WO2019119166A1 - Method for bioleaching sulfur-containing copper minerals using a consortium of microorganisms comprising iron-oxidising bacteria and the fungus acidomyces acidophilus he17 in an inorganic medium at a ph of less than 2, promoting bacterial growth and increasing extraction of the metal from the mineral - Google Patents

Method for bioleaching sulfur-containing copper minerals using a consortium of microorganisms comprising iron-oxidising bacteria and the fungus acidomyces acidophilus he17 in an inorganic medium at a ph of less than 2, promoting bacterial growth and increasing extraction of the metal from the mineral Download PDF

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WO2019119166A1
WO2019119166A1 PCT/CL2018/050134 CL2018050134W WO2019119166A1 WO 2019119166 A1 WO2019119166 A1 WO 2019119166A1 CL 2018050134 W CL2018050134 W CL 2018050134W WO 2019119166 A1 WO2019119166 A1 WO 2019119166A1
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bioleaching
acidophilus
bacteria
fungus
medium
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Spanish (es)
French (fr)
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Mario ESPARZA MANTILLA
Ronald Eleazar HUARACHI OLIVERA
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Universidad De Antofagasta
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    • C—CHEMISTRY; METALLURGY
    • C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
    • C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
    • C22B15/00—Obtaining copper
    • C—CHEMISTRY; METALLURGY
    • C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
    • C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
    • C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
    • C22B3/18—Extraction of metal compounds from ores or concentrates by wet processes with the aid of microorganisms or enzymes, e.g. bacteria or algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P10/00—Technologies related to metal processing
    • Y02P10/20—Recycling

Definitions

  • the present invention relates to the area of mining.
  • a method for the bioleaching of minerals Even more particularly, to a method for bio-lixivizing sulfur-containing minerals from a consortium of microorganisms comprising ferrooxidizing bacteria - access number of the Regional Microbial Genetic Resources Bank of Research Qullamapu, Chillán, Chile, RGM 2527 and the fungus Acidomyces acidophilus HE17 - Access number of the Regional Microbial Genetic Resources Bank of Research Qullamapu, Chillán, Chile, RGM 2451, in an inorganic medium free of ferrous sulphate and pH ⁇ 2, favoring bacterial growth and increasing the extraction of the metal from the ore.
  • Bioleaching is the process by which iron and sulfur oxidizing microorganisms catalyze the extraction of metals from minerals and industrial waste (Krebs).
  • bioleaching Compared to chemical extraction processes, bioleaching has the advantage of being friendly to the environment; besides having a low implementation cost. However, it is not without problems, since during the process an inhibition of microbial growth can occur due to the presence of chlorine, heavy metals, metalloids and dissolved organic matter (MOD). This results in a low recovery of the metal of interest (Hansford, GS 1997. Recent developments in modeling the kinetics of bioleaching, In: Rawlings, DE (Ed.), Biomining: Theory, Microbes and Industrial Processes.) Springer Verlag, Berlin, pp 153-175; Schrenk, MO, Edwards, KJ, Goodman, RM, Hamers, RJ, Banfield, JF 1998.
  • Heterotrophic microorganism f ⁇ hodotorula mucilaginosa R30 improves tannery sludge bioleaching through elevating dissolved C0 2 and extracellular polymeric substances in bioleach solution as well as scavenging toxic DOM to Acidithiobacillus species, Water fies 44, 5423-5431, Zheng, G., Zhou, L., Wang, S. 2009. An acid tolerant heterotrophic microorganism role in improv ing tannery sludge bioleaching conducted in successive multibatch reaction systems. Environ. Sci. Technol. 43, 4151-4156; Zheng, G., Wang, Z., Wang, D., Zhou, L. 2016.
  • CN106190871 (A) which describes a method for bioleaching in soil contaminated with heavy metals through filamentous fungi compound taking straw as carbon source.
  • the filamentous fungus is classified and named as Penicillium simplicissiumum and is kept at the General Center for Collection of Microbiological Crops of China on June 25, 2015 with the accession number CGMCC No.10990.
  • the invention further describes a composite filamentous fungal inoculant formed by the isometric spore mixture of Penicillium simplicissiumum NAU-12 (1 x 10 8 spores / mL at 2 x 10 8 spores / mL) and Aspergillus niger A80 (1 x 10 7 spores). / mL at 2 x 10 7 spores / mL).
  • the invention further describes a method for carrying out the bioleaching treatment in soils contaminated with heavy metals through compound filamentous fungi that grow rapidly in the soil, high in resistance to heavy metals and capable of using straw subjected to alkaline pretreatment with heat. alkaline as a carbon source for growth and have a high production of multiple small molecular organic acids. The method is adopted to treat soil contaminated with heavy metals, the removal efficiency of heavy metals is high and the cost is low.
  • TW201429883 (A) describes a method of bioleaching metals and their system using fungi for sludge.
  • the metal bioleaching system includes a bioleaching unit, a mud supply unit and a supply unit mushroom.
  • the bioleaching unit is provided to process a metal bioleaching treatment.
  • the mud supply unit is provided to supply sludge containing metals.
  • the fungal supply unit is provided to supply fungi to the bioleaching unit and then the fungi generate a metabolic material. The metabolic material is applied to leach the metals contained in the mud to remove the metals from the sludge.
  • AU2010288177 (B2) describes an additive for the bioleaching that makes it possible to increase the recovery of copper from sulfide minerals.
  • this additive is substantially constituted by the lipoprotein Llcanantase and a solution of sulfuric acid with a pH of 0.8 to 3.
  • the lipoprotein of Llcanantase having an amino acid sequence with at least 50% homology with respect to the sequence defined in the sequence SEQ ID No. 1 or is the translation product of a nucleotide sequence with at least 50% homology with respect to the sequence defined in the sequence SEQ ID NO. It also protects the improved bioleaching process that includes adding the additive during the mineral bioleaching process and continuing with the usual process, obtaining copper recoveries increased from 5 to 20%.
  • Llcanantasa is the predominant protein in the secretome of Acidithiobacillus thiooxidans when it is cultivated with elemental sulfur and is capable of increasing copper recovery from chalcopyrite when used as an additive for bioleaching.
  • the present invention describes the isolate and characterize the fungus Acidomyces acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chlllán, Chile, RGM 2451, obtained from acid mine drainage, and evaluate its
  • RECTIFIED LEAVES (RULE 91) addition to a bacterial consortium to improve the bioleaching process.
  • a bacterial consortium that may or may not include the fungus A. acidophilus HE17- Access number of the Genetic Resources Bank Regional Microbial Research Quilamapu, Chlllán, Chile, RGM 2451, demonstrates the importance of adding this fungus in the growth of leaching bacteria and the result of the recovery of copper from copper sulfide ores.
  • the present invention makes it possible to overcome the aforementioned difficulties by including the process of leaching sulfur minerals, the fungus Acidomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank Quilamapu Research, Chlllán, Chile, RGM 2451 as an additive of natural origin
  • This additive allows to reduce the bioleaching times of sulphided minerals to months or even weeks, and increases microbial cell growth by more than 20%, allowing an increase of more than 10%, in the recovery of copper from sulphide minerals, in particular, from solutions, whether they come from batteries, reactors, concentrates or other equipment or operations necessary for the extraction of the mineral.
  • the aforementioned value is an estimated value for recovery with the additive.
  • the present invention provides a method for bio-lixivizing copper sulphide minerals using a consortium of microorganisms comprising ferroxidant bacteria - access number of the Regional Microbial Genetic Resource Bank Quilamapu, Chlllán, Chile, RGM 2527 and Acidomyces acidophilus fungus HE17 - number of access of the Regional Microbial Genetic Resources Research Bank Quilamapu, Chlllán, Chile, RGM 2451 in a
  • Rhodotorula mucilaginosa R30 the activity of the fungus Rhodotorula mucilaginosa R30 has been reported, which allowed the increase of the bacterial biomass of Acidithiobacillus ferrooxidans in sludge with a high concentration of organic matter (Wang, S., Zheng, G., & Zhou, L. 2010. Heterotrophlc mlcroorganlsm Rhodotorula mucilaginosa R30 mproves tannery sludge bioleachlng through elevating dissolved C02 and extracellular polymerlc levels in bioleach solution as well as scavenglng toxic DOM to Acidithiobacillus species, Water Res. 44, 5423-5431). A bacterial consortium that adds A. acidophilus for bioleaching purposes has not been reported.
  • the method of the present invention is conducted in inorganic media at low pH ( ⁇ 2), and does not refer to bioleaching in sludge with heavy metals with a high concentration of dissolved organic matter using fungi and bacteria. It has been studied that the inoculation of the fungus Gaiactomyces sp. Z3 improves the bioleaching processes in sludge with heavy metals when two strains Acidithiobacillus ferrooxidans LX5 and Acidithiobacillus thiooxidans TS6 are used, decreasing the period required for the process, with greater efficiencies in the solubilization of copper (Zhou, J., Zheng, G., Wong, JW, & Zhou, L. 2013.
  • bioleaching processes with filamentous fungi in a static system in inorganic medium 9K without ferrous sulphate, most of the work in bioleaching with fungi was done in organic media at neutral pH in agitation systems (e e ⁇ b ⁇ like the research carried out by Mehta, KD, Das, C., Pandey, BD 2010.
  • s of a contaminated soil in an industrial area uses metabolites, mainly weak organic acids, produced by the fungus Aspergillus niger obtaining a recovery of 56% copper in the agitation system at 120 rpm at 30 e C at regular time intervals of 15 days .
  • metabolites mainly weak organic acids
  • the fungus Aspergillus niger obtaining a recovery of 56% copper in the agitation system at 120 rpm at 30 e C at regular time intervals of 15 days .
  • the present invention demonstrates that the fungus A. acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank of Research Quilamapu, Chillón, Chile, RGM 2451, exhibits mlxotroph and acidotolerant activity with bacteria of the genus Acidithiobacillus and Acidiphilium.
  • the fungus is able to favor bacterial growth and increase the recovery of copper from chalcopyrite, both in BE and in BA. Therefore, A.
  • the biolixivlation method is carried out with a consortium of microorganisms that also comprises the fungus A. acidophilus HE17 - Access number of the Regional Microbial Genetic Resources Bank of
  • RGM 2451 a bacterium selected from bacteria of the genus Acidithiobacillus, Acidiphilium or a combination thereof.
  • the present invention provides a way to isolate and characterize extremoacidiophilic fungi for its application in biolixivlation of copper sulphide minerals. First fungi are isolated and
  • RECTIFIED LEAVES (RULE 91) acidophilus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Quilamapu, Chillán, Chile, RGM 2451, enhances the process of bioleaching of chalcopyrite, compared to bacterial bioleaching, being the first research that describes the usefulness of a fungus filamentous acldotolerante in the bioleaching of sulfurized minerals in Inorganic medium. Studies were carried out with A. acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, to confirm viability, development and improvements in metal extraction processes.
  • the present invention proposes the use of Acidomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, as a bioleaching additive since it is capable of growing in an industrial mining ecosystem that has of an inorganic environment, which gives it a very relevant advantage in addition to the fact that it allows the fastest growth of leaching bacteria - access number of the Regional Microbial Genetic Resources Bank of Research Quilamapu, Chillán, Chile, RGM 2527, those that oxidize quickly Iron or sulfur, and in
  • RECTIFIED LEAVES (RULE 91) consequently, they allow to recover more copper in less time and in greater quantity with respect to bacterial systems that do not use this fungus.
  • Figures 1A and 1B Growth of the fungal strain in inorganic and organic culture medium.
  • Figure 1 A Growth of the fungal strain in Sabouraud organic solid medium and PDA at pH 6.49.
  • Figure 1 B Growth of the fungal strain in inorganic solid 9K-Fe medium pH 1.8; in the presence and absence of iron oxidizing bacteria.
  • Figures 2A-2M Morphology of the fungal strain.
  • Figure 2A Fungal colony in solid 9K-Fe medium, pH 1.8 after 28 days of incubation at room temperature. A halo of degradation surrounding the colony is observed.
  • Figure 2E F ⁇ alo of degradation observed in confocal microscopy. The presence of iron oxidizing bacteria is appreciated.
  • Figure 2B Fungal colony in solid 9K-Fe pH 1, 8 medium after 30 days of incubation at room temperature.
  • Figure 2C and Figure 2D Fungal colony in Sabouraud agar medium and PDA pH 6.49 after 17 days.
  • FIG. 1 Microscopic view (100X) of septate filaments of A. acidophilus, with methylene blue staining. Swollen intercalary and terminal cells (red arrows) are observed.
  • Figure 2J Microscopic view (100X) of the septate hyphae of A. acidophilus, with lugol staining.
  • Figure 2K Filaments of A. acidophilus HE17 from cultures in liquid medium 9K-Fe, pH 1.8 in observed confocal microscopy.
  • Figure 3 Phylogenetic analysis of Acidomyces acidophilus HE17 based on the sequences of the ITS region of the 5.8S rDNA and the D1 / D2 domains of the 28S rDNA. The phylogeny was reconstructed using parsimony Bootstrap, and the tree was constructed using maximum likelihood. The scale bar indicates 0.5 substitutions per site. Teratosphaeria micromaculata was used as an external root group.
  • FIGS 5A-5G Architecture of the biofilms of Acidomyces acidophilus HE17 in the presence of copper.
  • the fungus was grown in liquid medium 9K-Fe pH 1, 8 supplemented with 0 mM (negative control), 200 mM and 400 mM
  • FIGS 5A, 5B and 5C Photographs showing the biofilms of A. acidophilus in liquid medium 9K-Fe pH 1.8; to the different concentrations of CuS0 4 x5H 2 0.
  • Figures 5D, 5E and 5F bi-dimensional image of filaments biofilms of A. acidophilus.
  • Figures 5G, 5H and 5I three-dimensional image of filament biofilms of A. Acidophilus
  • FIGs 6A-6H Experiments of static bioleaching (BE) and bioleaching in airlift bioreactor (BA). An iron oxidizing bacterial community was used with or without the incorporation of the fungus Addomyces acidophilus HE17, and recovery of copper from chalcopyrite was evaluated.
  • Figure 6A Growth curve of iron oxidizing bacteria in BA.
  • Figure 6B Growth curve of iron oxidizing bacteria in BE.
  • Figure 6C Redox potential (ORP) in BE.
  • Figure 6D ORP in BA.
  • Figure 6E Iron production in BE.
  • Figure 6F Production of iron in BA.
  • T1 negative control
  • medium 9K + chalcopyrite T2, medium 9K + chalcopyrite + bacteria
  • Figures 7A and 7B Characterization of the bacterial consortium present in the experiments of static bioleaching and bioleaching in airlift bioreactor, by means of analysis of the 16S rDNA.
  • Figure 7A DGGE Gel 30-60% urea-formamide, in which the fragments of the 16S rDNA amplified were separated by PCR. The gel was stained with GeIRed. The bands were designated C1, C2 and C3.
  • Figure 7B Phylogenetic tree based on the three 16S rDNA sequences obtained from DGGE analysis. The tree was built by Maximum Likelihood analysis. The values of Bootstrap> 50% (100 replicas used) are shown. The scale bar indicates 0.01 substitutions per site. Acidiphilium cryptum JF-5 was used as an external root group. Detailed description of the invention
  • the bioleaching of copper and other metals usually uses oxidizing iron and sulfur bacteria, whose growth can be seen inhibited by the presence of heavy metals, dissolved organic matter, and others. This affects the recovery efficiency of the metal of interest. It has been seen that in blorremedlaclon in organic medium certain species of fungi contribute to improve the efficiency of recovery of metals and to counteract the inhibition of bacterial growth. However, no studies are reported on the process of bioleaching with fungi in Inorganic media.
  • the present invention provides as an additive to bioleaching, a filamentous fungus from an acid mine drainage.
  • the present invention demonstrates the utility of an aforementioned acidotolerant filamentous fungus in the bioleaching of sulfurized minerals in inorganic medium.
  • the bacteria are preferably selected from bacteria of the genus Acidithiobacillus, Acidiphilium or a combination thereof.
  • said bacteria are selected from the group consisting of Acidithiobacillus ferridurans strain YNTR1 -41, Acidiphilium sp. strain MPLK-613, Acidithiobacillus ferrooxidans strain HBDY3-5, or a combination thereof.
  • Example 1 Methodology to isolate the fungal strain and the bacterial consortium
  • the fungal strain was isolated from a microbial culture from an acid mine drainage. To isolate the fungal strain and characterize its growth, it was cultivated in four different culture media, at room temperature: First Culture Medium, culture (I): liquid 9K medium supplemented with FeS0 4 (9K-Fe, 0.4 g MgS0 4 x7H 2 0, 0.04 g K 2 HP0 4 , 0.1 g NH 4 S0 4 , 33.33 g FeS0 4 x 7H 2 0 per liter, pH 1, 8). Second Culture Medium, culture (I): Solid 9K-Fe (10 g NH 4 S0 4 ; 1.5 g MgSO 4 x 7H 2 0; 0.5 g
  • the bacterial community to be used in the bioleaching experiments was obtained from a sample of chalcopyrite from a mine, which was inoculated (10%, 10 6 cel / ml) in liquid 9K medium without the addition of FeS0 4 * 7H 2 0, pH 1, 8, Temperature 20 ⁇ 2 e C. Bacterial growth was evaluated every 7 days by cell counting in a Petroff-Hausser chamber.
  • Example 2 Composition of the mineral used
  • the ore was pulverized to a particle size of 38 to 150 ⁇ m, using a spray (Rocklabs, Auckland, New Zealand) with sieve N e 100 and 400, sterilizing it before experimentation.
  • the mineralogical analysis was carried out with a Siemens D5000 X-ray diffractometer (Siemens, Munich, Germany), whereby the mineral was found to correspond to copper sulphide, the following content being determined: 80.18% chalcopyrite (CuFeS); 4.52% trolita (FeS); 6.61% covelite (CuS); 1, 15% magnetite (Fe 2 0 3 ); and 7.54% quartz (Si0 2 ).
