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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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
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
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,
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
< 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
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
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
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
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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