WO2018119541A1 - Method for eliminating suspended dust originating from particulate tailings generated by means of wind erosion, comprising obtaining a biological composition, applying the biological composition, and stabilising the particulate matter, as well as the resulting biological composition and the application thereof - Google Patents

Method for eliminating suspended dust originating from particulate tailings generated by means of wind erosion, comprising obtaining a biological composition, applying the biological composition, and stabilising the particulate matter, as well as the resulting biological composition and the application thereof Download PDF

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Publication number
WO2018119541A1
WO2018119541A1 PCT/CL2017/050092 CL2017050092W WO2018119541A1 WO 2018119541 A1 WO2018119541 A1 WO 2018119541A1 CL 2017050092 W CL2017050092 W CL 2017050092W WO 2018119541 A1 WO2018119541 A1 WO 2018119541A1
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medium
biological composition
tailings
particulate
samples
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PCT/CL2017/050092
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Spanish (es)
French (fr)
Inventor
Claudia Andrea ORTIZ CALDERÓN
Marcela Andrea WILKENS ANWANDTER
Daniel BARROS VÁSQUEZ
Jaime PIZARRO KONCZAK
Alejandro MUÑOZ ROJAS
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Universidad De Santiago De Chile
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Priority to AU2017385419A priority Critical patent/AU2017385419B2/en
Priority to BR112019013552-9A priority patent/BR112019013552A2/en
Publication of WO2018119541A1 publication Critical patent/WO2018119541A1/en

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
    • C09K8/42—Compositions for cementing, e.g. for cementing casings into boreholes; Compositions for plugging, e.g. for killing wells
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
    • C09K8/42—Compositions for cementing, e.g. for cementing casings into boreholes; Compositions for plugging, e.g. for killing wells
    • C09K8/46—Compositions for cementing, e.g. for cementing casings into boreholes; Compositions for plugging, e.g. for killing wells containing inorganic binders, e.g. Portland cement
    • C09K8/467—Compositions for cementing, e.g. for cementing casings into boreholes; Compositions for plugging, e.g. for killing wells containing inorganic binders, e.g. Portland cement containing additives for specific purposes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention in general terms, discloses a method for suppressing suspended dust from particulate material, especially tailings, and biological compositions, comprising mixtures of cyanobacteria and microalgae obtained from deposits under the numbers KCTC13158BP and KCTC13159BP.
  • US 2013/0196419 describes a composition for reducing particulate matter suspended in air or in a liquid comprising a source of exopolysaccharides selected from strains of slime-producing microorganisms, a microorganism with ureolytic activity and a culture medium, where the silt producing microorganisms are selected from microalgae and the microorganism with ureolytic activity is a culture of Bacillus pasteurii.
  • the application CL 0241 -2012 of the same applicant describes a method for reducing the particulate material suspended in air or water comprising agglomerating the particulate material suspended in air or water with negative charge exopolysaccharides (EPS), where the microorganism that produces the Negative charge EPS is a bacterium or a microalgae.
  • EPS negative charge exopolysaccharides
  • Kalita describes the effect of exopolysaccharides (EPS ) of 10 strains of cyanobacteria during photoautotrophic culture.
  • EPS exopolysaccharides
  • the present invention provides the application of a novel biological composition comprising mixtures of cyanobacteria and microalgae This invention has the advantage of stabilizing the dust in suspension from the particulate tailings malt, which minimizes water infiltration and wind erosion in mining tailings.
  • the present invention relates to a method for suppressing suspended dust from particulate tailings material caused by wind erosion.
  • Said method comprises obtaining biological compositions in a suitable liquid culture medium; apply a certain volume of the biological composition to the tailings or substrate to be treated; and stabilize the particulate material.
  • compositions comprising mixtures of microorganisms that are obtained from the KCTC13158BP and KCTC13159BP deposits, suspended in a suitable culture medium.
