximum rate of metal extraction can be achieved if the environmental conditions
correspond with bacterial growth conditions.

2.5.3.1 Composition of Leaching Medium

Thiobacillus femoxidans is markedly affected by variation in ammonium sulfate and
dipotassium hydrogen phosphate concentrations of the leach medium [81]. Other nutrients
such as nitrate, calcium, potassium and chloride ions are present in sulfide-bearing
substrates. Best concentrations for ammonium sulfate were 3 gIL, dipotassium hydrogen
phosphate, 0.5 glL, sulfate at 2 glL, magnesium from 2 mglL to 12 mg1L and ammonium
and phosphate 9.6 mglL [83].
There has been a wide variation of opinion and experience on the importance of the
amount of ferrous ion in the bioleaching medium. Lakeview Research, Peterborough,
Ontario has been including FeSO, in the leaching medium when performing the BC
Research Confirmation test (see Section 2.6.1.2)with adapted bacterial cultures. McGoran
et al [76]achieved the highest growth rates and minimum generation time of Thiobacillus
ferooxidans when using Fe2+as substrate in 9.0 glL concentration. Barron and Lueking
conducted a detailed study of the growth and maintenance of Thiobacillos ferrooxidans
cells [84]. They found that bacterial growth was significantly influenced by the
concentration of FeSO,, with maximal growth rates in the presence of 2 to 3 g of Fe2+per
liter. However, in the BC Research Confirmation Test, FeSO, was withheld from the culture
media since it was expected that the required iron can be obtained by the bacteria from the
sample [67].This may be a critical issue for the survival of the bacteria throughout the
duration of this test since there is no soluble iron present initially. The samples to be
tested usually contain iron sulfides that are insoluble and need to be oxidized to more
soluble sulfates through bacterial catalysis. However, without bacterial maintenance, this
reaction may not occur.

2.5.3.2 Oxidation-Reduction Potential

The ferrous-ferric system requires an oxidation-reduction potential (ORP) of 747 mV
at 25 OC.The experimental Eh values from +220 mV to +540 mV have been observed
during the oxidation of metal sulfides as substrate [75,851. This is an important parameter
to measure since it is an indicator of the progress of reaction within the system. An
increasing positive Eh value is a sign of oxidation primarily the ferrous-ferric redox reaction.
Highly effective leach solutions are produced when the Eh is maintained at around +750
mV [86].
This ideal situation may be difficult to attain due to formation of iron precipitates
and poor aeration.

2.5.3.3 Temperature
Maximum leaching of metal sulfide ores and oxidation of ferrous ion by iron-
oxidizing bacteria such as Thiobaciiius femoxidans has been deterrnined to occur between
25-35 OC while 55OC is the limiting temperature for biological oxidation. Only chernical
oxidation occurs above this temperature [37]. The downside to using controlled
temperature for bioleaching is the cost of the energy required to operate the systern.
Boogerd et al as cited by Bos [87]reported that at around 30-35 OC,the contribution to the
overall kinetics of the oxidation of metal sulfide by ferric ion (indirect method, Equation 3)
is negligible. Only in thermophilic temperatures such as 45-70 OC will there be an increase
in the overall kinetics [87].

2.5.3.4 Hydrogen Ion Concentration

ldeal pH is between 1.O and 2.5 for the oxidation of ferrous ions and rnetal sulfides
[37]. Thiobacillus ferrooxidans is active in the pH range of 1.5-5.0 but will survive up to a
maximum of 6.0and minimum of 1 .Owithout adaptation of culture. Better results on metal
solubilization have been obtained by lowering the initial pH to 2.0 [37]and up to 3.5 [76,
771. Maximum bacterial activity can be attained at around pH 3.2 whereas minimum
activity can be reached at approxirnately pH 1.5 and pH 5 [88]. Also, at lower pH, the
formation of the orange brown precipitate jarosite or ferric hydroxide is rninimized.

2.5.3.5 Agitation and Oxygen Transfer

The effects of mixing have been studied in bioleaching by several researchers [33,
481. Leaching rates were increased by agitation but only with gentle mixing (100-250 rpm).
Rapid mixing (300 rpm and above) resulted in lowering the metal solubilization rates. It
should be noted that Thiobacillus femoxidans requires a minimum oxygen concentration
of 2.5% of saturation in coal mines as reported by Bos [87].However for sewage sludge
as a substrate, a minimum of oxygen concentration of 35% of the saturation is required by
Thiobacillusfemoxidans. Lower oxygen levels resulted in reduced rnicrobial activity [37].
At 23 OC, the oxygen solubility in pure water is 8.56 mglL and will decrease to 6.60 mg11
in the presence of 45 ppt salinity (ORION oxygen probe manual) and at higher temperature
[130].
The amount of oxygen that must be made available for a satisfactory bioleaching
process can be roughly estimated using the simplified equation:
bacteria
MS+20, -t MSO,

where M is a bivalent metal. According to the above equation, to convert 1 mole of the
metal sulfide to sulfate, 2 moles of oxygen are consumed [48]. Liu et al 189)has compared
the solubility values of oxygen in aqueous solutions and culture media used in bioleaching
of metal sulfide ores and concluded that it is reasonable to use the saturation solubitity of
oxygen in water (6.68 mglL) for the culture media in bioleaching process [89].

2.5.3.6 Particle Size and Substrate Concentration

Several researchers have utilized various particle sizes when carrying out AMD
potential studies. Lakefield Research, Peterborough, Ontario have been grinding samples
with a mortar and pestle achieving particle sizes of 150-180 Fm for their BC Research
Confirmation Test. University of Waterloo in their AMD kinetic studies used particle size
fractions between 90 to 125 pm from mine tailings 1901. Lawrence et al used -75 pm as
particle sample size in evaluating various AMD potential prediction procedures of mine
tailings [91]. Although it is preferable to use -45 pm which is the average size of mine
tailings, it is difficult to achieve this using mortar and pestle especially if the sample
contains high amounts of silica. It is also not advisable to use the mechanical grinder
especially for small quantity of sarnples since contamination from other users is bound to
be a problem.
Nicholson as cited by Ferguson and Erickson [66]gave a good explanation for why
particle size is an important physical factor that affects the AMD process. Coarse-grained
mining wastes allow greater oxygen advection and hence active acid generation can occur
to a greater depth in a waste heap than fine-grained waste. In coane metal mine waste
rock dumps, air convection is promoted by wind action, barometnc pressure changes, and
interna1 dump heating from the exothermic oxidation reactions. Under these conditions,
active acid generation rnay occur throughout the dump rather than being limited to the
surface zone, as in fine-grained mining wastes such as tailings [66].With this assessrnent,
it rnay be more advantageous not to have very fine grain particles for the AMD potential
test.

2.6 Prediction of Acid Mine Drainage Potential

In mineral mining, the prediction of acid mine drainage (AMD) is needed to find out
if the quality of waters draining from a mine site will exceed environmental regulatory
standards, and if sa, what mitigation measures have to be provided at the outset. Accurate
prediction of AMD is required both to protect the environment and to ensure that resources
are expended wisely to prevent or control AMD. The experience in the mining industry on
prediction of acidification potential and metal release wiII be useful also to the geothermal
industry where solid residues with components (silica and metal sulfides) similar with mine
tailings are produced [28, 921.

2.6.1 Prediction Procedures

Ten prediction techniques were evaluated by Lawrence et al [ Q I ] and Ferguson and
Erickson [93]. Of these. four were static tests and six were kinetic tests. Calow et al [94]
compared two common static tests: the BC Research Initial Test (BCRIT) from Canada
and the Acid-Base Accounting (ABA) from the US. The BCRlT was favored over ABA after
testing eight mine tailings samples [94]. The static and kinetic procedures are listed in
Tables 2-5 and 2-6. The USEPA and CANMET defined 'static tests' as methods
performed in a few hours or one day to determine initial acid producing potential [67,68].
On the other hand, 'kinetic tests' involve predicting the long-term weathering characteristics
of a waste material as a function of time hence of longer duration from weeks to months
and even years. Kinetic tests are usually carried out only if static test evaluation indicates
AMD potential. The tenn 'static' was used since the tests do not consider the relative rates
of acid production and consumption.
Several studies have shown that AMD predictions using static and kinetic
techniques correlated well with actual mine water quality [66, 91, 951. The uncertainty over
AMD prediction results c m be overcome through verifkation of test results with field
experience.

2.6.1.1 Static (Initial) Tests

There is no standard AMD potential test but the most widely used especially in
Canada is the 6.C Research Initial Test (BCRIT) which corresponds to the Acid-Base
Accounting of the US [94]. It was found to be reproducible, less prone to operator error,
conservative and more representative of the natural AMD problern.
The BCRIT determines two parameters : the neutralization capacity of the sample
and its acid producing potential. In cornparing the two values. if the acid production
potential (APP) exceeds the acid consumption (AC) expressed in kg H2S04per tonne of
material, the sample is classified as a potential acid producer and confirmation testing is
recommended. The tests are chemical rather than biochemical in nature but correspond
to the acidity contribution from H2S04as a result of its formation due to oxidation of the
sulfides to sulfates. The acid production potential was calculated from the percent sulfur
in the sample converted to kg H,SO, by a conversion factor (APP = 30.6 x %S) whereas
the acid consumption was computed from the volume of acid used to reach the endpoint
of pH 3.5 [67].This calculation is further explained in Appendix C.

2.6.1.2 Kinetic (Confirmation) Tests

Several researchers have found that static tests were oh'en accurate in predicting
drainage quality and were particularly valuable as screening tests to determine if more
sophisticated procedures should be used [93, 941. Table 2-6 inciudes a summary of kinetic
acid mine drainage prediction techniques as reported by CANMET (671, USEPA [68],
Lawrence et al [91] and Ferguson and Erickson [93].Of the many AMD kinetic tests. the
B. C. Research Confirmation Test, is widely used at base rnetal and gold mines in Canada
and even in USA [68,91, 93, 961. Positive results from this test are considered
confirmation that the microbiologically catalysed reactions can become self-sustaining [97].
The B.C. Research Confirmation Test requires inoculation with T. ferrooxidans to
stimulate the rapid stage of oxidation [91, 971. The sample (10-20 g depending on S
content) is placed in 250 rnL Erlenmeyer flask with 70 mL nutrient media, 5-10 mL culture
of T. ferooxidans at pH 2.2 - 2.5. The flask is placed on gyratory shaker at 35 OC in a CO, -
enriched atmosphere, pH is monitored and additional sarnple provided. If the pH rises
substantially, then the sample is considered nonacid producer. If the pH remains low, then
sample is a potential acid producer [97]. The limitations of this procedure are : (a) there is
no specified procedure to spawn an acclirnatized bacteria culture, (b) there is no
assurance of bacterial growth as FeSO, was withheld from the culture media and bacterial
viability was only checked at the onset and not periodically during the test, and (c) redox
potential (Eh) and metal concentration of solution are not measured to indicate chemical
reaction and rnetal release.

2.6.2 Laboratory Scale Bioleaching Techniques

Two types of taboratory scale bioleaching methods were discussed thoroughly by
Rossi [48]. The first type involves a qualitative or semiquantitative assessrnent of the
amenability of a material (ore or residue) to biodegradation by a well specified bacterial
strain. This class includes manometric, stationary flask and air sparging techniques. The
second class, in contrast, provides quantitative evaluation of parameters in an analytical
approach using kinetic simulation model. These methods are air-lift percolator, shake flask
and pressure bioleaching. In this study, the two techniques representative of both classes
are the stationary flask technique and the shake flask technique. Below is an almost
verbatim description'based on Rossi [48].

2.6.2.1 Stationary Flask Technique

This technique has been judged to be the simplest but rnost effective rnethod of
microbial culture due to modest cost of equipment and experimental simplicity [48]. The
experimental procedure is quite simple: culture medium, substrate and inoculum are
introduced into Erlenmeyer or Florence flasks, which are then plugged with adsorbent
cotton and placed in the cabinet or on the bench for the duration of the test. The flask is
plugged with adsorbent cotton to allow air to enter while filtering out airborne contaminants.
The area of air-liquid contact should be maximized to favor the diffusion of air into the liquid
mass. This is important in the case of stationary flasks where the liquid surface is still and
the rate of air transfer to the culture medium is controlled by Fick's law:
Table 2-5 Summary of Static Test Methods, Costs, Advantages, and Disadvantages
I Acid Base Accounting
Acid ProducingFotential =
30.6 Total S
60 rnesh (240 urn)
sample
add HCI as indicated by tin test.
boil one minute
then cool
titration endpt pH 7.0
duration: 1-2 hours
b cost: USS 34-1 10
-simple and short time
-no special equipment
-easy interpretation
many sarnples can be
tested
-does not relate to kineiic
-assumes parallel acid
alkaline release
-if APP and NP are close.
hard to interpret
-ditYerent particle size not
1 retlected
Source: This table was compiled from USEPA, 1994; CANMET, 1991; Lawrence, 1989; Ferguson, 1988;
Ferguson, 1987; Bruynesteyn, 1984; and Sobek, 1978.
Modiiicd Acid Base
Accounting
ACID PRODUCTION DETERMINATION
Acid Producing Potcntial =
30.6 Total S
60 mesh (240 um)
sample
add HCI as indicated by tiu
test agilate for 23
hou6 at room
temperature
-
pH 1.4 2.0 required
afier six hours agitation
titration endpt pH 8.3
duration: 24 hours
cost: USS 34-1 10
ADVANl LGES AND DISADVANT, GES
-simple
-fairly short time
-no special equipment
-easy interpretation
-does not relate io kinetic
-assumes parallel acidl
alkaline rctease
-ifAP and NP are close,
hard to interpret
-different particle sizc not
reflected
I BC RESEARCH
Initial
Total Acid Production =
30.6 Total S
ON POTENTlAL DETEF
300 rnesh (380 um)
sarnplc
iitrate sarnple to pH 3.5
with 1 .O N H2S0,
titration endpt not
applicable
luration: 5-8 hours
OS^: USS 65-170
-simple
-shon time
-no special equipment and
-casy interpretation
-rnany sarnples can bc
tested
-assumes parallel acidl
alkaline releasc
different partide size not
reflected
-if APP and NP are close.
hard to interpret
I Alkaljnc Production
Potcatiil: Sulfur
I Net Acid Production
Total S used as indicator 3 0 0 m l H20,added to
5 g rock to directly
230 um sample paniclc size not
presentcd
20 mL 0.1 N HCI to 0.4g acid produccd by iron sultide
solid for 2 hours at oxidation dissolves buffering
roorn temperature minenils
iimtion endpt pH 4.0 titration endpt pH 7.0
duration: 2 houn duration: 2 houn
cost: US$ 34-1 10 cost: US$ 25-68
.simple -simple
-short timc -short time
-no special equipment -no special equipment
easy interpretation
-moderate interpretation -1irnited reproducibility
-uncemin if extent of
suIfide oxidation
simulates that i n tield
Table 2-6 Summary of Sorne Kinetic Test Methods, Costs, Advantages, and Disadvantages
I HUMlDlTY CELLS
- - - --
2.38 mm particle size
I
ZOOg of rock exposed to three days dry air.
three days humidified air. and rinscd with
200 mL on day sevcn
duration: 8- 10 weeks
cost: US6 425-850
-models AP and NP well
-rnodels weddry
-approximates field conditions and
rate of acidity per unit of samplc
moderate to use
-results take long time
-sorne special equipment
-moderate ease of interpretation
-large data set generated
I BC RESWRCH CONFIRMATION
I 400 merh particle rire
15-30g added to bacteriolly active solution
at pH 2.2 to 2.5. T=35"C
if pH increases. sample is non acid
producer
if pH decreases. Il2 original sarnple mass
is added in each of two increments
duration: 3-4 weeks
cost: USSI70-330
-simple to use
-1ow cost
-assesses potential for biological leaching
-moderate to use
-longer time needed
-some special equipment needed
-difficult interpretation if pH change small
-does not rnodel initial AP step
Source: This table was cornpiled from USEPA, 1994; CANMET, 1991; Lawrence, 1989; Ferguson,
1988; Ferguson, 1987; Bruynesteyn, 1984; and Sobek, 1978.
1 SOXHLET EXTRACTION
SUMMARY OF TEST METHOD
panicle size not presented
T=70°C and at T=2S O C
water passed through sample is distilled
and recycled through sample
duration: 3-8 days
cost: USS 212-425
ADVANTACES AND DISADVANTACES
-simple
-results in shon time
-assessrnent of interaction behveen AP and
NP
-moderate to use
-need special equiprnent
-moderate interpreration
in developmental stage and
relationship to naiural processes not clear
1 BATCH REACTORJSHAKE F U S K S
iMETHOD
l 200 mesh panicle size
sarnpleiwater slurry is agitated
200g.500 rnL
duration : 3 rnonths
COSI:USS425-850
ADVANTACES AND DISADVANTACES
-able to examine many samples
sirnultaneously
-relativeiy simple equipment
-subject to large sampling errors
4ack of precision
I COLUMN TESTS
I variable particle sizc
colurnns containing mine waste are leached
wirh discrete volumes or recirculating
solutions
duration: 3-9 rnonths
-
cost: USS2000 4000
-models AP and NP
-modds cffect of different rock types
-models wetldry
-rnodels diffennt grain sizcs
difficult interpretation
-no1 practical for large numbcr of samples
-large volume of sample
-lots of data gcnenited
-long timc
-wtential oroblems: uneven leachate
applicatibn, channelization
1 FIELD TESTS
field scalc panicles
800 to 1300 metric ton test piles
consmcted on liners flow and water
quality data collectcd
tesu began in 1977 and are ongoing
duration : at least 1 year
COS!:usa 10000-40000
-uses actual mine waste under
environmentiil conditions
c a n be used to determine drainage volume
mitigation methods can be tested
-expensive initial construction
-long time
a=-DA&
dt dx
where dQldt is the rate of transport across the liquid surface, D is the diffusion coefficient,
dddx is the air concentration gradient across the liquid surface and A is the surface area
of the liquid phase. This technique is useful in identifying the amenability of rninerals to
bioleaching and the influence of physicochemical parameters in the process.

2.6.2.2 Shake Flask Technique

The two main shortcomings of the stationary flask technique : the time-dependent
heterogeneity of the suspension and the slowness of the gas diffusion (0, and CO,), are
overcome in the shake flask technique [48]. Test equiprnent consists of Pyrex Erlenmeyer
flask and a shaker consisting of a platform with several flask clamps moving in a
reciprocating or rotary plane motion. The device is called a "reciprocating shaker" when the
platform motion is reciprocating and a "rotary action shaker" when the motion is rotary.
Both devices shake the suspension, ensuring thorough mixing and homogeneity as well
as agitation of its surface, thereby enhancing the dissolution of atmospheric oxygen and
carbon dioxide needed by the microorganisms for their metabolism. A rotary shaker is
preferred over the reciprocating shaker since the suspension is uniformly agitated in al1
directions.
Rossi had described this procedure thoroughly 1481. At the beginning of the test,
after pH determination, the weight of flask and its content is measured. The flasks are
clamped to the shaker and the apparatus is started up. At regular intervals, the agitation
is interrupted and the solution allowed to rest, to measure flask weight and other
parameters. The initial weight is restored by adding distilled water to compensate for
evaporation estimated to be between 0.6 and 0.7 glday. A 1 mL aliquot from the
supernatant is obtained for chemical analysis. The results are recorded and used to plot
a metal leached vs time or pH vs time which usually exhibits a more or less pronounced
"Snshape curve corresponding to the lag, exponential and asymptotic growth phases of
bacteria. According to Rossi, the following operating conditions are considered appropriate:
250 mL Erlenmeyer fiasks, 1 to 10 g of ground sample, 1 mL of inoculum, 75-100 mL of
solution (culture medium plus inoculum) and 200-300 rpm. In order to shorten testing
times, the sample is finely ground from -40 pm to -200 pm.
2.7 Geochemical Equilibriurn Modeling
A variety of mathematical models have evolved through the yean which attempt to
predict the behavior of pollutants at equilibrium under various environmental settings.
MINTEQA2, a geochemical equilibrium program developed by the US Environmental
Protection Agency, is one of the most popular models [98]. The principle uses the
"equilibriurn constant method" which is simultaneous solution of nonlinear mass action
expressions and linear mass balance relationships. A brief description of MINTEQA2 is
presented in Appendix D.

