1. 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
fhcarrion@utpl.edu.ec
1

2. 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,
2

3. INTRODUCTION
In Ecuador, a lot of wastes with some Some bacteria have been discovered which
refractory characteristics have been are able of rusting from the elemental
accumulated for several industrial plants sulphur to the sulphuric acid (14) and the
and for mining craftsmen. The contents of influence of certain species of bacteria in
gold of these wastes are, in some cases, the oxidation and decomposition of
more than of 20 gram per ton sulphuric minerals, in particular the pyrite
(PRODEMINCA, 2001 Programa de (14). These methods have been determined
desarrollo minero y capacitaciòn as processes of catalytic action in the
ambiental del Ecuador). Moreover, with dissolution of mineral components through
these characteristics, these deposits are not the direct and indirect action of bacteria.
feasible of benefiting by traditional
processes of concentration or dissolution One of the micro-organisms that have
which impede to get bigger percentages of favoured these studies is the
recovering. Chemolithotrophic mesophilus
Thiobacillus ferrooxidans. It possess the
The problem of refractivity has placed the capability of catalyse reduced components
pyrite and the arsenopyrite as the most of sulphur and ferric ion, using oxygen as
important minerals that encapsulate and electric acceptor and generating sulphuric
make refractivity on some metals such as acid as a final product. (12).
gold (3). This makes the method of
recovering through cyanide a little The microbiologic leaching is a natural
optimum and it applies only the recovering process of dissolution that results from the
of native gold and electrum (1) action of a group of bacteria (basically
bacteria from Thiobacillus), with
The transcendence of the micro-organisms capability of rusting sulphuric minerals,
in the physical and chemical formation which allow to release the metallic values
and transformation of the minerals with an contented. (7)
enormous interest in those which present
natural oxidations and dissolutions The selected samples for the essay were
provoked by the action of the samples that taken from galleries of closed mines in the
obtain the energy for their metabolism 50s in the place Zoroche Unificado of
rusting the present iron and the sulphur. Portovelo and new deposit of the mine of
3

4. San Antonio in San Gerardo. The stones weathering of the mineralised stones,
present high degrees of weathering especially in the plains of structural
(oxidation) in these places. contact, characteristic for the presence of
stalactites and stalagmites.
The craft auriferous Ecuadorian mining
uses inappropriate quantities of cyanide, Some samples were taken from water and
without any technical principle rocks that were placed in sterile flasks of
(Prodeminca, 2001). This process 120 ml. They were carried in isolated
generates highly dangerous levels of soil thermic boxes.
and water pollution on these areas. A
bacterial screening in the studied areas The solid culturing was done in Petri
allowed demonstrating the growth of spp. dishes, using volume 125ml. of FeTSB
pseudomonas with an important rate of medium (11). And the pH was adjusted to
survival in the water containing important 2 with concentrated sulphuric acid.
amounts of cyanide.
Erlenmeyer flasks of 125 ml were used in
the liquid cultivations. A volume of 50 ml
The present study will permit to determine
of 9K medium (11) and the pH was
the bacterial behaviour at different
adjusted at 2, likewise with concentrated
concentrations of mineral, for verifying its
sulphuric acid. It was agitated at 150
growing, the oxidation degree from Fe+2 to
Fe+3 and the appropriate solid-liquid rev/min in orbital shaker
(THERMOLYNE) during 15 days.
percentage of pulp for the bio-oxidation as
a pre-treatment to the lixiviation with
cyanide, in addition, the capability of the The cultivation in both cases was done in
bacteria found when degrading important aseptic conditions using the laminar
quantities of dissolved cyanide. flowing chamber (ESCO) and the
materials were sterilized (121 ºC, 20
minutes) and subjected at 20 minutes of
MATERIALS AND METHODS
radiation ultra violet light before the
Isolation, cultivation and conservation inoculation.
The samples were taken form 50 and 100
The conservation of selected frozen
meters deep at those zones of mineral
samples is done in cryotubes. A
galleries that show high degrees of
cryoprotector solution of glycerol at 10%
4

