2. 482 Biohydrometallurgy 2009
Enrichment of microbial consortia. The liquid samples taken in areas of weathered mining shafts
were cultured in 250 ml flasks containing 90 ml of the medium Norris [3] and 10 % v/v of the
culture to pH 1.8. The specific growth rate was determined the oxidation, performance, and
productivity of Fe2. When the bacterial culture reached a constant of µm, it was replicated in a
medium of Norris modified with 2 g.L-1 for Fe2+ and 2% w/v ore. In all cases the flasks were
incubated at 220 rpm and 30 °C.
16S rR A Sequencing.
For DNA extraction Wizard ® Genomic DNA Purification Kit was used. The amplification was
done by PCR, using a combination of universal bacterial primers 27F (5'-
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5-GGTTACCTTGTTACGACTT-3) [1]. The
products were purified and sequenced by Macrogen in Seoul, Korea. The experiment was
conducted with the program Sequencher 4.6 Gene Codes, in Ann Arbor, MI. To identify published
sequences similar to the bacterial 16S rRNA, the NCBI BLAST was utilized. The sequences
obtained were aligned with the program MAFFT v.6 [2] through the interactive strategy G-INS-i.
Phylogenetic calculations were performed in PAUP using a variant of the neighbor-joining method
called Bio-Neighbor-Joining. The genetic tree was rooted with two sequences of the bacteria
Leptospirillum ferriphilum (Genbank AF356830) and Leptospirillum ferrooxidans (Genbank
AF356834).
Analytical determinations. The ferrous ion was determined by the Muir method based on the
formation of a colored complex between Fe2+ and 1.10-fenantrolyne and measured at 510 nm. The
total iron was measured by reduction of Fe3+ with hydroxylamine hydrochloride by measuring the
resulting Fe2+. The sulfate ion in solution was determined by turbidimetric method, by precipitation
in an acid medium and barium chloride. The mixture produced uniform barium sulfate crystals,
measured at 520 nm.
Biooxidation Assays. The batch biooxidation was conducted in 250 ml flasks at various pulp
densities (5-10-15-20-25% w / v). All assays were performed with a particle size of 74 µm for the
mineral. A Norris medium was modified with 2 g. L-1 for Fe2+ at initial pH 1.8 and inoculated at 10
% v/v. The vials were incubated at 220 rpm and 30 ºC.
Results and Discussion
Mineralogical Composition of Mineral Concentrate. The concentrate obtained by gravimetry and
milling was 15 % of the weight of the total sample with a D80 of 74 µm particle size. The presence
of copper sulfides such as chalcopyrite (1.88 %), chalcocite (0.01 %), covellite (0.06 %), bornite
(0.01 %), and gray copper type tenantita (1.10 %) were observed. The pyrite content reached 66.63
%, 0.59 % of pyrrhotite, galena 0.71 % and 4.77 % sphalerite. The presence of the oxidized iron
limonite (1.96 %) was also detected. These results are characteristic of gold and silver deposits
composed of copper, lead, and zinc based metals. The significant presence of pyrite and other
sulfides ensured that the sources of typical energy of microorganisms (iron(II) and reduced sulfur
compounds) were available. The high amount of pyrite also suggested that the encapsulation of the
refractory gold could largely be found in the pyrite and its dissolution by iron-oxidizing
microorganisms that use the mechanism by thiosulphate [3], could contribute to the release of the
microcrystal of gold and increase the dissolution during the process of cyanidation. The presence of
heavy metals, which could partially or totally inhibit microbial activity, was also a significant
reason to use native microorganisms adapted to the mineral.
Enrichment and physiological characterization. During the enrichment experiments and
physiological analyses of the isolated cultures using iron (II) as energy source, it was reached a
3. Advanced Materials Research Vols. 71-73 483
constant µm of 0,131 h-1 (characteristic of Acidithiobacillus ferrooxidans) with a productivity of iron
(III) of 0,298 g/h and a doubling time of 5.3 h. Figure 1 (a and b) shows typical kinetics of growth
and consumption of iron obtained in these trials.
a b c
Fig.1. (a and b) Kinetics of cell growth, consumption of Fe2+ and generation of Fe3+ of bacterial
consortium in axenic Norris medium. (c) Concentration of Fe2+, Fe3+ and sulfate during adaptation
phase of the Portovelo consortium in Norris medium with 2% [w/v] mineral density pulp.
Fig. 1c shows the iron and sulfate concentrations in solution during the adaptation phase to the
mineral. The iron(III) and sulfate concentrations reached maximum values of 8 and 37 g/l
respectively. Iron (III) and sulfate productivities were 0.27 g/d and 0.93 g/d respectively during this
phase. A high consumption of protons was observed, probably due to the presence of minerals such
as calcite and sulfide, which are relatively soluble in an acid medium. This led to controlling the pH
at 1.8 to prevent precipitation of jarosite [4].
