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 1 O4 a, t''" mol.L'' .h-' . A batch reactor technique

(AMDP test) using the iron and suifur oxidizing bacteria, Thiobaci//us ferrooxidans, 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 i i

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 I O

2.3 Techniques for Waste Characterization I O

2.3.1 Chemical Analysis I O

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

3.8.3 Acid 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 Microstructural Analysis

3.10.1 Light Microscopy with Image Analysis

3.1 0.2 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 Solu bilization 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 Proced ure 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

Fig. 2.2

Fig. 4.1

Fig. 4.2

Fig. 4.3

Fig. 4.4

Fig. 4.5

Fig. 4.6

Fig. 4.7

Fig. 4.8

Fig. 4.9

Fig. 4.1 O

Fig. 4.1 1

Fig. 4.12

Fig. 4.13

Fig. 4.14a

Fig. 4.14b

Fig. 4 .14~

Fig. 4.14d

Fig. 4.15

Fig. 4.16

Fig. 4.17

Fig. 4.18

Fig. 4.19

Fig. 4.20

Fig. 4.21

Fig. 4.22

Fig. 4.23

Fig. 4.24

Fig. 4.25

Fig. 4.26

Schematic model of a hot-water geothermal system

Schematic drawing showing the leaching of an ore particle

Photomicrographs of Philippine scale, PSC (1 OOx, 250x)

Photomicrograph of Philippine sludge, PSL (50x)

Photomicrograph of American scale, ASC (50x)

Photomicrograph of Mexican drilling mud, MDM (50x)

Photomicrograph of Mexican scale, MSC (1000~)

Photomicrograph of Mexican sludge, MSL (1 OOx, 50x)

Comparison of extent of leaching between LEP and TCLP

Comparison of extended TCLP leaching of Pb coarse and fine

Dissolution behavior in the oxic extended TCLP

Dissolution behavior in the anoxic extended TCLP

Sequential extraction results for PSC and PSL

Sequential extraction results for ASC and MDM

Sequential extraction results for MSC and MSL

Photomicrograph of T. ferooxidans : high density (800x)

Photomicrograp h of T. ferooxidans : medium density (800x)

Photomicrograph of T. fenooxidans : low density (800x)

Photomicrograph of T. ferooxidans with a cloud-like capsule

TEM photomicrograph of various sizes of T. ferrooxidans (60K x)

TEM photomicrograph of T. fenooxidans about to partition (99K x)

pH-Eh change over tirne in AMD experiments

Leaching in agitated AMD experiments : PSC, PSL and MSC

Leaching in stationary AMD experiments : PSC, PSL and MSC

Variations in pH over time for batch kinetic AMD experiments

Variations in Eh over time for batch kinetic AMD experiments

Effect of sterilization on metal bioleaching : PSC

Effect of sterilization on metal bioleaching : PSL

Effect of sterilization on metal bioleaching : MSC

Effect of agitation and temperature on metal bioleaching: PSC

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 1 O0

Fig. 4.31 Dissolution kinetics of Pb in Mexican scale 102

Fig. 4.32 Overall metal bioleaching, percent over time for MSC 1 03

Fig. 4.33 Solubilization rate for Cu, Zn, and Pb in the Mexican scale 1 04

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Average abundance of the elements in crustal rocks

Leachate quality criteria

Limits of heavy metal concentration in sludges for agriculture

Allowable values for metals in the European Union

Summary of sorne static AMD test methods

Summary of some kinetic AMD test methods

Summary proced ure for sequential chemical extraction

Comparison of protocol leaching tests

Culture medium for Thiobaci//us fenooxidans

Input data for modeling protocol leach tests

Chernical analysis of selected geothermal samples

Crustal abundance ratio of selected geothermal residues

Concentrations of radionuclides in the geothermal residues

X-ray diffraction data of selected geothermal residues

Preliminary acid mine drainage potential test results

Maximum solubilization rate in batch process

Comparison between BC Research Confirmation and AMDP Tests

Sumrnary of results from geochemical modeling of the Mexican scale 109

Hazard and risk rating for Mexican geothermal residues 114

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 ATCC APHA BNL CANMET EU ICRP UNSCEAR Uoff USEPA WHO W C

- Atomic Energy Control Board - American Type Culture Collection - American Public Health Association - Brookhaven National Laboratory - Canada Centre for Mineral and Energy Technology - European Union (formerly European Community) - International Commission on Radiological Protection - United Nations Scientific Cornmittee on the Effects of lonizing Radiation - University of Toronto - United States Environmental Protection Agency - World Health Organization - 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 - 4 m m = 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

important environmental 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 (1 978).