2.7.7 Different Thermodynamic Models

Precursors of existing equilibrium rnodels such as MINTEQAZ are the EQ316,
PHRQPITZ, SOLMNEQ, REDEQL. MINEQL, GEOCHEM [99-IOI]. EQ3NR and MICROQL
11[98], and PHREEQE [85] . They were al1 written in FORTRAN language and used a
solution algorithm based on the Newton-Raphson technique.
MINTEQA2 has several limitations. Firstly. to be able to simulate the actual system,
al1 the complex solids that need to be modeled have to be known. Secondly, the
MINTEQA2 thermodynamic database is not complete and may not contain al1 these solids
and thirdly, it has a maximum iterations of 200 to reach equilibrium. However, several
researchers have found MINTEQA2 to be less tedious than other thermodynamic models
[98,102-1051.

2.7.2 Applications
Several researchers have used MINTEQA2 to sirnulate solid-phase dissolutionfrom
coal fly ash and incinerator residues as well as to compare with the controlling solids
observed with experimental leaching methods 198,lO3,lO5]. Only a few like van der Sloot
[IO51 had found reasonably good agreement between model-predicted equilibriurn
aqueous phase concentrations and laboratory data. He recommended thermodynamic
modelling to supplement regulatory protocol tests 11051. MINTEQA2 was used extensively
by researchers from the University of Waterloo in Ontario, Canada under the acid mine
drainage program [go, 106, 1071. To date, there is no reported application in modelling
behavior of geothermal wastes.
CHAPTER 3 METHODS AND PROCEDURES

The geothermal residues were obtained from three geotherrnal fields : (a) Bulalo,
Philippines, (b) Cerro Prieto, Mexico, and (c) Dixie Valley, USA. They were al1 examined
on an as-received basis since they were relatively dry with an average moisture content
of less than 5% measured by drying overnight in a 105 OC oven. The samples were air
dried at ambient temperature and stored in polyethylene bottles. Each sample was
assigned the following acronyms to facilitate their processing and subsequently the
presentation of results and discussion. The code is based on the first letter of the country
of origin and the next two letters are descriptors of the samples: PSC - Philippine scale,
PSL - Philippine sludge, ASC - American scale. MOM - Mexican drilling mud, MSC -
Mexican scale, and MSL - Mexican sludge. PSC, PSL, MSL, and MDM have fine or flaky
particles below 9.5 mm in size (15% were below 125 Pm, by weight) while MSC and ASC
are hard and rock-like composed mostly of big particles ranging from 1 to 15 cm in size
(2%were below 125 Mm, by weight). For the procedures requiring fine particles (-125 prn),
the samples were ground in rnortar and pestle and sieved in Canadian Tyler standard
screen (120 mesh). All chemicals, salts, acids, and pH buffers used were of analytical
grade, while al1 solutions, standards, and dilutions were prepared using deionized water.
The following laboratories performed some of the procedures and analyses reported
in this work: at the University of Toronto: Centre for Nuclear Engineering for the
radioactivity counting, Department of Microbiology for the toxicity testing, Department of
Chemistry for the X-Ray diffraction, Faculty of Medicine for the transmission electron
microscopy, and at XRAL Laboratories (SGS Canada) for the whole rock analysis and
leachate analysis.

3.1 Waste Characterization

3.1.1 Chernical Analysis
Approximately 10 g each of the air-dried geothermal samples were used for
multielement whole rock analysis, a method commonly employed for detenining the
chemical composition of geochemical samples. The analytical techniques used were :
Leco sulfur analyzer for sulfur, cold vapour spectrometiy for mercury, selective ion
electrode for chlorine, X-Ray fluorescence spectrometry for the major species and
inductively coupled plasma emission spectrometry for the trace elements. The loss on
ignition (LOI) was determined at 950 OC. These analyses were perfomed by XRAL
Laboratories.

3.1.2 Radioactivity Counting

About 5 g per sample was used to detect radioactivity using a hyperpure germanium
(HPGe) well-type detector (with cryostat well dimensions H=40 mm, D=15 mm), 10%
relative efficiency, a full-width-half-maximum resolution of 2.60 keV at 1332 keV and a 5
cm lead shield giving a background of 8.1 pis in the energy range 35 keV-1780 keV.
Measuring time was 60,000 s using as reference standard a soi1 sample with known values
of U, Ra, and daughter's activities. Since MSC showed unusual radioactivity levels, four
confirmation runs were carried out with measuring time between 70,000 to 413,000
seconds (1 to 5 days). Spectrum analysis and activity calculations were derived using
equations from OSQIPIus Manual [108]. These analyses were carried out by the Centre
for Nuclear Engineering, U of T.

3.1.3 X-Ray Diffraction

Powder X-Ray diffraction (XRD) was used to identify minerals or crystalline
compounds. Each sample was ground in acetone using a mortar and pestle and spread
thinly on a glass slide. A Siemens MO00 diffractometer system having CuKa (A =
' to 6 6 with a scan speed of 1
1.542A) radiation at 40 kV, 15 mA and scanning from 5
degree 28 per min was used. Phase identification was carried out manually using 1989
Hanawalt lndex of the Joint Cornmittee on Powder Diffraction Standards JCPDSIPDF-2
Data Set. These analyses were undertaken by the Department of Chemistry, PXRD
Analyses and Services, U of T.

3.1.4 Optical Microscopy

For light microscopy, the powders were mounted in resin and polished to reveal the
interna1 structure and morphology of the particles. Photographs were taken using an
Olympus Vanox C-35 camera at various magnifications.
3.2 Toxicity Testing
Powder samples for toxicity tests were prepared using (a) solid extraction with 10%
dimethylsulfoxide (DMSO) + 10% methanol and (b) direct sediment testing procedure
(DSTP) [log]. Both bacteria, E. colistrain PQ37 and E. coli K12 OR85 were obtained from
Environmental Bio-detection Products, Inc., Brampton, Ontario. These tests were carried
out at the Department of Microbiology, U of T.
The SOS-Chromotest was perfonned using 100 PL of an exponential growth phase
culture of E. coli strain PQ37 in al1 the wells in a standard 96-well microtitration plate.
Following a two-hour sample incubation at 37 OC, 100 pL of blue chromogen was added
to the wells and reincubated for another hour. Genotoxic activity was noted by the
presence of a distinctive blue colour in the wells. A relative measure of genotoxicity was
determined by measuring the intensity of the blue color using a spectrophotometer.
For the Toxi-Chromotest, serial two-fold dilutions of the samples were prepared in
the microplate. A via1 of E. coli strain K I 2 OR85 was rehydrated and mixed with the
reaction mixture. To each well in the microplate, 100 PL of this mixture were added.
Following a 90 min incubation at 37 OC,blue chrornogen was added and reincubated for
another 90 min. Toxic activity was noted by the absence of blue color 11IO].

3.3 Sequential Chemical Extraction

A series of sequenüal extractions by Gupta [54] and Tessier 1551 as shown in Table
3-1 was used on the geothermal residues. Fractions A and B correspond to the Canadian
LEP and the US EPA's TCLP described below. Since the samples were mainly inorganic
in nature, the last fraction E, the residual phase, was revised by using HN03/HF/HCIfor
digestion without HCIO,. The samples were pulverized with a mortar and pestle and 0.5
g each of the six air-dried samples were placed in 15 rnL polypropylene centrifuge tubes
prior to extraction.
Between each successive extraction, separation was effected by centrifuging for
5 min. The supernatant was removed with a pipet and transferred to a 50 mL centrifuge
tube and diluted with deionized water and acidified to pH<2 with concentrated HNO, prior
to analysis. The residue was washed with deionized water, centrifuged for 3 min and the
washing was discarded. The five extraction steps were performed under a fumehood using
full precaution specified in the material safety data sheets of the reagents.
Table 3-1 Summary Procedure for Sequential Chernical Extraction

Fraction Extracted Procedure

A. Exchangeable 1 M sodium acetate (4 mL), pH 8.2, I h , 20°C, continuous agitation

B. Carbonate 1 M sodium acetate (4 mL), pH 5 (adjusted with HAc) , 5h, 20°C,
continuous agitation
C. Fe-MnOxides 0.04 M NH,OH.HCI in 25% HAc (10 ml), 6h, 96%, occasional
agitation
D. Sulfide 0.02 M HN03 (1.5 mL) and 30% H,O, (2.5 mL), pH 2, 2 h, 8S°C,
occasional agitation; further 30% H,O, (1.5 mL), pH 2, 3h, 85'C,
occasional agitation; then 3.2 M NH,OAC in 20% HNO, (2.5 mL), 0.5
h, Ca. 20°C.continuous agitation
E. Residual 70% HNO, (6 mL), 40% HF (4 mL) to near dryness; further 40% HF
(4 mL), 175OC; residue dissolved in 12 N HCI (5 mL) and diluted to
25 mL

3.4 Accelerated Weathering Test

In a 250 mL Erlenmeyer flask containing 70 rnL bacteria nutrient media [l a
II],
10 g pulverized sample (100 mesh) was shaken continuously inside an incubator-shaker
(Lab-Line Instruments) for 3 months at 150 rpm and 35 OC 167, 961. The pH was monitored
and the flasks and contents were weighed weekly. Deionized water was added to make
up for weight loss due to evaporation. At the end of the shaking, a 15 mL aliquot was
obtained from each flask,centrifuged, filtered, and analyzed by inductively coupled plasma
spectometry (ICP).

3.5 Protocol Leach Tests

Out of several leaching tests available, h o protocol tests that are widely used in
Canada and USA have been selected in this study (491. These are the Leachate Extraction
Procedure of Ontario, Canada and the Toxicity Characteristics Leaching Procedure of the
USEPA which are compared in Table 3-2.

3.5.1 Leachate Extraction Procedure (LEP)

The LEP (Ontario, Canada) was used to investigate the leaching potential of toxic
cornponents to the environment by extraction with an acidic medium [35]. A 50 g dry
sample (9.5 mm or less particle size) was tumbled continuously for 24 hours in a 1 L
polyethylene bottle containing 800 mL of deionized water in a rotary extractor (KBU
Environmental Technologies VS309) at room temperature. A pH of 5î0.2 was maintained
throughout the extraction by adding 0.5N acetic acid at 1, 3, 6, 22 h from starting time. No
more than 200 mL of acetic acid may be added. After completion of the extraction, the
slurry was centrifuged and the supernatant leachate was vacuum filtered through 0.45 pm
cellulose acetate filter paper. The chemical composition of the leachate was analyzed
using a Fisons ARL 3560 inductively coupled plasma atomic emission spectrometer (ICP-
AES).
The effect of particle size on metal leaching was investigated by carrying out the
LEP test on the Mexican scale using three different particle sizes : -125 pm , - 4 mm and
-9.5 + 6 mm. MSC was of particular interest since chemical analysis showed it had the
highest levels of Cu, Zn, and Pb among the samples which were associated with particles
less than 100 prn in size.

3.5.2 Toxicity Characteristic Leaching Procedure (TCLP)

Similar to the LEP, the TCLP maintains a 20:1 liquid to solid ratio and requires
particle size of 9.5 mm or less [36].Extraction was carried out continuously in a rotary
extractor at 30 rpm for 18 hours. The choice of extraction fluid was dependent on the initial
pH of the sample (taken after 5 minutes of magnetic stirring in deionized water). If the pH
was <5, extraction fluid #1, composed of a buffer at pH 4.7 of dilute acetic acid and 1N
NaOH, was used. On the other hand, if the pH was >5,extraction fiuid #2,composed
mainly of dilute acetic acid (pH 2.8) was used instead. After the 18-hour extraction, the
supernatant was filtered with a 0.45 pm membrane filter and analyzed for 33 elements
using ICP-AES.
Table 3-2 Comparison of Protocol Leaching Tests

Leachate Extraction Procedure (LEP) Toxicity Characteristic Leaching

of Ontario, Canada Procedure (TCLP) of USA
- 20: 1 liquid to solid ratio, 50 glL - 20:l liquid to solid ratio, 50 g1L
- leachant : 800 rnL deionized water - leachant : buffered acetic acid
- pH of solution : 510.2 if pH of sampIe6, use pH = 4.7
- particle size : 5 9.5 mm if pH of sarnple>b, use pH = 2.8
- extraction time : 24 hours with - particle size : s 9.5 mm
pH adjustment @ 1, 3, 6, 22 h with - extraction time : 18 h, without pH
0.5N acetic acid (lirnit of 200 mL) adjustment
- room temperature - room temperature
- rotary extraction at I O rpm - rotary extraction at 30 rpm
Extraction fluid #1 is prepared by mixing 5.7 mL glacial acetic acid into 500 mL of
deionized water and then adding 64.3 mL of I N NaOH (dissolve 40 g NaOH in 1 L
deionized water) and diluted to 1 L. When correctly prepared the pH of this solution should
be 4.93 I0.05. Extraction fluid #2 is prepared by adding 5.7 rnL of glacial acetic acid to
deionized water and.diluting to 1 L. When correctly prepared. the pH of this duid should be
2.88 & 0.05.
As in LEP, the effect of particle size on metal leaching was studied by using the
TCLP test on MSC using three different particle sizes: -125 Pm, 4 mm, and -9.5 + 6 mm.

3.6 Extended Leach Tests

The terms oxic and anoxic as used in this study conform to the definition by Berner
[27]and adopted by Appelo and Postma [85]referring to groundwater environment. A
distinction was made between oxic and anoxic conditions based on the rneasureable
amounts of dissolved 0,( 2 I O 6 mollL). An anoxic environment will have a dissolved O, of
0.032 mglL or less. This value is very much lower compared to O, solubility of 8.56 mglL
in pure water at 23 OC where oxic conditions prevail.
3.6.7 Oxic Conditions
A two-part experiment using TCLP described above was carried out for the Mexican
scale and Philippine sludge and scale by extending the duration of extraction from 18 h
to 96h. Sampling of aliquots for metaf analysis was done every 1,2, 3,6,9, 18, 24, 48,
72 and 96 h. For the oxic TCLP, an aliquot of 5 mL was obtained and replaced with the
extracting fluid at the specified monitoring intervals. The aliquots were centrifuged for 10
min, diluted to 10 mL, and acidified to p W 2 with concentrated HNO, prior to analysis.

3.6.2 Anoxic Conditions

For the anoxic TCLP. the extracting fluid in a polyethylene container was sparged
with N, gas overnight at a flowrate of 225 mumin. In duplicate, 10 bottles were prepared
to correspond to each sampling time. About 5 g of sodium sulfite was also added to each
bottle as anti-oxidant. Based on the results of the one-day TCLP test where only Pb
leaching occurred, the fine particles of the Mexican scale (-125 pm) were used with a
duplicate for the coarse size (-9.5 + 6 mm). The pH was checked at the end of the
extraction before taking aliquots. A 15-ml aliquot was obtained every 1, 2, 3, 6, 9, 18, 24,
48, 72 and 96 h, centrifuged for 10 min and acidified to pH*2.

3.7 Preliminary Acid Mine Drainage Potenüal Test

The B.C. Research Initial Test (BCRIT) was used to rneasure acid-consuming and
acid-producing components of the residues [67].In a 250 mL beaker containing 100 mL
of deionized water, a 10 g pulverized sample (100 mesh) was stirred continuously with a
magnetic stirrer. The natural pH was measured after 15 minutes. While stirring, the
sample slurry was titrated with I N H2S04to an endpoint of pH 3.5. Acid was introduced
slowly from a titration pipette until the acid addition over a 4 hour period was 0.1 mL or
less. The volume of acid consumed was noted and used for calculation of acid production
potential. The sulfur content of the sample must be known from the chernical analysis to
be used in estimating the acid potential of the sample. The choice of the endpoint of pH
3.5 is based on the assurnption that this represents the limit above which iron and sulfur
oxidizing bacteria such as Thiobacillus feffooxidans are no longer active. Therefore, if the
theoretical acid production is not sufficient to lower the pH to below 3.5, then biochemical
oxidation of the wastes will not occur and the formation of acid mine drainage is unlikely.
3.8 Acld Mine Drainage Confirmation Test
In addition to confirrning the acid mine drainage potential of geothermal residues,
a series of experiments was carried out also to detenine (a) the best growth environment
and medium for the Thiobacillus ferrooxidans, (b) the appropriate procedure for the
geothermal wastes, and (c) kinetic performance of the AMD procedure. The B.C.
Confirmation Test [67,97] which was found deficient in its method and monitoring scheme
as discussed in Section 2.6.1, was modified and an acid mine drainage potential (AMDP)
test for geothermal residues was developed. The effect of agitation, temperature, and
sterilization on metal leaching and bacterial growth was investigated using this AMDP test.
Supplementary techniques such as transmission electron microscopy (TEM) and light
microscopy with image analysis were also performed in relation to bacterial analysis.

3.8.1 Bacteria Culture and Medium

The growth medium for the Thiobacillus femoxidans (ATCC 19859) was modified
from the standard laboratory technique of the American Public Health Association [l
II].
This was popularly known as the 9K medium developed by Silverman and Lundgen in
and adopted by the APHA. The two modifications made in this work were the
1959 [77]
reduction of the FeSO, content to half the original formula and use of membrane filtration
(0.45 pm pore size cellulose acetate) for sterilization of solution instead of autoclaving.
The detailed procedure is described in Appendix G. The modified medium had the
following constituents as shown in Table 3-3 below. Add 5 mL of Thiobacillus femoxidans
inoculum to a 100 mL fresh culture medium in a 250 mL Erlenmeyer flask. Initial pH
should be 2.8 - 2.9, if not add 10N H,SO, until stable. The culture is allowed to grow at
room temperature without agitation. Growth of the organism can be detected by a decrease
in pH, increase in Eh, and an increase in the concentration of oxidized iron as orange-
brown or deep amber color of solution. Bacterial viability was checked under a light
microscope with at least 800x magnification. It is not advisable to withhold completely the
FeSO, from the leaching medium since the bacteria require at least 2-3 glL to 9 g/L to
survive [37, 73, 77, 78, 841. The modified medium below contained 4.5 glL which was
observed to be providing good bacterial growth from various trial experiments listed in
Appendix A. An excess FeSO, can trigger increased formation of jarosite and iron
oxyhydroxides hence shouid be avoided.
3.8.2 Acclimation of lnoculum
A critical stage of the AMDP procedure is the acclimatization of the pure bacteria
culture to the specific samples to be tested. To prepare a viable culture as inoculum and
which will survive throughout the duration of the test, a series of acclimation steps was
designed at room temperature (23-25 OC)and without agitation. This acclimation procedure
is described further in Appendix G. For each culture, 5 mL inoculum was used per 100 mL
of the medium. At the onset, a medium shown in Table 3-3 but using 44.22 g/L of ferrous
I l ] was used on the pure culture (Bo) inoculum of Thiobacillus ferrooxidans
sulfate [l
(ATCC 19859). Afterwards, the resulting culture (B,) was used as inoculum to a 100 mL
fresh medium with the addition of 2 g ground sample (120 mesh) to be tested to obtain an
acclimatized culture (83.Finally, B2was used on a freshly made culture media containing
22.1 1 g/L ferrous sulfate and 2 g sample to produce 8,culture that is ready as inoculum
for the AMDP test. Prior to this, several experiments were performed to obtain the best
conditions for high bacterial density and motility. These experiments, listed in Appendix A
were carried out with and without agitation, room temperature (23 OC) and inside incubator
(35 OC),various amounts of ferrous sulfate in medium as well as several pulp densities
(weight of sample over volume of solution). The success of each experiment was
determined qualitatively through bacterial viability (density and motility).