  • Example 3 Extraction of genomic DNA and amplification of bacterial and fungal ribosomal DNA
  • the 16S ribosomal DNA was amplified using the universal oligonucleotides 1492R / 27F and the GoTaq® Green Master Mix PCR kit (Promega, Madison, Wl, USA).
  • the amplification program was as follows: initial denaturation at 94 ° C for 5 min, followed by 35 cycles at 94 ° C for 45 s, 57 ° C for 45 s, and 72 ° C for 1 min 30 s; with a final extension at 72 e C for 5 min.
  • the region of the internal transcribed spacer (ITS) of the 5.8S rDNA was amplified, using the oligonucleotides ITS1 and ITS4 (White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: MA lnnis, DH Gelfand, JJ Sninsky, & TJ White (ed.), PCR Protocols: A Guide to the Methods and Applications, New York: Academic Press, 315-322); and the variable D1-D2 domains of 28S rDNA, using oligonucleotides NL1 and NL4 (O'Donnell K. 1993.
  • ITS1 and ITS4 White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: MA lnnis, DH Gelfand, JJ Sninsky,
  • the amplification reaction contained 13.2 pL of milliQ water; 5 pL Buffer PCR; 2 pL MgCl 2 ; 0.5 pL of dNTPs (Kapa Biosystems, Wilmington, MA, USA), 1 pL of each oligonucleotide; 0.25 pL GoTaq DNA Polymerase kit reagent (Promega / USA) and 2 pL tempering.
  • the amplification program was as follows: initial denaturation at 94 ° C for 5 min, followed by 35 cycles at 95 C for 30 s and 55 ° C for 1 min, and 72 ° C for 1 min.
  • the PCR products were visualized on a 1% agarose gel, and were purified with the GFX PCR DNA and Gel Band Purification Kit (General Electric Healthcare, Buckinghamshire, UK). They were stored at -20 ° C. Gel electrophoresis with denaturing gradient (DGGE).
  • the amplified fragments of the 16S rDNA were used as annealed in a new amplification with the specific oligonucleotides for bacteria 341 F-GC and 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments Arch. Microbiol. 164, 165-171).
  • the following program was used: an initial denaturation of 94 ° C for 5 min, followed by 35 cycles at 94 ° C for 30 s, 50 ° C for 45 s, and 72 ° C for 1 min, with a final extension of 72 ° C for 3 min.
  • the resulting PCR products were subjected to DGGE, based on the protocol described by Demergasso, C., Galleguillos, P., Escudero, L., Zepeda, A., Castillo, D., Casamayor, E. 2005.
  • Molecular characterization of microbial populations in a low-grade copper ore bioleaching test heap Hydrometallurgy. 80, 241-253.
  • the products were loaded on a 6% polyacrylamide gel containing a denaturing gradient of 30-60% urea-formamide (100% of the gradient was defined as 7 M urea and 40% formamide).
  • the gel was run in a BioRad D Gene electrophoresis chamber (BioRad, Hercules, CA, USA) at 60 ° C and 100 V for 7 h.
  • the gel was stained in a 0.5% GeIRed solution (Biotium, Fremont, CA, USA) for 1 h in darkness, and the bands were visualized under UV light in transilluminator.
  • the bands were cut and purified using the E.Z.N.A.TM. Gel Extraction Kit (OMEGA), to then re-amplify the PCR products with the 341 F-GC / 907R primers, under the same conditions of the aforementioned PCR, visualizing in agarose gel the
  • the bacterial PCR products were sequenced by Macrogen, Korea.
  • the sequences were manually edited using the ChromasPro 2.1 program (Technelysium Pty Ltd, Brisbane, Australia), and compared with the GenBank database (http://blast.ncbi.nlm.nih.gov), using the BLASTn algorithm ( - Altschul, SF, Gish, W., Miller, W., Myers, EW 1990. Basic local alignment search tool, J. Mol. Biol. 215, 403-410). Subsequently, the sequences were aligned using the MUSCLE tool (- Edgar, R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32, 1792-1797).
  • the phylogenetic tree was constructed using the statistical model Maximum Likelihood and the Bootstrap robustness test, using the program MEGA7 (Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 201 1.
  • MEGA5 Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol. 28, 2731 -2739).
  • the fungal PCR products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit's robust, and the ABI PRISM 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The same oligonucleotides used in the initial amplification of the 5.8S and 18S rDNAs were used. Consensus sequences were obtained using the AutoAssembler programs (Thermo Fisher Scientific, Waltham, MA, USA) and Lasergene Seqman (Dnastar, Madison, Wl, USA).
  • thermoacidophilic archaeon Sulfolobus metallicus possible role of polyphosphate metabolism.
  • Thiobacillus ferrooxidans response to copper and other heavy metais growth, protein synthesis and protein phosphorylation, Antonie van Leeuwenhoek, 77, 187-95).
  • the inhibitory effect of copper on the growth of the fungus was determined by measuring the diameter of the colony (in solid medium), and quantifying the difference of dry mass at the beginning and end of the experiment (in liquid medium).
  • Example 5 Methodology Experiments of Static Bioleaching (BE) and Bioleaching in Airlift Bioreactor (BA).
  • Static bioleaching was carried out in 250 mL Erlenmeyer flasks containing 100 mL of 9K saline medium without FeS0 4 x7H 2 0 and chalcopyrite concentrate at a pulp density of 4%.
  • Bioleaching bioreactor airlift was performed in a concentric tube 15 cm high, with a lower diameter of 9.5 cm and an upper diameter of 15 cm, which has an electromagnetic air compressor (model ACQ-001 , Boyu, China) with an air flow of 5 L / min (Fig. 1).
  • BA 1.8 L of 9K saline medium without FeS0 4 x7H 2 0 and the same pulp density as in BE were used.
  • T1 negative control
  • medium 9K + chalcopyrite T2, medium 9K + chalcopyrite + bacterial consortium
  • T2 and T3 an initial inoculum of 10 6 bacteria / mL was placed.
  • T3 and T4 an initial inoculum measured in equivalence to dry biomass of 0.025 mg / pL of A. acidophilus was placed.
  • ORP redox potential
  • Example 6 Methodology for the measurement of biofilms of A. acidophilus and visualization of its cellular structure.
  • biofilms of A. acidophilus was analyzed in cultures in liquid 9K-Fe-Cu medium with 0, 200 and 400 mM CuS0 4 x5H 2 0 and in the BA experiments (T3 and T4). In the latter, once the experiment was finished, the thickness of the biofilm of the fungus was measured and a sample of filaments was obtained. These were mounted on a Petri dish, collected with slides, stained with acridine orange, and observed in a confocal microscope. The filaments were also analyzed in a scanning electron microscope (7582, series A3038-5350-TV3-1841, England), for which they were mounted with Dako brand fixative.
  • the cellular structure of the fungus was studied from samples of culture medium 9K-Fe, PDA and Sabouraud, which were visualized in an inverted CS SP8 confocal microscope, with a compact Argon laser feeding unit at 488 nm and targets of 63X with immersion oil (Leica, Wetzlar, Germany).
  • Example 8 Morphological characterization of the fungal strain
  • Penidiella tenuiramis strain CBS124993 (KF442523.1) ( Figure 3).
  • Example 10 Growth and formation of biofilms of A. acidophilus HE17 in the presence of copper.
  • the diameter of the fungal colony was analyzed for 40 days. days of culture in solid medium 9K-Fe (negative control) and 9K-Fe-Cu, with 200 or 400 mM of CuS0 4 * 5H 2 0. The greatest growth of the fungus was in the negative control, with a diameter of 60 , 8 mm; while at 200 mM Cu the diameter was reduced to 33.8 mm; and 400 nM Cu was reduced to 20.5 mm (Fig. 4A).
  • T2 and T3 were analyzed at 68 days of experimentation.
  • the bacterial growth in T2 increased from 1.1 x 10 5 to 2.5 x 10 6 cells / mL; while in T3, a greater increase was observed, from 1.1 x 10 5 to 2.9 x 10 ® cells / mL (Fig. 6A, Fig. 6B).
  • Genomic DNA was extracted from the samples obtained from T2E, T3E, T2R, and T3R; and the 16S rDNA was amplified with the universal oligonucleotides 1492R / 27F.
  • the PCR products were re-amplified with the specific oligonucleotides for bacteria 341 F-GC and 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetics Relatlonships of Thiomicrosplra specles and thelr Identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresls of 16S rDNA fragments Arch. Microblol 164, 165-171).
  • the products resulting from the PCR were subjected to DGGE. Three bands were visualized for T2R and T3R, and four bands for T2E and T3E (Fig. 7A).
  • the bands were purified, the PCR products were re-amplified with the oligonucleotides 341 F-GC / 907R, and sequenced. Sequences of acceptable quality were obtained for three of the bands (C1, C2 and C3).
  • a fllogenetic tree was constructed from the sequences with the MEGA7 program, using sequences similar to ours, obtained from the database (Fig. 7B).
  • the analysis of the 16S rDNA sequences revealed the presence of two bacterial genera: Acidithiobacillus and Acidiphilium.
  • Table 1 shows the closest relative for each of the species identified by sequencing, corresponding to homologous fllotlpos to Acidithiobacillus ferridurans strain YNTR1 -41 (C3), Acidiphilium sp. strain MPLK-613 (C2) and Acidithiobacillus ferrooxidans strain HBDY3-51 (C1). It is interesting to note that A. ferridurans strain YNTR1 -41 was only found in BE experiments.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to the field of mining. Specifically, to a method for bioleaching minerals. Even more specifically, to a method for bioleaching sulfur-containing minerals using a consortium of microorganisms comprising iron-oxidising bacteria (Regional Microbial Genetic Resources Research Bank Quilamapu, Chillán, Chile, accession number RGM 2527) and the fungus Acidomyces acidophilus HE17 (Regional Microbial Genetic Resources Research Bank Quilamapu, Chillán, Chile, accession number RGM 2527) in a ferrous sulfate-free inorganic medium at pH < 2, promoting bacterial growth and improving extraction of the metal from the mineral.

Description

MÉTODO PARA BIOLIXIVIAR MINERALES SULFURADOS DE COBRE USANDO UN CONSORCIO DE MICROORGANISMOS QUE COMPRENDE BACTERIAS FERROOXIDANTES Y EL HONGO ACIDOMYCES ACIDOPHILUS HE17 EN UN MEDIO INORGÁNICO LIBRE DE SULFATO FERROSO Y PH MENOR QUE 2. FAVORECIENDO EL CRECIMIENTO BACTERIANO Y AUMENTANDO LA EXTRACCIÓN DEL METAL DESDE EL MINERAL.  METHOD FOR BIOLIXIVING COPPER SULFURATED MINERALS USING A MICROORGANISM CONSORTIUM COMPRISING FERROOXIDANT BACTERIA AND THE HONGO ACIDOMYCES ACIDOPHILUS HE17 IN A FERROUS SULFATE-FREE INORGANIC MEDIUM AND PH LOWER THAN 2. FAVORING BACTERIAL GROWTH AND INCREASING THE EXTRACTION OF METAL FROM THE MINERAL.
CAMPO DE LA INVENCION FIELD OF THE INVENTION
La presente invención se refiere al área de la minería. En particular, a un método para la biolixiviación de minerales. Aún más particularmente, a un método para biolixiviar minerales sulfurados a partir de un consorcio de microorganismos que comprende bacterias ferrooxldantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2527 y el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2451 , en un medio Inorgánico libre de sulfato ferroso y pH < 2, favoreciendo el crecimiento bacteriano y aumentando la extracción del metal desde el mineral.  The present invention relates to the area of mining. In particular, to a method for the bioleaching of minerals. Even more particularly, to a method for bio-lixivizing sulfur-containing minerals from a consortium of microorganisms comprising ferrooxidizing bacteria - access number of the Regional Microbial Genetic Resources Bank of Research Qullamapu, Chillán, Chile, RGM 2527 and the fungus Acidomyces acidophilus HE17 - Access number of the Regional Microbial Genetic Resources Bank of Research Qullamapu, Chillán, Chile, RGM 2451, in an inorganic medium free of ferrous sulphate and pH <2, favoring bacterial growth and increasing the extraction of the metal from the ore.
ANTECEDENTES BACKGROUND
Uno de los problemas que enfrenta la minería de sulfuras, en la actualidad, es que los procesos de biolixiviación microbiana en los minerales sulfurados tardan mucho tiempo Incluso años debido a que las bacterias crecen lentamente y su actividad metabóllca es muy débil. En ausencia de aditivos que aceleren el proceso de crecimiento bacteriano, y en particular, cuando ocurre la oxidación de Fe o S, y se favorece la lixiviación del mineral sulfurado. One of the problems facing sulphide mining today is that microbial bioleaching processes in sulphide minerals take a long time, even years because the bacteria grow slowly and their metabolic activity is very weak. In the absence of additives that accelerate the bacterial growth process, and in particular, when the oxidation of Fe or S occurs, and the leaching of the sulphide mineral is favored.
La biolixiviación es el proceso por el cual microorganismos oxidantes de hierro y azufre catalizan la extracción de metales a partir de minerales y residuos Industriales (Krebs Bioleaching is the process by which iron and sulfur oxidizing microorganisms catalyze the extraction of metals from minerals and industrial waste (Krebs).
HOJAS RECTIFICADAS (REGLA 91) W., Brombacher C., Bosshard P.P., Bachofen R., Brandl H. 1997. Microbial recovery of metáis from solids. FEMS Microbiol Rev. 20, 605-17). Es un proceso que resulta particularmente útil en la extracción de metales presentes en muy baja cantidad, que no son rentables de extraer mediante procesos químicos. En la biolixiviación se utilizan tanto cepas únicas como cultivos mixtos de microorganismos, siendo algunos de los más utilizados las bacterias acidófilas Acidithiobacillus ferrooxidans, Acidithiobacillus caldus, Acidithiobacillus thiooxidans y Leptospirillum ferrooxidans; y la arquea Sulfolobus acidocaldarius (Harrison, A. P., Norris, P. R. 1985. Leptospirillum ferrooxidans and similar bacteria: some characteristics and genomic diversity. FEMS Microbiol. Lett. 30, 99-102; Temple, K. L, Colmer, A. R. 1951 . The autotrophic oxidation of ¡ron by a new bacterium: Thiobacillus ferrooxidans. J. Bacteriol. 62, 605- 1 1 ). RECTIFIED LEAVES (RULE 91) W., Brombacher C., Bosshard PP, Bachofen R., Brandl H. 1997. Microbial recovery of metais from solids. FEMS Microbiol Rev. 20, 605-17). It is a process that is particularly useful in the extraction of metals present in very low amounts, which are not profitable to extract by chemical processes. In the bioleaching, both single strains and mixed cultures of microorganisms are used, being some of the most used the acidophilic bacteria Acidithiobacillus ferrooxidans, Acidithiobacillus caldus, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans; and the archaea Sulfolobus acidocaldarius (Harrison, AP, Norris, PR 1985. Leptospirillum ferrooxidans and similar bacteria: some characteristics and genomic diversity, FEMS Microbiol.Lett.30, 99-102; Temple, K.L., Colmer, AR 1951. autotrophic oxidation of rum by a new bacterium: Thiobacillus ferrooxidans, J. Bacteriol 62, 605-1 1).