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 (61 and 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

fluids [Il, 121. For a 433 MW geothermal power plant at lrnperial Valley, California, an

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 m3 while

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

power supply and capital investment [26, 271.

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

Elements Average, %

Minor Crustal

Elernents Average, ppm

2.2.2 Leacha te 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 Hea vy 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 u ptake. 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~uent waste 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.

2.3.2 X-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 interna1 structure 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 Evalua fion 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 lig ht.

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 (1 0 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 tem peratures. 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

metal sulfides) to mine tailings [28, 921 and therefore experience from the mining industry

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 cm 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, + 2H+ 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 Fe2++2 H2S04 (9)

bacteria

2 CuFeS, + 8.50, + H,S04 -+ 2 CuSO, + Fe,(SO,), + H,O (1 0)

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 6.0 and minimum of 1 .O without 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 (1 00-250 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

Thiobacillus femoxidans. 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 150-1 80 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 H2S04 per 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 H2S04 as 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

II Acid Base Accounting Modiiicd Acid Base I BC RESEARCH I Alkaljnc Production I Net Acid Production Accounting Initial Potcatiil: Sulfur

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

ACID PRODUCTION DETERMINATION

cost: USS 34-1 10 b -simple and short time -no special equipment -easy interpretation many sarnples can be

tested

Total S used as indicator Acid Producing Fotential = 30.6 Total S

-does not relate to kineiic -assumes parallel acid alkaline release

- i f APP and NP are close. hard to interpret

-ditYerent particle size not

1 retlected

Acid Producing Potcntial = 30.6 Total S

60 mesh (240 um) sample

Total Acid Production = 30.6 Total S

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

-simple -fairly short time -no special equipment -easy interpretation

-does not relate io kinetic -assumes parallel acidl alkaline rctease -if AP and NP are close, hard to interpret

-different particle sizc not reflected

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

LGES AND DISADVANT,

-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

230 um sample

20 mL 0.1 N HCI to 0.4g solid for 2 hours at roorn temperature

iimtion endpt pH 4.0

duration: 2 houn cost: US$ 34-1 10

GES

.simple -short timc -no special equipment

-moderate interpretation

3 0 0 m l H20, added to 5 g rock to directly

paniclc size not presentcd

acid produccd by iron sultide oxidation dissolves buffering

minenils

titration endpt pH 7.0

duration: 2 houn cost: US$ 25-68

-simple -short time -no special equipment easy interpretation

-1irnited reproducibility -uncemin if extent of suIfide oxidation simulates that in tield

Source: This table was compiled from USEPA, 1994; CANMET, 1991; Lawrence, 1989; Ferguson, 1988; Ferguson, 1987; Bruynesteyn, 1984; and Sobek, 1978.

Table 2-6 Summary of Sorne Kinetic Test Methods, Costs, Advantages, and Disadvantages

II HUMlDlTY CELLS 1 SOXHLET EXTRACTION I COLUMN TESTS

- - - --

SUMMARY OF TEST METHOD

2.38 mm particle size panicle size not presented I II ZOOg of rock exposed to three days dry air.

three days humidified air. and rinscd with 200 mL on day sevcn

T=70°C and at T=2S O C water passed through sample is distilled

and recycled through sample

ADVANTACES AND DISADVANTACES

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

duration: 3-8 days cost: USS 212-425

-results take long time -sorne special equipment -moderate ease of interpretation -large data set generated

-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

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

II BC RESWRCH CONFIRMATION 1 BATCH REACTORJSHAKE F U S K S 1 FIELD TESTS

II 400 merh particle rire l 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

iMETHOD

200 mesh panicle size

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

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

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

Source: This table was cornpiled from USEPA, 1994; CANMET, 1991; Lawrence, 1989; Ferguson, 1988; Ferguson, 1987; Bruynesteyn, 1984; and Sobek, 1978.

a = - D A &

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

"Sn shape 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, 102-1 051.

2.7.2 Applications

Several researchers have used MINTEQA2 to sirnulate solid-phase dissolution from

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 (1 5% 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 C haracterization

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 =

1.542A) radiation at 40 kV, 15 mA and scanning from 5' to 66 with a scan speed of 1

degree 28 per min was used. Phase identification was carried out manually using 1989

Hanawalt l ndex 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 Op tical 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 11 IO].

