study of native micro organisms in bioleaching processes of refractory auriferous
TRANSCRIPT
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Study of Native Micro organisms in Bioleaching Processes of Refractory Auriferous
Minerals and its Use as a Tool for Bio-regeneration
Francisco Gordillo Espinosa, Víctor Sanmartín, José Torracchi. Fabián Carrión.
Bac-Min 2004 Congress.
Contact: Fabián Carrión.
Zip. Cod: 11-01-608
Tlf: 593-7-2570275
Fax: 593-7-2584893
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Study of Native Micro organisms in Bioleaching Processes of Refractory Auriferous
Minerals and its Use as a Tool for Bio-regeneration
Francisco Gordillo Espinosa, Víctor Sanmartín, José Torracchi, Fabián Carrión.
Universidad Técnica Particular de Loja. San Cayetano s/n
Zip.code: 11-01-608 Loja-Ecuador
ABSTRACT
The broad biodiversity of our country is manifested through several different biological
forms. From the villages of Portovelo and San Gerardo in the southern part of Ecuador,
some samples of water and rocks were taken in order to identify the native micro-organisms
which take part in the natural processes of leaching of sulphurous minerals.
It’s been achieved to determine the presence of Spp. Thiobacillus ferrooxidans and a fungi
sample which has not been determined yet. These have been isolated, grown, experimented
and conserved in appropriate cryogenic environments.
The present research intends to study the individual and group adaptation of the bacteria and
the fungi upon suitable systems of agitation and ventilation, in which has been placed
several different concentrations of refractory auriferous samples. These come from the
recovery processes of gold through traditional methods and test their affectivity as pre-
treatments upon the cyaniding processes.
Also the capability of these bacteria to develop in minerals with high concentrations of
cyanide has been studied for the possibility of using the bacteria as a method for the
biodegradation of cyanide.
The presence of Thiobacillus ferrooxidans in acid conditions has already been tested in
advance, however, the presence of fungi species in these conditions are studied to prove
their efficiency as another alternative for the bioleaching of refractory auriferous minerals,
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INTRODUCTION
In Ecuador, a lot of wastes with some
refractory characteristics have been
accumulated for several industrial plants
and for mining craftsmen. The contents of
gold of these wastes are, in some cases,
more than of 20 gram per ton
(PRODEMINCA, 2001 Programa de
desarrollo minero y capacitaciòn
ambiental del Ecuador). Moreover, with
these characteristics, these deposits are not
feasible of benefiting by traditional
processes of concentration or dissolution
which impede to get bigger percentages of
recovering.
The problem of refractivity has placed the
pyrite and the arsenopyrite as the most
important minerals that encapsulate and
make refractivity on some metals such as
gold (3). This makes the method of
recovering through cyanide a little
optimum and it applies only the recovering
of native gold and electrum (1)
The transcendence of the micro-organisms
in the physical and chemical formation
and transformation of the minerals with an
enormous interest in those which present
natural oxidations and dissolutions
provoked by the action of the samples that
obtain the energy for their metabolism
rusting the present iron and the sulphur.
Some bacteria have been discovered which
are able of rusting from the elemental
sulphur to the sulphuric acid (14) and the
influence of certain species of bacteria in
the oxidation and decomposition of
sulphuric minerals, in particular the pyrite
(14). These methods have been determined
as processes of catalytic action in the
dissolution of mineral components through
the direct and indirect action of bacteria.
One of the micro-organisms that have
favoured these studies is the
Chemolithotrophic mesophilus
Thiobacillus ferrooxidans. It possess the
capability of catalyse reduced components
of sulphur and ferric ion, using oxygen as
electric acceptor and generating sulphuric
acid as a final product. (12).
The microbiologic leaching is a natural
process of dissolution that results from the
action of a group of bacteria (basically
bacteria from Thiobacillus), with
capability of rusting sulphuric minerals,
which allow to release the metallic values
contented. (7)
The selected samples for the essay were
taken from galleries of closed mines in the
50s in the place Zoroche Unificado of
Portovelo and new deposit of the mine of
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San Antonio in San Gerardo. The stones
present high degrees of weathering
(oxidation) in these places.
The craft auriferous Ecuadorian mining
uses inappropriate quantities of cyanide,
without any technical principle
(Prodeminca, 2001). This process
generates highly dangerous levels of soil
and water pollution on these areas. A
bacterial screening in the studied areas
allowed demonstrating the growth of spp.
pseudomonas with an important rate of
survival in the water containing important
amounts of cyanide.