En comparación a los procesos de extracción química, la biolixiviación tiene la ventaja de ser amigable con el medio ambiente; además de tener un bajo costo de implementación. Sin embargo, no está exenta de problemas, ya que durante el proceso puede ocurrir una inhibición del crecimiento microbiano debido a la presencia de cloro, metales pesados, metaloides y materia orgánica disuelta (MOD). Esto repercute en una baja recuperación del metal de interés (Hansford, G.S. 1997. Recent developments in modeling the kinetics of bioleaching. In: Rawlings, D.E. (Ed.), Biomining: Theory, Microbes and Industrial Processes. Springer Verlag, Berlín, pp. 153-175; Schrenk, M.O., Edwards, K.J., Goodman, R.M., Hamers, R.J., Banfield, J.F. 1998. Distribution of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans : implications for generation of acid mine drainage. Science. 279, 1519-1522; Rawlings, D.E. 1999. The molecular genetics of mesophilic, acidophilic, chemolithotrophic, iron- or sulfur-oxidizing microorganisms. Process Metallurgy. 9, 3-20; Rawlings DE. 2005. Characteristics and adaptability of ¡ron- and sulfure-oxidizing microorganisms used for the recovery of metáis from minerals and their concentrates. Microb. Cell Fact. 4, 13.). La eficiencia de la biolixiviación se puede mejorar con la incorporación de hongos heterotróficos, tales como Aspergillus spp. y Penicillium spp. Esto se debe a que los hongos excretan ácidos orgánicos (como ácido cítrico y ácido oxálico) durante el proceso de acidólisis, lo cual en ciertos minerales permite la solubilización de los metales por medio de la formación de complejos con estos ácidos (Bosecker, K. (1997). Bioleaching: metal solubilization by microorganisms. FEMS Microbiology reviews, 20(3-4), 591 -604; Bosshard, P., Bachofen, R., Brandl, H. 1996. Metal leaching of fly ash from municipal waste incineration by Aspergillus niger. Environ. Sci. Technol. 30, 3066-3070; Torre, M. A., Gomez-Alarcon, G., Vizcaíno, C., García, M. T. 1992. Biochemical mechanisms of stone alteration carried out by filamentous fungí living in monuments. Biogeochemistry. 19, 129-147). Además, Aspergillus spp. y Penicillium spp., que han sido aplicados mayoritariamente en la biolixiviación de materia orgánica, son muy eficientes en la degradación de MOD. Por lo tanto, la incorporación de estos hongos a los procesos de biolixiviación favorece el crecimiento bacteriano, reduciendo el tiempo requerido para la solubilización del metal de interés (Gu, X., Wong, J.W.C. 2004. Identification of inhibitory substances affecting bioleaching of heavy metáis from anaerobically digested sewage sludge. Environ. Sci. Technol. 38, 2934-2939; Gu, X.Y., Wong, J.W.C. 2007. Degradation of inhibitory substances by heterotrophic microorganisms during bioleaching of heavy metáis from anaerobically digested sewage sludge. Chemosphere. 69, 31 1 -318; Wang, S., Zheng, G., & Zhou, L. 2010. Heterotrophic microorganism fíhodotorula mucilaginosa R30 improves tannery sludge bioleaching through elevating dissolved C02 and extracellular polymeric substances levels in bioleach solution as well as scavenging toxic DOM to Acidithiobacillus species. Water fíes. 44, 5423-5431 ; Zheng, G., Zhou, L., Wang, S. 2009. An acid- tolerant heterotrophic microorganism role in improving tannery sludge bioleaching conducted in successive multibatch reaction systems. Environ. Sci. Technol. 43, 4151 - 4156; Zheng, G., Wang, Z., Wang, D., Zhou, L. 2016. Enhancement of sludge dewaterability by sequential inoculation of filamentous fungus Mucor circinelloides ZG- 3 and Acidithiobacillus ferrooxidans LX5. Chem. Eng. J. 284, 216-223; Ngom, B., Liang, Y., Liu, Y., Yin, H., Liu, X. 2015. Use of an acidophilic yeast strain to enable the growth of leaching bacteria on solid media. Arch. Microbiol. 197, 339-346; Zhou, J., Zheng, G., Wong, J. W., & Zhou, L. 2013. Degradation of inhibitory substances in sludge by Gaiactomyces sp. Z3 and the role of its extracellular polymeric substances in improving bioleaching. Bioresour. Technol. 132, 217-223). Compared to chemical extraction processes, bioleaching has the advantage of being friendly to the environment; besides having a low implementation cost. However, it is not without problems, since during the process an inhibition of microbial growth can occur due to the presence of chlorine, heavy metals, metalloids and dissolved organic matter (MOD). This results in a low recovery of the metal of interest (Hansford, GS 1997. Recent developments in modeling the kinetics of bioleaching, In: Rawlings, DE (Ed.), Biomining: Theory, Microbes and Industrial Processes.) Springer Verlag, Berlin, pp 153-175; Schrenk, MO, Edwards, KJ, Goodman, RM, Hamers, RJ, Banfield, JF 1998. Distribution of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans: implications for generation of acid mine drainage, Science 279, 1519-1522; Rawlings, DE 1999. The molecular genetics of mesophilic, acidophilic, chemolithotrophic, iron-or sulfur-oxidizing microorganisms, Process Metallurgy, 9, 3-20, Rawlings DE, 2005. Characteristics and adaptability of protein and sulphide-oxidizing microorganisms used for the recovery of metais from minerals and their concentrates, Microb.Cell Fact., 4, 13.). The bioleaching efficiency can be improved with the incorporation of heterotrophic fungi, such as Aspergillus spp. and Penicillium spp. This is because the fungi excrete organic acids (such as citric acid and oxalic acid) during the acidolysis process, which in certain minerals allows the solubilization of metals through the formation of complexes with these acids (Bosecker, K. (1997) Bioleaching: metal solubilization by microorganisms FEMS Microbiology reviews, 20 (3-4), 591-604; Bosshard, P., Bachofen, R., Brandl, H. 1996. Metal leaching of fly ash from municipal waste incineration by Aspergillus niger, Environ.Sci.Technol.30, 3066-3070; Tower, MA, Gomez-Alarcon, G., Vizcaino, C., Garcia, MT 1992. Biochemical mechanisms of stone alteration carried out by filamentous fungí living in monuments, Biogeochemistry, 19, 129-147). In addition, Aspergillus spp. and Penicillium spp., which have been applied mainly in the bioleaching of organic matter, are very efficient in the degradation of MOD. Therefore, the incorporation of these fungi to bioleaching processes favors bacterial growth, reducing the time required for the solubilization of the metal of interest (Gu, X., Wong, JWC 2004. Identification of inhibitory substances affecting bioleaching of heavy metal from anaerobically digested sewage sludge, Environ. Sci. Technol 38, 2934-2939; Gu, XY, Wong, JWC 2007. Degradation of inhibitory substances by heterotrophic microorganisms during bioleaching of heavy metais from anaerobically digested sewage sludge. 1 -318; Wang, S., Zheng, G., & Zhou, L. 2010. Heterotrophic microorganism fíhodotorula mucilaginosa R30 improves tannery sludge bioleaching through elevating dissolved C0 2 and extracellular polymeric substances in bioleach solution as well as scavenging toxic DOM to Acidithiobacillus species, Water fies 44, 5423-5431, Zheng, G., Zhou, L., Wang, S. 2009. An acid tolerant heterotrophic microorganism role in improv ing tannery sludge bioleaching conducted in successive multibatch reaction systems. Environ. Sci. Technol. 43, 4151-4156; Zheng, G., Wang, Z., Wang, D., Zhou, L. 2016. Enhancement of sludge dewaterability by sequential inoculation of filamentous fungus Mucor circinelloides ZG- 3 and Acidithiobacillus ferrooxidans LX5. Chem. Eng. J. 284, 216-223; Ngom, B., Liang, Y., Liu, Y., Yin, H., Liu, X. 2015. Use of an acidophilic yeast strain to enable the growth of leaching bacteria on solid media. Arch. Microbiol. 197, 339-346; Zhou, J., Zheng, G., Wong, JW, & Zhou, L. 2013. Degradation of inhibitory substances in sludge by Gaiactomyces sp. Z3 and the role of its extracellular polymeric substances in improving bioleaching. Bioresour. Technol. 132, 217-223).
En cuanto a documentos de patente es posible mencionar a CN106190871 (A) que describe un método para biolixiviación en suelo contaminado con metales pesados a través de hongos filamentosos compuestos tomando paja como fuente de carbono. El hongo filamentoso se clasifica y nombra como Penicillium simplicissiumum y se conserva en el Centro General de Recolección de Cultivos Microbiológicos de China el 25 de junio de 2015 con el número de acceso CGMCC No.10990. La invención describe adicionalmente un inoculante de hongos filamentosos compuesto formado por la mezcla de esporas isométricas de Penicillium simplicissiumum NAU-12 (1 x 108 esporas/mL a 2 x 108 esporas/mL) y Aspergillus niger A80 (1 x 107 esporas/mL a 2 x 107 esporas/ mL). La invención describe adicionalmente un método para llevar a cabo el tratamiento de biolixiviación en suelos contaminados con metales pesados a través de hongos filamentosos compuestos que crecen rápidamente en el suelo, altos en resistencia a metales pesados y capaces de utilizar paja sometida a pretratamiento alcalino con calor alcalino como fuente de carbono para el crecimiento y tener una alta producción de múltiples ácidos orgánicos moleculares pequeños. El método se adopta para tratar el suelo contaminado con metales pesados, la eficiencia de eliminación de metales pesados es alta y el costo es bajo. Regarding patent documents it is possible to mention CN106190871 (A) which describes a method for bioleaching in soil contaminated with heavy metals through filamentous fungi compound taking straw as carbon source. The filamentous fungus is classified and named as Penicillium simplicissiumum and is kept at the General Center for Collection of Microbiological Crops of China on June 25, 2015 with the accession number CGMCC No.10990. The invention further describes a composite filamentous fungal inoculant formed by the isometric spore mixture of Penicillium simplicissiumum NAU-12 (1 x 10 8 spores / mL at 2 x 10 8 spores / mL) and Aspergillus niger A80 (1 x 10 7 spores). / mL at 2 x 10 7 spores / mL). The invention further describes a method for carrying out the bioleaching treatment in soils contaminated with heavy metals through compound filamentous fungi that grow rapidly in the soil, high in resistance to heavy metals and capable of using straw subjected to alkaline pretreatment with heat. alkaline as a carbon source for growth and have a high production of multiple small molecular organic acids. The method is adopted to treat soil contaminated with heavy metals, the removal efficiency of heavy metals is high and the cost is low.
TW201429883 (A) describe un método de biolixiviación de metales y su sistema utilizando hongos para lodos. El sistema de biolixiviación de metales incluye una unidad de biolixiviación, una unidad de suministro de lodo y una unidad de suministro de hongos. La unidad de biolixiviación se proporciona para procesar un tratamiento de biolixiviación de metales. La unidad de suministro de lodo se proporciona para suministrar lodos que contienen metales. La unidad de suministro de hongos se proporciona para suministrar hongos a la unidad de biolixiviación y luego los hongos generan un material metabólico. El material metabólico se aplica para lixiviar los metales contenidos en el lodo para eliminar los metales de los lodos. TW201429883 (A) describes a method of bioleaching metals and their system using fungi for sludge. The metal bioleaching system includes a bioleaching unit, a mud supply unit and a supply unit mushroom. The bioleaching unit is provided to process a metal bioleaching treatment. The mud supply unit is provided to supply sludge containing metals. The fungal supply unit is provided to supply fungi to the bioleaching unit and then the fungi generate a metabolic material. The metabolic material is applied to leach the metals contained in the mud to remove the metals from the sludge.
AU2010288177 (B2) describe un aditivo para la biolixiviación que hace posible aumentar la recuperación de cobre a partir de minerales de sulfuro. En el que este aditivo está constituido sustancialmente por la lipoproteína Llcanantasa y una solución de ácido sulfúrico con un pH de 0,8 a 3. La lipoproteína de Llcanantasa que tiene una secuencia de aminoácidos con al menos 50% de homología con respecto a la secuencia definida en la secuencia SEQ ID N°1 o es el producto de traducción de una secuencia de nucleótidos con al menos 50% de homología con respecto a la secuencia definida en la secuencia SEQ I D N^. Tamb ién protege el proceso mejorado de biolixiviación que incluye añadir el aditivo durante el proceso de biolixiviación de mineral y continuando con el proceso habitual, obteniendo recuperaciones de cobre aumentó de 5 a 20%. Llcanantasa es la proteína predominante en el secretoma de Acidithiobacillus thiooxidans cuando es cultivada con azufre elemental y es capaz de Incrementar la recuperación de cobre desde calcopirita cuando se utiliza como aditivo para la biolixiviación. AU2010288177 (B2) describes an additive for the bioleaching that makes it possible to increase the recovery of copper from sulfide minerals. Wherein this additive is substantially constituted by the lipoprotein Llcanantase and a solution of sulfuric acid with a pH of 0.8 to 3. The lipoprotein of Llcanantase having an amino acid sequence with at least 50% homology with respect to the sequence defined in the sequence SEQ ID No. 1 or is the translation product of a nucleotide sequence with at least 50% homology with respect to the sequence defined in the sequence SEQ ID NO. It also protects the improved bioleaching process that includes adding the additive during the mineral bioleaching process and continuing with the usual process, obtaining copper recoveries increased from 5 to 20%. Llcanantasa is the predominant protein in the secretome of Acidithiobacillus thiooxidans when it is cultivated with elemental sulfur and is capable of increasing copper recovery from chalcopyrite when used as an additive for bioleaching.
Aunque ciertos hongos heterotróficos puedan ser buenos candidatos para mejorar la eficiencia de la biolixiviación de minerales. No hay reportes del uso de hongos en procesos de biolixiviación en un medio inorgánico. La presente Invención describe el aislar y caracterizar el hongo Acidomyces acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 , obtenido a partir de drenaje ácido de mina, y evaluar su Although certain heterotrophic fungi may be good candidates to improve the efficiency of mineral bioleaching. There are no reports of the use of fungi in bioleaching processes in an inorganic medium. The present invention describes the isolate and characterize the fungus Acidomyces acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chlllán, Chile, RGM 2451, obtained from acid mine drainage, and evaluate its
HOJAS RECTIFICADAS (REGLA 91) adición a un consorcio bacteriano para mejorar el proceso de biolixiviación. Al analizar la recuperación de cobre a partir de calcopirita en biolixiviación estática y en un blorreactor de flujo ascendente o airlift, en presencia de un consorcio bacteriano que además puedo o no Incluir el hongo A. acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 , se demuestra la importancia de agregar este hongo en el crecimiento de las bacterias lixiviantes y en el resultado de la recuperación de cobre desde minerales de sulfuros de cobre. La presente Invención permite salvar las dificultades al comienzo mencionadas, al incluir el proceso de lixiviación de minerales sulfurados, el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 como un aditivo de origen natural. Este aditivo permite reducir los tiempos de biolixiviación de los minerales sulfurados a meses incluso semanas, e Incrementa el crecimiento celular microbiano en más de un 20%, permitiendo un Incremento mayor al 10%, en la recuperación de cobre desde minerales sulfurados, en particular, desde soluciones ya sea provengan de pilas, reactores, concentrados u otros equipos u operaciones necesarias para la extracción del mineral. El valor antes mencionado es un valor estimado para la recuperación con el aditivo. RECTIFIED LEAVES (RULE 91) addition to a bacterial consortium to improve the bioleaching process. When analyzing the recovery of copper from chalcopyrite in static bioleaching and in an uplift or airlift, in the presence of a bacterial consortium that may or may not include the fungus A. acidophilus HE17- Access number of the Genetic Resources Bank Regional Microbial Research Quilamapu, Chlllán, Chile, RGM 2451, demonstrates the importance of adding this fungus in the growth of leaching bacteria and the result of the recovery of copper from copper sulfide ores. The present invention makes it possible to overcome the aforementioned difficulties by including the process of leaching sulfur minerals, the fungus Acidomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank Quilamapu Research, Chlllán, Chile, RGM 2451 as an additive of natural origin This additive allows to reduce the bioleaching times of sulphided minerals to months or even weeks, and increases microbial cell growth by more than 20%, allowing an increase of more than 10%, in the recovery of copper from sulphide minerals, in particular, from solutions, whether they come from batteries, reactors, concentrates or other equipment or operations necessary for the extraction of the mineral. The aforementioned value is an estimated value for recovery with the additive.
Breve Descripción de la Invención Brief Description of the Invention
La presente Invención proporciona un método para biolixiviar minerales sulfurados de cobre usando un consorcio de microorganismos que comprende bacterias ferroxidantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2527 y el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 en un  The present invention provides a method for bio-lixivizing copper sulphide minerals using a consortium of microorganisms comprising ferroxidant bacteria - access number of the Regional Microbial Genetic Resource Bank Quilamapu, Chlllán, Chile, RGM 2527 and Acidomyces acidophilus fungus HE17 - number of access of the Regional Microbial Genetic Resources Research Bank Quilamapu, Chlllán, Chile, RGM 2451 in a
HOJAS RECTIFICADAS (REGLA 91) medio inorgánico libre de sulfato ferroso y pH <2, de preferencia, pH = 1 ,8, favoreciendo el crecimiento de los microorganismos lixiviantes y aumentando la extracción del metal desde el mineral. Se realizaron cultivos de la cepa fúnglca Acidomyces acidophylus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 , en forma aislada con bacterias ferrooxidantes en medio sólido inorgánico agarosa 9K-Fe, pH=1 ,8, lo que favoreció el crecimiento tanto del hongo como de las bacterias. En el estado del arte, se han reportado hongos en consorcios bacterianos para recuperar metales pero en medios orgánicos a pH neutros. En particular Ngom, B., Llang, Y., Liu, Y., Yin, H., Liu, X. 2015. Use of an acidophlllc yeast strain to enable the growth of leachlng bacteria on solid media. Arch. Mlcrobiol. 197, 339-346 reporta el crecimiento de bacterias lixiviantes en medios sólidos 9K suplementado con glucosa a pH=5, se inoculó Candida digboiensis NB en la capa Inferior del gel, demostrando ser eficaz en la activación y mejoramiento del crecimiento de las bacterias autótrofas lixiviantes oxidantes de hierro y azufre. Asi mismo, se ha reportado la actividad del hongo Rhodotorula mucilaginosa R30 que permitió el Incremento de la biomasa bacterial de Acidithiobacillus ferrooxidans en lodos con alta concentración de materia orgánica (Wang, S., Zheng, G., & Zhou, L. 2010. Heterotrophlc mlcroorganlsm Rhodotorula mucilaginosa R30 ¡mproves tannery sludge bioleachlng through elevating dissolved C02 and extracellular polymerlc substances levels in bioleach solution as well as scavenglng toxic DOM to Acidithiobacillus species. Water Res. 44, 5423-5431 ). No se ha reportado entonces un consorcio bacteriano que adicione A. acidophilus con fines de biolixiviación. RECTIFIED LEAVES (RULE 91) inorganic medium free of ferrous sulfate and pH <2, preferably pH = 1.8, favoring the growth of leaching microorganisms and increasing the extraction of the metal from the ore. Cultures of the Acidomyces acidophylus fungal strain HE17 - Access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chlllán, Chile, RGM 2451, in isolation with ferrooxidant bacteria in solid 9K-Fe agarose inorganic solid medium, pH = 1 , 8, which favored the growth of both the fungus and the bacteria. In the state of the art, fungi have been reported in bacterial consortiums to recover metals but in organic media at neutral pH. In particular Ngom, B., Llang, Y., Liu, Y., Yin, H., Liu, X. 2015. Use of an acid and strain strain to enable the growth of bacterium on solid media. Arch. Mlcrobiol. 197, 339-346 reports the growth of leaching bacteria in 9K solid media supplemented with glucose at pH = 5, Candida digboiensis NB was inoculated in the lower layer of the gel, proving to be effective in activating and improving the growth of autotrophic leaching bacteria oxidizers of iron and sulfur. Likewise, the activity of the fungus Rhodotorula mucilaginosa R30 has been reported, which allowed the increase of the bacterial biomass of Acidithiobacillus ferrooxidans in sludge with a high concentration of organic matter (Wang, S., Zheng, G., & Zhou, L. 2010. Heterotrophlc mlcroorganlsm Rhodotorula mucilaginosa R30 mproves tannery sludge bioleachlng through elevating dissolved C02 and extracellular polymerlc levels in bioleach solution as well as scavenglng toxic DOM to Acidithiobacillus species, Water Res. 44, 5423-5431). A bacterial consortium that adds A. acidophilus for bioleaching purposes has not been reported.