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/HCI for

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-Mn Oxides 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 II], a

10 g pulverized sample (1 00 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 (TCL P)

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 1 N

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 1 8-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

- 20: 1 liquid to solid ratio, 50 glL

- leachant : 800 rnL deionized water

- pH of solution : 510.2

- particle size : 5 9.5 mm

- extraction time : 24 hours with

pH adjustment @ 1, 3, 6, 22 h with

0.5N acetic acid (lirnit of 200 mL)

- room temperature

- rotary extraction at I O rpm

Leachate Extraction Procedure (LEP)

of Ontario, Canada

- 20:l liquid to solid ratio, 50 g1L

- leachant : buffered acetic acid

if pH of sampIe6, use pH = 4.7

if pH of sarnple>b, use pH = 2.8

- particle size : s 9.5 mm

- extraction time : 18 h, without pH

adjustment

- room temperature

- rotary extraction at 30 rpm

Toxicity Characteristic Leaching

Procedure (TCLP) of USA

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 I 0.05. 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 H2S04 to 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

1959 [77] and adopted by the APHA. The two modifications made in this work were the

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

sulfate [l I l ] was used on the pure culture (Bo) inoculum of Thiobacillus ferrooxidans

(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, B2 was 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 Di potassium 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 g lasswares 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 incubatorlshaker at 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 111 11. 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 Microstructural Analysis

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. Photomicrographs were 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 fi ne-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 0.01 20

Concentration of acetic acid: moVL LEP (0.5 N) 0.0025 TCLP (0.1 N) O. 1 O00

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 1 0-l6 molal) 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 (1 0%) 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 (351 0

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-21 0 -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-4 O U estimate*

PSC ~ 7 5 0 990 120 110 I I O 42 I 22 360 * 33 ~ 9 . 4

PSL ~ 3 9 0 300190 80110 83120 240,:lO ~ 5 . 1

ASC ~ 3 5 0 420 180 530 I 20 550 * 73 310 * 10 ~ 4 . 8

MDM ~ 2 2 0 4 3 0 9 4 i 1 0 67111 310112 6 . 2

MSC ~ 3 8 0 1300001400 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 S pecies'

PSC

PSL

ASC

MDM

MSC

MSL

Amorphous material maximum at 4.10 A Quartz, SiO, Magnetite, Fe,O, Hematite, Fe203 Jarosite, KFe3(S04), (OH),

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

Amorphous material maximum at 3.43 A

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,),

Galena, PbS Sphalerite, ZnS C halcopyrite, CuFeS, Cubanite, CuFe2S3 Amorphous material maximum at 3.80 A

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 n e Scale (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 (1 OOx, 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 num ber 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 pm) 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 (1 00x 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, 1281 were 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, 11 51 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 1 00 T ime, houm

I

p P b +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 al1 the 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 4.1 1 Sequential extraction results for Philippine scale and sludge.

ASC

MDM

Figure 4.1 2 Sequential extraction results for Amencan sale and

Mexican drilling mud.

MSC

MSL

Figure 4.1 3 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 .5 ppm). 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 1 O 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 106 cellsImL 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 1 OOOx 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

magn ification (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 p hotom icrograph 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 1 o6 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 1 O6 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) m issing in num ber only; text follows. Microfilmed as received.

. .

UMI

Figure 4.1 6 This is a TEM p hotornicrograph 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

precipitate was 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 (A MDP) 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 4.1 9. In general, the agitated flasks had leached out more metals by as much as 1 0%

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.

1 L ' 200 O 5 10 15 20 25 30

Time, dry8

PSL

1 1 200 O 5 10 15 20 25 30

Time, dayr

MSC

1 1 J 200 O 5 10 15 20 25 30

Time, days

Time, days

Time, d a p

O 5 10 15 20 25 30 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

Cu I

C 0 - 4Q s

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 T ime, 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 (1 3)

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, 1 OOx 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

MSC

O 6 10 15 20 2S 30 Time, days

1 0 O 10 1s 20 26 30

Time, days

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.

PSL

MSC

O 6 10 15 20 25 30 Time, days

0 0 O 5 10 15 20 PL 30

Time, days

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

identified to 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 1123-1 251.

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 Co was 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 1 O4 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 I O 15 20 26 Timr, 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.

100

80

8 a0 a = 40 8

20

C

0- ' h - v = w œ

- - O 5 10 i S 20 25

Time, days

Zr!