  • Figure 1 shows a photographic record of the typical performance of the specimens without treatment, during the wind tunnel tests at different times: 0 minutes (Fig. 1 a), 5 minutes (Fig. 1 b) and 20 minutes (Fig. 1 c).
  • Figure 2 shows a photographic record of the typical performance of the Ph1 treatment specimens, during the wind tunnel tests at different times: 0 minutes (Fig. 2a), 10 minutes (Fig. 2b), 20 minutes ( Fig. 2c), 60 minutes (Fig. 2d), 120 minutes (Fig. 2e) and 180 minutes (Fig. 2f).
  • Figure 3 shows a photographic record of the typical performance of the specimens treated with the cyanobacterial mixture 1, during the wind tunnel tests at different times: 0 minutes (Fig. 3a), 10 minutes (Fig. 3b) , 20 minutes (Fig. 3c), 60 minutes (Fig. 3d), 180 minutes (Fig. 3e) and 360 minutes (Fig. 3f).
  • Figure 4 shows the weights (Fig. 4A) and percentages of tailings loss (Fig. 4B) by wind effect, recorded in wind tunnel tests for tailings samples without treatment. The black bars indicate the initial dry weight; the white bars indicate the final dry weight.
  • FIG. 5 shows the weights (Fig. 5A) and percentages of tailings loss
  • Figure 6 shows the weights (Fig. 6A) and percentages of tailings loss (Fig. 6B) by wind effect, recorded in wind tunnel tests for tailings samples treated with cyanobacterial mixture 1.
  • the black bars indicate the initial dry weight; the white bars indicate the final dry weight.
  • Figure 7 shows the weights (Fig. 6A) and percentages of tailings loss (Fig. 6B) by wind effect, recorded in wind tunnel tests for tailings samples treated with the mixture 2 of cyanobacteria and microalgae.
  • the black bars indicate the initial dry weight; the white bars indicate the final dry weight.
  • the present invention relates to a method and a composition for suppressing suspended dust from particulate tailings material, wherein said method comprises the steps of:
  • suitable liquid culture medium composed of a mixture of microorganisms, obtained from the KCTC13158BP and KCTC13159BP tanks;
  • the biological compositions were obtained by cultivating samples of soil scabs in suitable sterile liquid medium.
  • the complete protocol includes:
  • Solid sterile medium BG1 1;
  • Means for defrosting the vials nutritive medium containing N: P: K and trace elements Ca, Fe, Mg, Mn and S.
  • compositions comprising:
  • the microorganisms of the biological composition can be selected, but they are not limited to the genera of the Leptoiyngbya and Trichocoleus cyanobacteria. These can be selected, but are not limited to the species Leptoiyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptoiyngbya boryana, Leptoiyngbya sp.
  • Microalgae can be selected, but are not limited to species of the genus Chlorella. In addition, it is possible to use combinations of said cyanobacteria and microalgae.
  • cyanobacteria and microalgae have the ability to grow in granulometry soils that range from 1 to 125 pm, forming biocostras that facilitate the recovery of impoverished soils, as these favor the growth of plants and vegetables on fine granulometry soils.
  • the method and biological composition described in the present invention are aimed at stabilizing the suspended dust from the particulate tailings malt, which minimizes water infiltration and erosion caused by wind in mining tailings.
  • cyanobacteria and microalgae are used for the purpose described in the present invention.
  • Example No. 1 obtaining the crops used in the stabilization tests
  • the initial samples were obtained from the soil of the IV Region of Coquimbo located in the semi-arid zone of western South America, south of the great Atacama Desert (29 ° 00'S; 32 ° 10'S). In 6 different sectors within the Fourth Region of Coquimbo, 34 soil samples were extracted. Sampling sites were chosen based on color and apparent texture. For the extraction of samples, the first layer of soil (approximately 1 cm deep) was carefully removed, collecting 8 to 15 g of soil, which was stored in sterile Falcon tubes. Once all samples were collected, they were stored at 4 ° C.