5. vacuum filtrated, plus a solution of 9K Mineralogical characterization
medium, the glycerol-9Kmedium solution
were sterilized (121ºC, 15 minutes). The mineralogical composition was
determined by optic microscopy (NIKON
The micro-organisms for freezing are EPIPHOT) of reflected light in polished
cultivated in leaning agar tubes. The sections as seen on the Table 1.
cryoprotector solution is added in the agar
tubes and liquid medium. The cultivation Chemical analysis
is re-suspended by scraping the colonies
and agitating respectively. The reading for each basic metal was done
for Spectrophotometry of atomic
Determining bacterial growing absorption (PHILIPS-PYE-UNICAM).
A permanganate solution of potassium The precious metals were determined by
was prepared for determining the fire assay, acid desegregation of the gold
transformation from Fe+2 to Fe+3. It titrates pearl and the reading for
over 5 ml. of extracted solution from the spectrophotometer of atomic absorption.
tests of examination. The results as seen on the Table 2
The bacterial growing is determined taking Physical analysis
15 µl of culture. It is added 5 µl of blue
lacto phenol. For the bacteria of the The specific weight was determined by the
mineral pulp and blue of methylene for method of pycnometer and the control of
those which grow on cyanide. They are pH with pH meter (THERMO ORION).
mixed in a micro tube. The solution is The results are shown on the Table 3. The
placed in a chamber of re-counting granulometric analysis was done by dry
(NEUBAUER) and the bacteria are and wet via in sieve shaker (RETSCH)
counted in five fields. with a passing from the 80 to 190 mesh.
The resulting re-estimating value gives us Experimenting
the approximated number of bacteria per
millilitre of cultivation. Fifty samples were done for evaluating the
ranges of concentration of pulp, from
5

6. which 3 were selected of better growing (BURRELL) to 220 rev/min during 10
and were tested again but duplicated. days (14).
RESULTS
The samples were processed in containers
of precipitation of 2000 ml at Bacterial growing
concentrations from 5 to 60% respectively.
The bacterial growing in the studied
It was placed homogenized refractory
minerals in three different concentrations
mineral with a granulometry of 190 mesh
(fig 1, 2, 3) is observed in a similar
(0.78m). Distilled and demineralized water
relationship until the second week, from
was added to get a total solution of 1000
which it is distinguished differences in the
ml from the isolated cultures were taken
growing of the sample of San Gerardo.
100 µl of the sample with bigger kinetic of
Possibly by the influence of the
growing, to which was inoculated in the
mineralogical characteristics. On the last
solution. It was stirred at 175 rev/min an
week, there is an accelerated growing until
flasks shaker (PHIPPS & BIRD
the day 21.
STIRRER), the pH regulated periodically
at 2 with concentrated sulphuric acid and
pH Variation
the temperature of the growing chamber
was of 22ºC. Each sample was maintained
The consume of sulphuric acid to regulate
in agitation during 21 days (2).
the pH is in direct function of the
Degradation Test of Cyanide mineralogical composition, evidencing its
stabilization from the second week with an
The tests were done by triplication on average value of 2.5 as it is shown on the
cyanide dissolution to evaluate the figure 4.
adapting capability of the samples of
bacterial broth that was found in the Iron concentration
mining deposits. In Erlenmeyer flask, it
was prepared 250 ml of cyanide The titling of iron for the indirect
dissolution to 25 ppm at a pH of 9. The determination of the bacterial growing is
temperature of the chamber of growing shown in the figures 5 and 6, in which
was 22ºC and inoculated 500 µl of culture. from the second week its values increase
It was stirred in an agitator of arms proportionally due to the time as well as
the increasing the kinetic of bacterial
6