L

p 1*5 I L

r

O O 5 10 15 20 25

Time, days

P 1 A A v w A

a# v -

2

O 1.5 Q) r P 1 0, œ

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.

I

* a i

I I -

0.5 I

O O 5 10 15 20 25

Time, days

I - - I I m

ae 0.5 A 7 T A .- A ' b /

O I O 5 10 15 20 25

Tirne, days

Tirne, days

O 5 10 15 20 25 Time, days

+ Agitated, 35 OC &tationary, 25 OC

2

1.5 aa C

Pb I l a 0 ' 0.5

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.

I

C

0- - 9 I

I I

w I I 9 - O 5 10 15 20 25

Time, days

Cs!

O 5 10 15 20 25 Time, days

O 5 I O 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 1 a9 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

2

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 Ag itated , 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+and then 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 1 L 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 (c2 ppm) 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 4.1 8, 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/wellfield sites, 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

lnoculum

Nutrient Media

Particle size

Solid concentration

Agitation

Temperature

Sterilization

Measurements

B.C. Research Confirmation Test

no specific guideline

only basal salts, no FeSO,

400 mesh

15-30 gRO mL (amount depends on S content)

With agitation, shaker speed not specified

lnside incubator at 35 OC

Media to be autoclaved

pH only, metals are optional

Uoff- Acid Mine Drainage Potential Test

-

reproducible method of bacteria culture

basal saits arrd 50% of the standard 9.0 glL Fe "

120 rnesh

With or without agitation, (1 75 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 0.1 3 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.

4.1 0 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 C haracteristic Leachate Procedure (TCLP)

Parameters Experimental Model Dissolved Species

Concn, mol/L Activity, molIL* Cu2+ 1.8E-05 1.838E-17 Cu2' (82.1 %), CuAcetate (1 5.8%) Fe2+ 1.1 E-03 1.176E-06 Fe2'(97.2%), FeAcetate (2.8%) Zn2+ 1.2E-04 4.834E-06 Zn2'(95.8%), ZnAcetate (4.1 %) P b2+ 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 (0.01 ppm) for Cu and Zn and 1.5 x 1 ObmoVL (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 kP values 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

important factors. Laboratory experiments 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 intenpreted from 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

Parameter

Chernical content

Radioactivity

Toxicity

Leachability

AMD potential

MDM MSC 1 MSL

Very Low Low

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.

Medium High VeryLow

Table 4-10 Hazard and risk rating for American and Philippine geothermal residues -

Parameter

Chernical content

Radioactivity

Toxicity

Leachability

AMD potential

Low

-

PSC

High

J

Low

PSL

High

J

ASC

Very Low

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

SI Cu, 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

(1 75 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~osai of 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)

Hand book of Microbiological Media [Atlas, 19931

No FeSO,

50% of required FeSO,

100% of required FeSO,

10-1 5 911 00 ml

5 g/100 mL

2 gI100 mL

Nutrient Concentration

Solid Concentration

Agitation

Temperature

Sterilization

lnside incubator-shaker (agitated)

On laboratory bench (stationary)

lnside incu bator-shaker (35 OC)

Room temperature (23-25 OC)

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 discriminate the 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 mm2 where 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~

Bacterial density, cells1mm3 = 15.75~ = 1 5 . 7 5 ~ = 0.788~

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

6 d tf.xls 23 18000 1.8 x 10'

bacteria.xls 40 32000 3.2 x 10'

atcc2.xls 27 21 O00 2.1 x 106

atccl .xls

13 d civl .xls

civ2.xls

7 d asc2.xls

mdm2.xls

msc2.xls

msl2.xls

psc2.xls

psl2.xls

7 d t f l .tif

tf4.tif

W . tif

il d 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 l N acid x ka11000 q

2 x sample weight, g x tonne

1000 g

AC = mL 1 N H,SOI x 0.049 x 100Q

Sample weight in g

kg H,SO, per tonne

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 g11O6 x 50 g/L x 1 mole163.5 g = 0.0071

moles Pb = 1 1,600 uglg x 1 911 O6 x 50 g/L x 1 mole1207.2 g -0.0028

moles Zn = 15,900 uglg x 1 g1106 x 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 = 0.01 20

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

0.01 20 - - 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) :0.1 N 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) : 25 .00 U n i t s of concentration: MOLAL Ionic 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 30% P r e c i p i t a t i o n was a l l o w e d only f o r thrse so l i d s specified as ALLOWED

i n the i n p u t f i l e (if any) . T h e maximum number of i t e r a t i o n s is: 200 The method used t o c o m p u t e activity coeff ic ients is: Davies equation Intemediate output f i l e