Table 3-3 Culture Medium for Thiobaciilus fenooxidans

Basal salts: in a 1 L Erlenmever flask

Ammonium sulfate (NH,),SO,

Potassium chloride KCI
Dipotassium hydrogen phosphate K,HPO,
Magnesium sulfate MgS0,.7H20
Calcium nitrate Ca(NO,),
Sulfuric acid, 10 N H2S04
Distilled water

Enerav source: in a 500 mL Erlenmeyer flask

Ferrous sulfate FeS04.7H20

Distilled water
3.8.3 Acid Mine Drainage Potential Test
After evaluation of available literature and preliminary testing, the following acid
mine drainage potential (AMDP) procedure was designed to further study the geothermal
residues' amenability to land disposal. Ail glasswares were cleaned in detergent, rinsed
with tap water two tirnes, soaked in 20% HNO, overnight, rinsed with tap water two times
and finally rinsed with deionized water. Once dry, the Erlenmeyer flasks were covered with
aluminurn foi1 prior to use. The bacteria culture medium was prepared as described in
Appendix G. The dry samples were pulverized in a rnortar and pestle to pass a 120 mesh
Tyler screen and stored in air tight bottles prior to use. To 2 g of ground sample in a
labelled 250 mL Erlenmeyer flask, 100 mL of culture medium was poured slowly. The flask
was plugged with nonadsorbent cotton wrapped with gauze. The flask was swirled
manually and the pH was checked. If the pH was above 2.8, a few drops of ION H,SO,
were added until stable. Once the pH was stable, the flask was inoculated with an active
acclimatized culture of Thiobaciilus femoxidans prepared as in Appendix G. The weight
of flask with its contents without the cotton plug was taken initially to be able to monitor
weight loss due to evaporation. The flask was placed at room temperature (at least 23-25
OC) with adequate ventilation. The flask was manually shaken every determination. Prior
to each measurement, the fiask and contents (without plug) were weighed and deionized
water was added to replace loss by evaporation. Around 1 mL aliquot was obtained and
centrifuged at 1200 rpm for 10 min to separate solid from the supernatant. The supernatant
was removed with a pipet and transferred to another clean 15 mL centrifuge tube, diluted
to 5 mL with deionized water, acidified to pHc2 with -0.05 mL conc HNO,, and stored at
4% while waiting to be analyzed. Meanwhile 1 mL deionized water was added to ail the
flasks to replace the 1 mL aliquot sarnple.
Monitoring and sampling schedules are similar to that discussed in Section 3.9.1
below. The parameters monitored regularly were pH, Eh, bacterial growth, motility and
density, color of solution, dissolved oxygen, and metals in leachate.
When oxidativelbacterial activity had ceased as observed from the microscope and
a stable pH has formed, the test was terminated. If the pH is below 3.5 and metals in the
leachate were above regulatory limits, the sample is classified as having acid mine
drainage potential or potential for bioleaching treatment. This test can be completed within
3 4 weeks following inoculation.
3.9 Batch Kinetic Experiments
3.9.7 Effects of Agitation, Temperature, and Sterilzation
In order to obtain kinetic information about the acidification potential of the samples,
another set of experiments using the AMDP test were undertaken. To observe the effects
of agitation and increased temperature on the sarnples, the flasks and contents were
placed inside an incubatorlshaker (Lab-line Instruments) which operated continuously at
175 rpm and 35 OC. To detemine the effect of sterilization, control samples were prepared
whereby the flasks and dry samples were sterilized inside the oven at 120 OC for 1 day and
aftenvards covered with aluminum foi1 and cooled completely before use. In total, there
were five simultaneous batch tests with the following designation: (A) with agitation and
bacteria, inside the incubatorlshakerat 35 OC and 175 rpm, (B) stationary and with bacteria
placed on laboratory bench at room temperature (23-25 OC), (C) sterile conditions : similar
to B but with the sample and fiask sterilized at 120 OC inside oven for 1 day, (D) similar to
C with oven sterilized samples and flasks but without any bacteria, and (E) nonsterile
conditions, unsterilized medium and sarnple inoculated with acclimatized bacteria.
Experiments B to E were al1 stationary experiments at room temperature. These
experiments were designed to determine proper environment to be able to carry out the
AMD potential test, in particular, in a laboratory with limited equipment such as in minesite,
field laboratories, plant sites or in laboratories found in developing countries.

3.9.2 Monitoring and Sampling

Every 3 days, the following parameters were monitored : pH (Corning pH meter
model 7),Eh (Fisher Accumet pHlEh meter model 820) , bacterial growth, motility and
density (MEF3 Reichert-Jung Microscope with Image analysis Hitachi KP-MIU CCD
Camera at 800x magnification), color of solution, and dissolved metals (inductively coupled
plasma spectrometry). Dissolved oxygen (ORION oxygen meter model 860) was also
rneasured randomly in the solution to see if adequate oxygen was available for oxidation
(oxygen solubility at 23 OC is 8.5 mglL from the ORION oxygen probe manual). The pH
rneter was calibrated with pH 4 and 7 standards and al1 Eh readings were verified with
ZoBell's solution 11111. Utmost care was taken to avoid contamination among the
replicates from the various meter probes. Each probe was rinsed thoroughly with
deionized water spray and wiped with clean paper towel before doing any measurernent.
3.1 0 MicrostructuralAnalysis
3. IO. 1 Light Microscopy with Image Analysis
For routine bacterial monitoring, one drop (-20 PL) of sample taken at the surface
layer of the solution and another drop taken near the bottom of the flask were both placed
side by side on a labelled microscope slide each with a 22 x22 mm cover glass. These
were examined at 800x magnification using a MEF3 Reichert-Jung Microscope with a
Hitachi KP-Ml U CCD Camera connected to a Sony 20" television for image enhancement
and attached to a Panasonic video cassette recorder. The bacterial growth and
characteristics were observed visually and qualitatively as required by the AMDP procedure
and noted as very high, high, medium, or low to describe density and slow, fast, and very
fast for motility. A video of the bacteria at various stages of their growth as seen through
the light microscope was recorded showing their motility and density. The bacterial motility
was noted as motile or nonmotile, fast or slow since it was difficult to measure.
For bacterial count, one drop (-20 p l ) aliquot was placed on a microscope slide
with a 22 x 22 mm cover glass and examined under a light microscope at 1000x
magnification. Three to four fields per sample were photographed and stored in computer
format as an image file using an Olympus Vanox C-35 carnera with a CCD X-77 video
camera attached to a Macintosh Quadra 650 computer with an Image Scion 1.SI software.
Direct bacteria ceIl count from the images was carried out to calculate the total cell count.
The calculation of bacterial density is presented in Appendix B.

3.10.2 Transmission Electron Microscopy

Bacteria from the flask experiments were haivested by vortexing -1 0 mL of solution
to loosen bacteria adhered to particles for 10 min, centrifuging at slow speed for another
10 min and finally centrifuging at high speed for 15 min to form a white pellet and fixing
overnight in 2% gluteraldehyde (vlv). The samples were later embedded in Epon 812 resin
with the addition of osmium tetroxide and uranyl acetate. Thin sections (-60 nm) were cut
and mounted on carbon and Fornivar-coated Ni grids and were viewed on a Hitachi H7000
transmission electron microscope operating at 75 kV. Photomicrographswere taken using
a range of 24,000 to 99,000~magnification.
3 1 Geochemical Modeling
The geochemical themodynamic model MINTEQAZ (version 3.1 1) [98] was used
to determine equilibrium conditions and solid phase dissolution behavior in the TCLP for
the fine-sized Mexican scale (-125 pm). All the other samples did not provide significant
leaching hence the modeling was focused on the Mexican scale. Table 3-4 below lists the
input data used in modeling of a closed systern. Four major mineral phases (pyrite,
chalcopyrite, galena, and sphalerite) identified by XRD and microscopy were inputed as
concentrations of solid phases. Since this is a complex system, only the major species
identified in the chernical analysis, XRD and microscopy were included. Essentially this
involved ignoring the major complex silicate phases that are substantially inert and the
minor species as they were not detected in the ieachate analysis. After the first few trials,
covellite showed up as a supersaturated solid with a positive saturation index. It was
included in subsequent initial input. Appendix E shows the calculation for the
Table 3 4 Input Data for Modeling Protocol Leach Tests
- -- -- - - -

Parameters
- -
-
Values
--

Concentration of major rninerals: mol/L

Pyrite, FeS, 0.0120
Chalcopyrite, CuFeS, 0.0035
Covellite, CuS O.0036
Galena, PbS 0.0028
Sphalerite, ZnS 0.0120

Concentration of acetic acid: moVL

LEP (0.5 N) 0.0025
TCLP (0.1N) O. 1O00

concentrations of these minerals while Appendix F is a sample of the model output with the
input data on the first page. The cornponents (cations) were included as aqueous species
at very low concentrations (1 x 10-l6molal) to increase degrees of freedom. The pH was
not fixed but allowed to reach an equilibrium value and was compared with the
experirnental pH. Precipitation of solids was allowed only for those specified in the input
file and Davies equation was used to calculate ion activity coefficients. The calculated
concentrations of the major ionic species Fe ', Cu 2', Zn '+, and Pb 2+ were compared
with the actual leachate concentrations observed in the laboratory.
CHAPTER 4 RESULTS AND DISCUSSION

4.1 Waste C haracterization

4.1.7 Chernical Analysis
Whole rock analysis in Table 4-1 revealed that the geothermal residues are
composed mainly of silica ranging from 66-82% by weight. The Philippine samples (PSL
and PSC) contain higher levels of iron and aluminum content compared to the Mexican
samples (MSC and MDM). The American scale (ASC) is essentially an aluminosilicate due
to its high levels of alumina (10%) and silica (67%) with ail the rest of the elements in trace
quantities. MSL is predominantly silica (82%) with little contamination from trace elements.
From the chemical analyses, ASC and MSL will be less of a concern while the rest contain
above normal crustal levels of S, Cu, Zn, As, Ba, Hg and Pb. In particular, MSC is
concentrated with Cu, Zn, and Pb with around 1%each. Both PSC and PSL have the
most As content.
MSC, PSC, and PSL have sulfur content similar to mine tailings as shown also
midway in Table 4-1. These geothermal residues may also be susceptible to producing
acid mine drainage. These values are comparable to the S content of some mine tailings
in Canada such as Equity Silver (3.40%) and Noranda Bell (2.99%) in British Columbia as
well as Elliot Lake Quirke (3.79%) and INCO (0.69) in Ontario [91, 1121. In Canada, about
$3-6 billions will be required in the near future for remediation of mine sites where 500
million wet tonneslyear of acid generating tailings are produced.
In Table 4-2 is shown the crustal abundance ratio of selected species in PSC, PSL
and MSC which were calculated by dividing the values in Table 4-1 to the crustal averages
in Table 2-1 to get an abundance ratio. In these three geothermal residues, the levels of
S,Cu, Zn, As, Ba, Hg, and Pb are elevated compared to normal earth's crust which is why
they have been subjected to environmental regulations. More importantly, Pb in MSC has
the highest abundance ratio at 900 times the average crustal concentration. Several
techniques in this study have examined the availability of these elements to leach out to
the environment and determined their true waste category.
Table 4-1 Chernical Analysis of Selected Geothermal Sarnples

Species Units PSC PSL ASC MDM MSC MSL

SiO,
TiO,

Fe203
Mn0
Mg0
Ca0
Na,O
K P

Cr203
S
CI
Co
Ni
Cu
Zn
As
Sb
Cd
Ba
Hg
Pb
LOI
CO3
 Table 4-2 Crustal Abundance Ratio of
Selected Geothermal Residues

Elements PSC PSL MSC

4.1.2 Radioactivity
The radionuclides detected from the geothermal residues were Th-230, Pb-210. Ra-
226, Ac-228, K-40 and total U as summarized in Table 4-3. The most important
radionuclide to monitor is Ra-226 since it decays to radon which is toxic when inhaled. All
the activities were in the range of NORM (naturally occurring radioactive materials) with the
exception of Pb-210 (t,,, = 22 y) in the MSC sarnple. The validation counting for MSC at
longer duration of up to 5 days gave an average measurement of 130,000 Bqlkg (3510
pCilg) for Pb-210 at 90% confidence level. This Pb-210 activity was equivalent to a
radiation dose of 32.5 mSvly received via ingestion (40 Bqlg of Pb-210 -1 OpSvly). This
was 14 times the total annual effective dose equivalent from al1 natural sources of 2.4 mSv
[114]. Nevertheless, it was still lower than the current occupational dose limit of 50 mSv1y
[113, 1141 but higher than the Canadian public regulatory dose limit of 5 mSvly [39]. Gallup
and Featherstone reported 250-400 pCi/g in the Salton Sea geothermal brines in
southeastern California where the anticipated NORM regulation for solid wastes was 5
pCi/g 1381.
UNSCEAR and ICRP reported that there are regions in the world where outdoor
terrestrial background radiation levels appreciably exceed the NORM at 2-6 times the
average natural background of 1 mSv/y : Guarapari, Brazil; Kerala, India; and Yanjiang
County, Guangdong, People's Republic of China. This was due to the presence of
monazite sands with high levels of thorium, uranium and radium. The inhabitants in these
areas were studied between 1970-1985 and it was obsewed that there was no increase
in the frequency of cancer among the population [113, 114].
Table 4-3 Concentrations of radionuclides in the geothermal residues (in Bqikg)

Th-230 Pb-210 Ra-226 Ac-228 K-4O U estimate*

PSC ~ 7 5 0 990 120 110 I I O 42 I22 360 * 33 ~9.4

PSL ~ 3 9 0 300190 80110 83120 240,:lO ~5.1
ASC ~ 3 5 0 420 180 530 I20 550 * 73 310 * 10 ~4.8
MDM ~ 2 2 0 4 3 0 94i10 67111 310112 6.2
MSC ~ 3 8 01300001400 50110 36111 300*12 ~4.8
MSL ~ 6 1 0 <450 190*50 28110 900*60 20.0

* ppm. from activity of Th-234

< MDA (minimum detectable activity) with 90% level of confidence

4.1.3 X-ray Diffraction

Several dominant phases were detected by XRD in the samples as shown in Table
4-4. The mineral name and formula are listed in order of abundance. There may be other
phases present but they were not detected if they were less than 1- 2% by weight hence
will not give detectable diffraction peaks. Alrnost al1 of the samples, except MDM, contain
an amorphous silicate phase with a broad maximum at around 4 A. ASC does not contain
significant crystalline material; it was mostly amorphous silicate, possibly aluminosilicate.
MSL has halite and sylvite while MSC contains the minerals galena, sphalerite,
chalcopyrite, and cubanite. PSC contains the largest amount of amorphous material. 60th
PSL and PSC contain quartz, magnetite, and hernatite. MDM is a complex multi-mineral
sample with little amorphous material. The important phases such as sulfides in MSC will
be used as input data in geochemical modelling. In terms of their possible environmental
impacts, the natural minerals and layer silicates are relatively inert while halite and calcite
can dissolve and sulfides may oxidize releasing heavy metals.
Table 4-4 X-ray Diffraction Data of Selected Geothermal Residues

Sample Species'

PSC Amorphous material maximum at 4.10 A

Quartz, SiO,
Magnetite, Fe,O,
Hematite, Fe203
Jarosite, KFe3(S04),(OH),

PSL Albite, NaAISi,O,

Homblende, NaCaMg,FefitSi7O2,(OH)
Quartz, SiO,
Hematite, Fe203
Magnetite, Fe30,
Gypsum, CaS0,.2H20
Kaolinite, AI,Si,O,(OH),
Amorphous material maximum at 4.04 A

ASC Amorphous material maximum at 3.43 A

MDM Quartz, SiO,

Calcite, CaCO,
Halite, NaCl
Albite, NaAISi,O,
Microcline, KAISi,O,
Pyrite, FeS,
Mica, K(AI,(Si,AI)O,,(OH),
Montmorillonite, KAI,Si1,O,,(OH),
Chlorite, (Mg,Fe),(Si,AI),Olo(OH),
Dolomite, CaMg(CO,),
Monticellite, CaMgSiO,
Diopside, CaMg(SiO,),

MSC Galena, PbS

Sphalerite, ZnS
Chalcopyrite, CuFeS,
Cubanite, CuFe2S3
Amorphous material maximum at 3.80 A

MSL Halite, NaCl

Amorphous material maximum at 4.04 A
Sylvite, KCI

'Listed in order of abundance

4.1.4 Optical Microscopy
The scale, sludge and drilling rnud samples were physically and chernically cornplex
containing mostly particles of silica and iron and aluminum oxides. Detailed descriptions
and findings are discussed further below.

P h i l i ~ ~ i Scale
n e (PSC)
Most particles showed a banded, oriented texture characteristic of cyclic deposition
frorn a passing fluid on to the surface of the scale. The contrasting composition of
successive layers reflects changing chemical composition, reduction potential and
temperature in the fluid. Some of the unoriented particles are agglornerations of smaller
fragments cemented together within a silicate matrix.
Example of fragments with a layered texture can be seen in Figure 4. i where there
are alternating bands of magnetite and silicate, with larger masses of magnetite. The
particle at the top is 830 Pm while the particle in lower photograph is 330 @m. Some of the
cracks have been partially filled with deposited pyrite. Scale deposition could have started
at the top (pipe wall) following the sequence magnetite, silicate, pyrite, iron silicate.

Philippine Sludae [PSL)

The sludge shown in Figure 4.2 is composed of very srnall, often colloidal sized
particles of precipitate - primarily silica but including iron oxides and sulphide. These
particles easily agglomerate, due to their small size and the sticky nature of hydrous silica.
The particles making up the agglomerates are mainly submicron and porous, although
srnaIl solid fragments and particles can range in size up to 30 Pm.The small particles
resemble fine grained silt. The agglomerates are rounded equant lumps, that are highly
porous and quite friable, easily disintegrating into finer particles.

American Scale (ASC)

This particle in Figure 4.3 has a width of about 2.7 mm and is mainly silica with bits of
magnetite (black color) and a vermicullar form indicating high porosity. It is composed
largely of amorphous rnaterial with no distinct crystalline pattern.
Mexican Drillina Mud (MDM)
Particles shown in Figure 4.4 had.a variety of sires, textures, morphologies and
phases mostly of equant grains from 20 prn to 180 Pm. The black spots were iron oxides
and the white spots were iron sulfides surrounded by silicates.

Mexican Scale (MSC)

As shown in Figure 4.5, the important minerals were occluded inside the silicate
matrix. The top particle (120 hm) contains three important phases : the yellow was
chalcopyrite, the greyish green was sphalerite and the off white in cubic form was galena.
The lower particle (50 pm) was another silicate with pyrite at the center.

Mexican Sludae (MSL)

Figure 4.6 shows the vermicullar structure of the particles indicating high porosity.
The black spots were magnetite which were more obvious in top photograph depicting its
formation. This indicates that silica was fonned first and the rnagnetite developed later as
an envelope to the nucleated silica. The lower photograph was an overview of these
porous silica particles with milky white background.