Se demostró que A. acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chlllán, Chile, RGM 2451 , puede crecer en medio sólido 9K-Fe-Cu pH 1 ,8 con 400 mM de CuS04 (Fig. 7), It was demonstrated that A. acidophilus HE17- Access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chlllán, Chile, RGM 2451, can grow in solid medium 9K-Fe-Cu pH 1, 8 with 400 mM CuS0 4 ( Fig. 7),
HOJAS RECTIFICADAS (REGLA 91) condiciones que no se han utilizado con anterioridad a la presente invención. Para aislados de los géneros Aspergillus, Penicillium y Fusarium, se ha descrito una concentración inhibitoria mínima de cobre de 15-20 mM; 7,5-20 mM, y 12,5 mM, respectivamente (Ezzouhri, L., Castro, E., Moya, M., Espinóla, F., Lairini , K. 2009. Heavy metal tolerance of fllamentous fungí isolated from polluted sites ¡n Tangier, Morocco. Afr. J. Microbiol. Res. 3, 35-48). El hongo Antrodia vaillantii fue capaz de tolerar hasta 40 mM de cobre (Collett, O. 1992. Comparative tolerance of the brown-rot fungus Anthrodia vaillantii (DC.Fr.) Ryv. isolates to copper. Holzforschung. 46, 293- 298). Por lo tanto, en comparación a otros hongos descritos en la literatura, A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , posee una gran tolerancia al cobre, lo que permite pueda ser usado en procesos de biolixiviación que involucran este metal. En procesos de biolixiviación en biorreactor airlift usando hongos, sólo se ha reportado la publicación de trabajos de Sabra, N., Dubourguier, H. C., Hamieh, T. 2012. Fungal leaching of heavy metáis from sediments dredged from the Deúle Canal, France. Advances ¡n Chemical Engineering and Science. 2, 1 -8, donde se utilizan los hongos filamentosos Aspergillus niger y Penicillium chrysogenum para lixiviar metales pesados de sedimentos dragados en biorreactores airlift a escala semi-piloto usando medios orgánicos con sacarosa obteniendo una recuperación de 12% de cobre en 40 días. Comparado a los resultados de la presente invención, se obtuvo una mayor recuperación de cobre en biorreactores airlift (BA) con el hongo A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , usando medio inorgánico 9K, a partir del mineral calcopirita durante 68 días, conduciéndose la biolixiviación en BA con hongos lixiviando minerales en medios inorgánicos. RECTIFIED LEAVES (RULE 91) conditions that have not been used prior to the present invention. For isolates of the genera Aspergillus, Penicillium and Fusarium, a minimum copper inhibitory concentration of 15-20 mM has been described; 7.5-20 mM, and 12.5 mM, respectively (Ezzouhri, L., Castro, E., Moya, M., Spinola, F., Lairini, K. 2009. Heavy metal tolerance of fllamentous fungi isolated from polluted sites Tangier, Morocco, Afr. J. Microbiol, Res. 3, 35-48). The fungus Antrodia vaillantii was able to tolerate up to 40 mM of copper (Collett, O. 1992. Comparative tolerance of the brown-rot fungus Anthrodia vaillantii (DC.Fr.) Ryv. Isolates to copper., Holzforschung., 46, 293-298) . Therefore, in comparison to other fungi described in the literature, A. acidophilus - Access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, has a high tolerance to copper, which allows be used in bioleaching processes that involve this metal. In bioleaching processes in airlift bioreactor using fungi, only the publication of works by Sabra, N., Dubourguier, HC, Hamieh, T. 2012 has been reported. Fungal leaching of heavy metal from sediments dredged from the Deúle Canal, France. Advances n Chemical Engineering and Science. 2, 1 -8, where filamentous fungi Aspergillus niger and Penicillium chrysogenum are used to leach heavy metals from dredged sediments in semi-pilot airlift bioreactors using organic media with sucrose obtaining a recovery of 12% copper in 40 days. Compared to the results of the present invention, a greater recovery of copper was obtained in airlift bioreactors (BA) with the fungus A. acidophilus - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, using 9K inorganic medium, from the mineral chalcopyrite for 68 days, conducting bioleaching in BA with fungi leaching minerals in inorganic media.
HOJAS RECTIFICADAS (REGLA 91) En cuanto a operaciones de biolixiviación estática (BE) y biorreactor airlift (BA) de calcopirita en medio inorgánico 9K libre de sulfato ferroso a pH=1 ,8 sólo con bacterias ferrooxldantes, se observó una menor recuperación de cobre; comparado a la biolixiviación con bacterias ferrooxldantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2527 y la cepa fúngica A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , se mostró una mayor recuperación de cobre con una mayor eficiencia en biorreactores airlift, demostrando que la inoculación del hongo A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 mejora los procesos de biolixiviación. RECTIFIED LEAVES (RULE 91) Regarding static bioleaching operations (BE) and airlift bioreactor (BA) of chalcopyrite in 9K inorganic medium free of ferrous sulphate at pH = 1.8 only with ferrooxidizing bacteria, a lower recovery of copper was observed; compared to bioleaching with ferrooxaldantes bacteria - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2527 and the A. acidophilus fungal strain - access number of the Regional Microbial Genetic Resources Bank Quilamapu Research, Chillán, Chile, RGM 2451, showed a greater recovery of copper with higher efficiency in airlift bioreactors, demonstrating that the inoculation of the fungus A. acidophilus - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451 improves bioleaching processes.
El método de la presente Invención se conduce en medios inorgánicos a pH bajo (< 2), y no se refiere a biolixiviación en lodos con metales pesados con una alta concentración de materia orgánica disuelta usando hongos y bacterias. Se ha estudiado que la inoculación del hongo Gaiactomyces sp. Z3 mejora los procesos de biolixiviación en lodos con metales pesados cuando se usan dos cepas Acidithiobacillus ferrooxidans LX5 y Acidithiobacillus thiooxidans TS6 disminuyendo el período requerido para el proceso, con mayores eficiencias en la solubilización del cobre (Zhou, J., Zheng, G., Wong, J. W., & Zhou, L. 2013. Degradation of ¡nhlbitory substances in sludge by Gaiactomyces sp. Z3 and the role of its extracellular polymerlc substances in ¡mprovlng bioleachlng. Bloresour. Technol. 132, 217-223). La inoculación de Pichia spartinae mejoró la biolixiviación de lodos de curtiduría usando dos cepas bacterianas A. ferrooxidans LX5 y A. thiooxidans TS6 con la mayor solubilización de otro metal como el cromo (Zheng, G., Zhou, L., Wang, S. 2009. An acid-tolerant heterotrophlc mlcroorganlsm role in ¡mprovlng tannery sludge bioleachlng The method of the present invention is conducted in inorganic media at low pH (<2), and does not refer to bioleaching in sludge with heavy metals with a high concentration of dissolved organic matter using fungi and bacteria. It has been studied that the inoculation of the fungus Gaiactomyces sp. Z3 improves the bioleaching processes in sludge with heavy metals when two strains Acidithiobacillus ferrooxidans LX5 and Acidithiobacillus thiooxidans TS6 are used, decreasing the period required for the process, with greater efficiencies in the solubilization of copper (Zhou, J., Zheng, G., Wong, JW, & Zhou, L. 2013. Degradation of nhlbitory substances in sludge by Gaiactomyces sp.Z3 and the role of its extracellular polymerlc substances in microprovlng bioleachlng, Bloresour, Technol. 132, 217-223). The inoculation of Pichia spartinae improved the bioleaching of tannery sludge using two bacterial strains A. ferrooxidans LX5 and A. thiooxidans TS6 with the highest solubilization of another metal such as chromium (Zheng, G., Zhou, L., Wang, S. 2009. An acid-tolerant heterotrophlc mlcroorganlsm role in mprovlng tannery sludge bioleachlng
HOJAS RECTIFICADAS (REGLA 91) conducted in successive multibatch reaction systems. Environ. Sci. Technol. 43, 4151 - 4156). La inoculación del hongo filamentoso Mucor circinelloides ZG-3 mejoró de biolixiviación de lodos con A. ferrooxidans reduciendo el periodo de oxidación del hierro ferroso (Zheng, G., Wang, Z., Wang, D., Zhou, L. 2016. Enhancement of sludge dewaterability by sequential inoculation of filamentous fungus Mucor circinelloides ZG- 3 and Acidithiobacillus ferrooxidans LX5. Chem. Eng. J. 284, 216-223). RECTIFIED LEAVES (RULE 91) conducted in successive multibatch reaction systems. Environ. Sci. Technol. 43, 4151-4156). The inoculation of the filamentous fungus Mucor circinelloides ZG-3 improved the bioleaching of sludge with A. ferrooxidans reducing the oxidation period of ferrous iron (Zheng, G., Wang, Z., Wang, D., Zhou, L. 2016. Enhancement of sludge dewaterability by sequential inoculation of filamentous fungus Mucor circinelloides ZG-3 and Acidithiobacillus ferrooxidans LX5, Chem. Eng. J. 284, 216-223).
En cuanto al tipo de sistema de biolixiviación, no se reportan trabajos sobre procesos de biolixiviación con hongos filamentosos en sistema estático en medio inorgánico 9K sin sulfato ferroso, la mayor parte de los trabajos en biolixiviación con hongos se hicieron en medios orgánicos a pH neutro en sistemas de agitación (e eΐ bή como la investigación realizada por Mehta, K.D., Das, C., Pandey, B.D. 2010. Leaching of copper, nickel and cobalt from Indian Ocean manganese nodules by Aspergillus niger. Hydrometallurgy. 105, 89-95, donde la biolixiviación usa medios orgánicos en sistema de agitación a 120 rpm en 30 días, a partir de nodulos de manganeso polimetálicos del Océano índico usando un hongo Aspergillus niger se obtuvo una recuperación de 97% de cobre. En la investigación de Ren, W. X., Li, P. J., Geng, Y., Li, X. J. 2009. Biological leaching of heavy metáis from a contaminated soil by Aspergillus niger. J. Hazard. Mat. 167, 164-169, para la biolixiviación de metales pesados de un suelo contaminado en un área industrial usa metabolitos, principalmente ácidos orgánicos débiles, producidos por el hongo Aspergillus niger obteniendo una recuperación del 56% de cobre en sistema de agitación a 120 rpm a 30eC a intervalos de tiempo regulares de 15 días. Por otra parte Deng, X., Chai, L, Yang, Z., Tang, C., Tong, H., Yuan, P. 2012. Bioleaching of heavy metáis from a contaminated soil using indigenous Penicillium chrysogenum strain F1 . J. Hazard. Mater. 233, 25-32 para la biolixiviación también usa medio orgánico para remediar el suelo contaminado por metales pesados usando el hongo Penicillium Chrysogenum obteniendo una recuperación del 34,89% de Cu en agitador orbital a 120 rpm durante 15 días. Por tanto, la biolixiviación estática de calcopirita a diferentes tratamientos con el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillón, Chile, RGM 2451 , en medio inorgánico 9K sin sulfato ferroso de la presente Invención, es más eficiente. Regarding the type of bioleaching system, no reports are reported on bioleaching processes with filamentous fungi in a static system in inorganic medium 9K without ferrous sulphate, most of the work in bioleaching with fungi was done in organic media at neutral pH in agitation systems (e eΐ bή like the research carried out by Mehta, KD, Das, C., Pandey, BD 2010. Leaching of copper, nickel and cobalt from Indian Ocean manganese nodules by Aspergillus niger Hydrometallurgy 105, 89-95, where the bioleaching system uses organic media in an agitation system at 120 rpm in 30 days, a 97% recovery of copper was obtained from the polymetallic manganese nodules of the Indian Ocean using an Aspergillus niger fungus. Li, PJ, Geng, Y., Li, XJ 2009. Biological leaching of heavy metal from a contaminated soil by Aspergillus niger, J. Hazard, Mat 167, 164-169, for heavy metal bioleaching. s of a contaminated soil in an industrial area uses metabolites, mainly weak organic acids, produced by the fungus Aspergillus niger obtaining a recovery of 56% copper in the agitation system at 120 rpm at 30 e C at regular time intervals of 15 days . On the other hand Deng, X., Chai, L, Yang, Z., Tang, C., Tong, H., Yuan, P. 2012. Bioleaching of heavy metais from a contaminated soil using indigenous Penicillium chrysogenum strain F1. J. Hazard. Mater. 233, 25-32 for the bioleaching also uses organic medium to remedy the soil contaminated by heavy metals using the fungus Penicillium Chrysogenum obtaining a recovery of 34.89% Cu in orbital shaker at 120 rpm for 15 days. Therefore, the bioleaching of static of chalcopyrite to different treatments with the fungus Acidomyces acidophilus HE17 - Access number of the Regional Microbial Genetic Resources Bank of Research Quilamapu, Chillón, Chile, RGM 2451, in inorganic medium 9K without ferrous sulfate of the present invention, is more efficient.
La presente Invención demuestra que el hongo A. acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillón, Chile, RGM 2451 , presenta actividad mlxotrofa y acidotolerante con bacterias del género Acidithiobacillus y Acidiphilium. El hongo es capaz de favorecer el crecimiento bacteriano y de aumentar la recuperación de cobre a partir de calcopirita, tanto en BE como en BA. Por lo tanto, A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillón, Chile, RGM 2451 , potencia la solubilización de minerales sulfurados, probablemente - sin consentir con ninguna teoría, mediante la excreción de ácidos orgánicos ayudando a aumentar la biomasa de bacterias lixiviantes debido a una posible degradación de materia orgánica disuelta (MOD). The present invention demonstrates that the fungus A. acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank of Research Quilamapu, Chillón, Chile, RGM 2451, exhibits mlxotroph and acidotolerant activity with bacteria of the genus Acidithiobacillus and Acidiphilium. The fungus is able to favor bacterial growth and increase the recovery of copper from chalcopyrite, both in BE and in BA. Therefore, A. acidophilus - Access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillón, Chile, RGM 2451, potentiates the solubilization of sulphide minerals, probably - without consent with any theory, through the excretion of organic acids helping to increase the biomass of leaching bacteria due to a possible degradation of dissolved organic matter (MOD).
Por lo que en la presente Invención, el método biolixivlación se realiza con un consorcio de microorganismos que comprende además del hongo A. acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional deTherefore, in the present invention, the biolixivlation method is carried out with a consortium of microorganisms that also comprises the fungus A. acidophilus HE17 - Access number of the Regional Microbial Genetic Resources Bank of
Investigación Quilamapu, Chillón, Chile, RGM 2451 , una bacteria seleccionada de bacterias del género Acidithiobacillus, Acidiphilium o una combinación de las mismas. Research Quilamapu, Chillón, Chile, RGM 2451, a bacterium selected from bacteria of the genus Acidithiobacillus, Acidiphilium or a combination thereof.
Se han reportado pocos estudios que revelen el uso de hongos en medios extremadamente ácidos (pH < 2). Tampoco se conoce su función en relación a procesos de biolixivlación de minerales. Por tanto, la presente Invención proporciona una forma de aislar y caracterizar hongos extremoacidófilos para su aplicación en biolixivlación de los minerales de sulfuras de cobre. Primero se aíslan hongos y Few studies have been reported revealing the use of fungi in extremely acid media (pH <2). Neither is its function known in relation to mineral biolixivlation processes. Therefore, the present invention provides a way to isolate and characterize extremoacidiophilic fungi for its application in biolixivlation of copper sulphide minerals. First fungi are isolated and
HOJAS RECTIFICADAS (REGLA 91) bacterias desde cultivos lixiviantes extremadamente ácidos (pH 1 ,8), luego se analizan por: métodos mlcrobiológicos, a saber, microscopía óptica, confocal y electrónica; métodos de biología molecular, a saber PCR, DGGE y secuenclaclón de ADN; y métodos blolnformátlcos para análisis f ilogenéticos. Tanto los hongos como las bacterias Identificadas son sometidas a procesos de biolixiviación con mineral calcopirita 4% en sistema estático y en blorreactores airlift de 2L a 22 +.213 durante 68 días, usando 4 tratamientos: Tratamiento 1 (T1 ) = Control negativo; Tratamiento 2 (T2) = Bacterias; Tratamiento 3 (T3) = Bacterias + Hongo y Tratamiento 4 (T4)= Hongo. El hongo fue Identificado como Acidomyces acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , mostrando tener un metabolismo mixótrofo siendo capaz de crecer en quimilitoautotrofia en ambientes extremadamente ácidos en presencia de hierro 33,3 g/L o cobre 200-400 mM (9K-Fe-Cu, pH 1 ,8) y en organografía (agar sabouraud y PDA a pH=6,49 . A. acidophilus- número de acceso del Banco de Recursos Genético Microbianos Regional de InvestigaciónRECTIFIED LEAVES (RULE 91) bacteria from extremely acid leaching cultures (pH 1, 8), then analyzed by: microbiological methods, namely, optical, confocal and electronic microscopy; molecular biology methods, namely PCR, DGGE and DNA sequencing; and blolymphatic methods for phylogenetic analyzes. Both the fungi and the identified bacteria are subjected to bioleaching processes with mineral chalcopyrite 4% in static system and in airlift blorreactors from 2L to 22 +.213 for 68 days, using 4 treatments: Treatment 1 (T1) = Negative control; Treatment 2 (T2) = Bacteria; Treatment 3 (T3) = Bacteria + Fungus and Treatment 4 (T4) = Fungus. The fungus was identified as Acidomyces acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, showing to have a mixotrophic metabolism being able to grow in chemoatotropy in extremely acidic environments in the presence of iron 33 , 3 g / L or copper 200-400 mM (9K-Fe-Cu, pH 1, 8) and in organography (Sabouraud agar and PDA at pH = 6.49 A. acidophilus - access number of the Genetic Resources Bank Regional Microbial Research
Quilamapu, Chillán, Chile, RGM 2451 tiene la función de potenciar el crecimiento de las bacterias ferrooxldantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2527 siendo mayor la tasa de crecimiento en T3 en biolixiviación estática (T2 = 0,045/día; T3=0, 048/día) y en blorreactor airlft, (T2 = 0,034/día; T3 = 0,037/día). En el consorcio bacteriano - en procesos de biolixiviación, se identificaron especies del género Acidithiobacillus y Acidiphilium. Quilamapu, Chillán, Chile, RGM 2451 has the function of promoting the growth of ferrooxaldante bacteria - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2527, with the highest rate of growth in T3 in bioleaching. static (T2 = 0.045 / day, T3 = 0.048 / day) and in airlft burearector, (T2 = 0.034 / day, T3 = 0.037 / day). In the bacterial consortium - in bioleaching processes, species of the genus Acidithiobacillus and Acidiphilium were identified.