------------------------------------------------------------------------------- 330 1.000E-01 -1.00 H+ 992 1.000E-01 -1 .00 A c e t a t e 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, Chalcopyri te 1060001 15 .1320 -19.4000 2.800E-03 PbS G a l e n a 1095001 I l . 6180 -8.2500 1.200E-02 ZnS S p h a l e r i te 1023101 23.0380 -24.0100 3.6003-03 CuS C o v e l l i t e

INPUT DATA BEFORE TYPE MODIFICATIONS

NAME H+l A c e t a t e HS-1 E-1 Fe+2 Pb+2 Zn+2 Cu+2 H20

ACTIVITY GUESS 1.000E-01 1.000E-01 1.000E-16 1.000E-16 1.000E-16 1.000E-16 1.000E-16 1.000E-16 1.000E+00

LOG GüESS -1.000 -1.000

-16.000 -16.000 -16.000 -16.000 -16.000 -16.000

o . O00

ANAL TOTAL 1.000E-O1 1.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01

Charge Balance : UNSPECIATED Sum of CATIONS= 1.000E-01 Sum of ANIONS = 1.000E-01 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 : 2 7 : 2 2

PARAMETERS OF THE COMPONENT MOST OUT OF BALANCE:

ITER O H S - 1 1 HS-1 2 H S - 1 3 H S - 1 4 H S - 1 5 H S - 1 6 HS-1 7 H S - 1 8 HS-1 9 HS-1

1 0 HS-1 11 HS-1 1 2 H S - 1 13 HS-1 1 4 HS-1 15 H S - 1 1 6 HS-1 1 7 HS-1 1 8 HS-1 1 9 HS-1 20 HS-1 2 1 HS-1 22 HS-1 23 HS-1 25 HS-1 2 6 HS-1

NAME H+l Acetate HS-1 II20 E-1 Fe+2 Cu+2 Zn+2 Pb+2

NAME TOTAL MOL 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 o . 0003-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.300E-Ci 0.000E-01 0.000E-01

ANAL MOL 1.000E-01 1.OOOE-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01

CALC MOL 1.3583-03 1.3723-03 6.770E-10

- l . 2 5 6 E - l l 9 .3033-01 1.389E-06 2.1723-17 5.7123-06 1.7493-09

LOG ACTVTY -2.88524 -2.88063 -9.18752 -0.00148 -0.02764 -5.92972

-16.73572 -5.31572 -8.82 972

DIFF FXN 2.9003-09 3.1063-09 4.386E-10 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01 0.000E-01

Type 1 - COMPONENTS AS SPECIES I N SOLUTION

NAME H+l Acetate HS-1 Cu+2 Fe+2 Pb+2 Zn+2

CALC MOL ACTïVITY 1.3583-03 1.3023-03 1.3723-O3 1.3163903 6.770B-10 6.4943-10 2.1723-17 1.8383-17 1.3893-06 1.1763-O6 3.7493-09 1.48OE-09 5.7123-06 4.0343-06

,----------------------

LOG ACTVTY GAbMA -2.88524 0 .95912 -2.08063 0 .95912 -9.18752 0 .95912

-16.73572 0 .84625 -5.92972 0 .84625 -8.82972 0 .84625 -5.31572 0 .84625

--CI------------------

NEW LOGK O . 018 O . O18 0.018 O . 073 0.073 O . 073 O . 073 ----------

Type II - OTHER SPECIES IN SOLUTION OR ADSORBED

NAME ZN ACETATE4 FeACETATE OH- FeOH + FeOH3 -1 ~ 8 0 ~ 2 AQ Fe(HS) 2 AQ Fe(HS) 3 - Cu ACETATE CuOH + Cu(0H) 2 AQ Cu(0H) 3 - Cu(OH)4 -2 Cu2 (OH) 2+2 Cu(HS) 3 - ZnOH + Zn(0H) 2 AQ Zn(0H) 3 - Zn(0H) 4 -2 Zn(HS) 2 A(;Z Zn(HS) 3 - PbOH + Pb(0H) 2 AQ Pb(0H) 3 - Pb20H +3 Pb(HS) 2 AQ Pb(HS) 3 - Pb3 (OH) 4+2 Pb(0H) 4 -2 H2S AQ S -2 . H ACETATE CuACETATE 2 CuACETATE 3 CuACE TATE 4 PBACETATE PBACE TATE 2 PBACETATE 3 PBACE TATE 4 ZN ACETATE ZN ACETATE2 ZNACETATE 3