The present study will permit to determine
the bacterial behaviour at different
concentrations of mineral, for verifying its
growing, the oxidation degree from Fe+2 to
Fe+3 and the appropriate solid-liquid
percentage of pulp for the bio-oxidation as
a pre-treatment to the lixiviation with
cyanide, in addition, the capability of the
bacteria found when degrading important
quantities of dissolved cyanide.
MATERIALS AND METHODS
Isolation, cultivation and conservation
The samples were taken form 50 and 100
meters deep at those zones of mineral
galleries that show high degrees of
weathering of the mineralised stones,
especially in the plains of structural
contact, characteristic for the presence of
stalactites and stalagmites.
Some samples were taken from water and
rocks that were placed in sterile flasks of
120 ml. They were carried in isolated
thermic boxes.
The solid culturing was done in Petri
dishes, using volume 125ml. of FeTSB
medium (11). And the pH was adjusted to
2 with concentrated sulphuric acid.
Erlenmeyer flasks of 125 ml were used in
the liquid cultivations. A volume of 50 ml
of 9K medium (11) and the pH was
adjusted at 2, likewise with concentrated
sulphuric acid. It was agitated at 150
rev/min in orbital shaker
(THERMOLYNE) during 15 days.
The cultivation in both cases was done in
aseptic conditions using the laminar
flowing chamber (ESCO) and the
materials were sterilized (121 ºC, 20
minutes) and subjected at 20 minutes of
radiation ultra violet light before the
inoculation.
The conservation of selected frozen
samples is done in cryotubes. A
cryoprotector solution of glycerol at 10%
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vacuum filtrated, plus a solution of 9K
medium, the glycerol-9Kmedium solution
were sterilized (121ºC, 15 minutes).
The micro-organisms for freezing are
cultivated in leaning agar tubes. The
cryoprotector solution is added in the agar
tubes and liquid medium. The cultivation
is re-suspended by scraping the colonies
and agitating respectively.
Determining bacterial growing
A permanganate solution of potassium
was prepared for determining the
transformation from Fe+2 to Fe+3. It titrates
over 5 ml. of extracted solution from the
tests of examination.
The bacterial growing is determined taking
15 µl of culture. It is added 5 µl of blue
lacto phenol. For the bacteria of the
mineral pulp and blue of methylene for
those which grow on cyanide. They are
mixed in a micro tube. The solution is
placed in a chamber of re-counting
(NEUBAUER) and the bacteria are
counted in five fields.
The resulting re-estimating value gives us
the approximated number of bacteria per
millilitre of cultivation.
Mineralogical characterization
The mineralogical composition was
determined by optic microscopy (NIKON
EPIPHOT) of reflected light in polished
sections as seen on the Table 1.
Chemical analysis
The reading for each basic metal was done
for Spectrophotometry of atomic
absorption (PHILIPS-PYE-UNICAM).
The precious metals were determined by
fire assay, acid desegregation of the gold
pearl and the reading for
spectrophotometer of atomic absorption.
The results as seen on the Table 2
Physical analysis
The specific weight was determined by the
method of pycnometer and the control of
pH with pH meter (THERMO ORION).
The results are shown on the Table 3. The
granulometric analysis was done by dry
and wet via in sieve shaker (RETSCH)
with a passing from the 80 to 190 mesh.
Experimenting
Fifty samples were done for evaluating the
ranges of concentration of pulp, from
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which 3 were selected of better growing
and were tested again but duplicated.
The samples were processed in containers
of precipitation of 2000 ml at
concentrations from 5 to 60% respectively.
It was placed homogenized refractory
mineral with a granulometry of 190 mesh
(0.78m). Distilled and demineralized water
was added to get a total solution of 1000
ml from the isolated cultures were taken
100 µl of the sample with bigger kinetic of
growing, to which was inoculated in the
solution. It was stirred at 175 rev/min an
flasks shaker (PHIPPS & BIRD
STIRRER), the pH regulated periodically
at 2 with concentrated sulphuric acid and
the temperature of the growing chamber
was of 22ºC. Each sample was maintained
in agitation during 21 days (2).
Degradation Test of Cyanide The tests were done by triplication on
cyanide dissolution to evaluate the
adapting capability of the samples of
bacterial broth that was found in the
mining deposits. In Erlenmeyer flask, it
was prepared 250 ml of cyanide
dissolution to 25 ppm at a pH of 9. The
temperature of the chamber of growing
was 22ºC and inoculated 500 µl of culture.
It was stirred in an agitator of arms
(BURRELL) to 220 rev/min during 10
days (14).