4.7.5 Toxicity Testing

Toxi-Chromotest and SOS-Chromotest can provide an indication of the sensitivity
of bacteria to toxic and genotoxic elements. From the results and interpretation of these
tests provided in Appendix H, the samples did not appear to exhibit any toxicity or
genotoxicity. This may also indicate that the genotoxins and toxins were bound within the
silicate matrix or were insoluble in the extractant which makes them inaccessible to the
microorganisms. The results of leaching tests and sequential extractions to be discussed
in the following sections had confirmed that most of the heavy metals were in the residual
phases. The XRD results in Table 4-4 also indicated that Cu, Zn, and Pb especially in MSC
were present as insoluble sulfides or bound with silicates. Therefore as long as the
samples were disposed and maintained in a stable or inert state, they probably would not
exhibit toxicity or genotoxicity.
 NOTE TO USERS

Page(s) missing in number only; text follows. Microfilmed âs

received.

UMI
Figure 4.1 This is a particle (830 pm) of Philippine scale (PSC) showing a
layered structure detailed on the lower photograph (330 pm). Note
each particle is surrounded by a silicate matrix (1OOx, 250x).
Figure 4.2 This is the Philippine sludge (PSL) showing agglomerates of fine particles
made up of iron oxide and sulfide. The white background resembles fine
grained silt. The two particles on the left are about 500 pm while the bigger
particle on the right is 830 pm (50x).
Figure 4.3 The American scale (ASC) has a vermicullar structure indicating
high porosity with no distinct crystalline pattern. The black lines
are magnetite in a silica background (50x).
 NOTE TO USERS

Page(s) missing in number only; text follows. Microfilmed as

received.

. .

UMI
Figure 4.4 The Mexican drilling mud (MDM) contains a variety of textures and
phases with particle size from 20 pm to 180 ,m.The black spots are
iron oxides and the white spots are iron sulfides surrounded by
silicates (50x).
Figure 4.5 The Mexican scale (MSC) has minerals inside a silicate matrix. The top
particle (120pm) has chalcopyrite (yellow), sphalerite (greyish green) and
galena (white cubic) while the bottom particle (50 p m ) has pyrite at its center.
Figure 4.6 The top photograph of the Mexican sludge (MSL) shows silica surrounded
by black magnetite. The bottom particles have verrnicullar structure indicating
high porosity with milky white silica background (100x and 50x, respectively).
4.1.6 Weatherfng test
Several minerals with solubility in water which are expected to be easily weathered
such as halite, gypsum, calcite, dolomite, hornblende, primary layer silicates and albite [46,
47, 85, 1281were also found present in the geothermal samples by XRD (Table 44). Only
about 5% of the original amount of Al from its silicates and 30% of the transition metals Mn,
Fe, Cu and Zn (probably from their suifides) were found in the leachate after three months
of continuous agitation. However, none of the TCLP or LEP regulated elements (As, Cr,
Cd, Ba, Hg, and Pb) were detected in the leachate in any sample. This may indicate that
agitation does not contribute significantly to leaching of geothermal residues as will be
shown in subsequent test results involving agitation.

4.2 Protocol Leaching Tests

With the protocol particle size of 9.5 mm or less in the LEP and TCLP tests, the
regulated elernents in al1 the samples were below the limits for leachate quality criteria in
Table 2-2. This may be an indication that the heavy metals exist as insoluble species or
may be trapped in the silicate matrix and were unable to leach within the one-day tests.
However, in a worst case scenario of a finer particle size of -125 pm for the
Mexican scale, Pb leached out above regulatory limits of 5 ppm. With increased surface
area exposed to oxidation, about 2% (12 ppm) of the total Pb leached out in the LEP while
18% (105 ppm) was found in the TCLP leachate as shown in Figure 4.7a. All values
plotted in this figure were the averages of three duplicate experiments with less than 5%
standard deviation. ihere was little leaching in both TCLP and LEP for the coarse sizes
(-9.5 + 4 mm) but was greater for the fine sizes (-125 pm). From these data, the TCLP can
be considered a more aggressive test perhaps due to the presence of more acetic acid in
the extracting fluid. In the LEP, 0.0025 mol1L of acetic acid were required to bring the pH
to 5 whereas in TCLP, the leachant had 40 times more acid concentration (0.1 moVL acetic
acid) at pH 3. These two procedures will be compared in detail in Section 4.9.1. TCLP has
three times greater rotation speed compared to LEP which can contribute to the dissolution
enhancement. Stumm suggested that faster diffusion controlled dissolution can be
achieved with increased flow velocities or increased stirring [128]. However, the extended
 -125 um -4 mm
Particle size

-125 um 4 mm -9.5 + 6 mm
Particle size

Figure 4.7 Cornparison of extent of Ieaching between LEP and TCLP for
Pb and Zn in the Mexican scale at various particle sizes.
agitation in the weathering tests (Section 4.1.6) did not show any significant increase in
leaching. Hence the difference between the LEP and TCLP must be the acid content of the
leachant. The same trend was shown for Zn in Figure 4.7b where leaching was observed
to increase by a factor of 3 in TCLP. Cu data was not shown since only <2 ppm leached
out in both coarse and fine fractions. Based on the protocol size of -9.5 mm, the
geotherrnal residues passed both Canadian and Amencan regulatory leach tests indicating
they can be disposed of at bulk sizes in a secure landfill. However, the fine sized Mexican
scale (-125 pm) may require treatment using appropriate technology prior to disposal.
Concern over the long-term fate of the residues had prompted the investigation of the
extended leaching behavior of both coarse and fine fractions of the Philippine scale and
sludge and the Mexican scale. The TCLP was used but the duration of the test was
increased from 18 h to 96 h.

4.3 Extended Leach Tests

4.3.7 Oxic Conditions
As in the one-day TCLP test, there was no significant leaching of trace metals frorn
the Philippine scale and sludge in either the coarse and fine fractions even after 96 h of
continuous extraction. Therefore subsequent discussions on extended leaching results will
focus on the fine sized Mexican scale (MSC) where leachate concentrations were found
above the regulatory limits. In Figure 4.8 is shown a comparison of the leaching behavior
of Pb from the coarse and fine particles of MSC. Each value in this figure was an average
of duplicate experiments with less than 5% standard deviation. The Pb leach curves of
coarse (Pb-C) and fine samples (Pb-F) shown in Figure 4.8a probably indicate a diffusion-
controlled dissolution of a phase that did not reach constant solubilization of metals within
the duration of the experiment as the metal concentration in the leachate was increasing
over time. This typifies the general dissolution behavior in plots of concentration versus
time shown in Figure 2.2, also observed by other researchers in municipal solid waste fly
ash [104, 1151 and more evidently in silicates [116-1181. The leaching kinetics of Pb will
be investigated further in Section 4.8.1 with supporting data for Fe and Zn in Appendix 1.
As shown in Figure 4.8a, it was indicated that only about 35% of the total Pb can be
leached out within one week. The unleached fraction may be assumed to be Pb existing
either as an insoluble phase or trapped inside the silicate rninerals.
Figure 4.8 Cornparison of extended TCLP leaching of Pb for coane
PM= (- 9.5 + 6 mm) and fine particles Pb4 (-125 pm).
The sequential extraction tests to be discussed in Section 4.4 also suggested that 65%
of the W was bound as residuals in the silicate rnatrix and only 35% of the total Pb can be
considered available. Figure 4.9 presents the dissolution behavior of Cu, Zn, Pb, Fe and
Na in MSC. At longer times, the dope of Pb appeared similar to the slope of Zn (and
possibly Cu but cannot be detected in this graph) which indicates that they may be
associated together and were thus released at the same tirne. From the photomicrograph
in Figure 4.5, galena, chalcopyrite and sphalerite were shown to be bound together in the
same silicate matrix.

O 20 40 60 80 100
Time, houm
I
pPb +Zn *Fe *Na +Cu

Figure 4.9 Dissolution behavior of major elements in the extended

oxic TCLP for MSC fines (-125 pm).
4.3.2 Anoxic Conditions
The anoxic extended TCLP leach test, perfomed on the Mexican scale fines, did
not produce any signifiant leaching of the regulated elements including Pb. Likewise, the
coarse samples leached out only a small fraction ( 4 % ) of Fe, Cu, Zn and Pb after 24 h
of leaching (data not shown). As shown in Figure 4.10 below for the fine sized samples,
Cu, Zn, and Pb leached out considerably less (-1%) during anoxic conditions. This
somehow supports their association in the same silicate matrix. The low rnetal solubility of
these sulfides will probably result in sequesteing of metals as long as reducing conditions
prevail. Thus the leaching of Cu, Zn, and Pb may be attributed to oxidative dissolution and
that, under anoxic conditions or where oxygen is depleted such as in a municipal solid
waste landfill, the Mexican scale is likely to be stable.

Figure 4.10 Dissolution behavior of major elements in the extended

anoxic TCLP for MSC fines (-125 pm).
4.4 Sequential Chemical Extraction
The sequential extraction results for the geothermal residues are shown in Figures
4.1 1 to 4.13. They have been plotted in stack-bar graphs as the percent leached in each
extraction stage (A to E) for the six regulated elements (Cr, As, Cd, Ba, Hg and Pb).
Overall, little extraction was found in either exchangeable or carbonate phase (Fraction A
and B) which were equivalent to LEP or TCLP protocol leach tests. The six elements were
found associated with the phases involving Fe-Mn oxides and sulfides (Fractions C and D,
respectively) with the highest values in Fraction El the residual silicate phase. Pb was the
metal of importance that was associated with the non-residual phases in al1 samples.
Below is a discussion of each element and their phase association.

Chromium
In the Mexican drilling mud and scale, more than 60% of Cr was extracted as easily
exchanged, also associated with carbonates and oxides. In the Mexican sludge, Philippine
scale and sludge, less than 40% belonged to the non-residual phases (A-D). There was
a significant fraction of this element associated with the silicate matrix of al1the geothemal
wastes. The proportion ranged from 95% of the American scale to 35% of the Mexican
drilling mud.

Arsenic
Only in Mexican scale and Philippine sludge was As found to be associated with
the non-residual fractions with 40-60% released in the non-residual phases as Fractions
A-C. All the other samples hold the As in their silicate lattice and hence were safely
immobilized over time. Arsenic was generally bound in siliceous or carbonaceous material
that was indigenous to the geothermal reservoir fluid [12].

Cadmium
Present in small quantity, Cd was primarily solubilized in four of the six samples as
easily exchanged or extracted where dissolution was greater than 80%. ASC had Cd
associated with the carbonate phase (Fraction B) and the MSC with only the sulfide and
silicate phases (Fractions D and E).
 PSC

Figure 4.11 Sequential extraction results for Philippine scale and sludge.
 ASC

MDM

Figure 4.12 Sequential extraction results for Amencan sale and

Mexican drilling mud.
 MSC

MSL

Figure 4.13 Sequential extraction results for the Mexican scale and
Mexican studge.
Barium
In al1 the samples, Ba was found mainly in the silicate phase and wiII probably not
pose any danger of eventual release to the environment.

Mercury
The Hg content of al1 of the geothermal wastes was generally low ( ~ 0 . 5ppm). It
was found associated with the silicate phase in five samples and leachable in the
carbonate phase in only one sarnple (Mexican sludge).

Lead
More than half of the Pb content of al1 samples was associated with the silicate
phase and will not be available to leach into the environment. No more than 40% of Pb
was extracted in the Philippine scale and sludge and American scale while around 50%
was found in the residual phases of Mexican sludge and drilling mud. In the Mexican scale,
the leachable Pb (35%) was associated with carbonates, oxides, and suifides and 65%
was lodged in the residual phase. There is agreement with protocol TCLP leaching results
in Figure 4.8a where the maximum leaching for Pb reached 32% after one week and was
increasing at a very slow rate thereafter.

4.5 Preliminary Acid Mine Drainage Potential Test

The static test BC Research Initial Test provided a preliminary indication that 3 of
the 6 samples had AMD potential. A calculation of the acid consumption (AC) and the acid
production potential (APP) was illustrated in Appendix C. The APP was based on the %
S which can be obtained from chemical analysis as in Table 4-1. The difference between
the APP and AC in kg H2S04/tonne material, determines the AMD potential. If the value
was positive then the waste was classified to have acidification potential which required
further testing. If it was negative, it was classified as a nonacid producer.
In Table 4-5 is shown that ASC, MSL and MDM can be considered as nonacid
producer since they produced negative values of AMD potential. This may be due to their
low S content and high pH with sufficient alkaline buffering capacity. On the other hand,
MSC, PSC, and PSL were found to have acid mine drainage potential with positive values.
Although these figures were low relative to mine heaps or dumps where AMD potential
were in the order of 1O to 100 times more [66. QI],
it was necessary to verify their long-tenn
acidification potential involving microbial mediation of the iron and sulfur oxidizing bacteria,
Thiobacillus ferrooxidans, present in the natural environment.

Table 4-5 Preliminary acid mine drainage potential test results

Sample PH %S APP* AC* APP - AC*

MSC 7.2 3.40 100.00 5.40 +98.0

PSC 3.5 1.70 51 .O0 0.98 +50.0
PSL 4.5 0.46 14.00 0.98 +13.0
MSL 7.8 0.01 0.31 2.90 -2.6
ASC 9.7 0.03 0.92 6.90 -5.9
MDM 8.3 0.10 3.10 86.00 -83.0
- ..

* in kg H,SO, per tonne of sample

4.6 Confirmation of Acid Mine Drainage Potential

4.6.7 Bactehal G r o W and Acclimation
Based on the trial expariments found in Appendix A, the best Thiobacillus
fenooxidans culture and medium providing highest bacterial density and motility were the
pure culture ATCC 19859 and APHA media, respectivety. The pure culture was purchased
from the Arnerican Type Culture Collection in Maryland, USA and the composition of the
APHA medium is given in Table 3-3. These were used in acclimatization of the bacteria to
the samples to be tested. A five-minute video (not submitted with this thesis) was produced
which documented bacterial growth from the lag phase to the endogenous phase. In Figure
4.14a is shown a photomicrograph of viable acclimatized bacteria taken from a light
microscope at 800x magnification. The bacteria were observed to be at the end of the
logarithmic phase and entering the stationary phase after one week with a density of 2 x
10 cells per mL. A sample calculation for estimating bacterial density is shown in
Appendix B. In the stationary phase as shown in Figure 4.14b, the bacterial population
density remained steady at 1 to 2 x 106cellsImL for about two more weeks until they begin
to die (Figure 4.14~).Also during this period, they were observed to have greater motility
existing as single, in pairs or even short chains. These three figures represent high,
medium and low bacteria density which can be used as a benchmark when monitoring
bacterial growth using a light microscope.
Precipitation and biofilm formation had been strongly linked with the tendency of
Thiobacillus ferooxidans to grow on surfaces. Figure 4.14d was taken from a light
microscope at 1OOOx magnification from a two-week old bacteria culture. It shows bacteria
interacting with solid particles and the presence of an organic capsule surrounding the solid
samples. According to Rojas-Chapana et al, this capsule may contain phosphorus and
colloidal sulfur particles that act as a reactive medium for the sulfur metabolism [119].
Atkinson and Fowler as cited by Rossi [48] rnaintained that the biofilm or capsule which
was pervious to air and nutrients for depths ranging from 50 to 150 pm, was a
biochemically inert organic polymer rnatrix where inorganic material such as iron
oxyhydroxides rnay also be trapped. The TEM photomicrographs (60,000~)in Figure 4.1 5
showed the intimate association of bacteria with amorphous and crystalline minerals and
soma globules. The fine crystalline particles surrounding the bacteria could be ferric ion
precipitates such as ferric oxyhydroxide or jarosite. The average size of each bacteria was
0.5 l m . There was an apparent shrinkage or distortion due to air drying and vacuum
collapse of the cell wall as well as the absence of fiagellum since these bacteria were
obtained at the end of a three-week leaching which was nearly the end of the stationary
phase. A dark outline of the cell wall rnay indicate extracellular adsorption of heavy metals
such as Cu, Zn or Pb onto the bacteria such as that reported by Davis et al [120]. However
this couid not be confirmed by TEM-EDX since the concentrations were less than 1%.
In Figure 4.16 is shown the Thiobacillus fenooxidans cell at higher TEM
magnification (99,000~).Cell division of T. ferrooxidans is mostly by constriction but
occasionally by partitioning [12 11 and this was captured in this photomicrograph. The celi
about to divide into two cells is about 1.7 pm in length with a 0.5 pm diameter. There were
two globules on each side of the bacteria each about 0.2 Pm in diameter. These globules,
according to Rojas-Chapana et al could be colloidal sulfur, a source of energy for the
bacteria, that act as energy reservoir for later use [Il
91.However, these globules could
also be cell remnants or section of dead cells.
Figure 4.14a A photomicrograph of acclimatized Thiobacillus fenooxidans
taken from a light microscope with an estimated high density
of 2 x I O 7 cellslmL (800~).
Figure 4.14b A photomicrograph of acclimatized Thiobacillus ferrooxidans
taken from a light microscope with an estimated medium density
of 2 x 1o6 cellslmL (800~).
Figure 4 . 1 4 ~ A photomicrograph of acclimatized Thiobacillus ferrooxidans
taken from a light microscope with an estimated low density
of 1.O x 1O6 cells/mL (800x).
Figure 4.14d This is a photomicrograph of Thiobacillus ferooxidans taken from a light
microscope showing a two-week old culture that developed a cloud-like
capsule around the samples. Note the bacteria (white spots) inside the
capsule and also on the surface of the solid samples (1000~).
 NOTE TO USERS

Page(s) missing in number only; text follows. Microfilmed as

received.

. .
UMI
Figure 4.16 This is a TEM photornicrograph showing a Thiobacillus femoxidans cell
(1.7 pm by 0.5 pm) about to partition. Note the two globules (G) on each side
as well as the jarosite (J) precipitates (99,000~).
 Dissolved oxygen from ambient air was found sufficient in this study to sustain
microbial growth of Thiobacillus femoxidans. The dissolved oxygen (DO) measurements
near the surface of the liquid were in the range of 5-7 mglL throughout the experiment.
This amount appeared to have supplied the oxygen requirement of the bacteria which is
best maintained at close to oxygen saturation in water at 6.6 mglL 1891. (Refer to Section
2.5.3.5.) The DO was slightly lower in the agitated flasks inside the incubator at 35 OC
presurnably due to the lower solubility of oxygen at higher temperature. In Figure 4.21 is
shown that there was a progressive increase in redox potential (Eh) in al1 samples
throughout the experiment, indicating that oxidation was probably taking place [77]. It was
also seen in the videotape taken under the microscope that there was microagitation of
bacteria under the glass slide due to regular air movement in the room such as opening
or closing of the door. This may be related to possible agitation inside the stationary flasks.
Jarosite and iron oxyhydroxide formation is not desired in bacteria culture and
bioleaching as reported by several authors. The detrimental effects of jarosite formation
are : (a) it diminishes the available femc ion in solution; (b) it limits the amount of biomass
retention since ferric ion deposits occupy the bulk of the available space; and ( c) it creates
a kinetic barrier because of the slow diffusion of reactants and products through the
precipitation zone [73, 861.Jensen and Webb provided an excellent review of jarosite
formation linked to wall growth and biofilms [73]. They cited a pH range of 2 - 2.5 when
bacteria attain maximum growth and a range which inhibits ferric ion precipitation. Pesic
and Kim reported that bacteria cells serve as nucleation sites for the formation of jarosite
[122]. In this study, a yellow to orange brown solution color was observed after one week
when the pH was 2.5-3.0 for stationary flask and pH 2 for agitated fiask, and a fine yellow
precipitatewas visually obsewed only on the 4th week towards the end of the experiments
when the bacterial activity had declined. The yellow orange to orange brown precipitate
was determined by X-ray diffraction as jarosite (K)Fe, (SO,),(OH),. Barron and Lueking
reported that precipitates occurred at the stationary phase and color change was observed
during logarithrnic phase with no precipitate [84].
There was more precipitation found in stationary flasks than in shake flasks. This
was because the formation of ferric ion precipitates, especially jarosite, was dependent on
pH as can be seen from Equations 2 and 5. In Figure 4.17 is shown the pH-Eh versus
time graphs of the Philippine scale and sludge and the Mexican scale. These pH values
had a standard deviation of k0.05 units taken from three replicates. The pH's of the left
column (agitated flasks) had lower pH of 2 compared to pH 2.5 - 3 for the right column
(stationary flasks). In agitated flasks, there was probably increased oxidation of ferrous ion
and release of more acidity which lowered the pH. A pH of 2 was required to prevent
precipitation [73].Also in Figure 4.17 is shown a sudden rise in pH at the onset which was
also observed by several researchers [48, 731.This could be due to the consumption of
H,S04 during oxidation of Fe *' to Fe '+ catalyzed by Thiobacillus femooxidans (Equation
5). Afierwards the pH decreased as sulfur was oxidized to SO, 2' producing acidity
(Equation 4). The Eh profiles also in Figure 4.17, especially for the stationary flasks
increased steadily indicating progressive bacterial growth and metal solubilization [37,771.

