Advanced Materials Research Vols. 71-73 (2009) pp 481-484
online at
© (2009) Trans Tech Publicat...
482                                Biohydrometallurgy 2009

Enrichment of microbial consortia. The liquid samples taken...
Advanced Materials Research Vols. 71-73                                        483

constant µm of 0,131 h-1 (characteri...
484                                 Biohydrometallurgy 2009

phenomenon has been observed previously [5] except when suc...
Advanced Materials Research Vols. 71-73                         485

Biohydrometallurgy 2009
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  1. 1. Advanced Materials Research Vols. 71-73 (2009) pp 481-484 online at © (2009) Trans Tech Publications, Switzerland Online available since 2009/May/19 Mineralogical characterization of a polymetallic concentrate Portovelo mining district. Bioleaching by a native bacterial consortium F. Gordillo 1,a, J.P. Suarez 1,b, V. Sanmartín 1,c , P. Aguirre1,d , J.C. Gentina 2,e and E. Donati 3,f 1. Centro de Biología Celular y Molecular, UTPL, Loja, Ecuador 2. Escuela de Ingeniería Bioquímica, PUCV, Valparaíso, Chile 3. CINDEFI (CONICET, UNLP), Facultad de Ciencias Exactas, La Plata, Argentina a,,, d,, Keywords: mineralogy, polymetallic tails, A. ferrooxidans, biooxidation, 16S rRNA Abstract. The mining district of Zaruma-Portovelo of southern Ecuador is a deposit of polymetallic sulfides worked since 1908. Currently the small traditional mining operation processes 1800 tons of ore a day, generating environmental waste that is accumulated and systematically discharged into the Calera and Amarillo Rivers. In this watershed area of Puyango-Tumbes, where samples were taken and subjected to gravimetric concentration, a mineralogical analysis of the concentrate was made. Samples of sediments taken from areas of weathered mining shafts were used to isolate native microorganisms for subsequent molecular and physiological characterization. The mineral concentrate contains 66.63 % of pyrite, 175 ppm Ag and 6.9 ppm Au (with 80 % of refractory gold). This mineral was subjected to biooxidation by the isolated native organisms. In experiments with pulp density of 10%, the solubility of the sulfides was very significant, reaching concentrations of Fe3 + 30 g/L and sulfate 60 g/L. Introduction The use of inefficient technologies for leaching gold does not allow traditional mining to reach more than 70 % recovery of the metal. These product residuals generate approximately 600,000 tons of ore per year (still with high grade gold) which are stored in an incipient form on the banks of the rivers, directly affecting the Puyango-Tumbes watersheds of Ecuador and Peru. These residuals that are a potential source of contamination must be treated to recover metals that are present and to allow for their proper final disposal. Bioleaching of concentrates from these residuals using native microorganisms (usually adapted to the mineral species, and therefore often more efficient for the solubility of these minerals) could provide a technology suitable to address this serious environmental problem. For this reason, a gravimetric concentration analysis of a representative sample from the wastes was conducted, the mineralogical characterization of the concentrate was conducted, and physiological and molecular analyses of native microorganisms were isolated in order to later utilize them in biooxidation under different conditions. Materials and Methods Chemical and mineralogical analysis. A representative sample of the residuals was taken and processed in a gravimetric concentration table. The concentrate was milled until it reached a particle size of 74µm. By fire assay, acid disintegration of metal bead and readings by atomic absorption spectrophotometry were determined Au (6.9 ppm), Ag (175 ppm), Hg (2 ppm), Pb (0.6 %), Zn (3.2 %), S (36.4 %), Cu (1.3 %), As (0.2 %), and total Fe (33.4 %). The mineralogical composition was determined in refined sections using an optical microscope of 12.5 x with a lens of 16x network metrics with 400 points attached to the eye piece. The weight percentage of mineral grains was calculated using the specific gravity of each one and was then extrapolated with the minimal weight until they reached the values given by chemical analysis. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, (ID:,20:04:07)
  2. 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. 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. 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 [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. 5. Advanced Materials Research Vols. 71-73 485 Biohydrometallurgy 2009 doi:10.4028/ Mineralogical Characterization of a Polymetallic Concentrate Portovelo Mining District. Bioleaching by a Native Bacterial Consortium doi:10.4028/ 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 [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