RESULTS
Bacterial growing
The bacterial growing in the studied
minerals in three different concentrations
(fig 1, 2, 3) is observed in a similar
relationship until the second week, from
which it is distinguished differences in the
growing of the sample of San Gerardo.
Possibly by the influence of the
mineralogical characteristics. On the last
week, there is an accelerated growing until
the day 21.
pH Variation
The consume of sulphuric acid to regulate
the pH is in direct function of the
mineralogical composition, evidencing its
stabilization from the second week with an
average value of 2.5 as it is shown on the
figure 4.
Iron concentration
The titling of iron for the indirect
determination of the bacterial growing is
shown in the figures 5 and 6, in which
from the second week its values increase
proportionally due to the time as well as
the increasing the kinetic of bacterial
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growing. It was determined that the iron
percentage increases in the pulp from the
12th day for San Gerardo and the 14th for
Portovelo’s samples.
DISCUSSION.
Under controlled conditions the collected
bacterial samples adapt successfully in the
cultivation environments in vitro and in
the tests with mineral pulp. The statistic
results show that the bacterial growing is
directly proportional at the pulp
concentrations especially from the 4 7% to
51% in controlled conditions of
granulometry, pH, temperature and stirrer
(Fig. 8 and 9). However, when comparing
the figure 4 and the figure 9 are evident
that the bacterial growing tends to
decrease for the low transference of
oxygen in the environment for the major
pulp concentration.
During the first days, the non-metal
mineral dissolution increases the values of
pH which stabilizes with the time until
achieving a constant value of 2,5. This
suggests that microbiological
metabolism produces sulfuric acid for auto
regulation of the environment. (16)
The observed samples by microscopy of
transmitted light present a great quantity
of crystals of calcium sulphate that are
formed during the bio-oxidation process.
This constitutes like the indirect indicator
of the decomposition of calcite carbonated
minerals.
Likewise, a great adaptation of a
Penicillium species was observed during
the microscopy observation. It was very
sporulating and survives in strong acid
conditions and without the presence of
carbohydrates. This contrasts the normal
growing of this micro-organism that are
not even determined like direct
participants of the process of bioleaching.
The analysis of fig 5 determines the
bacterial capability to adapt approximately
in five days to cyanided solutions and an
exponential growth in ahead. It evidently
adjusts to the ranks found in the reading of
determination of ppm of dissolved cyanide
(fig 6) in which was found precisely
smaller cyanide levels when the growth
tends at the maximum level. It is estimated
that after 5 days the present cyanide levels
in the tested solutions are smaller than
0, 07 ppm.
REFERENCES
1. Avila, M, Díaz, Y, 2000.
Biodegradaciòn de cianuro: uso de
8
microorganismos inmovilizados,
Quito Ecuador, in Beneficio del
Oro y Tratamiento de Efluentes
Course, pp 1-12 (Universidad
Politécnica Nacional: Ecuador,
Universidad Católica de Lovaina:
Belgium)
2. Bañuelos, S, and Castillo, P, 1993.
Recuperación de metales preciosos
a partir de sulfuros minerales
refractarios, utilizando el proceso
de lixiviación bacteriológica.
Geomimet Magazine Nº 184, pp.
9-18.
3. Chapaca, G, Ávila, M, 2003.
Evaluación de las causas de
refractariedad de un mineral
aurífero de la zona de Bella Rica,
Seminario Internacional de
Minería, Metalurgia y Medio
Ambiente. pp. 113-123.
4. Chiacchiarini, P. Lavalle, L.
Tecnologías emergentes para la
bioremediación de metales y su
relación con la enseñanza de la
Química, Universidad Nacional de
Comahue. Facultad de Ingeniería,
Argentina.
5. Dercach, V, 1982. Métodos
especiales de enriquecimiento de
minerales pp 409 (Vneshtorgizdat:
Moscú).
6. Diaz, X, and Moya, L, 2003.
Recuperación de Oro mediante
biolixiviación y tiocianato, in
Seminario internacional de
minería, metalurgia y medio
asbiente, pp. 127-135 (Universidad
Politécnica Nacional: Ecuador).
7. Fowler, T, Holmes, P, Crundwell,
F, 1999. Mechanism of pyrite
dissolution in the presence of
Thiobacillus ferrooxidans, Applied
and Environmental Microbiology,
Vol. 65, pp 2987-2993.
8. Guerreo, J, 1998. Biotecnología en
la disolución y recuperación de
metales, in Primer Congreso
Peruano de Biotecnología y
Bioingeniería, (Trujillo Perú).