  • the tubes with liquid medium with a ratio of 1/10 and the seeded plates were incubated in a culture chamber, which had a photoperiod of 12 h light-12 h darkness, at a temperature of 28 ° C. Liquid cultures were left in the culture chamber until dark green filaments or light green spots were observed. An analysis was performed by Optical microscopy, in a Zeiss Axiostarplus® 100X microscope with a 10X magnification of the eyepiece, to confirm the presence of microorganisms and a portion was transferred to sterile liquid BG1 1 medium. In the case of the plates in solid medium with evident growth of pigmented colonies, an analysis by optical microscopy was carried out and seeded in plates of solid BG1 for the separation of colonies, as well as inoculated in liquid medium to obtain biomass .
  • the samples were identified by obtaining genomic DNA for which 200 ⁇ of the culture was taken, taking care that filaments were obtained in the collection; in the case of the most compact microorganisms, they were removed from the culture medium and a carefully removed portion was obtained with a sterile scalpel. For cultures in solid medium, sections were cut with a sterile scalpel. All samples were introduced into the respective collection tubes and, if necessary, flush up to 200 ⁇ with sterile water.
  • the FavorPrep kit, Soil DNA isolation minikit from Favorgen Biotech corp® was used for the extraction of genomic DNA. DNA was quantified and amplification of DNA fragments was performed to determine the presence of microorganisms in the samples.
  • each sequence was processed with the Geneious software in relation to the presence of the primers and the quality of the sequencing delivered by the electropherograms of the sequencing performed. An alignment of each sequence was then performed with the BLASTn tool aimed at each genre of microorganism. Subsequently, the list of significant results obtained and the results of the distance tree delivered by the same tool were analyzed, identifying the most probable genus to which the microorganism could belong.
  • a 10% v / v inoculation was made from the cryopreserved culture of the isolated cyanobacterial and microalgae species, and the samples were grown for two weeks in nutrient medium containing a proportion of N: P: K and trace elements: Ca, Fe, Mg, Mn and S.
  • the growth conditions corresponded to 3,000 lux from fluorescent light (40.5 pmol m-2 s-1), orbital agitation with constant speed at 120 rpm, light cycle of 14/12 hours, humidity between 40% and 60%.
  • the new suspension was cooled for 5 minutes at 4 ° C, 30 minutes at -20 ° C and finally the culture was brought to -86 ° C.
  • Samples obtained from soil crusts have been deposited under the specifications of the Budapest Treaty at the international deposit authority "Korean Collection for Type Cultures", under the numbers KCTC 13158BP and KCTC 13159BP. Said deposited samples contain the cyanobacterial species Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp. and microalgae of the genus Chlorella.
  • Example No. 3 Evaluation of cell viability of cryopreserved samples
  • the cultures maintained at -86 ° C were thawed, keeping each sample at 4 ° C, after which they were centrifuged at 4,000 rpm for 5 minutes at 4 ° C. After this, the supernatant was removed and all the precipitated biomass was inoculated on liquid or solid medium (BG1 1) at a concentration of 10% v / v.
  • BG1 1 liquid or solid medium
  • the cryopreserved isolates were inoculated on solid nutrient culture medium (BG1 1 with 1% agar) and kept for 7 days at 3,000 lux from fluorescent light (40.5 pmol m "2 s " 1 ), cycle light of 12/14 hours, humidity between 40% and 60%. In this case the viability was verified by plaque growth.
  • cryopreserved cyanobacteria and microalgae biomass was inoculated on nutrient culture medium (BG1 1) and the culture was maintained for 30 days under the following growth conditions: 3,000 lux from fluorescent light (40, 5 pmol m "2 s " 1 ), light cycle of 14/12 hours, humidity between 40% and 60%, and orbital agitation at a constant speed of 120 rpm.
  • BG1 1 nutrient culture medium
  • the viability is verified by growth, through the observation of the biomass grown after one and two weeks.
  • Example No. 4 Geotechnical characterization of tailings material
  • Table N ° 2 Granulometry by hydrometer method (ASTM standard)
  • ASTM standard the parameters of specific gravity (Table No. 3), maximum compacted density (Table No. 4) and bulk bulk density (Table No. 5) were measured to the tailings samples.