Durante el proceso de biolixiviación estática (tratamientos T2 y T3) se recuperó la siguiente cantidad de cobre, en mg/L: 476,09 (T2 = 56,68%); 554 (T3 = 65,97%). En biolixiviación con blorreactor airlift, la cantidad en mg/L, fue: 9623,16 (T2 = 63,65 %) y 1 1 120,28 (T3 = 73,55%) respectivamente. De esta forma, el hongo filamentoso A. During the static bioleaching process (treatments T2 and T3), the following amount of copper was recovered, in mg / L: 476.09 (T2 = 56.68%); 554 (T3 = 65.97%). In bioleaching with airlift, the amount in mg / L was: 9623.16 (T2 = 63.65%) and 1120.28 (T3 = 73.55%) respectively. In this way, the filamentous fungus A.
HOJAS RECTIFICADAS (REGLA 91) acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , potencia el proceso de biolixiviación de calcopirita, en comparación a la biolixiviación bacteriana, siendo la primera Investigación que describe la utilidad de un hongo filamentoso acldotolerante en la biolixiviación de minerales sulfurados en medio Inorgánico. Se realizaron estudios con A. acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , para confirmar viabilidad, desarrollo y mejoras en procesos de extracción de metales. Para las dificultades mencionadas al comienzo de esta solicitud, no se han reportado soluciones de ningún tipo, menos aún el uso de la adición de hongos para mejorar la recuperación de cobre desde minerales sulfurados. Cabe destacar que los procesos de biolixiviación industrial son ecosistemas extremoacidófilos en un ambiente estrictamente inorgánico (aguas cloruradas, minerales inorgánicos y H2SO4). Sin embargo en otros sectores como medio ambiente - un sector diferente al sector minero, se aplican hongos en aplicaciones de biorremediación usando ecosistemas orgánicos, donde se ha usado hongos como Aspergillus niger y Penicillium Chrysogenum para extraer cobre presente en pasivos ambientales con una alta cantidad de materia orgánica disuelta. RECTIFIED LEAVES (RULE 91) acidophilus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Quilamapu, Chillán, Chile, RGM 2451, enhances the process of bioleaching of chalcopyrite, compared to bacterial bioleaching, being the first research that describes the usefulness of a fungus filamentous acldotolerante in the bioleaching of sulfurized minerals in Inorganic medium. Studies were carried out with A. acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, to confirm viability, development and improvements in metal extraction processes. For the difficulties mentioned at the beginning of this application, no solutions of any kind have been reported, let alone the use of the addition of fungi to improve the recovery of copper from sulphide minerals. It should be noted that the industrial bioleaching processes are extreme-hydrophilic ecosystems in a strictly inorganic environment (chlorinated waters, inorganic minerals and H 2 SO 4 ). However, in other sectors such as the environment - a sector different to the mining sector, mushrooms are applied in bioremediation applications using organic ecosystems, where fungi such as Aspergillus niger and Penicillium Chrysogenum have been used to extract copper present in environmental liabilities with a high amount of dissolved organic matter.
La presente invención propone entonces el uso de Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , como aditivo de biolixiviación ya que es capaz de crecer en un ecosistema minero industrial que dispone de un ambiente inorgánico, lo que le confiere una ventaja muy relevante sumado al hecho que permite el crecimiento más rápido de bacterias lixiviantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2527, las que oxidan rápidamente Hierro o azufre, y en The present invention then proposes the use of Acidomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, as a bioleaching additive since it is capable of growing in an industrial mining ecosystem that has of an inorganic environment, which gives it a very relevant advantage in addition to the fact that it allows the fastest growth of leaching bacteria - access number of the Regional Microbial Genetic Resources Bank of Research Quilamapu, Chillán, Chile, RGM 2527, those that oxidize quickly Iron or sulfur, and in
HOJAS RECTIFICADAS (REGLA 91) consecuencia, permiten recuperar más cobre en menos tiempo y en mayor cantidad con respecto a sistemas bacterianos que no usan este hongo. RECTIFIED LEAVES (RULE 91) consequently, they allow to recover more copper in less time and in greater quantity with respect to bacterial systems that do not use this fungus.
Breve Descripción de las Figuras Brief Description of the Figures
Figuras 1A y 1 B. Crecimiento de la cepa fúngica en medio de cultivo inorgánico y orgánico. Figura 1 A Crecimiento de la cepa fúngica en medio sólido orgánico Sabouraud y PDA a pH 6,49. Figura 1 B Crecimiento de la cepa fúngica en medio sólido inorgánico 9K-Fe pH 1 ,8; en presencia y ausencia de bacterias oxidantes de hierro. Figures 1A and 1B. Growth of the fungal strain in inorganic and organic culture medium. Figure 1 A Growth of the fungal strain in Sabouraud organic solid medium and PDA at pH 6.49. Figure 1 B Growth of the fungal strain in inorganic solid 9K-Fe medium pH 1.8; in the presence and absence of iron oxidizing bacteria.
Figuras 2A-2M. Morfología de la cepa fúngica. Figura 2A Colonia fúngica en medio sólido 9K-Fe, pH 1 ,8 después de 28 días de incubación a temperatura ambiente. Se observa un halo de degradación que rodea a la colonia. Figura 2E Fíalo de degradación observado en microscopía confocal. Se aprecia la presencia de bacterias oxidantes de hierro. Figura 2B Colonia fúngica en medio sólido 9K-Fe pH 1 ,8 después de 30 días de incubación a temperatura ambiente. Figura 2C y Figura 2D. Colonia fúngica en medio agar Sabouraud y PDA pH 6,49 después de 17 días. Figura 2F, Figura 2G, Figura 2H. Crecimiento de A. acidophilus en medio sólido agarosa 9K-Fe-Cu, pH= 1 ,8 aFigures 2A-2M. Morphology of the fungal strain. Figure 2A Fungal colony in solid 9K-Fe medium, pH 1.8 after 28 days of incubation at room temperature. A halo of degradation surrounding the colony is observed. Figure 2E Fíalo of degradation observed in confocal microscopy. The presence of iron oxidizing bacteria is appreciated. Figure 2B Fungal colony in solid 9K-Fe pH 1, 8 medium after 30 days of incubation at room temperature. Figure 2C and Figure 2D. Fungal colony in Sabouraud agar medium and PDA pH 6.49 after 17 days. Figure 2F, Figure 2G, Figure 2H. Growth of A. acidophilus in solid 9K-Fe-Cu agarose medium, pH = 1.8
0 mM, 200 mM y 400 mM Cu después de 40 días. Figura 2I Vista microscópicas (100X) de filamentos septados de A. acidophilus, con tinción de azul de metileno. Se observan células hinchadas intercalares y terminales (flechas rojas). Figura 2J Vista microscópica (100X) de las hifas septadas de A. acidophilus, con tinción de lugol. Figura 2K Filamentos de A. acidophilus HE17 de cultivos en medio líquido 9K-Fe, pH=1 ,8 en observado microscopía confocal. Figura 2L Imágenes en 3D de los filamentos de A. acidophilus observado en microscopía confocal de fluorescencia. Figura 2M Micrografía electrónica del filamento de A. acidophilus. 0 mM, 200 mM and 400 mM Cu after 40 days. Figure 2I Microscopic view (100X) of septate filaments of A. acidophilus, with methylene blue staining. Swollen intercalary and terminal cells (red arrows) are observed. Figure 2J Microscopic view (100X) of the septate hyphae of A. acidophilus, with lugol staining. Figure 2K Filaments of A. acidophilus HE17 from cultures in liquid medium 9K-Fe, pH = 1.8 in observed confocal microscopy. Figure 2L 3D images of the filaments of A. Acidophilus observed in fluorescence confocal microscopy. Figure 2M Electronic micrograph of the filament of A. acidophilus.
Figura 3. Análisis filogenético de Acidomyces acidophilus HE17 basado en las secuencias de la región ITS del rDNA 5.8S y de los dominios D1/D2 del rDNA 28S. La filogenia se reconstruyó utilizando la parsimonia Bootstrap, y el árbol se construyó usando máxima verosimilitud. La barra de escala indica 0,5 sustituciones por sitio. Se utilizó Teratosphaeria micromaculata como grupo raíz externo. Figure 3. Phylogenetic analysis of Acidomyces acidophilus HE17 based on the sequences of the ITS region of the 5.8S rDNA and the D1 / D2 domains of the 28S rDNA. The phylogeny was reconstructed using parsimony Bootstrap, and the tree was constructed using maximum likelihood. The scale bar indicates 0.5 substitutions per site. Teratosphaeria micromaculata was used as an external root group.
Figura 4. Crecimiento de Acidomyces acidophilus HE17 en presencia de cobre. Figura Figure 4. Growth of Acidomyces acidophilus HE17 in the presence of copper. Figure
4A Diámetro de la colonia fúngica durante 40 días de incubación en medio sólido 9K-Fe pH 1 ,8 suplementado con 0 mM (control negativo), 200 mM y 400 mM de CuS04x5Fl20. Figura 4B Peso seco de A. acidophilus HE17 medido en las tres condiciones evaluadas (0 mM, 200 mM y 400 mM de4A Diameter of the fungal colony during 40 days of incubation in solid medium 9K-Fe pH 1.8, supplemented with 0 mM (negative control), 200 mM and 400 mM of CuS0 4 x5Fl 2 0. Figure 4B Dry weight of A. acidophilus HE17 measured in the three conditions evaluated (0 mM, 200 mM and 400 mM
CUS04X5H20) en medio líquido 9K-Fe-Cu, pH 1 ,8. CUS0 4 X5H 2 0) in liquid medium 9K-Fe-Cu, pH 1.8.
Figuras 5A-5G. Arquitectura de las biopelículas de Acidomyces acidophilus HE17 en presencia de cobre. El hongo se creció en medio líquido 9K-Fe pH 1 ,8 suplementado con 0 mM (control negativo), 200 mM y 400 mM deFigures 5A-5G. Architecture of the biofilms of Acidomyces acidophilus HE17 in the presence of copper. The fungus was grown in liquid medium 9K-Fe pH 1, 8 supplemented with 0 mM (negative control), 200 mM and 400 mM
CUS04X5H20. A los 40 días de cultivo, las biopelículas se tiñeron con naranja de acridina para la visualización de las hifas mediante microscopía confocal. Figuras 5A, 5B y 5C Fotografías que muestran las biopelículas de A. acidophilus en medio líquido 9K-Fe pH 1 ,8; a las distintas concentraciones de CuS04x5H20. Figuras 5D, 5E y 5F imagen bidimensional de biopelículas de filamentos de A. acidophilus. Figuras 5G, 5H y 5I imagen tridimensional de biopelículas de filamentos de A. acidophilus. Figuras 5J, 5K y 5L Imagen en 3D de biopelículas de filamentos de A. acidophilus mostrando su espesor Barra: 40pm. CUS0 4 X5H 2 0. At 40 days of culture, the biofilms were stained with acridine orange for the visualization of the hyphae by confocal microscopy. Figures 5A, 5B and 5C Photographs showing the biofilms of A. acidophilus in liquid medium 9K-Fe pH 1.8; to the different concentrations of CuS0 4 x5H 2 0. Figures 5D, 5E and 5F bi-dimensional image of filaments biofilms of A. acidophilus. Figures 5G, 5H and 5I three-dimensional image of filament biofilms of A. Acidophilus Figures 5J, 5K and 5L 3D image of filament biofilms of A. acidophilus showing its thickness Bar: 40pm.
Figuras 6A-6H. Experimentos de biolixiviación estática (BE) y biolixiviación en biorreactor airlift (BA). Se utilizó una comunidad bacteriana oxidante de hierro con o sin la incorporación del hongo Addomyces acidophilus HE17, y se evaluó la recuperación de cobre a partir de calcopirita. Figura 6A Curva de crecimiento de bacterias oxidantes de hierro en BA. Figura 6B Curva de crecimiento de bacterias oxidantes de hierro en BE. Figura 6C Potencial redox (ORP) en BE. Figura 6D ORP en BA. Figura 6E Producción de hierro en BE. Figura 6F Producción de hierro en BA. Figura 6G Recuperación de cobre en BE. Figura 6H Recuperación de cobre en BA. Tratamientos (T): T1 (control negativo), medio 9K + calcopirita; T2, medio 9K + calcopirita + bacterias; T3, medio 9K + calcopirita + bacterias + A. acidophilus HE17; T4, medio 9K + calcopirita + A. acidophilus HE17. Figures 6A-6H. Experiments of static bioleaching (BE) and bioleaching in airlift bioreactor (BA). An iron oxidizing bacterial community was used with or without the incorporation of the fungus Addomyces acidophilus HE17, and recovery of copper from chalcopyrite was evaluated. Figure 6A Growth curve of iron oxidizing bacteria in BA. Figure 6B Growth curve of iron oxidizing bacteria in BE. Figure 6C Redox potential (ORP) in BE. Figure 6D ORP in BA. Figure 6E Iron production in BE. Figure 6F Production of iron in BA. Figure 6G Recovery of copper in BE. Figure 6H Recovery of copper in BA. Treatments (T): T1 (negative control), medium 9K + chalcopyrite; T2, medium 9K + chalcopyrite + bacteria; T3, medium 9K + chalcopyrite + bacteria + A. acidophilus HE17; T4, medium 9K + chalcopyrite + A. acidophilus HE17.
Figuras 7A y 7B. Caracterización del consorcio bacteriano presente en los experimentos de biolixiviación estática y biolixiviación en biorreactor airlift, mediante análisis del rDNA 16S. Figura 7A Gel de DGGE al 30-60% urea- formamida, en el cual se separaron los fragmentos del rDNA 16S amplificados por PCR. El gel se tiñó con GeIRed. Las bandas se denominaron C1 , C2 y C3. Figura 7B Árbol filogenético basado en las tres secuencias del rDNA 16S obtenidas del análisis por DGGE. El árbol se construyó mediante análisis Máximum Likelihood. Se muestran los valores de Bootstrap > 50% (100 réplicas utilizadas). La barra de escala indica 0,01 substituciones por sitio. Se utilizó Acidiphilium cryptum JF-5 como grupo raíz externo. Descripción Detallada de la Invención Figures 7A and 7B. Characterization of the bacterial consortium present in the experiments of static bioleaching and bioleaching in airlift bioreactor, by means of analysis of the 16S rDNA. Figure 7A DGGE Gel 30-60% urea-formamide, in which the fragments of the 16S rDNA amplified were separated by PCR. The gel was stained with GeIRed. The bands were designated C1, C2 and C3. Figure 7B Phylogenetic tree based on the three 16S rDNA sequences obtained from DGGE analysis. The tree was built by Maximum Likelihood analysis. The values of Bootstrap> 50% (100 replicas used) are shown. The scale bar indicates 0.01 substitutions per site. Acidiphilium cryptum JF-5 was used as an external root group. Detailed description of the invention
La biolixiviación de cobre y otros metales suele utilizar bacterias oxidantes de hierro y azufre, cuyo crecimiento se puede ver Inhibido por la presencia de metales pesados, materia orgánica disuelta, y otros. Esto afecta la eficiencia de recuperación del metal de Interés. Se ha visto que en blorremedlaclon en medio orgánico ciertas especies de hongos contribuyen a mejorar la eficiencia de recuperación de metales y a contrarrestar la Inhibición del crecimiento bacteriano. Sin embargo, no se reportan estudios sobre el proceso de biolixiviación con hongos en medios Inorgánicos. La presente Invención proporciona como aditivo a la biolixiviación, un hongo filamentoso proveniente de un drenaje ácido de mina. Mediante secuenclaclón del rDNA 5.8S y de la reglón ITS, se determinó que el hongo correspondía a Acidomyces acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chlllán, Chile, RGM 2451 . Se demostró la capacidad de este hongo para promover la recuperación de cobre a partir de calcopirita, en biolixiviación estática (BE) y biolixiviación en blorreactor airlift ( BA). Para estos experimentos se utilizó un consorcio bacteriano- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chlllán, Chile, RGM 2527, que mediante secuenclaclón del rDNA 16S, se determinó que estaba formado por las especies Acidithiobacillus ferridurans straln YNTR1 -41 , Acidithiobacillus ferrooxidans straln HBDY3-51 y Acidiphilium sp. straln MPLK-613, entre otras especies que la técnica usada no permitió detectar. Tanto en los experimentos de BE como de BA, la presencia A. acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chlllán, Chile, RGM 2451 , provocó un aumento en la recuperación de cobre, en el potencial redox oxldatlvo, y en la cantidad de hierro disuelto.  The bioleaching of copper and other metals usually uses oxidizing iron and sulfur bacteria, whose growth can be seen inhibited by the presence of heavy metals, dissolved organic matter, and others. This affects the recovery efficiency of the metal of interest. It has been seen that in blorremedlaclon in organic medium certain species of fungi contribute to improve the efficiency of recovery of metals and to counteract the inhibition of bacterial growth. However, no studies are reported on the process of bioleaching with fungi in Inorganic media. The present invention provides as an additive to bioleaching, a filamentous fungus from an acid mine drainage. By sequencing the 5.8S rDNA and the ITS region, it was determined that the fungus corresponded to Acidomyces acidophilus HE17- access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chlllán, Chile, RGM 2451. The ability of this fungus to promote the recovery of copper from chalcopyrite, in static bioleaching (BE) and bioleaching in airlift (BA) blorreactor was demonstrated. For these experiments, a bacterial consortium-access number of the Regional Microbial Genetic Resources Bank of Qullamapu Research, Chlllán, Chile, RGM 2527, was used. By sequencing the 16S rDNA, it was determined that it was formed by the species Acidithiobacillus ferridurans straln YNTR1 - 41, Acidithiobacillus ferrooxidans straln HBDY3-51 and Acidiphilium sp. straln MPLK-613, among other species that the technique used did not detect. In both the BE and BA experiments, the presence of A. acidophilus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chlllán, Chile, RGM 2451, caused an increase in copper recovery, in the potential oxidized redox, and in the amount of dissolved iron.