9. Guevara, A, De la Torre, E. 2003.
Importancia de los estudios
mineralógicos en el procesamiento
de minerales auríferos refractarios,
in Seminario Internacional de
Minería, Metalurgia y Medio
Ambiente 2003, pp 99-110
(Universidad Politécnica Nacional:
Ecuador)
9
10. González, M, 2004.
Biorremediación y
tratamiento de efluentes,
Monografías.Com, Lucas
Morea/Sinexi S.A.
11. Hartikainen, T. Ruuskanen, J.
Raty, K. Von Wright, A. and
Martikainen, P, 2000. Physiology
and taxonomy of Thiobacillus
strain TJ330, which oxidizes
carbon disulphide (CS2), Journal of
Applied Microbiology, vol. 89, pp.
580-586.
12. Hernández, R., Fernández, C. y
Baptista, P, 1998. Metodología de
la Investigación, pp 105 – 112, 376
– 395 (McGraw Hill: México).
13. Johnson, B. Macvicar, J. Rolfe, S,
1987. A New Solid Medium for
the Isolation and Enumeration of
Thiobacillus ferrooxidans and
Acidophilic bacteria. Journal of
Microbiological Methods. pp. 7-
18..
14. Noel, D M, Fuerstenau, M C and
Hendrix, J L, 1991. Degradation of
cyanide utilizing facultative
anaerobic bacteria, Department of
Chemical and Metallurgical
Engineering University of Nevada ,
(Reno: Nevada).
15. Razo, I, Lopez, S, Lara, C and
Monrroy, M. Study on the ability
of isolated and collection strains to
degrade cyanide: an application of
heap-leaching residues and
effluents, Instituto de Metalurgia,
U.A.S. L.P., San Luis Potosì,
Mexico.
16. Rossi, G. 2001. The design of
Bioreactor. Hidrometallurgy. Vol
59.
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chemistry and treatment of
cyanidation wastes, pp 219-237
(Mining Journal books limited:
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18. Susuki, N. Asai, S. Konoshi, Y.
Tokushige, M, 2001. Cooper
Recovery from chalcopyrite
concentrates by acidophilic
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TABLES LIST
Table 1. Mineralogical analysis of the wastes
Table 2. Chemical analysis of wastes
Table 3. Physical analysis of the wastes
Quantity, % Minerals
Formula Portovelo San Gerardo
Pyrite Chalcopyrite Esfalerita Galena Arsenopyrite Ganga
FeS2 CuFeS2 (ZnFe)S
PbS FeAsS
--
19.1 0.44 0.95 0.4 --
79.11
15 6.1 -- -- 10
68.9
Concentration Element
Portovelo San Gerardo
Cu (%) Fe (%) Pb (%) Zn (%) As (%) S (%)
Au (g/ton)
0.14 9.5
0.45 0.62 0.08 12.1 9.2
2.5
15.3 0.03 0.03 7.84 8.9
19.98
Values Parameter
Portovelo San Gerardo
Specific weight, g/cm3
2.65
2.92
pH
( 35% solids)
6.5
7.5
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FIGURES LIST
Figure 1. Bacterial growing at 25% of pulp
Figure 2. Bacterial growing at 30% of pulp
Figure 3. Bacterial growing at 35% of pulp
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Figure 4. Bacterial growing at different concentrations of pulp
Figure. 5 Bacterial growing in cyanide
Figure. 6 . Degradation of the potassium cyanide
[days]
14
Figure 7. Variation of pH in the pulp at 35% of concentration
Figure 8. Redox in Portovelo`s wastes
Figure 9. Redox in San Gerardo wastes
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Figure 8. Pearson Correlation and regression analysis of bacterial growing on the pulp
concentration
Regression95% confid.
CRECIMI vs. CONCENTRCONCENTR = .11182 + 0.0000 * CRECIMI
Correlation: r = .92098
CRECIMI
CO
NC
ENTR
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
3e7 5e7 7e7 9e7 1.1e8 1.3e8 1.5e8
Figure 9. Curve Adjustment to relate mathematically the pulp concentration in the solution
with bacterial growing
Scatterplot (biolix.STA 7v*11c)y=-3.199e9+4.508e10*x-2.447e11*x^2+6.454e11*x^3-8.21e11*x^4+4.035e11*x^5+eps
CONCENTR
CR
ECIM
I
3e7
5e7
7e7
9e7
1.1e8
1.3e8
1.5e8
0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65