  • Wind tunnel tests were carried out under the Chilean tailings deposit standard NCh 3266 - 2012, simulating wind speed and speed conditions that are registered at sites of mining sites affected by wind erosion, so that the results are extrapolated to ground conditions.
  • cryopreserved samples were prepared 2 mixtures of microorganisms: mixture 1 contains Leptolyngbya badia, Trichocoleus sociatus, and mixture 2 contains Trichocoleus desertorum, Leptolyngbya boryana and Chlorella.sp.
  • Figures 1 -3 show a photographic record of the typical performance of the specimens without treatment (Figure 1), treatment with the mitigating agent Ph1 ( Figure 2), and treatment with the mixture 1 ( Figure 3), at different times during the realization of the tests subjected to the wind effect in the wind tunnel.
  • Figure 1 For the test with mixture 2, we do not provide photographs.
  • the weights and percentages of tailings loss due to wind effect recorded in the tests performed, are presented in table No.

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Abstract

The present invention relates to a method for eliminating suspended dust originating from particulate tailings generated by means of wind erosion, comprising obtaining a biological composition, applying the biological composition, and stabilising the particulate matter. The invention also relates to the resulting biological composition and to the application thereof to particulate tailings.

Description

MÉTODO PARA SUPRIMIR EL POLVO EN SUSPENSIÓN PROVENIENTE DE MATERIAL PARTICULADO DE RELAVES GENERADO POR EROSIÓN EÓLICA, QUE COMPRENDE OBTENER UNA COMPOSICIÓN BIOLÓGICA, APLICAR DICHA COMPOSICIÓN BIOLÓGICA Y ESTABILIZAR EL MATERIAL PARTICULADO, ASÍ COMO LA COMPOSICIÓN BIOLÓGICA OBTENIDA Y SU  METHOD FOR SUPPRESSING THE POWDER IN SUSPENSION FROM PARTICULATED RELAY MATERIAL GENERATED BY WIND EROSION, WHICH INCLUDES OBTAINING A BIOLOGICAL COMPOSITION, APPLY SUCH BIOLOGICAL COMPOSITION AND STABILIZE THE PARTICULATED ASTIMATED BIOLOGICAL COMPOSITION
APLICACIÓN  APPLICATION
CAMPO DE APLICACIÓN SCOPE
La presente invención en términos generales, divulga un método para suprimir el polvo en suspensión proveniente de material particulado, especialmente de relaves, y composiciones biológicas, que comprenden mezclas de cianobacterias y microalgas obtenidas de depósitos bajo los números KCTC13158BP y KCTC13159BP.  The present invention, in general terms, discloses a method for suppressing suspended dust from particulate material, especially tailings, and biological compositions, comprising mixtures of cyanobacteria and microalgae obtained from deposits under the numbers KCTC13158BP and KCTC13159BP.
ANTECENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Es conocido en el estado del arte que las actividades industriales, de transporte, y fuentes naturales como la erosión eólica conllevan a la emisión de material particulado desde la superficie de depósitos mineros; lo anterior constituye un problema medioambiental pues este material particulado puede portar elementos tóxicos y peligrosos, como es el caso de los relaves mineros. Estos sustratos corresponden a una suspensión fina de sólidos en líquido, constituidos fundamentalmente por el mismo material presente en el yacimiento, al cual se le ha extraído la fracción con mineral valioso. Estos relaves están conformados principalmente por partículas de tamaño menor o igual a 10 micrones, con un alto y variado contenido mineralógico. Al ser dispersadas por el viento, estas partículas afectan el medio ambiente, generando un alto impacto ambiental. It is known in the state of the art that industrial activities, transportation, and natural sources such as wind erosion lead to the emission of particulate material from the surface of mining deposits; This constitutes an environmental problem because this particulate material can carry toxic and dangerous elements, such as mining tailings. These substrates correspond to a fine suspension of solids in liquid, consisting essentially of the same material present in the reservoir, to which the fraction with valuable mineral has been extracted. These tailings are mainly made up of particles smaller than or equal to 10 microns, with a high and varied mineralogical content. When dispersed by the wind, these particles affect the environment, generating a high environmental impact.