HOJAS RECTIFICADAS (REGLA 91) Así, la presente invención demuestra la utilidad de un hongo filamentoso acidotolerante antes mencionado en la biolixiviación de minerales sulfurados en medio inorgánico. La presente Invención proporciona un método para biolixiviar minerales sulfurados a partir de un consorcio de microorganismos que comprende bacterias ferroxldantes, preferentemente, bacterias ferroxldans que se encuentran en el mineral sulfurado; y además el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2451 , en un medio Inorgánico libre de sulfato ferroso y pH < 2, de preferencia, pH = 8, favoreciendo el crecimiento de los microorganismos lixiviantes y aumentando la extracción del metal desde el mineral. donde las bacterias se seleccionan preferentemente de bacterias del género Acidithiobacillus, Acidiphilium o una combinación de las mismas. Preferentemente, dichas bacterias se seleccionan del grupo consistente de Acidithiobacillus ferridurans cepa YNTR1 -41 , Acidiphilium sp. cepa MPLK-613, Acidithiobacillus ferrooxidans cepa HBDY3-5, o una combinación de las mismas. Ejemplos RECTIFIED LEAVES (RULE 91) Thus, the present invention demonstrates the utility of an aforementioned acidotolerant filamentous fungus in the bioleaching of sulfurized minerals in inorganic medium. The present invention provides a method for bio-lixivizing sulphided minerals from a consortium of microorganisms comprising ferroxldante bacteria, preferably, ferroxldans bacteria found in the sulphide mineral; and also the fungus Acidomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Bank of Research Qullamapu, Chillán, Chile, RGM 2451, in an Inorganic medium free of ferrous sulfate and pH <2, preferably, pH = 8, favoring the growth of leaching microorganisms and increasing the extraction of the metal from the ore. wherein the bacteria are preferably selected from bacteria of the genus Acidithiobacillus, Acidiphilium or a combination thereof. Preferably, said bacteria are selected from the group consisting of Acidithiobacillus ferridurans strain YNTR1 -41, Acidiphilium sp. strain MPLK-613, Acidithiobacillus ferrooxidans strain HBDY3-5, or a combination thereof. Examples
Ejemplo 1 : Metodología para aislar la cepa fúngica y del consorcio bacterianoExample 1: Methodology to isolate the fungal strain and the bacterial consortium
La cepa fúngica se aisló a partir de un cultivo microbiano proveniente de un drenaje ácido de mina. Para aislar la cepa fúngica y caracterizar su crecimiento, se cultivó en cuatro medios de cultivo distintos, a temperatura ambiente: Primer Medio de Cultivo, cultivo (I): medio 9K líquido suplementado con FeS04 (9K-Fe; 0,4 g MgS04 x7H20; 0,04 g K2HP04; 0,1 g NH4S04; 33,33 g FeS04 x 7H20 por litro, pH 1 ,8). Segundo Medio de Cultivo, cultivo (¡I): 9K-Fe sólido (10 g NH4S04; 1 ,5 g MgS04x 7H20; 0,5 g The fungal strain was isolated from a microbial culture from an acid mine drainage. To isolate the fungal strain and characterize its growth, it was cultivated in four different culture media, at room temperature: First Culture Medium, culture (I): liquid 9K medium supplemented with FeS0 4 (9K-Fe, 0.4 g MgS0 4 x7H 2 0, 0.04 g K 2 HP0 4 , 0.1 g NH 4 S0 4 , 33.33 g FeS0 4 x 7H 2 0 per liter, pH 1, 8). Second Culture Medium, culture (I): Solid 9K-Fe (10 g NH 4 S0 4 ; 1.5 g MgSO 4 x 7H 2 0; 0.5 g
HOJAS RECTIFICADAS (REGLA 91) K2HP04; 10 g agarosa y 33,3 g FeSC h^O por litro, pH 1 ,8). Tercer Medio de Cultivo, cultivo (iii): agar Sabouraud (10 g peptona de caseína; 40 g glucosa; 12 g agar por litro, pH 6,4), y Cuarto Medio de Cultivo, cultivo (iv): agar papa dextrosa (PDA, 250 g de trozos de papa; 20 g glucosa; 20 g agar agar por litro, pH=6,49). RECTIFIED LEAVES (RULE 91) K 2 HP0 4 ; 10 g agarose and 33.3 g FeSC h ^ O per liter, pH 1.8). Third Culture Medium, culture (iii): Sabouraud agar (10 g casein peptone, 40 g glucose, 12 g agar per liter, pH 6.4), and Fourth Medium Culture, culture (iv): potato dextrose agar ( PDA, 250 g of potato pieces, 20 g glucose, 20 g agar agar per liter, pH = 6.49).
La comunidad bacteriana a utilizar en los experimentos de biolixiviación se obtuvo a partir de una muestra de calcopirita proveniente de una mina, la cual fue inoculada (10 %, 106 cel/ml) en medio 9K líquido sin la adición de FeS04*7H20, pH 1 ,8, Temperatura 20 ^2eC. El crecimiento bacteriano se evaluó cada 7 días mediante recuento celular en una cámara de Petroff-Hausser. The bacterial community to be used in the bioleaching experiments was obtained from a sample of chalcopyrite from a mine, which was inoculated (10%, 10 6 cel / ml) in liquid 9K medium without the addition of FeS0 4 * 7H 2 0, pH 1, 8, Temperature 20 ^ 2 e C. Bacterial growth was evaluated every 7 days by cell counting in a Petroff-Hausser chamber.
Ejemplo 2: Composición del mineral utilizado Example 2: Composition of the mineral used
El mineral se pulverizó hasta un tamaño de partícula de 38 a 150 pm, utilizando un pulverizador (Rocklabs, Auckland, New Zealand) con tamiz Ne 100 y 400, esterilizándolo antes de la experimentación. El análisis mineralógico se realizó con un difractómetro de rayos X Siemens D5000 (Siemens, Munich, Germany), mediante el cual se comprobó que el mineral correspondía a sulfuro de cobre, determinándose el siguiente contenido: 80,18% calcopirita (CuFeS); 4,52% trolita (FeS); 6,61 % covelita (CuS); 1 ,15% magnetita (Fe203); y 7,54% cuarzo (Si02). The ore was pulverized to a particle size of 38 to 150 μm, using a spray (Rocklabs, Auckland, New Zealand) with sieve N e 100 and 400, sterilizing it before experimentation. The mineralogical analysis was carried out with a Siemens D5000 X-ray diffractometer (Siemens, Munich, Germany), whereby the mineral was found to correspond to copper sulphide, the following content being determined: 80.18% chalcopyrite (CuFeS); 4.52% trolita (FeS); 6.61% covelite (CuS); 1, 15% magnetite (Fe 2 0 3 ); and 7.54% quartz (Si0 2 ).
Ejemplo 3: Extracción de DNA genómico y amplificación de DNA ribosomal bacteriano y fúngico Example 3: Extraction of genomic DNA and amplification of bacterial and fungal ribosomal DNA
El DNA genómico bacteriano se extrajo de cuatro muestras líquidas de procesos de biolixiviación a partir de 1 mL en medio 9K mas calcopirita después de 68 días y el DNA genómico de hongos se extrajo directamente de cultivo de microorganismos lixiviantes a partir de 1 mL de medio 9K-Fe, pH=1 ,8 después de 60 días, realizando la purificación con el PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) de acuerdo a las instrucciones del fabricante. El DNA purificado se cuantificó en un espectrofotómetro Epoch (Bio-Tek Instruments Inc, Winooski, VT, USA), y su pureza se comprobó mediante la relación de absorbancia a 260 nm y a 280 nmBacterial genomic DNA was extracted from four liquid samples of bioleaching processes from 1 mL in 9K medium plus chalcopyrite after 68 days and fungal genomic DNA was extracted directly from culture of leaching microorganisms from 1 mL of 9K medium -Fe, pH = 1, 8 after 60 days, performing the purification with the PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions. The purified DNA was quantified in an Epoch spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA), and its purity was checked by the absorbance ratio at 260 nm and 280 nm
(A260/280) · Para la identificación de especies bacterianas, se amplificó el DNA ribosomal (rDNA) 16S utilizando los oligonucleótidos universales 1492R/27F y el kit de PCR GoTaq® Green Master Mix (Promega, Madison, Wl, USA). El programa de amplificación fue el siguiente: una desnaturalización inicial a 94°C por 5 min, seguida de 35 ciclos a 94°C por 45 s, 57°C por 45 s, y 72°C por 1 min 30 s; con una extensión final a 72eC por 5 min. (A260 / 280) · For the identification of bacterial species, the 16S ribosomal DNA (rDNA) was amplified using the universal oligonucleotides 1492R / 27F and the GoTaq® Green Master Mix PCR kit (Promega, Madison, Wl, USA). The amplification program was as follows: initial denaturation at 94 ° C for 5 min, followed by 35 cycles at 94 ° C for 45 s, 57 ° C for 45 s, and 72 ° C for 1 min 30 s; with a final extension at 72 e C for 5 min.
Para la identificación de la cepa fúngica se amplificó la región del espaciador transcrito interno (ITS) del rDNA 5.8S, utilizando los oligonucleótidos ITS1 e ITS4 (White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: M. A. lnnis,D. H. Gelfand, J. J. Sninsky, & T. J. White (ed.), PCR Protocols: A Guide to the Methods and Applications. New York: Academic Press. 315-322); y los dominios variables D1 -D2 del rDNA 28S, utilizando los oligonucleótidos NL1 y NL4 (O’ Donnell K. 1993. Fusarium and its near relatives. In: D. R. Reynolds J. W. Taylor (eds), The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Wallingford: CAB International: 225- 233). La reacción de amplificación contuvo 13,2 pL de agua miliQ; 5 pL de Buffer PCR; 2 pL de MgCI2; 0,5 pL de dNTPs (Kapa Biosystems, Wilmington, MA, USA), 1 pL de cada oligonucleótido; 0,25 pL GoTaq DNA Polymerase kit reagent (Promega/USA) y 2 pL de templado. El programa de amplificación fue el siguiente: una desnaturalización inicial a 94°C por 5 min, seguida de 35 ciclos a 95eC por 30 s, 55°C por 1 min, y 72°C por 1 min. Los productos de PCR fueron visualizados en un gel de agarosa al 1 %, y se purificaron con el lllustra GFX PCR DNA and Gel Band Purification Kit (General Electric Healthcare, Buckinghamshire, UK). Se almacenaron a -20°C. Electroforesis en gel con gradiente desnaturalizante (DGGE). For the identification of the fungal strain, the region of the internal transcribed spacer (ITS) of the 5.8S rDNA was amplified, using the oligonucleotides ITS1 and ITS4 (White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: MA lnnis, DH Gelfand, JJ Sninsky, & TJ White (ed.), PCR Protocols: A Guide to the Methods and Applications, New York: Academic Press, 315-322); and the variable D1-D2 domains of 28S rDNA, using oligonucleotides NL1 and NL4 (O'Donnell K. 1993. Fusarium and its near relatives., In: DR Reynolds JW Taylor (eds), The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics, Wallingford: CAB International: 225-233). The amplification reaction contained 13.2 pL of milliQ water; 5 pL Buffer PCR; 2 pL MgCl 2 ; 0.5 pL of dNTPs (Kapa Biosystems, Wilmington, MA, USA), 1 pL of each oligonucleotide; 0.25 pL GoTaq DNA Polymerase kit reagent (Promega / USA) and 2 pL tempering. The amplification program was as follows: initial denaturation at 94 ° C for 5 min, followed by 35 cycles at 95 C for 30 s and 55 ° C for 1 min, and 72 ° C for 1 min. The PCR products were visualized on a 1% agarose gel, and were purified with the GFX PCR DNA and Gel Band Purification Kit (General Electric Healthcare, Buckinghamshire, UK). They were stored at -20 ° C. Gel electrophoresis with denaturing gradient (DGGE).
Los fragmentos amplificados del rDNA 16S se utilizaron como templados en una nueva amplificación con los oligonucleótidos específicos para bacterias 341 F-GC y 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164, 165-171 ). Se utilizó el siguiente programa: una desnaturalización inicial de 94°C por 5 min, seguida por 35 ciclos a 94°C por 30 s, 50°C por 45 s, y 72°C por 1 min, con una extensión final de 72°C por 3 min. Los productos resultantes de PCR se sometieron a DGGE, basándose en el protocolo descrito por Demergasso, C., Galleguillos, P., Escudero, L., Zepeda, A., Castillo, D., Casamayor, E. 2005. Molecular characterization of microbial populations in a low- grade copper ore bioleaching test heap. Hydrometallurgy. 80, 241 -253. Brevemente, se cargaron los productos en un gel de poliacrilamida al 6% conteniendo un gradiente desnaturalizante de 30-60% urea-formamida (el 100% del gradiente se definió como 7 M urea y 40% formamida). El gel se corrió en una cámara de electroforesis BioRad D Gene (BioRad, Hercules, CA, USA) a 60 °C y 100 V durante 7 h. Posteriormente, el gel se tiñó en una solución de 0,5% GeIRed (Biotium, Fremont, CA, USA) durante 1 h en oscuridad, y las bandas se visualizaron bajo luz UV en transiluminador. Las bandas se cortaron y purificaron usando E.Z.N.A™ Gel Extraction Kit (OMEGA), para luego re- amplificar los productos de PCR con los partidores 341 F-GC/907R, bajo las mismas condiciones del PCR mencionado anteriormente, visualizándose en gel de agarosa al The amplified fragments of the 16S rDNA were used as annealed in a new amplification with the specific oligonucleotides for bacteria 341 F-GC and 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments Arch. Microbiol. 164, 165-171). The following program was used: an initial denaturation of 94 ° C for 5 min, followed by 35 cycles at 94 ° C for 30 s, 50 ° C for 45 s, and 72 ° C for 1 min, with a final extension of 72 ° C for 3 min. The resulting PCR products were subjected to DGGE, based on the protocol described by Demergasso, C., Galleguillos, P., Escudero, L., Zepeda, A., Castillo, D., Casamayor, E. 2005. Molecular characterization of microbial populations in a low-grade copper ore bioleaching test heap. Hydrometallurgy. 80, 241-253. Briefly, the products were loaded on a 6% polyacrylamide gel containing a denaturing gradient of 30-60% urea-formamide (100% of the gradient was defined as 7 M urea and 40% formamide). The gel was run in a BioRad D Gene electrophoresis chamber (BioRad, Hercules, CA, USA) at 60 ° C and 100 V for 7 h. Subsequently, the gel was stained in a 0.5% GeIRed solution (Biotium, Fremont, CA, USA) for 1 h in darkness, and the bands were visualized under UV light in transilluminator. The bands were cut and purified using the E.Z.N.A.TM. Gel Extraction Kit (OMEGA), to then re-amplify the PCR products with the 341 F-GC / 907R primers, under the same conditions of the aforementioned PCR, visualizing in agarose gel the
1 %. Secuenciación de los productos de PCR one %. Sequencing of PCR products
Los productos de PCR bacterianos fueron secuenciados por Macrogen, Korea. Las secuencias se editaron manualmente utilizando el programa ChromasPro 2.1 (Technelysium Pty Ltd, Brisbane, Australia), y se compararon con la base de datos GenBank (http://blast.ncbi.nlm.nih.gov), usando el algoritmo BLASTn (- Altschul, S.F., Gish, W., Miller, W., Myers, E.W. 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403-410). Posteriormente se realizó el alineamiento de las secuencias mediante la herramienta MUSCLE (- Edgar, R. C. 2004. MUSCLE: múltiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797). El árbol filogenético se construyó usando el modelo estadístico Máximum Likelihood y el test de robustez Bootstrap, utilizando el programa MEGA7 (Tamura K.; Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 201 1 . MEGA5: Molecular evolutionary genetics analysis using máximum likelihood, evolutionary distance, and máximum parsimony methods. Mol. Biol. Evol. 28, 2731 -2739).  The bacterial PCR products were sequenced by Macrogen, Korea. The sequences were manually edited using the ChromasPro 2.1 program (Technelysium Pty Ltd, Brisbane, Australia), and compared with the GenBank database (http://blast.ncbi.nlm.nih.gov), using the BLASTn algorithm ( - Altschul, SF, Gish, W., Miller, W., Myers, EW 1990. Basic local alignment search tool, J. Mol. Biol. 215, 403-410). Subsequently, the sequences were aligned using the MUSCLE tool (- Edgar, R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32, 1792-1797). The phylogenetic tree was constructed using the statistical model Maximum Likelihood and the Bootstrap robustness test, using the program MEGA7 (Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 201 1. MEGA5 : Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol. 28, 2731 -2739).