4.6.2 Acid Mine Drainage Potential (AMDP) Test

After 23 days of bioleaching using the AMDP test, Pb was not found in the leachate
in any sample or experimental test conditions. For the Philippine scale and sludge, less
than 2% leached out for both Cu and Zn with or without bacteria. On the other hand, for
the Mexican scale, almost 700% of Cu and Zn were released in the leachate both for
agitated and stationary experiments, respectiveîy. These results are shown in Figures 4.18
and 4.19. In general, the agitated flasks had leached out more metals by as much as 10%
for MSC, 0.3% for PSL and 0.5% for PSC. Since Pb is the regulated element (and Cu and
Zn are not), al1 the samples pass the regulatory requirement for leachate quality criteria in
Table 2-2 and therefore should not be classified as hazardous wastes. Also, PSC and PSL
leachate quality were within the limits for disposal to agricultural lands listed in Tables 2-3
and 2-4 while MSC requires special attention.
As shown in Table 4-5. MSC, PSC and PSL were found to have AMD potential
based on the preliminary static test. Further investigation using this kinetic test confirmed
that they al1 had acidification potential since the final pH of al1 the solutions was below 3.5
as illustrated in Figure 4.20. However, in terrns of releasing heavy metals, only MSC has
a confirmed acid mine drainage potential for Cu and Zn but not for Pb. In spite of the acid
generation which was supposed to promote breakdown of the sulfide mineral lattice hence
more dissolution of metals, the bioleachate only contained the soluble metal sulfates of Cu
and Zn and not the insoluble lead sulfate.
 1L
O 5 10 15 20 25
'
30
200

Time, dry8 Time, days

PSL

11 200
O 5 10 15 20 25 30
Time, dayr Time, d a p

MSC

11 J 200
O 5 10 15 20 25 30 O 5 10 15 20 25 30
Time, days Time, days

Figure 4.17 These graphs show the inverse relationship of pH-Eh change over tirne. The
graphs on the left column (a, c, e) were agitated experiments at 35 OC while
graphs on the right column (b, d, f) were stationary experirnents at 25 OC.
 PSC

Time, days

PSC

O 5 10 15 20 25
Time, days

100

80

O
60
CuI C
-
0
s 4Q

20

O
O S 10 15 20 25
Time, days

Figure 4.18 Agitated experiments at 35 OC: overview of metal leaching over time
frorn PSC, PSL and MSC during acid mine drainage potential tests.
 PSC

O 10 15 20 25
Tims, d a p

O 5 10 15 20 25
Time, days

O S 10 15 20 25
Time, days
r Cu + Zn t Pb

Figure 4.19 Stationary experiments at 25 OC: overview of metal leaching over time
of PSC, PSL and MSC during acid mine drainage potential tests.
 Pb, however, which forrned an insoluble sulfate was not found in solution since it
could have precipitated immediately and associated with the other precipitates such as
ferric sulfates and oxyhydroxides. Silver and Tonna [144] and Tomizuka as cited by Rossi
[48] detected lead sulfate (anglesite) through X-ray diffraction analysis of the insoluble
residual rnatter from bioleaching of galena or lead sulfide concentrate containing 42% Pb
and 30% S. Two methods as described in Section 2.5.2 can produce lead sulfate. In the
presence of ferric ion. the oxidation can be expressed by Equation 13 as follows

PbS + Fe,(SO,), -+ PbSO, + 2FeS0, + S0 (13)

While with direct biological mediation, lead sulfate can be formed via Equation 14, with the
Thiobacillus ferrooxidans also involved in the oxidation of elemental sulfur and ferrous ion
generated in Equation 13.

bacteria
PbS +20, -+ PbSO,

The low metal leaching in PSC and PSL can be due to a number of reasons. Firstly,
MSC has a much higher metal concentration (which were identified as metal sulfides by
XRD) than in PSC and PSL. On the average, MSC's original composition reported in
Table 4-1 had 50x more Cu, 1OOx more Zn and 70x more Pb. Secondly, the heavy metals
in PSC and PSL may not be in sulfide forrns which should be amenable to bioleaching.
Thirdly, as shown in the photomicrographs in Figures 4.1 and 4.2, they were not accessible
to the bacteria and to the leach solution since they were bound by strongly cemented
silicates.

4.7 Batch Kinetic Experiments

The original concentration of Cu, Zn, and Pb in MSC was about two orders of
magnitude higher than in PSC and PSL as shown in Table 4-1. Thus the low levels of Cu
and Zn in PSC and PSL rnay lower metal recovery cornpared to MSC. This will be reflected
in the following discussion where the overall fraction extracted for Cu and Zn is almost
100% in MSC with less than 2% for PSC and PSL. Based on sequential extraction, rnost
of the heavy metals were lodged in residual phases. According to X-ray diffraction. PSC
and PSL contain silicates and crystalline particles that are not easily weathered as was
reported also from the accelerated weathering test in Section 4.1 -6. The effects of
sterilization, agitation and temperature, and bacteria on metal leaching are presented
beloiiv. The results plotted in the graphs are averages of three replicates.

4.7.1 Effect of Sterilization

There is no marked difference in the overall leaching efficiency of Cu, Zn, and Pb
under both sterile and nonsterile conditions, as shown in Figures 4.22 - 4.24 for PSC, PSL
and MSC, respectively. Without agitation, around 80% of Cu and Zn had leached out from
MSC after 23 days. The leaching of Pb in MSC showed a decreasing pattern from 4% at
the start to near zero at the end of the test. There is evidence of secondary precipitation
of a Pb compound, possibly PbSO,, which is perhaps why it was not detected in the
leachate. Both Cu and Zn had less than 2% leached out in PSC and PSL in both sterile
and nonsterile conditions. These results could mean that sterilization of sample and the
leaching medium was not critical as far as the growth of Thiobacillus ferrooxidans was
concerned. This was also verified qualitatively under light microscope where the bacterial
density and rnotility appeared to be the same for both conditions. The cotton plug on the
Erlenmeyer flasks also served to filter any airborne contaminants and although spores and
other microorganisms can enter the flask, they will not likely survive the low operating pH
(c 3). The AMDP test can therefore be performed in nonsterile conditions.

4.7.2 Effect of Agitation and Temperature

The intent of these experiments was to compare whether the AMDP test can be
carried out using either stationary or shake flask technique. The results are shown in
Figures 4.25 - 4.27 for PSC, PSL and MSC. In the Mexican scale in Figure 4.27, the
maximum leaching efficiency (-100%) was attained for Cu and Zn within the first two
weeks in agitated experiments. On the other hand for the Philippine scale and sludge.
agitation and higher temperature did not appear to have any significant effect (~0.5%)on
metal leaching. With these results, the duration of the test could probably be shortened
if shaking technique was used at higher temperature (35 O C). The shake flask technique
appeared to be more aggressive as it leached out approximately 20% more than the
 PSC

O 6 10 15 20 2S 30
Time, days

1
0 O 10 1s 20 26 30
Time, days

MSC

O 5 10 15 20 26 30
Tirne, days

Figure 4.20 These three graphs show variations in pH over tirne for
the five sets of batch kinetic AM0 experiments. The agitateci
experiments were performed at 35 OC while the rest at 25 OC.
 O 6 10 15 20 25 30
Time, days

PSL

0 0
O 5 10 15 20 PL 30
Time, days

MSC

O 5 10 16 20 2 30
Time, days

Figure 4.21 These three graphs show variations in Eh over tirne for
the five sets of batch kinetic AMD experiments. The agitated
experiments were performed at 35 OC while the rest at 25 OC.
stationary flask in the MSC. Thus if there was no provision for an incubator-shaker in a
laboratory, the stationary flask technique can still be used but the duration of the test has
to be increased from three to four weeks. On the other hand, if the shake flask technique
was used, then the test could provide reasonably good results within two weeks.
Temperature may not be a concern in warm or tropical climates where the average
ambient temperature is 35 OC or higher especially in geothermal areas.
It was found also during bacterial acclimation that the culture of Thiobacillus
femoxidans adapted well at room temperature yielding higher density than those shaken
and incubated at 35 OC as shown in the bacteria growth curve in Appendix G. In addition,
the particle size could be decreased also from the present -125 pm to as low as possible,
perhaps -75 + 45 pm as suggested by several authors [48. 91, 971.

4.7.3 Effect of Bacteria

The contribution of bacteria to the overall leaching was found more significant in
MSC than PSC and PSL as shown in Figures 4.28-30. It can be observed from Figures
4.28 and 4.29 that the bacteria had no effect in the leaching of heavy metals Cu, Zn , and
Pb from PSC and PSL. From XRD data in Table 4-4, Cu, Zn and Pb sulfides were
identifiedto be present in the Mexican scale but not in PSC and PSL.From Figure 4.1, only
the iron sulfide pyrite (FeS,) was found present in PSC interspersed with magnetite and
silicate. As shown in Table 4-1, PSC and PSL contain smaller amounts of sulfur compared
to MSC which may not al1 be in sulfide form amenable to bacterial leaching. With rnicrobial
mediation in MSC, metal sulfides were oxidized either by oxygen (Equation 1) or ferric ion
(Equation 3) to form metal sulfates which were generally more soluble except for PbSO,
which is sparingly soluble (K, = 1.6 x 'IO9). Due to the formation of insoluble Pb species,
leaching of Pb ions to the environment may not occur. In addition, solubilized lead can
precipitate with ferric hydroxide or jarosite (orange brown color) which were common
indicators of acid mine drainage. Thus metal immobilization of Pb, the regulated element,
may be a beneficial consequence of microbial mediation, as reported also by several
authors 1123-1251.
Both bacterial rnotility and density were found to have direct effect on metal leaching
than bacterial density alone. In general, higher leaching efficiency was observed in agitated
experiments which yielded more active bacteria though lower density than the stationary
experiments. The bacterial motility was monitored visually as speed or activity was dimcult
to measure [126]. It was noted as very fast, fast or slow and was captured vividly in
videotape.

4.8 Reaction Rates and Mechanisms

4.8.7 Chernical Leaching Kinetics
A reaction rate equation has been developed for Pb which can be used in predicting
the rate of metal release over time if disposed in a landfill. Results reported in Section 4.3.1
on extended TCLP under oxic conditions have indicated that most of Pb extraction follow
a dissolution mechanism that was controlled by diffusion, Le., transport of the reactants
and products through a layer of the mineral surface as depicted in Figure 2.2. Initially,
rapid leaching occurs which is surface-controlled reaction at the exposed surface area of
the particles containing sulfides. As the inert or leached layer grows thicker, the distance
of diffusion is increased which in turn slows the rate of leaching. An excellent reference on
the theory and mathematics of diffusion is by Crank [145]. In the chernical leaching of the
Mexican scale, the rate equation corresponds to a diffusion-controlled reaction known as
the parabolic rate law. This was described by Stumm and Wollast [127-1291 as

r = dC/dt = kt -Il2 mol.L-'.h" (1-1)

where k is the reaction rate constant (mol.L''.h ""). By integration, the concentration in
solution, C (mollL), increases with the square root of time as such

mol.1-'

In Figure 4.31 is shown Pb concentration increasing linearly as a function of the square

root of time. The plot for the coarse fraction fits linearly through zero. However for MSC
fines, the intercept was displaced which indicates exposure of a more leachable Pb fraction
or species at the initial period of the leaching. The intercept Cowas the number of moles
rapidly exchanged between the mineral surface and the solution and in this case has a
value of 145 pmollL from regression analysis. The grinding appears to have liberated a
significant amount of highly leachable Pb at a rapid rate occurring within the first hour of
the experiment. Once this first phase has been exhausted, a parabolic dissolution rate
was observed which indicates a slow dissolution behavior of a less soluble or less
accessible phase toward equilibriurn. This was also observed in Figure 4.9 for the other
rnetals. Additional dissolution kinetics data for Fe and Zn may be found in Appendix 1. Cu
was not plotted due to its very low recovery (~0.5%).
The dope of the line represents k,the parabolic rate constant with values of k, .4.6
x I O 6 mo1.L-'.h for coarse particles and k, = 1.1 x 1O4 mol.L".h -'"for fine particles as
shown also in Figure 4.31. Thus to account for the dependence of the rate on particle size,
the rate expression for Pb involving both coarse and fine particles should include a term
a which represents the coarse fraction and fine fraction, a, and a, respectively with the
-
relationship {aa,=1 a, ). Equation 2-1 then becomes

and differentiating it gives

Thus the rate equation for the dissolution of Pb in the Mexican scale can be expressed as

The slower rate r, of the coarse fraction was obviousiy controlling the overall reaction rate.
Equation 5-1 confirms the relationship between leaching and particle size: as particles are
reduced ta finer sizes increasing the surface area, more leaching was expected to occur.
Thus, eliminating or isolating the fines from the coarse fraction prior to disposal can reduce
the leaching by a very wide margin.
 --
O 5 10 15 20 25
Time, days

O 5 10 15 20 25
Tirne, days

O 5 10 15 20 25
Time, days

Figure 4.22 The three graphs show the effect of sterilization on rnetal
bioleaching in the Philippine scale for Cu, Zn and Pb,
in stationary flasks at 25 OC.
 Tirne, days

Time, days

Figure 4.23 The three graphs show the effect of stenlization on metal
bioleaching in the Philippine sludge for Cu, Zn and Pb,
in stationary flasks at 25 OC.
 O 10 15 20 25
Time, days

O 5 IO 15 20 26
Timr, days

100

80

8 a0
C
=
a
8
40

20

0
- ' -
h
v = w
œ
--
O 5 10 iS 20 25
Time, days

Figure 4.24 The three graphs show the effect of sterilization on metal
bioleaching in the Mexican scale for Cu, Zn and Pb.
in stationary flasks at 25 OC.
 L

p 1*5 L
I

r
Pa# 1 A
I
-
A
v v
A w

*ai I

0.5

O
O 5 10 15 20 25
Time, days

O 1.5
Q)
r
Zr! P
0,
1
œ I
I
I
-
-
-
I
I m
ae 0.5 A
7
.-
A A
T
'b/
O
O 5 10 15 20
I
25
Tirne, days

O
O 5 10 15 20 25
Time, days

Figure 4.25 The three graphs show the effect of agitation and higher
temperature on metal bioleaching in the Philippine scale
for Cu, Zn and Pb.
 Tirne, days

O 5 10 15 20 25
Time, days

1.5
aa
C I

I l C
Pb a
'
0

0.5

0
-
-
9
I
I
I
w I
I
-
9

O 5 10 15 20 25
Time, days
+ Agitated, 35 OC &tationary, 25 OC

Figure 4.26 The three graphs show the effect of agitation and higher
temperature on metal bioleaching in the Philippine sludge
for Cu, Zn and Pb.
 Cs!

O 5 10 15 20 25
Time, days

O 5 IO 15 20 25
Tirne, days

100

80
'Lt
60
m
2 40
s 20

0 0
O 5 10 15 20 25
Time, days

+ Agitated, 35 OC + Stationary, 25 OC

Figure 4.27 The three graphs show the effect of agitation and higher
temperature on metal bioleaching in the Mexican scale
for Cu,Zn and Pb.
 2

= 1.5
Q, I
t
P
a9
1
I

0.5

O
O 5 10 15 20 25
Time, days

O 5 10 15 20 25
Time, days

O 5 10 15 20 25
Time, days

+ With bacteria Without bacteria

Figure 4.28 Effect of bacteria on metal bioleaching in the Philippine scale for
Cu, Zn and Pb in stationary experiments at 25 OC.
 O 5 10 15 20 25
Time, days

p IS
r
O
crr 1
a#
9

0.5
b
O 1 '
O 5 10 15 20 25
Time, days

O
O 5 10 15 20 25
Time, days

+ With bacteria Without bacteria

Figure 4.29 Effect of bacteria on metal bioleaching in the Philippine sludge for
Cu, Zn and Pb in stationary experiments at 25 OC.
 Time, days

'O0 0

O 5 10 15 20 25
Time, days

O 5 10 15 20 2s
Tirne, days

+ With bacteria Without bacteria

Figure 4.30 Effect of bacteria on metal bioleaching in the Mexican scale for
Cu. Zn and Pb in stationary experiments at 25 OC.
4.8.2 Metal Solubilization Rate
Leaching of Cu and Zn in MSC was almost at the same rate but ver- low or
negligible for Pb. In Figure 4.32, the extent of leaching was compared for these three
metals as a function of time. Of the five sets of experiments, the highest metal recovery
was observed in the agitated flasks more than the stationary ffasks except in the case of
Pb, as discussed earlier in Sections 4.6 and 4.7. Premuzic et al reported 85-90% recovery
for Cu and Zn from bioleaching of geothermal sludges at 55 OC and pH 1-2 after only one
day (21, 241. Similar recovery was obtained in this study for Cu and Zn but after one week
and at 35 OC and pH 2-2.5.
The rnetal dissolution rate for Cu, Zn, and Pb in the Mexican scale was at its
maximum within the fint week of the AMDP test. As shown in Figure 4.33, the
solubilization rate for Cu, Zn, and Pb was more enhanced in the agitated experiments at
the beginning. However, both agitated and stationary flasks approached the same rate at
the end of 23 days when the bacteria would have reached the end of their stationary
phase. In Table 4-6 is presented the maximum solubilization rate that occurred within the
first week especially with agitation. This indicates that as far as the AMDP test was
concerned, the agitated test is probably more aggressive than the stationary test by as
much as three tirnes.