16S rR A sequencing. DNA extraction was performed L P P
in the exponential phase of culture (approximately 2x
108 cells/ml). Extractions were performed with
universal primers generated by a fragment of
approximately 1500 bp of the 16S rRNA region and
were subsequently sequenced. Image 1 shows the
results of the amplification of PCR products in agarose
gel to 0.7. Comparing these with the sequences
available in GenBank showed a high degree of
Image1. PCR products of 1500bp in Agarose Gel l
similarity with sequences of Acidithiobacillus. The
0.7%. Issues of amplification of the region 16S
phylogenetic analysis was conducted using Bio- rDNA. (L) 1 Kb DNA Ladder; (P) Portovelo; (B)
Neighbor-Joining. The strain was grouped within a Blank.
clade (89% confidence in bootstrap analysis) with
strains of A. ferrooxidans (data not shown).
Biooxidation ores. Fig. 2 shows the evolution of some of the measured parameters (pH and
concentrations of iron(II), total iron and soluble sulfate) in the biooxidation experiments of the
concentrates at different pulp densities. Iron(II) concentration decreased in the first 25 days,
suggesting iron oxidizing microbial activity independent of pulp densities. Simultaneously there
was an increase in the total iron and sulfate concentrations, clear signs that pyrite was essentially
soluble. These results were relevant to both lower pulp densities, although final iron and sulfate
concentrations were greater for 10 % of pulp density (about 35 g/l of total iron and 60 g/l sulfates).
Extraction percentages were greatest for lower pulp density, almost 88 % of the total iron in the ore
which decreased to 31 % and 8 % for the pulp densities 10 % and 15 % respectively. For higher
pulp densities (20 % and 25 %) the iron extraction was not significant (1.9 % and 0.08 %
respectively) and similar to the values obtained for sterile systems. This sharp decrease in iron
extraction is probably due to the inhibition of microbial activity at high pulp densities. This
4. 484 Biohydrometallurgy 2009
phenomenon has been observed previously [5] except when successive adjustments or special
configurations for the reactors were conducted [6]. The iron and sulfate concentrations during the
experiments were similar to the expected stoichiometric relationship (1:2) for the first 20 days and
then increased probably due to the jarosite precipitation. At the end of the bioxidation, there was an
increase in the concentration of iron(II), especially in systems with lower density related to the
inhibition of iron(II) oxidation by A. ferrooxidans that occurs at pH lower than 1.3. This was
compatible with the values of pH found in these cultures, the pH drops due to the solubility of
pyrite, and the subsequent jarosite precipitation. The small initial rise in pH was possibly due to the
neutralization of basic species such as calcite and also to the metal sulfides (such as covellite,
chalcocite and sphalerite) diluted in acid. These metals were soluble in a wide range reaching
recoveries of 49%-96 for copper and 49%-48% for zinc.
Fig. 2. Results of bioleaching experiments at different pulp densities 5-10-15-20-25% [w/v]
Conclusions
Residual concentrates from the Portovelo area were efficiently leached with high levels of soluble
iron, copper and zinc by native strains that were characterized as A. ferrooxidans. However,
significant recoveries were made just at low pulp densities; therefore a microbial adaptation to high
pulp densities is necessary to allow the subsequent use of the biooxidation process for the recovery
of metals from residues. That process could be a suitable treatment to decrease the serious potential
risk of spreading contamination in certain areas around these sediments and mineral residues are
located.
Acknowledgments
We are particularly grateful to the BIORECA (CYTED) network for funding a grant at the School
of Biochemical Engineering at the Pontificia Universidad Católica in Valparaíso, Chile.
References
[1] T. Miyoshi, T. Iwatsuki and T. Naganuma: Appl. Environ. Microbiol. Vol. 71 (2005), p. 1084
[2] Information on http://align.bmr.kyushou-u.ac.jp/mafft/online/server/
[3] W. Sand, T. Gehrke, P.-G. Józsa and A. Schippers: Hydrometallurgy Vol. 59 (2001), p. 159
[4] F. Acevedo and J.C. Gentina: Bioprocess Eng. Vol. 4 (1989), p. 223
[5] P. Valencia and F. Acevedo: W. J. Microbiol. Biotechnol. Vol. 25 (2009), p. 101
[6] C. Astudillo and F. Acevedo: Hydrometallurgy Vol. 92 (2008), p. 11
5. Advanced Materials Research Vols. 71-73 485
Biohydrometallurgy 2009
doi:10.4028/www.scientific.net/AMR.71-73
Mineralogical Characterization of a Polymetallic Concentrate Portovelo Mining
District. Bioleaching by a Native Bacterial Consortium
doi:10.4028/www.scientific.net/AMR.71-73.481
References
[1] T. Miyoshi, T. Iwatsuki and T. Naganuma: Appl. Environ. Microbiol. Vol. 71 (2005), p.