Los productos de PCR fúngicos se secuenciaron utilizando el BigDye® Terminator v3.1 Cycle Sequencing Kit's robust, y el ABI PRISM 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Se usaron los mismos oligonucleótidos utilizados en la amplificación inicial de los rDNAs 5.8S y 18S. Las secuencias consenso se obtuvieron utilizando los programas AutoAssembler (Thermo Fisher Scientific, Waltham, MA, USA) y Lasergene Seqman (Dnastar, Madison, Wl, USA). The fungal PCR products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit's robust, and the ABI PRISM 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The same oligonucleotides used in the initial amplification of the 5.8S and 18S rDNAs were used. Consensus sequences were obtained using the AutoAssembler programs (Thermo Fisher Scientific, Waltham, MA, USA) and Lasergene Seqman (Dnastar, Madison, Wl, USA).
Ejemplo 4: Tolerancia de Acidomyces acidophilus HE17 al cobre Example 4: Tolerance of Acidomyces acidophilus HE17 to copper
Los ensayos de tolerancia al cobre se realizaron en medio 9K-Fe sólido y líquido pH 1 ,8, suplementado con 0, 200 y 400 mM de CuS04*5H20 (9K-Fe-Cu) (Remonsellez,The copper tolerance tests were carried out in solid 9K-Fe medium and liquid pH 1.8, supplemented with 0, 200 and 400 mM CuS0 4 * 5H 2 0 (9K-Fe-Cu) (Remonsellez,
F., Orell, A., Jerez, C.A. 2006. Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology. 152, 59-66; Novo, M.T.M., Da Silva A.C., Moreto, R., Cabral, P.C. P., Costacurta, A., García, O.J., Ottoboni, L.M.M. 2000. Thiobacillus ferrooxidans response to copper and other heavy metáis: growth, protein synthesis and protein phosphorylation, Antonie van Leeuwenhoek, 77, 187-95). El efecto inhibitorio del cobre sobre el crecimiento del hongo se determinó midiendo el diámetro de la colonia (en medio sólido), y cuantificando la diferencia de masa seca al principio y al final del experimento (en medio líquido). F., Orell, A., Jerez, CA 2006. Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 152, 59-66; Novo, MTM, Da Silva AC, Moreto, R., Cabral, PCP, Costacurta, A. García, OJ, Ottoboni, LMM 2000. Thiobacillus ferrooxidans response to copper and other heavy metais: growth, protein synthesis and protein phosphorylation, Antonie van Leeuwenhoek, 77, 187-95). The inhibitory effect of copper on the growth of the fungus was determined by measuring the diameter of the colony (in solid medium), and quantifying the difference of dry mass at the beginning and end of the experiment (in liquid medium).
Ejemplo 5: Metodología Experimentos de Biolixiviación Estática (BE) y Biolixiviación en Biorreactor Airlift (BA). Example 5: Methodology Experiments of Static Bioleaching (BE) and Bioleaching in Airlift Bioreactor (BA).
La biolixiviación estática (BE) se llevó a cabo en matraces Erlenmeyer de 250 mL conteniendo 100 mL de medio salino 9K sin FeS04x7H20 y concentrado de calcopirita a una densidad de pulpa del 4%. La biolixiviación en biorreactor airlift (BA) se realizó en un tubo concéntrico de 15 cm de altura, con un diámetro inferior de 9,5 cm y un diámetro superior de 15 cm, el cual posee un compresor de aire electromagnético (modelo ACQ-001 , Boyu, China) con un caudal de aire de 5 L/min (Fig. 1 ). Para BA se usaron 1 ,8 L de medio salino 9K sin FeS04x7H20 y a la misma densidad de pulpa que en BE. Static bioleaching (BE) was carried out in 250 mL Erlenmeyer flasks containing 100 mL of 9K saline medium without FeS0 4 x7H 2 0 and chalcopyrite concentrate at a pulp density of 4%. Bioleaching bioreactor airlift (BA) was performed in a concentric tube 15 cm high, with a lower diameter of 9.5 cm and an upper diameter of 15 cm, which has an electromagnetic air compressor (model ACQ-001 , Boyu, China) with an air flow of 5 L / min (Fig. 1). For BA, 1.8 L of 9K saline medium without FeS0 4 x7H 2 0 and the same pulp density as in BE were used.
En cada uno de los procesos de biolixiviación se analizaron los siguientes tratamientos (T): T1 (control negativo), medio 9K + calcopirita; T2, medio 9K + calcopirita + consorcio bacteriano; T3, medio 9K + calcopirita + consorcio bacteriano + A. acidophilus·, T4, medio 9K + calcopirita + A. acidophilus. En T2 y T3 se puso un inoculo inicial de 106 bacterias/mL. En T3 y T4 se puso un inoculo inicial medido en equivalencia a biomasa seca de 0,025 mg/pL de A. acidophilus. In each of the bioleaching processes, the following treatments were analyzed (T): T1 (negative control), medium 9K + chalcopyrite; T2, medium 9K + chalcopyrite + bacterial consortium; T3, medium 9K + chalcopyrite + bacterial consortium + A. acidophilus ·, T4, medium 9K + chalcopyrite + A. acidophilus. In T2 and T3, an initial inoculum of 10 6 bacteria / mL was placed. In T3 and T4 an initial inoculum measured in equivalence to dry biomass of 0.025 mg / pL of A. acidophilus was placed.
Cada tratamiento se evaluó por triplicado y se monitoreó durante 68 días. El potencial redox (ORP) de cada tratamiento se midió cada dos días, utilizando un medidor ORP Meter modelo Hl 9126 (Hanna Instruments, Woonsocket, Rl, USA). La concentración de cobre y hierro se analizó con un espectrofotómetro de absorción atómica con llama modelo Avanta P (GBC Scientific Equipment, Melbourne, Australia). Each treatment was evaluated in triplicate and monitored for 68 days. The redox potential (ORP) of each treatment was measured every two days, using an ORP Meter meter model Hl 9126 (Hanna Instruments, Woonsocket, Rl, USA). Concentration of copper and iron was analyzed with an atomic absorption spectrophotometer with flame model Avanta P (GBC Scientific Equipment, Melbourne, Australia).
Ejemplo 6: Metodología para la medición de biopelículas de A. acidophilus y visualización de su estructura celular. Example 6: Methodology for the measurement of biofilms of A. acidophilus and visualization of its cellular structure.
La formación de biopelículas de A. acidophilus se analizó en cultivos en medio 9K-Fe- Cu líquido con 0, 200 y 400 mM de CuS04x5H20 y en los experimentos de BA (T3 y T4). En estos últimos, una vez finalizado el experimento, se midió el espesor de la biopelícula del hongo y se obtuvo una muestra de filamentos. Estos se montaron en una placa de Petri, se colectaron con portaobjetos, se tiñeron con naranja de acridina, y se observaron en microscopio confocal. Los filamentos también se analizaron en un microscopio electrónico de barrido (7582, serie A3038-5350-TV3-1841 , England), para lo cual se montaron con fijador marca Dako. La estructura celular del hongo se estudió a partir de muestras de medio de cultivo 9K-Fe, PDA y Sabouraud, las cuales se visualizaron en un microscopio confocal CS SP8 invertido, con una unidad de alimentación compacta de láser de Argón a 488 nm y objetivos de 63X con aceite de inmersión (Leica, Wetzlar, Germany). The formation of biofilms of A. acidophilus was analyzed in cultures in liquid 9K-Fe-Cu medium with 0, 200 and 400 mM CuS0 4 x5H 2 0 and in the BA experiments (T3 and T4). In the latter, once the experiment was finished, the thickness of the biofilm of the fungus was measured and a sample of filaments was obtained. These were mounted on a Petri dish, collected with slides, stained with acridine orange, and observed in a confocal microscope. The filaments were also analyzed in a scanning electron microscope (7582, series A3038-5350-TV3-1841, England), for which they were mounted with Dako brand fixative. The cellular structure of the fungus was studied from samples of culture medium 9K-Fe, PDA and Sabouraud, which were visualized in an inverted CS SP8 confocal microscope, with a compact Argon laser feeding unit at 488 nm and targets of 63X with immersion oil (Leica, Wetzlar, Germany).
Las diferencias en la recuperación de cobre, concentración de hierro y ORP en los experimentos de biolixiviación se analizaron mediante ANOVA de dos vías, utilizando los softwares SPSS v. 20 y Sigma plot v. 1 1 .  The differences in copper recovery, iron concentration and ORP in the bioleaching experiments were analyzed by means of two-way ANOVA, using the SPSS v. 20 and Sigma plot v. eleven .
Ejemplo 7: Crecimiento de la cepa fúngica Example 7: Growth of the fungal strain
Para evaluar los requerimientos nutricionales de la cepa fúngica, se comparó su crecimiento en medio inorgánico sólido 9K-Fe pH 1 ,8 con medio orgánico Sabouraud y PDA pH 6,49 (Tabla 1 ; Fig. 1 A). El mayor crecimiento se obtuvo en medio Sabouraud, en el cual la colonia fúngica obtuvo un diámetro máximo de 34 mm, mientras que en medio sólido 9K-Fe y PDA el diámetro alcanzó 37 mm y 21 ,67 mm, respectivamente. Estos resultados muestran que, si bien la cepa fúngica crece mejor en presencia de sustratos orgánicos, es capaz de crecer en un medio inorgánico y a pH ácido, lo cual indica que se trata de un hongo acidotolerante y mixótrofo. To evaluate the nutritional requirements of the fungal strain, its growth was compared in solid inorganic medium 9K-Fe pH 1.8 with Sabouraud organic medium and PDA pH 6.49 (Table 1, Fig. 1A). The highest growth was obtained in Sabouraud medium, in which the fungal colony obtained a maximum diameter of 34 mm, while in solid 9K-Fe and PDA the diameter reached 37 mm and 21, 67 mm, respectively. These results show that, although the fungal strain grows better in the presence of organic substrates, is able to grow in an inorganic medium and acidic pH, which indicates that it is an acidotolerant and mixotrophic fungus.
Tabla 1  Table 1
Figure imgf000026_0001
Figure imgf000026_0001
A continuación se evaluó si la presencia de bacterias oxidantes de hierro favorece el crecimiento de la cepa fúngica en el medio sólido 9K-Fe pH 1 ,8. La cepa se sembró en presencia y ausencia de bacterias, y se realizó un seguimiento de su crecimiento por 45 días. La colonia fúngica se hizo visible al séptimo día de cultivo, y su crecimiento fue mayor en presencia de bacterias, alcanzando un diámetro máximo de 57,5 mm; 1 ,55 veces más que en ausencia de bacterias (Tabla 2 y Fig. 1 B). Next, it was evaluated whether the presence of iron oxidizing bacteria favors the growth of the fungal strain in the solid 9K-Fe pH 1, 8 medium. The strain was planted in presence and absence of bacteria, and their growth was monitored for 45 days. The fungal colony became visible on the seventh day of culture, and its growth was greater in the presence of bacteria, reaching a maximum diameter of 57.5 mm; 1, 55 times more than in the absence of bacteria (Table 2 and Fig. 1 B).
Tabla 2.  Table 2
Figure imgf000027_0001
Ejemplo 8: Caracterización morfológica de la cepa fúngica
Figure imgf000027_0001
Example 8: Morphological characterization of the fungal strain
El análisis de la colonia fúngica por microscopía óptica mostró una morfología similar en medio sólido 9K-Fe, agar Sabouraud y PDA (Fig. 2A-D). En medio sólido 9K-Fe, la colonia fúngica creció de color blanco, con un crecimiento rápido del micelio, y presentó un halo de degradación de materia orgánica (Fig. 2A). Mediante microscopía confocal se pudo apreciar que este halo favoreció el crecimiento de bacterias productoras de ácido sulfúrico (Fig. 2E). En otros cultivos en medio sólido 9K-Fe no se observó halo de degradación, sólo colonias de color blanco (Fig. 2B). En medio Sabouraud y PDA las colonias crecieron de color negro (Fig. 2C, D).  The analysis of the fungal colony by optical microscopy showed a similar morphology in solid 9K-Fe medium, Sabouraud agar and PDA (Fig. 2A-D). In solid 9K-Fe medium, the fungal colony grew white, with rapid mycelial growth, and presented a halo of organic matter degradation (Fig. 2A). By confocal microscopy it was possible to appreciate that this halo favored the growth of sulfuric acid producing bacteria (Fig. 2E). In other cultures in solid 9K-Fe medium no halo of degradation was observed, only white colonies (Fig. 2B). In the middle Sabouraud and PDA the colonies grew black (Fig. 2C, D).
Un análisis microscópico más detallado mostró células hinchadas intercaladamente. A more detailed microscopic analysis showed swollen cells intercalately.
La presencia de filamentos de A. acidophilus se estudió por microscopía óptica con tinción de azul de metileno (Fig. 21) y lugol (Fig. 2J); y por microscopía confocal con tinción de naranja de acridina se observaron los filamentos de A. acidophilus de cultivos en medio liquido 9K.Fe, pH=1 ,8 (Fig. 2K), en imagen en 3D se observa la medida de los filamentos de A. acidophilus con longitud de 76-192 pm y un diámetro de 2-6 pm (Fig. 2L). The presence of filaments of A. acidophilus was studied by optical microscopy with methylene blue stain (Fig. 21) and lugol (Fig. 2J); and by confocal microscopy with acridine orange stain the filaments of A. acidophilus were observed from cultures in liquid medium 9K.Fe, pH = 1, 8 (Fig. 2K), in 3D image the measurement of the filaments of A. acidophilus with a length of 76-192 pm and a diameter of 2-6 pm (Fig. 2L).
La figura 2M muestra la estructura de un filamento de A. acidophilus en medio liquido 9K-Fe, pH=1 ,8 visualizado en microscopía electrónica de barrido. Figure 2M shows the structure of a filament of A. acidophilus in liquid 9K-Fe medium, pH = 1.8, visualized in scanning electron microscopy.
Ejemplo 9: Análisis filogenético de la cepa fúngica Example 9: Phylogenetic analysis of the fungal strain
Para identificar a qué especie pertenece la cepa fúngica aislada, se extrajo DNA genómico y se amplificaron los rDNAs 5.8S y 28S, utilizando los oligonucleótidos ITS1/ITS4 y NL1/NL4, respectivamente. La secuenciación de los productos de PCR reveló que el hongo corresponde a Addomyces acidophilus HE17— número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , perteneciente a la división Ascomycota. El árbol filogenético con soporte de bootstrap bajos mostró esta cepa como una especie distinta, agrupada con Mycospharaella sp. CPW22515 (DQ632682.1 ), Acidomyces acidophilus strain MH934 (JQ172742.1 ), Acidomyces acidothermus strain MH1 101 (JQ172743.1 ) yTo identify which species the isolated fungal strain belongs to, genomic DNA was extracted and the 5.8S and 28S rDNAs were amplified, using the oligonucleotides ITS1 / ITS4 and NL1 / NL4, respectively. Sequencing of the PCR products revealed that the fungus corresponds to Addomyces acidophilus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Quilamapu, Chillán, Chile, RGM 2451, belonging to the Ascomycota division. The phylogenetic tree with low bootstrap support showed this strain as a distinct species, grouped with Mycospharaella sp. CPW22515 (DQ632682.1), Acidomyces acidophilus strain MH934 (JQ172742.1), Acidomyces acidothermus strain MH1 101 (JQ172743.1) and
Penidiella tenuiramis strain CBS124993 (KF442523.1 ) (Figura 3). Penidiella tenuiramis strain CBS124993 (KF442523.1) (Figure 3).
Ejemplo 10: Crecimiento y formación de biopelículas de A. acidophilus HE17 en presencia de cobre. Example 10: Growth and formation of biofilms of A. acidophilus HE17 in the presence of copper.
Para determinar si la presencia de cobre en el medio afecta el crecimiento de A. acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , se analizó el diámetro de la colonia fúngica durante 40 días de cultivo en medio sólido 9K-Fe (control negativo) y 9K-Fe-Cu, con 200 o 400 mM de CuS04*5H20. El mayor crecimiento del hongo se dio en el control negativo, con un diámetro de 60,8 mm ; mientras que a 200 mM de Cu el diámetro se redujo a 33,8 mm; y a 400 nM de Cu se redujo a 20,5 mm (Fig. 4A). Adlclonalmente, a los 40 días, en medio liquido 9K-Fe-Cu, pH=1 ,8 se cuantificó el peso seco del hongo en las tres condiciones evaluadas, el cual fue 18,9 mg/100 mL en el control negativo, de 1 1 ,9 mg/100 mL a 200 mM Cu, y de 6,4 mg/100 mL a 400 mM Cu (Figura 4B). To determine if the presence of copper in the medium affects the growth of A. acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, the diameter of the fungal colony was analyzed for 40 days. days of culture in solid medium 9K-Fe (negative control) and 9K-Fe-Cu, with 200 or 400 mM of CuS0 4 * 5H 2 0. The greatest growth of the fungus was in the negative control, with a diameter of 60 , 8 mm; while at 200 mM Cu the diameter was reduced to 33.8 mm; and 400 nM Cu was reduced to 20.5 mm (Fig. 4A). Adclclonally, at 40 days, in liquid medium 9K-Fe-Cu, pH = 1.8, the dry weight of the fungus was quantified in the three conditions evaluated, which was 18.9 mg / 100 mL in the negative control, 1 1, 9 mg / 100 mL at 200 mM Cu, and from 6.4 mg / 100 mL to 400 mM Cu (Figure 4B).