Table 4-6 Maximum Solubilization Rate in Batch Process

Metal Agitated, Stationary, Rate

35 OC, 25 OC, Difference
mg/Ud mglUd
Figure 4.31 Dissolution kinetics for Pb in the coarse (-9.5 + 6 mm) and fine
(-125 pm) fractions of the Mexican scale. Data on concentrations
over tirne were obtained from extended TCLP under oxic conditions.
 O 5 10 15 20 25
Time, days

O S 10 15 20 25
Time, days

Time, days
+ ~gitsted + Statîonsy + Stedle
* Control -F Nonsterile
Figure 4.32 Overall percent leaching over time of Cu, Zn and Pb for the five
sets of AMD potential experirnents on the Mexican scale. The agitated
experiments were performed at 35 OC while the rest at 25 OC.
 O 4 8 12 16 20 24
Time, days

O 5 10 15 20 25
Time, days

O 5 10 15 20 25
Tirne, days

r Agitated, 35 OC 4 Stationary, 25 OC

Figure 4.33 Soiubilization rate for Cu, Zn, and Pb in the Mexican scale during the
AMD potential test for both agitated and stationary experiments.
4.9 Cornparison Between Acid Leaching and Bioleaching
4.9.1 Between TCLP and LEP
TCLP appean to be more aggressive than LEP since it leached out more metals.
A good example is illustrated for Zn and Pb in Figure 4.7. The concentration of Cu in the
leachate was less than 2 ppm in al1 samples and was not reported in the graphs. The major
component of the leachant was acetic acid (HAc) in both procedures. Of the three ions,
Pb2+has the highest affinity for acetate ion followed by Zn2+andthen Cu2+[130].Therefore
because the acetate concentrations were higher in the TCLP, it was most likely that lead
acetate would be in abundance in the leachate especially since this was a Pb species that
was highly soluble.
The TCLP extracting fluid in the Mexican scale was 1 L of acetic acid solution (0.1 N
HAc) at a pH of 3. The available {H'} and {Ac-} were 1.33 mmollL each. Whereas in the
LEP, the extracting fluid was 1L of deionized water with periodic addition of 0.5N H Ac to
the solution to maintain a pH <5 * 2. Total acid added for Mexican scale in LEP was
typically 5 mL rnaking the available {H+} and {Ac} as 0.2 mmol/L. The amount of {Az}
available in TCLP was therefore 7-fold more than in LEP. With this advantage of excess
concentration of {H'} and (Ad), the TCLP solution was a better buffer and was able to
maintain the H+ion demand during the test due to redox reactions or complexation. Since
thete was greater supply of {Ac3 ions in the TCLP extracting solution, there was more
acetate complexation with Cu2+,Zn2+,and pb2+which increased their solubility in solution
[131]. However, since Cu2' has the least affinity for acetate ions, its leaching was not
significant (c2ppm) compared to Zn2+and Pb2+. As shown in Figure 4.7, Zn2+and Pb2+
were released more in the TCLP leachate than in LEP leachate. In addition, the agitation
speed of the rotary extractor was three times more in the TCLP than in LEP (30 rpm
against 10 rpm) which could prornote weathering. Thus, in the case of the geothermal
residues, the American TCLP appears to be a more aggressive test for leachability than
the Canadian LEP.

4.9.2 Between TCLP and A MDP

In the case of the bioleaching, AMDP leached out more Cu and Zn than Pb, which
was the reverse behavior obsewed in TCLP. Only the agitated procedure of the AMDP was
used for cornparison with TCLP so that weathering of particles was a common process in
both tests. In the case of the Mexican scale in Figure 4.18, it was shown that AMDP was
a more aggressive test than TCLP (Figure 4.9a) since more metals were dissolved in the
leachate. However, almost none of the regulated element (Pb)leached out. The major
components in AMDP solution were H', SO, ", and Fe 24 with Cu ", Zn 2', and Pb 2+ from
the sample. Of the three metal ions, Pb 2' has the highest affinity for sulfate ion followed
by Zn2+and then Cu2' [130]. Therefore it is likely that PbSO, will precipitate first. PbSO,
which is a sparingly soluble to insoluble compound (K,= 1.6 x lob) will probably be in the
solid phase and hence will not be detected in the leachate. Though the AMDP test
produced more Cu and Zn, the regulated element Pb was found to have been immobilized
through bacterial action. From these results, it was not clear which was a better test in
determining the hazard potential of the residues. It would be most prudent to use them
conjunctively as they provide two different scenarios that may happen in field conditions.

4.9.3 Evaluation of the A MDP Procedure

Several criteria were used to evaluate laboratory tests that were most appropriate
for prediction of acid mine drainage potential. Most common evaluation criteria that were
mentioned in Tables 2-5 and 2-6 were simplicity, time required, equipment required, cost,
ease of interpretation, and correlation with field data [68,911. The AMDP procedure
developed at the University of Toronto for geothermal residues can be described as low
cost, simple, low technology, reproducible, requires little operator training, no specialized
equipment needed, tolerant of sample variations, and can be performed in a nonsterile
environment. Table 4-7 below shows a cornparison between the BC Research
Confirmation and AMDP test. It shows the advantages of the AMDP procedure since it has
the versatility to be used more widely in laboratories with limited equiprnent, in actual
mine/wellfieldsites, and especially in developing countries where use of geothermal energy
was rapidly increasing.
The major drawbacks of the BC Research Confirmation Test in relation to geothermal
residues were:
1) the pulp density was too high
2) no indication of redox reactions
3) no indication of bacterial viability
4) no FeSO, in the leaching medium.
Table 4-7 Cornparison between BC Research Confirmation and AMDP Tests

B.C. Research Uoff- Acid Mine Drainage

Confirmation Test Potential Test
-

lnoculum no specific guideline reproducible method of

Nutrient Media only basal salts, no FeSO,
Particle size 400 mesh
Solid concentration 15-30 gRO mL (amount
depends on S content)
Agitation With agitation, shaker
speed not specified
Temperature lnside incubator at 35 OC
Sterilization Media to be autoclaved
Measurements pH only, metals are
optional
bacteria culture
basal saits arrd 50% of the
standard 9.0 glL Fe "
120 rnesh
With or without agitation,
(175 rpm if shaker
available)
Room temperature
(between 23-25 OC)
Media to be filtered with
0.45 pm ceilulose acetate
pH, Eh, bacterial growth,
metals

There have been several studies to validate the results of AMD prediction tests and
most of thern provided good correlation with field conditions. Around 44 static and kinetic
tests were performed on 22 rock samples from seven rnetal mines in British Columbia and
Yukon, Canada [66,93, 951. Samples of tailings and waste rock were obtained from both
active and abandoned mine sites with sulfur tontent in the range of 0.13 to 49.2%. The
static and kinetic tests correctly predicted the formation of AMD in al1 but six cases: three
were incorrect and three were inconclusive due to inconsistent data. The Energy, Mines
and Resources Canada carried out a comprehensive study to evaluate AMD prediction
techniques used in Canada and USA [QI]. Eleven procedures such as those listed in
Tables 2-5 and 2-6 were undertaken on 4 waste rock samples and 8 tailings samples for
a total of 12 samples. The prediction of the AMD correlated well with field data in al1 but
one tailings in static procedures (positive prediction but there was no AMD in the site) and
two tailings in kinetic procedures (the reverse happened for both AMD predictions, one
positive and the other negative). In the US, seven of the 56 mining-related sites were
reviewed to determine if acid generation predictive tests were conducted at individual sites
and if so. compare with actual AMD situation [68]. This study is currently going on.
Although notning in literature has been reported on the occurrence of AMD from
geothemal residues, these examples provide a higher confidence put on AMDP prediction
techniques.
In this study, the acid mine drainage potential of geothemal residues was evaluated
using the AMDP test. It was found that the Mexican scale, Philippine scale and sludge
have the potential for acidification since pH at the end of the test was below 3.5. However,
on the basis of solubilizing metals and releasing them to the environment, only the Mexican
scale has the potential to do so for Cu and Zn but not for Pb. These results require further
verfication through testing of more samples and field observations.

4.10 Geochernical Modeling

The use of MINTEQA2 in geochemical modeling can provide concentrations and
speciation under equilibrium conditions. The interpretation of the results and comparison
with experimental data can be challenging. Table 4- 8 below shows the results of modeling
of the Mexican scale in TCLP under anoxic conditions, Le., closed system which closely
fit the experimental conditions (the bottles were completely sealed throughout the test). All
the other sarnples did not provide significant leaching hence modeling was focused on the
MSC.
The model outputs were verified by making material balance calculations and
verification whether the Y, and K,, values were similar to those reported in literature. From
Part 6 of Output File (Appendix F), the saturation indices of al1 input solids were zero which
means that they were the controlling solids allowed to precipitate. Shown in Table 4-8
were the activities of each ions as obtained from Part 3 of Output File as Type I and II
species. Type I are components as species in solution while Type II are complexes, free
ions, adsorbed species in solution. There was a variation in the Ksp values from different
sources [85,98, 130, 1431. The Ksp from Lindsay [143] appears to be close to the
calculated Ksp especially for H,O, CuS, ZnS, and PbS.
Table 4-8 Summary of Results from Geochemical Modeling of the Mexican Scale

Toxicity Characteristic Leachate Procedure (TCLP)

Parameters Experimental Model Dissolved Species

Concn, mol/L Activity, molIL*

Cu2+ 1.8E-05 1.838E-17 Cu2' (82.1%), CuAcetate (15.8%)
Fe2+ 1.1E-03 1.176E-06 Fe2'(97.2%), FeAcetate (2.8%)
Zn2+ 1.2E-04 4.834E-06 Zn2'(95.8%), ZnAcetate (4.1%)
Pb2+ 8.7E-06 1.480E-09 Pb2'(53.2%). PbAcetate (45.8%)

'obtained from Part 3 of Output File (Type I components), Appendix F,

y, activity coefficient : 0.84625

The detection limit for the elements of concern were 1.8 x I O 8 mollL (0.1 ppm) for
Fe, 1.5 x IO-' moVL (0.01ppm) for Cu and Zn and 1.5 x 1ObmoVL (0.3 ppm) for Pb, based
on the ICP analysis of the leachate. Any value below 1.O x IO'? was assumed to be below
detection limit and was not significant except for theoretical calculations.
The {H+) in the equilibrated mass distribution (Part 5 of Appendix F) does not
correspond to the equilibrium pH. The equilibrium pH was treated by MlNTEQA2 as the
hydrogen activity contributed by the H' species in solution (Part 3 of Output File, Type 1)
whereas the equilibrated total {H') was the sum of both dissolved and adsorbed species
(Part 4 of Output File). Any difference between the equilibrated concentration and the
dissolved concentration represents the H' concentration bound in adsorbed species (Part
4 of Output File) as acetate. Note that the activities were used instead of concentrations
since MINTEQA2 uses activity (a=y c where a is activity, yactivity coefficient and c molar
concentration) in its calculations.
Relative solubilities of metal sulfides can be predicted based on the kPvalues if
they produce the same total number of ions during dissolution as in the case of CuS, PbS
and ZnS which produce two ions each [130]. Thus among CUS (K,, = IO-^'), PbS (Y, =
) and ZnS (Ks, = 1 ), ZnS would be more soluble, followed by PbS and CUS,the
least soluble. At equilibrium, this prediction was validated by MINTEQA2 where CuZ+was
the least dissolved compared to Zn2' and pbZ'.
In Table 4-8 is shown that equilibrium behavior was obviously not realized in the
experimental tests since the concentrations obtained from the experiments did not
correspond to the model results. At equilibrium, the pH was predicted to decrease from
the experimental value of 6.5 to 2.8 since there will be more free H' and acetate ions in
solution due to the precipitation of the metals. The dissolved metal species were in their
ionic f o n s and as acetate compounds. In Sections 4.3 and 4.4, it was found that only
35% of the original concentrations of Pb in the Mexican scale would have been available
for leaching, which was not reflected in the model. This is the kinetic constraint of
thermodynamic models and without any cornplementary experimentation can lead to
misinterpretation.
The modeling results indicated that protocol leach tests do not represent long-term
leaching behavior of the Mexican geothermal scale under natural environment. The
inherent shortcoming of this modeling effort is the lack of accurate information on the other
important species in the complex geothermal samples which may be controlling the
solubility of the major species.

4.11 Summary
Geotherrnal residues are composed rnainly of silica (-70% by weight) with iron and
aluminum oxides and trace amounts of S, Cu, Zn,and Pb. Particte size varies from
submicron to 1 mm and above. They are made up of heterogeneous particles with each
particle having a complex microstructure and rnineralogy especially in the case of the
scale. The elements of environmental concern are mostly trapped inside a hard silicate
matrix. Sludges are porous and made up of agglomerates while drilling mud is a complex
mixture of secondary silicate minerals. These geothermal residues were studied for their
long-term leaching behavior in acidic medium and in the presence of bacteria.

4.71.1 Chernical Leaching Behavior in Protocol Tests

The process of leaching is affected by various parameters such as type of leachant,
dissolved oxygen, acidity, particle size, type of material, concentration of rninerals and to
a certain extent, temperature and agitation. In the case of mineral leaching from
geothenal residues, the following successive steps are postulated :
a) mass transport of dissolved reactants such as oxygen and acid from bulk solution
to the mineral surface,
b) transfer of species through the reactive zone.
c) chemical reactions of the sulfide minerals,
d) transport of products to the surface,
e) mass transport of species into the bulk of solution.
The concentration vs time profile indirectly indicated that diffusion through the reaction
zone is rate lirniting (Section 4.8.1).
In the case of the Mexican scale where significant amounts of Fe,Zn and Pb were
released, particle size, pH of the leachant, and oxygen availability were found to be most
importantfactors. Laboratoryexperiments demonstrated that leaching was enhanced when
particle size was reduced (Figures 4.7 & 4.81, oxygen was available (Figure 4.9), and pH
was low (Figure 4.7). On the other hand, leaching was found minimal or even insignificant
when oxygen was sparged from the system by nitrogen gas (Figure 4.10).
The leachant, in this case, acetic acid solution, has a number of roles such as
a) a carrier of dissolved oxygen and other reactants,
b) provision of acetate ions for solubilizing metals.
c) attack of mineral phases
Thus with more acidity such as in TCLP vs LEP, more metals were found dissolved
in the leachate. lncreased agitation could have contributed to a minor extent but in the long
run when diffusion was the rate limiting or dominant reaction, agitation did not appear to
have a marked effect on rnetal dissolution (Figure 4.9).

4.17.2 Bioleaching of Geothennal Residues

Geothermal residues have sulfur content in the forrn of rnetal sulfides that indicate
a potential in the long-terni for acid mine drainage (AMD). AMD is experienced in coal and
metal mines whereby pyrites and other sulfide minerals are oxidized releasing acidity and
metals to the environment. This is usually promoted by the presence of iron and sulfur
oxidizing bacteria such as Thiobacillus ferrooxidans as discussed in Section 2.5.2.
 For the geothennal residues. a new test called Uoff's acid mine drainage potential
test (Appendix G), was developed based on an existing procedure because the bacteria
had to be acclirnatized to this substrate and ferrous ion had to be added to the culture
media since the residues are low in Fe and S relative to mine tailings. From this test, only
the Mexican scale was found to have a slight AMD patential since Cu and Zn leached out
but not the regulated element Pb. Although less than 4% of Pb was in the leachate at the
beginning, its disappearance was probably due to precipitation as insoluble PbSO,
(Section 4. 6.2).
Since the mineral sulfides are inside a silicate matrix and are relatively inaccessible
to the bacteria, it is likely that enhanced leaching occurs primarily via indirect attack with
ferric ion as the oxidant. More leaching was observed in the presence of bacteria (Figure
4.30).
The results indicate that the geothermal residues will probably not pose a direct
threat to the environment as they al1 passed the acid leaching protocol tests and they do
not contain genotoxicants or radioactive materials beyond norm. In terms of acid mine
drainage potential, the geotherrnal residues will presumably m a t e a nuisance or aesthetic
pollution due to the formation of iron precipitates which are visible as rusty orange brown
solids. But since the geothermal residues have less iron content than mine tailings, there
will likely be less iron precipitation cornpared to coal or copper mines.
From the results of this work, it is likely that the geothermal residues can be safely
disposed in a landfill environment.

4.12 Risk Assessment of Geothermal Residues

This section will provide an overall risk assessment of landfill disposal of geothermal
residues. Discussions on environmental risk assessment have been covered thoroughly
by several authors [4, 132-1421. It was generally recognized that public perceptions of
risks relating to waste disposal activities often reflect general societal anxieties and fears
that were not necessarily supported by the results of a technical risk assessment. The
technique used in this study is a qualitative rating (low, medium, high as against 1 in 106
risk) but based on quantitative figures from analytical and experirnental results. This study
is a step further than existing desk top methodologies in that actual laboratory simulations
were carried out. There are risks that are very low and acceptable (de minimus in legal
 -
terms 'the law does not deal with trivialities') and are recognized to be true in certain
cases [135] as in this study.
A distinction can be made between hazard and risk. A hazard has the potential to
cause harm while risk is the likelihood of a hazard causing harm. As there are great
uncertainties, only a simple ranking or rating procedure can be used [133, 1351. In the case
of geothenal residues, the hazard rating was based on its chemical content, radioactivity,
and toxicity. On the other hand, the risk rating was derived from the leachability of the
toxic constituents and the probability of producing acid mine drainage. The route of entry
for these toxic elements to cause public health risk is ingestion via drinking water and food.
The designations used in rating can be intenpretedfrom the text box below.

- -

HAZARD AND RlSK RATING

Very low : has low hazard, no significant risk, de minimus
Low : has high hazard; within regulatory and normal levels
Medium : has moderate to high hazard; regulatory and normal
levels are partially met
High : has high hazard; regulatory and normal ievels are
exceeded, with significant risk.

A more realistic risk assessment must include the following analyses: a) site
evaluation, b) release analysis, c) intermediate transfers, d) fate and transport analysis,
e) exposure assessment, and finally, f) risk calculation 11391. Site evaluation alone will
require about 50 input data about climate, hydrology, landfill design, and geology which
were not offen easy to obtain if they were available. In this study, the scoping process had
identified groundwater contamination as the most important threat to the environment
through leachate production. It was also assumed from a practical point of view that the
release mechanisms, exposure pathways and intake or dose rates were similar to al1 the
geothenal wastes studied and that the landfill is secured with impervious liner. Thus what
will Vary were the intrinsic characteristics of the wastes along with their mobility from one
media to another. This simple but satisfactory approach can also be used in determining
whether a certain waste is suitable for landfill disposal or not.
 A high risk perception was attributed to MSC based on its chemical content as
shown in Table 4-9. However, as the level of screening becornes more sophisticated and
detailed, the actual risk decreases to low and medium. A medium rating was assigned to
MSC due to its low level radioactivity from Pb-210 and complete release of Cu and Zn
during the AMDP test. However, it can be recalled from Sections 4.2 and 4.3, that only the
powder sized paracles of -125 pm were leachable and that with 4rnm and above, the toxic
etements were not mobile. Also. the regulated element Pb which could be released rapidly
will form an insoluble precipitate at low pH and will not be available in solution. On the
other hand, MDM and MSL, being basically silica, were practically harmless at a de
minimus level thus the 'very low' rating.