CALC MOL 3.9293-16 4.053E-08 8.0153-12 2.9663-13 5.4913-29 1.8523-21 4.4173-16 3.2573-23 4.1863-18 1.4663-22 2.2473-25 1.0833-35 1.8703-45 1.0233-38 4.1583-19 4.2283-12 3.5703-17 9.0093-26 1.2383-35 1.7753-O9 1.7373-37 2.3023-14 6.5713-21 6.0213-29 1.0653-21 1.162E-12 1.5703-20 1.7313-39 1.1993-37 7.3903-06 7.1163-20 9.8633-02 1.3583-19 5.5023-23 5.1793-26 1.5063-09 3.0823-11 1.3693-14 1.3193-17 2.4653-07 6.6513-10 4.2713-13

ACTIVITY 3.3253-16 3.8879-08 7.6873-12 2.8453-13 5.2673-29 1.8533-23 4.4183-16 3.1243-23 4.015E-18 1.406E-22 2.2483-25 3.0393-35 1.5823-45 8.6613-39 3.9883-19 4.055E-12 3.5713-37 8.6413-2 6 1.048E-35 1.775E-Og 1.6663-17 2.2083-14 6.5743-21 5.7753-29 7.3173-22 1.1623-12 1.5063-20 1-4653-39 1.0153-37 7.3923-06 6.0223-20 9.866E-02 1.3583-19 5.2773-23 4.3833-26 1.4443-09 3.0833-11 1.3133-14 1.1163-17 2.3643-07 6.6533-10 4.096E-13

NEW LOGK 1.433 1.418

-13.980 -9.482

-30.982 -20.570 8.950 11.005 2.238 -7.982

-13.680 -26.881 -39.527 -10.286 25.917 -8.942

-16.899 -28.381 -41.126 14.940 16.118 -7.692 -17.120 -28.042 -6.197 lS.270 16.588

-23.807 -39.626 6.941

-12.845 4.760 3.630 3.118 2.973 2.888 4.080 3.608 3.473 1.588 1.900 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 S P W R I T E 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 FILE 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

A c e t a t e

PERCENT BOUND I N SPECIES # 330 PERCENT BOUND IN SPECIES #3309921

PERCENT BOüND I N SPECIES # 992 PERCENT BOUND IN SPECIES W3309921

PERCENT BOUND I N SPECIES W3307300

PERCENT BOUND IN SPECIES #3300020 PERCENT BOUND IN SPECIES a2803300 PERCENT BOUND IN SPECIES U9503300

PERCENT BOUND I N SPECIES # 280 PERCENT BOUND IN SPECIES #2809920

PERCENT BOUND I N SPECIES # 231 PERCENT BOUND I N SPECIES #2319921 PERCENT BOUND I N SPECIES #2317300

PERCENTBOUNDINSPECIES# 950 PERCENT BOUND I N SPECIES #9509921

PERCENT BOUND I N SPECIES # 600 PERCENT BOUND I N SPECIES #6009921

H+1 H ACETATE

A c e t a t e H ACETATE

OH- FeOH+ ZnOH+

Cu+2 Cu ACETATE Cu(HS)3 - Znt2 ZN ACETATE

Pbt2 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 = 1-64 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 al1 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.

GIA Short Version

1. Grind samples in a mortar and pestle to 120 mesh size (1 25 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 pm) 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 H2S04 until stable at pH 2.9 I 0.1.

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 alread y exists.

Resultina inoculum

Full media + pure culture B I

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

Association [Il 11. The major differences are the reduction of the FeSO, content and use

of membrane filtration for sterilization of solution instead of autoclaving.

Mod ified Growt h 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//us femoxidans 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 3.1 3%). In Table H1 below, a sample with a value of

3.1 3% 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 1 O]. 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 H 1 Toxi-Chromotest Results for Geothermal Samples

Sampfes - - - -

+ Control

- Control

PSC

PSL

ASC

Toxic at X %

3.13

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

Non-toxic (NT)

50

50

50

NT

NT

NT

NT

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.

Samples

+ Control

Toxic at X %

3.13

- Control

MDM

MSC

MSL

Non-toxic (NT)

NT

50

50

50

25

25

NT

APPENDIX I - ADDITIONAL DISSOLUT ION 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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