A continuación, se estudió la formación de biopelículas de A. acidophilus HE17- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , en medio líquido 9K-Fe y 9K-Fe- Cu pH 1 ,8 con 200 mM y 400 mM de CuS04x5H20. Se realizó tinción con naranja de acrldina para visualizar las hlfas bajo microscopía confocal de fluorescencia. Tanto en el control negativo como a 200 mM de Cu, se observó la formación de biopelículas ricas en hlfas, con una arquitectura homogénea (Figura 5A y 5B). A 400 mM de Cu se Next, the formation of biofilms of A. acidophilus HE17- access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, in liquid medium 9K-Fe and 9K-Fe-Cu pH 1 was studied. , 8 with 200 mM and 400 mM of CuS0 4 x5H 2 0. Staining was carried out with acrldine orange to visualize the leaves under fluorescence confocal microscopy. Both in the negative control and at 200 mM of Cu, the formation of horn-rich biofilms was observed, with a homogeneous architecture (Figure 5A and 5B). At 400 mM of Cu
HOJAS RECTIFICADAS (REGLA 91) observó una menor cantidad de hitas (Figura 5C). El espesor de las blopelículas se midió calculando las profundidades. En el control negativo la biopelícula tuvo una profundidad promedio aproximada de 216 pm (Figuras 5D, 5G y 5J), a 200 mM Cu se obtuvo una profundidad de 179 pm (Figuras 5E, 5H y 5K), y a 400 mM Cu una profundidad de 120 pm (Figura 5F, 5I y 5L). RECTIFIED LEAVES (RULE 91) observed a smaller number of hitas (Figure 5C). The thickness of the film was measured by calculating the depths. In the negative control the biofilm had an approximate average depth of 216 μm (Figures 5D, 5G and 5J), at 200 μM Cu a depth of 179 μm was obtained (Figures 5E, 5H and 5K), and at 400 μM Cu a depth of 120 pm (Figure 5F, 5I and 5L).
Ejemplo 11 : Experimentos de biolixiviación de calcopirita con Acidomyces adidophilus HE17 Example 11: Bioleaching Experiments of Chalcopyrite with Acidomyces adidophilus HE17
Para estudiar si A. acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2451 , aumenta la recuperación de cobre en un proceso de biolixiviación bacteriana, se realizaron experimentos de biolixiviación estática (BE) y de biolixiviación en biorreactor airllft (BA). Se utilizaron muestras de calcopirita a partir de las cuales se obtuvo la comunidad bacteriana naturalmente presente en el mineral. Los siguientes tratamientos se analizaron por triplicado en BE y BA: T1 (control negativo), medio 9K + calcopirita; T2, medio 9K + calcopirita + comunidad bacteriana; T3, medio 9K + calcopirita + comunidad bacteriana + A. acidophilus-, T4, medio 9K + calcopirita + A. acidophilus. Para evaluar la Influencia de A. acidophilus - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , en el crecimiento bacteriano, se analizaron T2 y T3 a los 68 días de experimentación. En BE, el crecimiento bacteriano en T2 aumentó de 1 ,1 x105 a 2,5x106 células/mL; mientras que en T3 se observó un Incremento mayor, de 1 ,1 x105 a 2,9x10® células/mL (Flg. 6A, Fig. 6B). En BA ocurrió una situación similar, el crecimiento bacteriano en T2 aumentó de 3,6x10s a 3,8x10® células/mL, y en T3 aumentó de 3,1 x10® a 3,9x10® células/mL (Flg. 6C, Flg. 6D). Por lo tanto, en ambos sistemas de biolixiviación, la adición de A. acidophilus HE17 - número de acceso del To study whether A. acidophilus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chillán, Chile, RGM 2451, increases the recovery of copper in a bacterial bioleaching process, static bioleaching experiments were carried out (BE) and of bioleaching in bioreactor airllft (BA). Chalcopyrite samples were used, from which the bacterial community naturally present in the mineral was obtained. The following treatments were analyzed in triplicate in BE and BA: T1 (negative control), medium 9K + chalcopyrite; T2, medium 9K + chalcopyrite + bacterial community; T3, medium 9K + chalcopyrite + bacterial community + A. acidophilus-, T4, medium 9K + chalcopyrite + A. acidophilus. To evaluate the influence of A. acidophilus - access number of the Regional Microbial Genetic Resources Bank of Quilamapu Research, Chillán, Chile, RGM 2451, in bacterial growth, T2 and T3 were analyzed at 68 days of experimentation. In BE, the bacterial growth in T2 increased from 1.1 x 10 5 to 2.5 x 10 6 cells / mL; while in T3, a greater increase was observed, from 1.1 x 10 5 to 2.9 x 10 ® cells / mL (Fig. 6A, Fig. 6B). In BA a similar situation occurred, the bacterial growth in T2 increased from 3.6x10 s to 3.8x10 ® cells / mL, and in T3 it increased from 3.1 x10 ® to 3.9x10 ® cells / mL (Flg. 6C, Flg. 6D). Therefore, in both bioleaching systems, the addition of A. acidophilus HE17 - accession number of the
HOJAS RECTIFICADAS (REGLA 91) Banco de Recursos Genético Microbianos Regional de Investigación Quilamapu, Chillán, Chile, RGM 2451 , tuvo un efecto positivo en el crecimiento bacteriano. RECTIFIED LEAVES (RULE 91) Regional Microbial Genetic Resource Bank Quilamapu, Chillán, Chile, RGM 2451, had a positive effect on bacterial growth.
Se midió el potencial redox (ORP) y la concentración de hierro disuelto en cada tratamiento. En T3 se encontraron los valores más altos de ORP, con 620,47 mV en BE y 640,9 mV en BA (Fig. 6E, Fig. 6F). En T2 se obtuvo un ORP de 581 ,9 mV en BE y de 616,8 mV en BA (Fig. 6E, Fig. 6F). T3 también presentó la mayor cantidad de hierro disuelto, con 250,19 mg/L en BE y 1435,18 mg/L en BA (Fig. 6G, Fig, 6H). En T2 se encontraron 238,49 mg/L de Fe en BE y 1324,84 mg/L de Fe en BA (Fig. 6G, Fig. 6H). Por lo tanto, se observa que la presencia de A. acidophilus HE17 aumenta el crecimiento del consorcio bacteriano aumentando el ORP, esto significa que existe un aumento en reacciones de oxidación y reducción de Fe con una mayor extracción de cobre a partir del mineral realizado por bacterias lixiviantes. La recuperación de cobre se midió a partir del día 1 1 en adelante. Como es de esperar, la menor recuperación se observó en T1 , con un 9,27% en BE y un 21 ,43% en BA; seguido de T4 con una recuperación del 13,14% en BE y del 23,3% en BA (Fig. 6I, Fig. 6J). En T2 la recuperación de cobre fue de un 35,94% en BE y de un 49,7% en BA; mientras que en T3 fue de un 47,4% en BE y de un 59,6% en BA (Fig . 6I, Fig. 6J). Según el análisis estadístico, las diferencias entre BE y BA son significativas (P<0.01 ). A. acidophilus HE17 creció de forma equivalente en ambos procesos de biolixiviación, alcanzando en BE un peso seco de 0,1735 mg/pL en T3 y de 0,1402 mg/pL en T4; y en BA un peso seco de 0,21 15 mg/pL en T3 y de 0,1688 mg/pL en T4. Ejemplo 12: Identificación de las bacterias presentes en los experimentos de biolixiviación The redox potential (ORP) and the concentration of dissolved iron were measured in each treatment. In T3 the highest ORP values were found, with 620.47 mV in BE and 640.9 mV in BA (Fig. 6E, Fig. 6F). In T2, an ORP of 581.9 mV was obtained in BE and of 616.8 mV in BA (Fig. 6E, Fig. 6F). T3 also showed the highest amount of dissolved iron, with 250.19 mg / L in BE and 1435.18 mg / L in BA (Fig. 6G, Fig. 6H). In T2 were found 238.49 mg / L of Fe in BE and 1324.84 mg / L of Fe in BA (Fig. 6G, Fig. 6H). Therefore, it is observed that the presence of A. acidophilus HE17 increases the growth of the bacterial consortium by increasing the ORP, this means that there is an increase in oxidation reactions and reduction of Fe with a higher extraction of copper from the mineral made by leaching bacteria. Copper recovery was measured from day 1 1 onwards. As expected, the lowest recovery was observed in T1, with 9.27% in BE and 21, 43% in BA; followed by T4 with a recovery of 13.14% in BE and 23.3% in BA (Fig. 6I, Fig. 6J). In T2, copper recovery was of 35.94% in BE and of 49.7% in BA; while in T3 it was 47.4% in BE and 59.6% in BA (Fig. 6I, Fig. 6J). According to the statistical analysis, the differences between BE and BA are significant (P <0.01). A. acidophilus HE17 grew in an equivalent way in both bioleaching processes, reaching in BE a dry weight of 0.1735 mg / pL in T3 and 0.1402 mg / pL in T4; and in BA a dry weight of 0.21 15 mg / pL in T3 and of 0.1668 mg / pL in T4. Example 12: Identification of the bacteria present in the bioleaching experiments
HOJAS RECTIFICADAS (REGLA 91) Se caracterizaron aquellas bacterias presentes en los experimentos de biolixiviación, en presencia y ausencia de A. acidophilus- número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2451 . Para esto, se tomaron muestras de los tratamientos T2 y T3 de BE (T2E, T3E) y T2 y T3 de BA (T2R, T3R). Una tinción Gram reveló la presencia de múltiples bacilos Gram- negatlvos. A continuación se detalla el proceso de Identificación de estas bacterias. RECTIFIED LEAVES (RULE 91) The bacteria present in the bioleaching experiments were characterized in the presence and absence of A. acidophilus - access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chillán, Chile, RGM 2451. For this, samples were taken from treatments T2 and T3 of BE (T2E, T3E) and T2 and T3 of BA (T2R, T3R). A Gram stain revealed the presence of multiple Gram-negative bacilli. The identification process of these bacteria is detailed below.
Electroforesis en gel con gradiente desnaturalizante (DGGE). Gel electrophoresis with denaturing gradient (DGGE).
Se extrajo DNA genómico de las muestras obtenidas de T2E, T3E, T2R, y T3R; y se amplificó el rDNA 16S con los ollgonucleótldos universales 1492R/27F. Los productos de PCR se re-amplificaron con los ollgonucleótldos específicos para bacterias 341 F- GC y 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetlc relatlonships of Thiomicrosplra specles and thelr Identification in deep-sea hydrothermal vent samples by denaturing gradlent gel electrophoresls of 16S rDNA fragments. Arch. Microblol. 164, 165-171 ). Los productos resultantes del PCR se sometieron a DGGE. Se visualizaron tres bandas para T2R y T3R, y cuatro bandas para T2E y T3E (Fig. 7A). Las bandas se purificaron, los productos de PCR se re amplificaron con los ollgonucleótldos 341 F-GC/907R, y se secuenclaron. Se obtuvieron secuencias de calidad aceptable para tres de las bandas (C1 , C2 y C3). A partir de las secuencias se construyó un árbol fllogenétlco con el programa MEGA7, utilizando secuencias similares a las nuestras, obtenidas de la base de datos (Fig. 7B). El análisis de las secuencias del rDNA 16S reveló la presencia de dos géneros bacterianos: Acidithiobacillus y Acidiphilium. La Tabla 1 muestra el familiar más cercano para cada una de las especies Identificadas por secuenclaclón, correspondiendo a fllotlpos homólogos a Acidithiobacillus ferridurans strain YNTR1 -41 (C3), Acidiphilium sp. strain MPLK-613 (C2) y Acidithiobacillus ferrooxidans strain HBDY3-51 (C1 ). Es Interesante destacar que A. ferridurans strain YNTR1 -41 sólo se encontró en los experimentos de BE.  Genomic DNA was extracted from the samples obtained from T2E, T3E, T2R, and T3R; and the 16S rDNA was amplified with the universal oligonucleotides 1492R / 27F. The PCR products were re-amplified with the specific oligonucleotides for bacteria 341 F-GC and 907R (Muyzer G., Teske A., Wirsen CO., Jannasch HW. 1995. Phylogenetics Relatlonships of Thiomicrosplra specles and thelr Identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresls of 16S rDNA fragments Arch. Microblol 164, 165-171). The products resulting from the PCR were subjected to DGGE. Three bands were visualized for T2R and T3R, and four bands for T2E and T3E (Fig. 7A). The bands were purified, the PCR products were re-amplified with the oligonucleotides 341 F-GC / 907R, and sequenced. Sequences of acceptable quality were obtained for three of the bands (C1, C2 and C3). A fllogenetic tree was constructed from the sequences with the MEGA7 program, using sequences similar to ours, obtained from the database (Fig. 7B). The analysis of the 16S rDNA sequences revealed the presence of two bacterial genera: Acidithiobacillus and Acidiphilium. Table 1 shows the closest relative for each of the species identified by sequencing, corresponding to homologous fllotlpos to Acidithiobacillus ferridurans strain YNTR1 -41 (C3), Acidiphilium sp. strain MPLK-613 (C2) and Acidithiobacillus ferrooxidans strain HBDY3-51 (C1). It is interesting to note that A. ferridurans strain YNTR1 -41 was only found in BE experiments.
HOJAS RECTIFICADAS (REGLA 91) Tabla 1. Banda de clones de bacterias lixiviantes representativos en DGGE RECTIFIED LEAVES (RULE 91) Table 1. Band of clones of representative leaching bacteria in DGGE
Banda de DGGE El familiar más cercano (closest relative) Score Máx. identidad E-value  DGGE band The closest relative (closest relative) Score Max. E-value identity
C3 Acidithiobacillus ferrídurans strain YNTR1 -41 94 99 C3 Acidithiobacillus ferrídurans strain YNTR1 -41 94 99
0.0 0.0
C2 Acidiphilium sp. strain MPLK-613 93 98 C2 Acidiphilium sp. strain MPLK-613 93 98
0.0 0.0
C1 Acidithiobacillus ferrooxidans strain HBDY3-51 84 99 0.0  C1 Acidithiobacillus ferrooxidans strain HBDY3-51 84 99 0.0

Claims

REIVINDICACIONES
1 . Método para biolixiviar minerales sulfurados de cobre aumentando el crecimiento del consorcio bacteriano biolixiviante caracterizado porque comprende conducir la biolixiviación con un consorcio de microorganismos que comprende bacterias ferroxldantes - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2527 y el hongo Acidomyces acidophilus HE17 - número de acceso del Banco de Recursos Genético Microbianos Regional de Investigación Qullamapu, Chillán, Chile, RGM 2451 , en un medio Inorgánico libre de sulfato ferroso y pH < 2. one . Method for bio-lixivizing copper sulphide minerals increasing the growth of the biolixiviante bacterial consortium characterized in that it comprises conducting the bioleaching with a consortium of microorganisms comprising ferroxldante bacteria - access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chillán, Chile, RGM 2527 and Acidomyces acidophilus fungus HE17 - access number of the Regional Microbial Genetic Resources Research Bank Qullamapu, Chillán, Chile, RGM 2451, in an inorganic medium free of ferrous sulphate and pH <2.
2. El método de la reivindicación 1 caracterizado porque las bacterias ferroxldans que se encuentran en el mineral sulfurado. 2. The method of claim 1, characterized in that the ferroxldans bacteria that are found in the sulphide mineral.
3. El método de la reivindicación 1 caracterizado porque el pH tiene valor 1 ,8. 3. The method of claim 1, characterized in that the pH has a value of 1.8.
4. El método de la reivindicación 1 caracterizado porque dichas bacterias se seleccionan preferentemente de bacterias del género Acidithiobacillus, Acidiphilium o una combinación de las mismas. 4. The method of claim 1 characterized in that said bacteria are preferably selected from bacteria of the genus Acidithiobacillus, Acidiphilium or a combination thereof.
5. El método de la reivindicación 1 caracterizado porque dichas bacterias se seleccionan del grupo consistente de Acidithiobacillus ferridurans cepa YNTR1 -41 , Acidiphilium sp. cepa MPLK-613, Acidithiobacillus ferrooxidans cepa HBDY3-5 o una combinación de las mismas. The method of claim 1 characterized in that said bacteria are selected from the group consisting of Acidithiobacillus ferridurans strain YNTR1 -41, Acidiphilium sp. strain MPLK-613, Acidithiobacillus ferrooxidans strain HBDY3-5 or a combination thereof.
6. El método de la reivindicación 1 caracterizado porque dicho medio Inorgánico libre de sulfato ferroso es un medio 9K. 6. The method of claim 1 characterized in that said ferrous sulfate-free Inorganic medium is a 9K medium.
HOJAS RECTIFICADAS (REGLA 91) RECTIFIED LEAVES (RULE 91)
7. El método de la reivindicación 1 caracterizado porque dicho mineral sulfurado de cobre es calcopirita. 7. The method of claim 1, characterized in that said sulphide copper mineral is chalcopyrite.
8. El método de la reivindicación 1 caracterizado porque dicha biolixiviación se realiza en bioreactor estático. 8. The method of claim 1, characterized in that said bioleaching is carried out in a static bioreactor.
9. El método de la reivindicación 1 caracterizado porque dicha biolixiviación se realiza en un bioreactor de flujo ascendente o bioreactor airlift. The method of claim 1, characterized in that said bioleaching is carried out in an upflow bioreactor or bioreactor airlift.
PCT/CL2018/050134 2017-12-19 2018-12-19 Method for bioleaching sulfur-containing copper minerals using a consortium of microorganisms comprising iron-oxidising bacteria and the fungus acidomyces acidophilus he17 in an inorganic medium at a ph of less than 2, promoting bacterial growth and increasing extraction of the metal from the mineral WO2019119166A1 (en)

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WO2019119166A1 (en) Method for bioleaching sulfur-containing copper minerals using a consortium of microorganisms comprising iron-oxidising bacteria and the fungus acidomyces acidophilus he17 in an inorganic medium at a ph of less than 2, promoting bacterial growth and increasing extraction of the metal from the mineral

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