Table 4-9 Hazard and risk rating for Mexican geothermal residues

MDM MSC 1 MSL

Parameter Very Low Low Medium High VeryLow
Chernical content
Radioactivity
Toxicity
Leachability
AMD potential

PSC and PSL have been found to have similar rating as shown in Table 4-10. Their
chemical content can provide significant hazard. However, closer examination will reveal
that they will only pose low risk as leaching was not expected to occur even with bacterial
mediation. A more cautious approach was to carry out treatability studies for PSC and PSL
to reduce the hazard content prior to landfill disposal. The risk rating for ASC was
practically the same for MSL and MDM as being very low and insignificant. Thus MSC
appears to have greater risk cornpared to the other residues and would require special
handling.
Table 4-10 Hazard and risk rating for American and Philippine geothermal residues
- -

PSC PSL ASC

Parameter Low High Low High Very Low
Chernical content J J
Radioactivity
Toxicity
Leachability
AMD potential

If more information was known about the wastes to be managed, then a reasonable
risk assessrnent can be made based on sound predictions. There had been debates on
the relative merit of static, kinetic and themodynamic approaches as well as errors made
in contaminant prediction. However, the growing experience of successful predictions
suggest that it can now be approached with confidence. Thus the prediction of risk may
closely approximate actual risk.
CHAPTER 5 CONCLUSIONS

Based on extensive characterization of the geothermal residues, experimental and

analytical results, and geochemical modelling, the following conclusions can be drawn from
the study:
All the geothermal residues tested were mainly silica (66-82%) with trace elements
SICu, Zn, and Pb at above earth's average crustal levels. They have varying
crystalline and amorphous character. Scale samples were not chemically
homogeneous but showed layered structures from the deposition process while the
sludge samples were agglomerations of submicron particles forming a porous
structure. The drilling mud was a complex mixture of secondary silicate rninerals.
The radioactive levels of al1 samples were within acceptable levels for naturally
occurring radioactive materials except for the Mexican scale which had an elevated
Pb-210 content. However, the radioactivity was still lower than the occupational
dose limit.
Toxicity tests did not indicate the presence of toxins or genotoxins in any of the
geothermal samples. The regulated elements (As, Ba, Cd, Cr, Hg, and Pb) were not
found above regulatory limits in the leachate after a three-month weathering test
(175 rpm, 35 OC).
Results of the regulation leaching procedures do not classify the geothermal
residues as hazardous since their leachate quality was below the regulatory limits.
They can be safely disposed in a landfill since the heavy metal content in their
leachate was sufficiently low to warrant an acceptable risk over a long period of
time. However, particle size was important in the Mexican scale as leaching is
increased considerably when the samples were ground from the protocol size of
9.5 mm to tess than 125 Pm. The powder sized particles, though present in small
amounts in the raw sample, should be isolated and treated prior to disposal.
Sequential chernical extraction indicated that As, Cd, and Pb from the Mexican
scale and Philippine scale and sludge could be available to the environment but
only under extreme conditions (pHs 2, 85-175 OC).
Geochemical modeling and extended leaching experiments showed that leaching
of Cu, Zn, and Pb were due to oxidative dissolution and that under anoxic
conditions, only a very small amount was released.
In the TCLP test, the rate mechanism for the chernical leaching of Pb in the
Mexican scale follows a parabolic rate law for diffusion-controlled dissolution which
is preceded by an initial, rapid leaching that is surface-controlled.
The sulfur and iron oxidizing bacteria, Thiobacillus femoxidans can be cultured and
acclimatized without agitation in nonsterile laboratory conditions.
Of the geothermal samples, only the Mexican scale was found to have acid mine
drainage potential and leaching of Cu and Zn was observed. The galena likely was
attacked but Pb was not found in the leachate at the end of the test and could have
precipitated as insoluble PbSO,. This may also indicate that Cu and Zn can be
reclaimed through microbial leaching as in mineral recovery of metals.
Results of the acid mine drainage potential (AMDP) test developed in this work
indicated that none of the geothermal residues tested should have significant
environmental impact in a landfill even with biological mediation.
Although certain hazards exist in some samples, the associated risk due to leaching
of the toxic cornponents to contaminate groundwater has been assessed to be low
to insignificant based on a qualitative hazard and risk rating for evaluating landfill
dis~osaiof aeothermal residues.
CHAPTER 6

The following research areas are suggested for future worù:

Column leaching on geothermal residues involving three types of leachant:
a) with and without bacteria using distilled water
b) without bacteria using weak acid
c) with bacteria using AMDP culture medium.
Rotary agitated protocol test at various pH (pH 3-10).
lncreased testing time (longer than one week) for extended TCLP for both oxic and
anoxic conditions.
Use of acid mine drainage potential test with a mixed culture of Thiobacillus
ferrooxidans and Thiobacillus thiooxidans.
Verification of the AMDP prediction through comparison with occurrence of acid
mine drainage in geothermal stockpile sites.
Expanded geochemical modelling to consider
a) simulation of leaching procedure with alt important species
b) microbial reactions in acid mine drainage.
More sampling and analysis to reflect temporal and spatial variation in
characteristics of geothermal residues.
Treatrnent of the Philippine scale and sludge and Mexican scale such as
solidification, thermal treatment or microbial minera1 recovery.
Waste disposal and utilization options for Mexican sludge and drilling mud and
American scale.
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APPENDIX A -Trial Experiments Prior to Procedure Development
To be able to select the appropriate AMD potential procedure for the geothermal
residues from those listed in Section 2.6, several trial experiments were performed within
the conditions and facilities available in a chemical/environrnental engineering laboratory.
Based on the laboratory results and literature, the acid mine drainage potential (AMDP)
test suitable for geothermal residues was developed. The success of each trial was based
on bacterial viability in terms of mobility and density at 800x magnification.
Table A-1 Series of Experiments Performed Prior to Test Development

Variables Experiments
Various Sources of American Type Culture Collection (ATCC 19859)
Thiobacillus ferrooxidans U of T Laboratories:
culture -Mine Drainage from Dept of Microbiology
-01d acclimatized culture from Dept of Civil Eng
Growth Medium Standard Method [APHA, 19921
American Type Culture Collection (ATCC, 1989)
Handbook of Microbiological Media [Atlas, 19931
Nutrient Concentration No FeSO,
50% of required FeSO,
100% of required FeSO,
Solid Concentration 10-15 91100 ml
5 g/100 mL
2 gI100 mL
Agitation lnside incubator-shaker (agitated)
On laboratory bench (stationary)
Temperature lnside incubator-shaker (35 OC)
Room temperature (23-25 OC)
Sterilization Culture media autoclaved
Culture media filtered
Samples oven-sterilized
Samptes not sterilized
BC Research Confirmation 150 m particle size
Test 20 g sample in 70 mL culture medium
150 rpm and 35 OC
Medium without FeSO,
6 weeks duration
 -
APPENDIX B Bacterial Density Estimation for Thlobacillus fenooxidans
In estimating bacterial density using image analysis, the image was viewed at 800-
1000x magnification in a light microscope as described in Section 3.10.1. The image was
captured in a computer format (1 image = 300 Kb) and saved with a graphics extension
such as .xls for Microsoft Excel or .tif for any graphics viewer such as Graphics Workshop
and ACDSee. Samples of such image are shown in Figure 4.14. The computer software
was unable to discriminatethe bacteria with the other particles in the sample hence manual
counting from the images was resorted to by drawing gridlines on the image and counting
the cells per grid.
The various steps in estimating the bacterial density are briefly described below.
The volume of one drop of liquid sample placed onto glass slide and covered with cover
glass was around 0.02 rnL or 20 mm3- This was measured by counting the number of
drops per known volume, Le. 1 mL and dividing it with the number of drops (50).
Volume of one drop of liquid sample, V = 20 mm3
Area of image = (640)(480) pixel or 30.72 mm2where 1 pixel = 0.01 mm
Area of cover glass = (22)(22) or 484 mm2
Number of bacterial cellslimage = x
Number of images/slide = 484130.72 = 15.75
Total cellslslide = 15.75~
= 1 5 . 7 5 =~ 0 . 7 8 8 ~
Bacterial density, cells1mm3= 1 5 . 7 5 ~
v 20
t, thickness between slide and cover = 20 mm3 = 0.040 mm = 40 pm
484 mm2
From Figures 15 and 16, the Thiobacillus ferooxidans bacteria has a diameter of
0.4 to 0.5 Fm and length of 1.O to 1.5 pm for rods and 0.5 to 0.7 for spheres whereas the
bacteria filled-liquid film under the cover glass has a thickness of 40 vm. It was therefore
possible that the image analysis can sense only part of actual bacterial density than what
was shown in the photograph. In practice, it was difficult to determine the number of viable
cells since mutated cells and non-dividing cells are always present. Furthermore, in the
case of Thiobacillus fenooxidans, other difficulties arise from the adhesion of a
considerable number of cells to the solid substrate as shown in Figure 14d. The heat and
light from the microscope could also make the bacteria retreat to a lower depth of the
sample thus decreasing the density as captured in the image. It was therefore necessary
to adjust the density to refiect actual bacterial count. From the TEM photos, the average
area of each bacteria was computed to be about 0.5 !m2. The volume of a Thiobacillus
femoxidans is around 0.23462 pm3 [48] hence the thickness was about 0.47 Fm. The
adjustment can be calculated as thickness of the slide divided by the thickness of the
bacteria : 4010.47 = 85. To account for the uncertainties, a factor of 100 was suggested.
Table B-1 was a sample estimation for various bacteria culture and acclirnatization stage.

Table 6-1 Typical Bacteria Counting and Calculation

--

Age of Filename Bacterial count, Bacterial Adjusted

culture .xls/.tif cellslimage, X density, bacterial
cells/rnL, density, çells/mL
788X = D 1000
6d tf.xls 23 18000 1.8 x 10'
bacteria.xls 40 32000 3.2 x 10'
atcc2.xls 27 21O00 2.1 x 106
atccl .xls
13 d civl .xls
civ2.xls
7d asc2.xls
mdm2.xls
msc2.xls
msl2.xls
psc2.xls
psl2.xls
7d t f l .tif
tf4.tif
W .tif
ild rb2.tif
rb8.tif
22 d tf8.tif
 -
APPEMDlX C Calculatioion for the Prelirninary Acid Mine Drainage Potential Results

Based on the data obtained from the BC Research Initial Test in Table 4-5, the acid
*
production potential (APP) and acid consumption (AC) were calculated as follows [67]:

Acid production potential = Percent sulfur x gB x IOOQ

32 IO0

APP= % S x 30.6 kg H,SO, per tanne

&id consumption = 98 x Vol acid. rpL x lN acid x ka11000 q

2 x sample weight, g x tonne
1000 g

AC = mL 1N H,SOI x 0.049 x 100Q kg H,SO, per tonne

Sample weight in g

The acid production potential and acid consumption values are cornpared. If the
APP exceeds the AC, the sample was classified as being a potential source of acid mine
drainage. It was recommended to confirm the results using kinetic tests such as those
listed in Table 2-6.
APPENDIX D - About the Geochemical Model MlNTEQA2
The basic solution scheme used in MINTEQA2, a Geochemicai kssessment Model
for Environmental Systems [Allison, 19931 is summarized as follows:

1. ldentify species of interest, choose a set of components, and set up a table.

2. Guess the concentration of each component.
3. Calculate the equilibrium composition of the system using the estimated component
concentrations in the rnass law equations.
4. Calculate the error in the mole balance equation for each component.
5. Obtain improved estimates for the component concentrations using the
multidimensional Newton-Raphson iteration technique on the mole balance errors.
6. Calculate a new equilibrium composition, the corresponding mole balance equation
errors, and obtain irnproved estimates for the component concentrations.
7. Continue the iterative procedure until the errors in the mole balance equations are
small.
The mode1 has its own thermodynarnic database so the primary information that
must be conveyed through the input file was the total dissolved concentration or fixed
activity of each component of the system. Solids are identified to PRODEFA2 by
specifying the component that represents the major cation and the main mineral group to
which the solid belongs (e.g. carbonate, sulfide). Alternatively, one may specify the 7sligit
ID number for any aqueous or solid species if it was known. Menus and prompts within
PRODEFA2 allow al1 of these things to be done with relative ease. MINTEQA2 solves the
equilibrium problem iteratively by computing mole balances from estimates of cornponent
activities. PRODEFAP makes this guess automatically for every component as equal to
the component total dissolved concentration but also provides the means for the user to
change the guess. It was possible for the user to insist that certain conditions prevail at
equilibrium for pH, pe, or gas partial pressure.
There are four choices for units of concentration for the input data: 1) Molal (moVkg,
same as molar for the dilute systems appropriate for MINTEQA2), 2) mgll, 3) ppm (parts
per million), or 4) meqA (milliequivalents per liter). Regardless of the units chosen for input
data, MINTEQA2 output data are always molal.
 -
APPENDIX E Input Data Derivation for Geochemical Model
The calculation of solid concentration for the important minerai species in the
Mexican scale was undertaken with the following steps. Only the major species such as
pyrite, chalcopyrite, galena, and sphalerite were considered in the modelling. Later on
covellite was added since it showed as a controlling solid with a positive saturation index
after the initial runs. To sirnplify calculations, amount for chalcopyrite was assumed to
include covellite, and the C concentration was split into two.
P = moles pyrite, FeS,
C = moles chalcopyrite, CuFeS,
G = moles galena, PbS
S = moles sphalerite, ZnS
v - moles covellite, CuS

Calculate the moles of the respective elements from Table 4-1:

moles S = 3.38 g1100g x 50 glL x 1 mole/32 g = 0.053

moles Cu = 9080 uglg x 1 g11O6x 50 g/L x 1 mole163.5 g = 0.0071
moles Pb = 11,600 uglg x 1 911O6 x 50 g/L x 1 mole1207.2 g -0.0028
moles Zn = 15,900 uglg x 1 g1106x 50 g/L x 1 mole165.4 g = 0.0120

S balance : 2P + 2C + G + S = moles S - 0.053

Cu balance: C -- moles Cu = 0.0071
Pb balance: G - moles Pb = 0.0028
Zn balance : S - moles Zn = 0.0120

Solving the equations, the following values were obtained:

0.0120 -
- moles pyrite, FeS,
O. 0035 - moles chalcopyrite, CuFeS,
0.0036 - moles covellite, CuS
O. 0028 = moles galena, PbS
0.0120 - moles sphalerite, ZnS
The moles of acetic acid added were calculated as follows:
Leachate Extraction Procedure(LEP): 0.5N x 5 mL = 0.0025 mol/L
Toxicity Characteristic Leaching Procedure (TCLP) :0.1N x 1 L = 0.1 mollL
 -
Appendix F Sample Output of Geochemical Modelling

PART 1 of OUTPUT FILE

PCMINTEQA'v3.10 DATE OF CALCULATIONS: 20-NOV-96 TIME: 0:27:21

CALCULATE THE EQUILIBRIUM CONDITIONS BY ADDING 0.1N HAC

TO MEXICAN SCÀLE I N TOXICITY CHARACTERISTIC LEACHING PROCEDURE

--------------------------------------------------------------------------------
T e m p e r a t u r e (Celsius): 2 5 . 0 0
U n i t s of concentration: MOLAL
I o n i c s t r e n g t h t o be c o m p u t e d .
If specified, carbonate c o n c e n t r a t i o n represents t o t a l inorganic carbon.
Do not a u t o m a t i c a l l y t e r m i n a t e i f charge imbalance exceeds 3 0 %
P r e c i p i t a t i o n w a s a l l o w e d only f o r thrse s o l i d s specified as ALLOWED
.
i n the i n p u t f i l e ( i f any)
T h e maximum number of i t e r a t i o n s i s : 200
The method used t o c o m p u t e a c t i v i t y c o e f f i c i e n t s is: Davies equation
Intemediate output f i l e
-------------------------------------------------------------------------------
330 1.000E-01 -1.00 H+
992 1.000E-01 -1.00 Acetate
730 0.000E-01 -16.00 HS-1
1 0.000E-01 -16.00 E-1
280 0.000E-01 -16.00 Fe+2
600 0.000E-01 -16.00 Pb+2
950 0.000E-01 -16.00 Zn+2
231 0.000E-01 -16.00 Cu+2

H20 has been inserted as a COMPONENT

4 5
1028003 18.4790 -11.3000 1.200E-02 FeS, Pyrite
1023102 35.2700 -35.4800 3.5003-03 CuFeS, Chalcopyrit e
1060001 15.1320 -19.4000 2.800E-03 PbS Galena
1095001 I l . 6180 -8.2500 1.200E-02 ZnS S p h a l e r ite
1023101 23.0380 -24.0100 3.6003-03 CuS Covellite

INPUT DATA BEFORE TYPE MODIFICATIONS

NAME ACTIVITY GUESS LOG GüESS ANAL TOTAL

H+l 1.000E-01 -1.000 1.000E-O1
Acetate 1.000E-01 -1.000 1.000E-01
HS-1 1.000E-16 -16.000 0.000E-01
E-1 1.000E-16 -16.000 0.000E-01
Fe+2 1.000E-16 -16.000 0.000E-01
Pb+2 1.000E-16 -16.000 0.000E-01
Zn+2 1.000E-16 -16.000 0.000E-01
Cu+2 1.000E-16 -16.000 0.000E-01
H20 1.000E+00 o . O00 0.000E-01

Charge Balance : UNSPECIATED

Sum of CATIONS= 1.000E-01 Sum of ANIONS = 1 . 0 0 0 E - 0 1
PERCENT DIFFERENCE = 0.000E-01 (ANIONS -
CATIONS) / (ANIONS + CATIONS)
 PART 3 of OUTPUT FILE
PC MINTEQAI! v3.10 DATE OF CALCULATIONS: 20-NOV-96 TIME: 0:27:22

PARAMETERS OF THE COMPONENT MOST OUT OF BALANCE:

ITER NAME TOTAL MOL

O HS-1 0.000E-01
1 HS-1 0.000E-01
2 HS-1 0.000E-01
3 HS-1 0.000E-01
4 HS-1 0.000E-01
5 HS-1 0.000E-01
6 HS-1 o. 0003-01
7 HS-1 0.000E-01
8 HS-1 0.000E-01
9 HS-1 0.000E-01
10 HS-1 0.000E-01
11 HS-1 0.000E-01
12 HS-1 0.000E-01
13 HS-1 0.000E-01
14 HS-1 0.000E-01
15 HS-1 0.000E-01
16 HS-1 0.000E-01
17 HS-1 0.000E-01
18 HS-1 0.000E-01
19 HS-1 0.000E-01
20 HS-1 0.000E-01
21 HS-1 0.000E-01
22 HS-1 0.000E-01
23 HS-1 0.300E-Ci
25 HS-1 0.000E-01
26 HS-1 0.000E-01

NAME ANAL MOL CALC MOL LOG ACTVTY DIFF FXN

H+l 1.000E-01 1.3583-03 -2.88524 2.9003-09
Acetate 1.OOOE-01 1.3723-03 -2.88063 3.1063-09
HS-1 0.000E-01 6.770E-10 -9.18752 4.386E-10
II20 0.000E-01 - l . 2 5 6 E - l l -0.00148 0.000E-01
E-1 0.000E-01 9.3033-01 -0.02764 0.000E-01
Fe+2 0.000E-01 1.389E-06 -5.92972 0.000E-01
Cu+2 0.000E-01 2.1723-17 -16.73572 0.000E-01
Zn+2 0.000E-01 5.7123-06 -5.31572 0.000E-01
Pb+2 0.000E-01 1.7493-09 -8.82 972 0.000E-01

Type 1 - COMPONENTS AS SPECIES I N SOLUTION

NAME CALC MOL ACTïVITY LOG ACTVTY GAbMA NEW LOGK

H+l 1.3583-03 1.3023-03 -2.88524 0.95912 O . 018
Acetate 1.3723-O3 1.3163903 -2.08063 0.95912 O . O18
HS-1 6.770B-10 6.4943-10 -9.18752 0.95912 0.018
Cu+2 2.1723-17 1.8383-17 -16.73572 0.84625 O . 073
Fe+2 1.3893-06 1.1763-O6 -5.92972 0.84625 0.073
Pb+2 3.7493-09 1.48OE-09 -8.82972 0.84625 O . 073
Zn+2 5.7123-06 4.0343-06 -5.31572 0.84625 O . 073
,---------------------- --CI------------------ ----------
 Type II - OTHER SPECIES IN SOLUTION OR ADSORBED