7. growing. It was determined that the iron formed during the bio-oxidation process.
percentage increases in the pulp from the This constitutes like the indirect indicator
12th day for San Gerardo and the 14th for of the decomposition of calcite carbonated
Portovelo’s samples. minerals.
DISCUSSION.
Likewise, a great adaptation of a
Penicillium species was observed during
Under controlled conditions the collected
the microscopy observation. It was very
bacterial samples adapt successfully in the
sporulating and survives in strong acid
cultivation environments in vitro and in
conditions and without the presence of
the tests with mineral pulp. The statistic
carbohydrates. This contrasts the normal
results show that the bacterial growing is
growing of this micro-organism that are
directly proportional at the pulp
not even determined like direct
concentrations especially from the 4 7% to
participants of the process of bioleaching.
51% in controlled conditions of
granulometry, pH, temperature and stirrer
The analysis of fig 5 determines the
(Fig. 8 and 9). However, when comparing
bacterial capability to adapt approximately
the figure 4 and the figure 9 are evident
in five days to cyanided solutions and an
that the bacterial growing tends to
exponential growth in ahead. It evidently
decrease for the low transference of
adjusts to the ranks found in the reading of
oxygen in the environment for the major
determination of ppm of dissolved cyanide
pulp concentration.
(fig 6) in which was found precisely
smaller cyanide levels when the growth
During the first days, the non-metal
tends at the maximum level. It is estimated
mineral dissolution increases the values of
that after 5 days the present cyanide levels
pH which stabilizes with the time until
in the tested solutions are smaller than
achieving a constant value of 2,5. This
0, 07 ppm.
suggests that microbiological
metabolism produces sulfuric acid for auto
regulation of the environment. (16)
REFERENCES
The observed samples by microscopy of
1. Avila, M, Díaz, Y, 2000.
transmitted light present a great quantity
Biodegradaciòn de cianuro: uso de
of crystals of calcium sulphate that are
7

8. microorganismos inmovilizados, minerales pp 409 (Vneshtorgizdat:
Quito Ecuador, in Beneficio del Moscú).
Oro y Tratamiento de Efluentes
Course, pp 1-12 (Universidad
Politécnica Nacional: Ecuador, 6. Diaz, X, and Moya, L, 2003.
Universidad Católica de Lovaina: Recuperación de Oro mediante
Belgium) biolixiviación y tiocianato, in
Seminario internacional de
2. Bañuelos, S, and Castillo, P, 1993. minería, metalurgia y medio
Recuperación de metales preciosos asbiente, pp. 127-135 (Universidad
a partir de sulfuros minerales Politécnica Nacional: Ecuador).
refractarios, utilizando el proceso
de lixiviación bacteriológica. 7. Fowler, T, Holmes, P, Crundwell,
Geomimet Magazine Nº 184, pp. F, 1999. Mechanism of pyrite
9-18. dissolution in the presence of
Thiobacillus ferrooxidans, Applied
3. Chapaca, G, Ávila, M, 2003. and Environmental Microbiology,
Evaluación de las causas de Vol. 65, pp 2987-2993.
refractariedad de un mineral
aurífero de la zona de Bella Rica, 8. Guerreo, J, 1998. Biotecnología en
Seminario Internacional de la disolución y recuperación de
Minería, Metalurgia y Medio metales, in Primer Congreso
Ambiente. pp. 113-123. Peruano de Biotecnología y
Bioingeniería, (Trujillo Perú).
4. Chiacchiarini, P. Lavalle, L.
Tecnologías emergentes para la 9. Guevara, A, De la Torre, E. 2003.
bioremediación de metales y su Importancia de los estudios
relación con la enseñanza de la mineralógicos en el procesamiento
Química, Universidad Nacional de de minerales auríferos refractarios,
Comahue. Facultad de Ingeniería, in Seminario Internacional de
Argentina. Minería, Metalurgia y Medio
Ambiente 2003, pp 99-110
5. Dercach, V, 1982. Métodos (Universidad Politécnica Nacional:
especiales de enriquecimiento de Ecuador)
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9. 10. González, M, 2004. Engineering University of Nevada ,
Biorremediación y (Reno: Nevada).
tratamiento de efluentes,
Monografías.Com, Lucas 15. Razo, I, Lopez, S, Lara, C and
Morea/Sinexi S.A. Monrroy, M. Study on the ability
of isolated and collection strains to
11. Hartikainen, T. Ruuskanen, J. degrade cyanide: an application of
Raty, K. Von Wright, A. and heap-leaching residues and
Martikainen, P, 2000. Physiology effluents, Instituto de Metalurgia,
and taxonomy of Thiobacillus U.A.S. L.P., San Luis Potosì,
strain TJ330, which oxidizes Mexico.
carbon disulphide (CS2), Journal of
Applied Microbiology, vol. 89, pp. 16. Rossi, G. 2001. The design of
580-586. Bioreactor. Hidrometallurgy. Vol
59.
12. Hernández, R., Fernández, C. y
Baptista, P, 1998. Metodología de 17. Smith, A and Mudder, T. The
la Investigación, pp 105 – 112, 376 chemistry and treatment of
– 395 (McGraw Hill: México). cyanidation wastes, pp 219-237
(Mining Journal books limited:
13. Johnson, B. Macvicar, J. Rolfe, S, London).
1987. A New Solid Medium for
the Isolation and Enumeration of 18. Susuki, N. Asai, S. Konoshi, Y.
Thiobacillus ferrooxidans and Tokushige, M, 2001. Cooper
Acidophilic bacteria. Journal of Recovery from chalcopyrite
Microbiological Methods. pp. 7- concentrates by acidophilic
18.. thermopile acidianus brierleyi in
batch and continuous flow stirrer
14. Noel, D M, Fuerstenau, M C and tank reactors. Hydrometallurgy.
Hendrix, J L, 1991. Degradation of Vol 59 Nº 2-3.
cyanide utilizing facultative
anaerobic bacteria, Department of 19. Thompson, L C. Developments in
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processes, Pintail Systems, Inc.
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10. 11801 E. 33rd Ave. Suite C.
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Beliaevskaya, L. 1981. Teoría de
los procesos metalúrgicos. pp. 208-
213 (Vneshtorgizdat: Moscú).
10