En el estado del arte encontramos distintas estrategias para controlar el material particulado de distinto origen, que de una u otra manera emplean elementos de origen biológico, sin embargo, requieren de múltiples aplicaciones, por lo tanto no logran proporcionar soluciones definitivas, aumentando los costos de mantenimiento.  In the state of the art we find different strategies to control the particulate material of different origin, which in one way or another use elements of biological origin, however, they require multiple applications, therefore they fail to provide definitive solutions, increasing the costs of maintenance.
Por ejemplo, el documento US 2013/0196419 describe una composición para disminuir el material particulado suspendido en aire o en un líquido que comprende una fuente de exopolisacáridos seleccionados de cepas de microorganismos productoras de limo, un microorganismo con actividad ureolítica y un medio de cultivo, donde los microorganismos productores de limo son seleccionados de microalgas y el microorganismo con actividad ureolítica es un cultivo de Bacillus pasteurii. La solicitud CL 0241 -2012 del mismo solicitante, describe un método para disminuir el material particulado en suspensión en aire o agua que comprende aglomerar el material particulado suspendido en aire o agua con exopolisacáridos (EPS) de carga negativa, donde el microorganismo que produce el EPS de carga negativa es una bacteria o una microalga.  For example, US 2013/0196419 describes a composition for reducing particulate matter suspended in air or in a liquid comprising a source of exopolysaccharides selected from strains of slime-producing microorganisms, a microorganism with ureolytic activity and a culture medium, where the silt producing microorganisms are selected from microalgae and the microorganism with ureolytic activity is a culture of Bacillus pasteurii. The application CL 0241 -2012 of the same applicant describes a method for reducing the particulate material suspended in air or water comprising agglomerating the particulate material suspended in air or water with negative charge exopolysaccharides (EPS), where the microorganism that produces the Negative charge EPS is a bacterium or a microalgae.
Respecto a las cianobacterias, existen documentos que describen la capacidad o la relación de estas con la remoción de contaminantes, tanto metálicos como orgánicos.  Regarding cyanobacteria, there are documents that describe their capacity or relationship with the removal of contaminants, both metallic and organic.
El documento no patente "Exopolysaccharide-producing cyanobacteria in heavy metal removal from water: molecular basis and practical applicability of the biosorption process", redactado por R. Philippis, G. Cólica, y E. Micheletti, describe que los microorganismos, específicamente los exopolisacáridos (EPS) de la superficie celular de cianobacterias, son capaces de remover metales desde ambientes contaminados a través de diversos mecanismos, tales como procesos mediados metabólicamente o por la adsorción de metales en macromoléculas con carga iónica de la superficie celular. En esta misma línea, el documento "Characterization and Optimization of Bioflocculant Exopolysaccharide Production by Cyanobacteria Nostoc sp. BTA97 and Anabaena sp. BTA990 in Culture Conditions" redactado por O. Tiwari y cois., describe experimentos donde se demostró la producción de exopolisacáridos (EPS) de 40 cepas de cianobacterias como biofloculantes para absorber o remover contaminantes orgánicos, y como alternativa de diversas aplicaciones biotecnológicas en el medio ambiente. Al igual que el documento anterior, la publicación científica titulada "Production of exopolysaccharides by the cyanobacterium Anabaena sp. BTA992 and application as bioflocculants" , redactada por R. Khangembam, O. Tiwari y M. Kalita, describe el efecto de los exopolisacáridos (EPS) de 10 cepas de cianobacterias durante su cultivo fotoautotrófico. Los resultados demostraron que cianobacterias seleccionadas de Anabaena sp., pueden ser un buen candidato para la producción comercial de EPS y pueden ser utilizadas en aplicaciones como alternativa a floculantes sintéticos. The non-patent document "Exopolysaccharide-producing cyanobacteria in heavy metal removal from water: molecular basis and practical applicability of the biosorption process", written