NAME CALCMOL ACTIVITY NEW LOGK

ZN ACETATE4 3.9293-16 3.3253-16 1.433
FeACETATE 4.053E-08 3.8879-08 1.418
OH- 8.0153-12 7.6873-12 -13.980
FeOH + 2.9663-13 2.8453-13 -9.482
FeOH3 -1 5.4913-29 5.2673-29 -30.982
~ 8 0 AQ
~ 2 1.8523-21 1.8533-23 -20.570
Fe(HS)2 AQ 4.4173-16 4.4183-16 8.950
Fe(HS)3 - 3.2573-23 3.1243-23 11.005
Cu ACETATE 4.1863-18 4.015E-18 2.238
CuOH + 1.4663-22 1.406E-22 -7.982
Cu(0H)2 AQ 2.2473-25 2.2483-25 -13.680
Cu(0H)3 - 1.0833-35 3.0393-35 -26.881
Cu(OH)4 -2 1.8703-45 1.5823-45 -39.527
Cu2 (OH)2+2 1.0233-38 8.6613-39 -10.286
Cu(HS)3 - 4.1583-19 3.9883-19 25.917
ZnOH + 4.2283-12 4.055E-12 -8.942
Zn(0H)2 AQ 3.5703-17 3.5713-37 -16.899
Zn(0H)3 - 9.0093-26 8.6413-2 6 -28.381
Zn(0H)4 -2 1.2383-35 1.048E-35 -41.126
Zn(HS)2 A(;Z 1.7753-O9 1.775E-Og 14.940
Zn(HS)3 - 1.7373-37 1.6663-17 16.118
PbOH + 2.3023-14 2.2083-14 -7.692
Pb(0H)2 AQ 6.5713-21 6.5743-21 -17.120
Pb(0H)3 - 6.0213-29 5.7753-29 -28.042
Pb20H +3 1.0653-21 7.3173-22 -6.197
Pb(HS)2 AQ 1.162E-12 1.1623-12 lS.270
Pb(HS)3 - 1.5703-20 1.5063-20 16.588
Pb3 (OH)4+2 1.7313-39 1-4653-39 -23.807
Pb(0H)4 -2 1.1993-37 1.0153-37 -39.626
H2S AQ 7.3903-06 7.3923-06 6.941
S -2 . 7.1163-20 6.0223-20 -12.845
H ACETATE 9.8633-02 9.866E-02 4.760
CuACETATE2 1.3583-19 1.3583-19 3.630
CuACETATE3 5.5023-23 5.2773-23 3.118
CuACETATE4 5.1793-26 4.3833-26 2.973
PBACETATE 1.5063-09 1.4443-09 2.888
PBACETATE2 3.0823-11 3.0833-11 4.080
PBACETATE3 1.3693-14 1.3133-14 3.608
PBACETATE 4 1.3193-17 1.1163-17 3.473
ZN ACETATE 2.4653-07 2.3643-07 1.588
ZN ACETATE2 6.6513-10 6.6533-10 1.900
ZNACETATE3 4.2713-13 4.096E-13 1.588

Type III - SPECIES WITH FIXED ACTIVITY

ID NAME: CALC MOL LOG MOL NEW LOGK DH

2 H20 -1.256E-11 + -10.901 O. 001 O. 000

Type IV - FINITE SOLIDS (present at equilibrium)

ID NAME CALC MOL LOG MOL NEW LOGK DH

1028003 PYRITE 1.2OOE-O2 O. O00 18.479 -11.300
1023101 COVELLITE 3.6013-03 -5.845 23.038 -24.010
1023102 CHALCOPYRXTE 3.4993-03 -5.845 35.270 -55.480
1095001 SPWRITE 1.199E-02 -5.225 11.618 -8.250
1060001 GALENA 2.8003-03 -8.483 15.132 -19.400

Type V I - EXCLUDED SPECIES ( n o t included i n mole balance)

ID NAME CALC MOL LOG MOL NEW LOGK DH

3300021 02 (g) 0.000E-01 -71.471 -83.120 133.830
1 E-1 9.3833-01 -0.028 0.000 0.000

PART 4 of OUTPUT F I L E
PC MINTEQA2 v3.10 DATE O F CALCIJIATIONS: 20-NOV-96 TIME: 0:27:22

PERCENTAGE DISTRIBUTION OF COMPONENTS AMONG

TYPE 1 and TYPE II (dissolved and adsorbed) species

PERCENT BOUND I N SPECIES # 330 H+1

PERCENT BOUND I N SPECIES #3309921 H ACETATE

Acetate PERCENT BOüND I N SPECIES # 992 Acetate

PERCENT BOUND I N SPECIES W3309921 H ACETATE

PERCENT BOUND I N SPECIES W3307300

PERCENT BOUND I N SPECIES #3300020 OH-

PERCENT BOUND I N SPECIES a2803300 FeOH+
PERCENT BOUND I N SPECIES U9503300 ZnOH+

PERCENT BOUND I N SPECIES # 280

PERCENT BOUND I N SPECIES #2809920

PERCENT BOUND I N SPECIES # 231 Cu+2

PERCENT BOUND I N SPECIES #2319921 Cu ACETATE
PERCENT BOUND I N SPECIES #2317300 Cu(HS)3 -
PERCENTBOUNDINSPECIES# 950 Znt2
PERCENT BOUND I N SPECIES #9509921 ZN ACETATE

PERCENT BOUND I N SPECIES # 600 Pbt2

PERCENT BOUND I N SPECIES #6009921 PBACE TATE
 PART 5 of OUTPUT FILE
PC MINTEQAZ v3.10 DATE OF CALCüIATIONS: 20-NOV-96 TIME: 0:27:23

IDX NAHE DISSOLVED SORBED PRECIPITATED

MOL/KG PERCENT MOL/KG PERCENT MOL/KG PERCENT

H+1
Acetate
HS-1
H20
E-1
Fe+2
Cu+2
Zn+2
Pb+2

Charge Balance : SPECIATED

Sum of CATIONS = 1.3723-03 Suni of ANIONS 1.3723-03

PERCENT DIFE'ERENCE = 2.3483-05 (ANIONS - CATIONS)/(ANIONS + CATIONS)

EQUILIBRIUM IONIC STRENGTH (m) = 1.3003-03

EQUILIBRIUM pH = 2.885

EQUILIBRIWM pe = 0.028 orEh = 1 - 6 4 mv

DATE ID NUMSER: 961120

TIME ID NUMBER: 272304

PART 6 of OUTPUT FILE

PC MINTEQA2 v3.10 DATE OF CALCULATIONS: 20-NOV-96 TLME:: 0:27:23

Saturation indices and stoichiometry of a l 1 minerals

ID # NAME Sat. Index Stoichiometry in [brackets]

1028000 FES PPT -8.317 [ -1.0001 330 [ 1.000] 280 1 1.0001 730

1028003 PYRITE

[ 2.0001 730
1023101 COVELLITE 0.000 [ -1 .OOO] 330 [ 1.0001 231 [ 1.000J 730
2023100 CU(0H)2 -19.608

2023101 TENORITE -18.507

1023102 CHALCOPYRITE 0.000

95000 ZN METAL
2095000 ZN(OH)2 (A)

2095001 ZN(OH)2 (C)

2095002 ZN(0H)2 (BI

2095003 ZN(0H)Z (G)

2095004 ZN(0H)2 (E)

2095005 ZNO(ACTIVE)

2095006 ZINCITE

1095000 ZNS (A)

1095001 SPHALERITE

1095002 WURTZITE

60000 PB METAL
2060000 MASSICOT

2060001 LITHARGE

2060002 PBO, .3H20

1060001 W N A

2060003 PLATTNERITE

3060001 MINIUM -77.048

2060004 PB(0H)2 (C) -11.212

2060005 PB20 (OH)2 -32.323

73100 SUL= -4.137

2028000 WUSTITE -11.533

 -
APPENDIX G UofT Acid Mine Drainage Potential Test

Introduction
The proposed Uoff Acid Mine Drainage Potential test (AMDP) for geothermal
residues is an irnprovernent of the BC Research Confirmation Test (BCRCT) which has
been developed and widely used in Canada and the US for the last 15 years. It is a
confirmation test to determine the acidification potential of a sample with biological
mediation. The BC Research Confirmation Test had a number of shortcomings because
of the nature of geothermal residues. The BCRCT was designed for mine tailings that are
high in sulfur content (up to 40%) and have smaller particle size (-50 pm) making them
more amenable to bacterial attack. Geothennal residues, on one hand, have less S content
(-4%) and are bulkier with particle size from 1 mm and higher. Due to these reasons, the
BCRCT was adjusted to the nature of geotherrnal residues. Barron and Lueking suggested
methods on how to maintain Thiobacillus fhooxidans [84]. Bruynesteyn and Hackl [97]
and CANMET 1671 were useful references on the evaluation of acid production potential
of rnining waste materiais. The rnost important modifications are the use of a lower solid
concentration (or pulp density), addition of FeSO, in culture media, regular measurement
of redox potential (Eh), pH, metals as well as monitoring of bacterial growth and viability.
The AMDP test can be performed in nonsterile conditions at room temperature even
without continuous agitation. Table 4-7 provides a cornparison between the old and the
new procedure.
This procedure is a confirmation of acid mine drainage potential after a preliminary
assessrnent has been made using the BC Research Initial Test described in Section 3.7.
Sample collection, preparation and storage were undertaken using standard methods
[ I l Il.

G I A Short Version

1. Grind samples in a mortar and pestle to 120 mesh size (125 pm).
2. Have ready an acclimatized culture of Thiobacillus ferrooxidans. Refer to
acclimatization of inoculum below (Section G1.3).
3. Prepare Thiobacillus femoxidans culture media using the formula in table beiow
(Section G1.4).
4. If a shaker is available, apply agitation at 150-175 rpm. Otherwise place flasks on
a laboratory bench or open shelf. Without agitation is acceptable but testing tirne will
increase one week more.
5. If an incubator-shaker is available, se? tzmperature to 35 OC at 150-175 rpm.
Otherwise place flasks on a laboratory bench or open shelf at room temperature of
23-25 OC. Monitor room temperature using a therrnometer. Lower temperature will
increase testing time.
6. Use solid concentration or pulp density of 2 % (2 g sample in 100 mL media).
7. Prepare inoculum according to proposed new method (Section G?.3). Use 5 mL
inoculum from logarithmic growth phase having a bacteria density of 2 x I O 7
cellslmL for every 100 mL media (Section G1.7).
8. Monitoring schedule is at the begiiining and every 3 days for 3-4 weeks.
9. Monitor pH, Eh, bacterial density and rnotility (Sections G1.6 and G1.7), and metals
(1 rnL aliquot diluted to 5 mL). Weigh flask and contents at the beginning and
replace water due to evaporation.

1. Clean al1 glassware to be used in detergent, rinse three times, soak in 20% HNO,
overnight, rinse with tap water three times and finally rinse with deionized water.
Once dry, cover the 250 mL Erlenmeyer flasks with aluminum foi1 prior to use.
2. Pulverize the sample to pass a 120 mesh Tyler screen (approximate particle size
-125 p m ) and store in air tight bottles prior to use.
3. Prepare the bacteria culture media using the media specified in Section G1.4 below.
The pH of the media must be 2.9.
4. In duplicate, weigh 2 g of ground sample into 250 mL Erlenmeyer flask. Label the
flasks accordingly. Slowly add 100 mL of culture media and cover flask with a plug
made of nonadsorbent cotton wrapped with gauze. Swirl manually and check pH.
If the pH is above 2.9, add ION H2S04until stable at pH 2.9 I0.1.
5. Inoculate flasks with an active culture of Thiobacillus femoxidans prepared
according to Section G1.3. Record weight of flask with its contents without the
cotton plug.
6. Place the flask on a laboratory bench or open shelf at room temperature (at least
23-25 OC) with adequate ventilation. If an incubatorhhaker is available, place flask
on a shaker at 175 rpm and 35 OC. With agitation, testing time is shorter by one
week.
7. Prior to each measurement every 3 days, weigh flask and contenis (without plug)
and add deionized water to replace loss by evaporation. Obtain 1 mL aliquot,
centrifuge for 10 min, and transfer supernatant to a clean 15 mL centrifuge tube.
Dilute to 5 m l with deionized water, acidify ta pH12 with -0.05 mL concentrated
HNO,, and store at 4OC while waiting to be analyzed. Add ImL deionized water to
ail the flasks to replace the 1 mL aliquot sample.
8. Monitor pH, Eh, bacterial growth (motility and density), color of solution, and
dissolved metals every 3 days. Clean the pH and Eh probes at every measurements
with water spray to avoid contamination from one flask to another. Manually shake
flask at every determination. The color of solution will progressively change from
light grey to yellow to deep amber or orange brown color which indicates iron
oxidation. The solution will also change from a clear solution to slightly turbid which
indicates bacterial growth and some precipitation.
9. Within the sampling period, monitor bacterial motility (Section G1.6) and density
(Section G1.7) under a light microscope at 800x - 1000x magnification. When
bacterial activity has ceased as observed from the microscope and a stable pH has
been achieved, teninate the test. Analyze the aliquots for regulated metals. If the
pH is below 3.5, and dissolved metals are present in the leachate above regulatory
limits, the sample is classified as having acid mine drainage potential (or potential
for bioleaching treatment).
10. This test can be completed within 3-4 weeks following inoculation.
G i .3 Acclimatization of lnoculum

Results of work on aeothermal residues

A critical stage of the procedure is the acclimatization of the pure bacteria culture to the
specific samples to be tested. A series of steps has been designed to assure that viable
culture was ready as inoculum for the UOT- AMDP test. The trial experiments had shown
that a half formula with 4.5 g/L Fe2+was providing the requirement for bacterial growth. It
was not advisable to withhold completely the FeSO, from the leaching medium since the
bacteria require from 2-3 g/L to 9 g/L Fe2+to survive [37,73, 77,78,84]. The BC Research
Confirmation Test does not include FeSO, in its growth medium since its premise was that
the bacteria will obtain FeSO, from the oxidation of pyrite from the mine tailings sample.
However for geothermal residues, without the initial seed FeSO,, the bacteria cannot
survive as some of the sulfides (including pyrite) are not readily accessible as they may be
bound in silicate matrix. An excess FeSO, however should be avoided as it can increase
the formation of a yellow precipitate called jarosite, KFe,(SO,),(OH), and iron
oxyhydroxides, Fe(OH), and FeOOH. To begin the procedure. a full media containing
100% of the required FeS04.7H,0 was used to provide rapid bacterial growth. A full media
on has 44.22 g/L FeSO4.7H,O while a half media has 22.1 1 g/L only. The bacteria growth
medium is described below. Aftelwards, a second culture is grown with the addition of
2 g geothermal sarnple using the half media described below (Section G1.4) and the
acclimatized bacteria to produce a culture that was ready as inoculum. A shorter step was
required if an acclimatized culture already exists.
Resultina inoculum
Full media + pure culture BI
Full media + pure culture (BI) + 2 g sarnple B2
Half media + acclimatized bacteria (82)+ 2 g sample 83
83 ready as inoculum for the AMDP test.

If a dormant B3 culture has to be revived,

Full media + acclimatized culture (83)
Half media + 84 + 2 g sample
B5 is ready as inoculum for AMDP test.
G i .4 Bacteria Culture Medium
Below is the proposed growth medium for the Thiobacillus femMxidans which was
modified from the standard laboratory technique developed by the Amencan Public Health
11. The major differences are the reduction of the FeSO, content and use
Association [Il
of membrane filtration for sterilization of solution instead of autoclaving.

Modified Growth Medium for Thiobacillus fen-ooxidans

Basal salts: in a 1 L Erienmeyer flask
Ammonium sulfate (NH4),S0, 3.0 g
Potassium chloride KCI 0.10 g
Dipotassiurn hydrogen phosphate K2HP0, 0.50 g
Magnesium sulfate MgS04.7H,0 0.50 g
Calcium nitrate Ca(NO,), 0.01 g
Sulfuric acid, 10 N H,S04 1.0 mL
Distilled water 700 mL

Energy source: in a 500 mL Erlenmeyer flask

Ferrous sulfate FeS04.7H,0

Distilled water

Separately filter using cellulose acetate (pore size 0.45 pm) the basal salts and
energy source and combine after filtration. The medium will be opalescent and green and
a precipitate will form (probably ferrous and ferric phosphates). The pH should be 2.9 with
the solution containing 4500 mglL ferrous ion. The medium can be stored for at least 2
weeks in the refrigerator.

G1.5 Cultivation
Add 5 mL of inoculum (American Type Culture Collection 19859) in a 250 mL
Erlenmeyer flask containing 100 mL of fresh bacteria culture medium found in Section
G1.4. Growth of the Thiobacillos femoxidans is manifested by a decrease in pH and an
increase in the concentration of oxidized iron as orange-brown or deep amber color. Check
under the microscope for bacterial motility and density with at least 800-1000x
magnification.
G1.6 Bacteria Moti!ity
Bacteria motility is difficult to quantify but it can be described following the bacterial
growth curve 148, 72, 1261. Thiobacillus femoxidans are very active and motile at the
logarithmic and stationary phases. They can corne as single, pairs or short chahs. At the
lag phase and death phase, they are dormant and nonmotile sometimes looking like white
spots. The rnotility can be reported simply as motile (slow, medium, fast) or nonmotile.

G1.7 Bacteria Density

Bacteria density can be estimated qualitatively by cornparhg what is observed in
the light microscope with the photographs of bacteria in Figures 4. M a to 4 . 1 4 ~
which
depict low, medium and high density (from I O 6 to 10' cellslmL). Bacteria density can be
calculated by following the steps in Appendix B. Below is a general bacteria cuwe for
Thiobaci//usfemoxidans in both agitated (35 OC)and stationary (25 OC)experiments. This
should serve as a guideline only. The y-axis was constructed with values from 1 to 5
corresponding to a range from low to high density with an F factor to cover the cell counts.

Bacteria Growth Cuwe for T.f.

6

O 5 10 15 20 25 30 35
f ime, days

Agitated, T=35 C + Stationary, T=2SC

 -
APPENDIX H RESULTS OF TOXlClTY TESTING

For the Toxi-Chromotest, the blue color developrnent signifies that the E. Coli were
alive and therefore the sample is non-toxic at the particular concentration. Conversely, if
there was no color development, it rneans the bacteria were dead and the sample was
toxic at the particular sample concentration. Non-toxic (NT) rneans the samples did not
exhibit toxicity at every concentration. Five sample concentrations (%wIv) were used
(50%. 25%. 12.5%, 6.25%. and 3.13%). In Table H1 below, a sample with a value of
3.13% was considered exhibiting toxicity since it required only a little amount to affect the
bacteria, i.e., the lower the percent of substrate concentration (3.13% and below), the
higher the degree of toxicity. A detailed explanation of this scheme can be found in several
references [4345, I O M 1O]. The results in Table H1 showed toxicity only at higher
concentrations : 50% for PSC and MDM as well as 25% for MSC while the rest (PSL. ASC,
and MSL) were classified as non-toxic. These results can classify the samples as generally
having negative toxicity.

Table H1 Toxi-Chromotest Results for Geothermal Samples

Sampfes Toxic at X % Samples Toxic at X %
- -- -

+ Control 3.13 + Control 3.13

- Control Non-toxic (NT) - Control Non-toxic (NT)
PSC 50 MDM NT
50 50
50 50
PSL NT MSC 50
NT 25
NT 25
ASC NT MSL NT

For the SOS-Chromotest, an indicator of genotoxicity is the presence of blue color

in the chromopads (the opposite of Toxi-Chromotest above). All the samples did not
produce blue color in the chromopads indicating they are negative for genotoxins.
 -
APPENDIX I ADDITIONAL DISSOLUTION KINETICS DATA

Below are two graphs showing dissolution kinetics of Fe and Zn in the oxic TCLP
test of the fine sized Mexican scale. Like Pb in Figure 4-31, there was an initial, rapid
leaching of Zn and Fe with intercept values of 80 prnol and 670 pmol, respectively. This
is presumed to be surface controlled followed by a slow diffusion reaction. A similar
leaching pattern also was observed for Cu, Zn. and Pb in Figure 4.9, which indicates their
common rate controlling mechanism. Fe could have leached out from chalcopyrite
(CuFeS,) and pyrite (FeS,).

O 2 4 6 8 10
Time, h1l2

Time, hl"
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