1084
doi:10.1128/AEM.71.2.1084-1088.2005
PMid:15691970 PMCid:546738
[2] Information on http://align.bmr.kyushou-u.ac.jp/mafft/online/server/
[3] W. Sand, T. Gehrke, P.-G. Józsa and A. Schippers: Hydrometallurgy Vol. 59 (2001), p.
159
doi:10.1016/S0304-386X(00)00180-8
[4] F. Acevedo and J.C. Gentina: Bioprocess Eng. Vol. 4 (1989), p. 223
doi:10.1007/BF00369176
[5] P. Valencia and F. Acevedo: W. J. Microbiol. Biotechnol. Vol. 25 (2009), p. 101
doi:10.1007/s11274-008-9866-4
[6] C. Astudillo and F. Acevedo: Hydrometallurgy Vol. 92 (2008), p. 11
![482 Biohydrometallurgy 2009
Enrichment of microbial consortia. The liquid samples taken in areas of weathered mining shafts
were cultured in 250 ml flasks containing 90 ml of the medium Norris [3] and 10 % v/v of the
culture to pH 1.8. The specific growth rate was determined the oxidation, performance, and
productivity of Fe2. When the bacterial culture reached a constant of µm, it was replicated in a
medium of Norris modified with 2 g.L-1 for Fe2+ and 2% w/v ore. In all cases the flasks were
incubated at 220 rpm and 30 °C.
16S rR A Sequencing.
For DNA extraction Wizard ® Genomic DNA Purification Kit was used. The amplification was
done by PCR, using a combination of universal bacterial primers 27F (5'-
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5-GGTTACCTTGTTACGACTT-3) [1]. The
products were purified and sequenced by Macrogen in Seoul, Korea. The experiment was
conducted with the program Sequencher 4.6 Gene Codes, in Ann Arbor, MI. To identify published
sequences similar to the bacterial 16S rRNA, the NCBI BLAST was utilized. The sequences
obtained were aligned with the program MAFFT v.6 [2] through the interactive strategy G-INS-i.
Phylogenetic calculations were performed in PAUP using a variant of the neighbor-joining method
called Bio-Neighbor-Joining. The genetic tree was rooted with two sequences of the bacteria
Leptospirillum ferriphilum (Genbank AF356830) and Leptospirillum ferrooxidans (Genbank
AF356834).
Analytical determinations. The ferrous ion was determined by the Muir method based on the
formation of a colored complex between Fe2+ and 1.10-fenantrolyne and measured at 510 nm. The
total iron was measured by reduction of Fe3+ with hydroxylamine hydrochloride by measuring the
resulting Fe2+. The sulfate ion in solution was determined by turbidimetric method, by precipitation
in an acid medium and barium chloride. The mixture produced uniform barium sulfate crystals,
measured at 520 nm.
Biooxidation Assays. The batch biooxidation was conducted in 250 ml flasks at various pulp
densities (5-10-15-20-25% w / v). All assays were performed with a particle size of 74 µm for the
mineral. A Norris medium was modified with 2 g. L-1 for Fe2+ at initial pH 1.8 and inoculated at 10
% v/v. The vials were incubated at 220 rpm and 30 ºC.
Results and Discussion
Mineralogical Composition of Mineral Concentrate. The concentrate obtained by gravimetry and
milling was 15 % of the weight of the total sample with a D80 of 74 µm particle size. The presence
of copper sulfides such as chalcopyrite (1.88 %), chalcocite (0.01 %), covellite (0.06 %), bornite
(0.01 %), and gray copper type tenantita (1.10 %) were observed. The pyrite content reached 66.63
%, 0.59 % of pyrrhotite, galena 0.71 % and 4.77 % sphalerite. The presence of the oxidized iron
limonite (1.96 %) was also detected. These results are characteristic of gold and silver deposits
composed of copper, lead, and zinc based metals. The significant presence of pyrite and other
sulfides ensured that the sources of typical energy of microorganisms (iron(II) and reduced sulfur
compounds) were available. The high amount of pyrite also suggested that the encapsulation of the
refractory gold could largely be found in the pyrite and its dissolution by iron-oxidizing
microorganisms that use the mechanism by thiosulphate [3], could contribute to the release of the
microcrystal of gold and increase the dissolution during the process of cyanidation. The presence of
heavy metals, which could partially or totally inhibit microbial activity, was also a significant
reason to use native microorganisms adapted to the mineral.
Enrichment and physiological characterization. During the enrichment experiments and
physiological analyses of the isolated cultures using iron (II) as energy source, it was reached a](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)