11. TABLES LIST
Table 1. Mineralogical analysis of the wastes
Quantity, %
Minerals Formula
Portovelo San Gerardo
Pyrite FeS2 19.1 15
Chalcopyrite CuFeS2 0.44 6.1
Esfalerita (ZnFe)S 0.95 --
Galena PbS 0.4 --
Arsenopyrite FeAsS -- 10
Ganga -- 79.11 68.9
Table 2. Chemical analysis of wastes
Concentration
Element
Portovelo San
Gerardo
Cu (%) 0.14 2.5
Fe (%) 9.5 15.3
Pb (%) 0.45 0.03
Zn (%) 0.62 0.03
As (%) 0.08 7.84
S (%) 12.1 8.9
Au (g/ton) 9.2 19.98
Table 3. Physical analysis of the wastes
Values
Parameter
Portovelo San
Gerardo
Specific weight, g/cm3 2.65 2.92
pH 6.5 7.5
( 35% solids)
11

12. 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
12

13. Figure 4. Bacterial growing at different concentrations of pulp
[days]
Figure. 5 Bacterial growing in cyanide
Figure. 6 . Degradation of the potassium cyanide
13

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
14

15. Figure 8. Pearson Correlation and regression analysis of bacterial growing on the pulp
concentration
CRECIMI vs. CONCENTR
CONCENTR = .11182 + 0.0000 * CRECIMI
Correlation: r = .92098
0.65
0.60
0.55
0.50
CONCENTR
0.45
0.40
0.35
0.30
0.25
Regression
0.20
3e7 5e7 7e7 9e7 1.1e8 1.3e8 1.5e8 95% confid.
CRECIMI
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
1.5e8
1.3e8
1.1e8
CRECIMI
9e7
7e7
5e7
3e7
0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65
CONCENTR
15