by R. Philippis, G. Cólica, and E. Micheletti, describes that microorganisms, specifically exopolysaccharides (EPS) of the Cyanobacterial cell surface, are capable of removing metals from contaminated environments through various mechanisms, such as metabolically mediated processes or by adsorption of metals in macromolecules with ionic charge of the cell surface. Along the same lines, the document "Characterization and Optimization of Bioflocculant Exopolysaccharide Production by Cyanobacteria Nostoc sp. BTA97 and Anabaena sp. BTA990 in Culture Conditions" drafted by O. Tiwari and cois., Describes experiments where the production of exopolysaccharides (EPS) was demonstrated ) of 40 strains of cyanobacteria as biofloculants to absorb or remove organic pollutants, and as an alternative to various biotechnological applications in the environment. Like the previous document, the scientific publication entitled "Production of exopolysaccharides by the cyanobacterium Anabaena sp. BTA992 and application as bioflocculants", written by R. Khangembam, O. Tiwari and M. Kalita, describes the effect of exopolysaccharides (EPS ) of 10 strains of cyanobacteria during photoautotrophic culture. The results showed that selected cyanobacteria of Anabaena sp., Can be a good candidate for commercial production of EPS and can be used in applications as an alternative to synthetic flocculants.
Todos los documentos citados utilizan exopolisacáridos como bio-floculantes y son aplicados para la remoción de metales pesados en líquidos contaminados, pero ninguno de ellos viene a solucionar el problema técnico de suprimir el polvo en suspensión generado por erosión eólica, proveniente de material particulado de relaves. Ante esta problemática, la presente invención proporciona la aplicación de una novedosa composición biológica que comprende mezclas de cianobacterias y microalgas. Esta invención tiene la ventaja de estabilizar el polvo en suspensión proveniente del matenal particulado de relaves, lo que minimiza la infiltración de agua y la erosión causada por el viento en relaves mineros. All the documents cited use exopolysaccharides as bio-flocculants and are applied for the removal of heavy metals in contaminated liquids, but none of them come to solve the technical problem of suppressing the suspended dust generated by wind erosion, from particulate tailings material . Faced with this problem, the present invention provides the application of a novel biological composition comprising mixtures of cyanobacteria and microalgae This invention has the advantage of stabilizing the dust in suspension from the particulate tailings malt, which minimizes water infiltration and wind erosion in mining tailings.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención se refiere a un método para suprimir el polvo en suspensión proveniente de material particulado de relaves causado por la erosión eólica. Dicho método comprende obtener composiciones biológicas en un medio de cultivo líquido adecuado; aplicar un volumen determinado de la composición biológica al relave o sustrato a tratar; y estabilizar el material particulado.  The present invention relates to a method for suppressing suspended dust from particulate tailings material caused by wind erosion. Said method comprises obtaining biological compositions in a suitable liquid culture medium; apply a certain volume of the biological composition to the tailings or substrate to be treated; and stabilize the particulate material.
Además, se reivindican composiciones biológicas de comprenden mezclas de microorganismos que se obtienen de los depósitos KCTC13158BP y KCTC13159BP, suspendidas en un medio de cultivo adecuado.  In addition, biological compositions are claimed comprising mixtures of microorganisms that are obtained from the KCTC13158BP and KCTC13159BP deposits, suspended in a suitable culture medium.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
La Figura 1 muestra un registro fotográfico del desempeño típico de las probetas sin tratamiento, durante la realización de los ensayos en túnel de viento a diferentes tiempos: 0 minutos (Fig. 1 a), 5 minutos (Fig. 1 b) y 20 minutos (Fig. 1 c).  Figure 1 shows a photographic record of the typical performance of the specimens without treatment, during the wind tunnel tests at different times: 0 minutes (Fig. 1 a), 5 minutes (Fig. 1 b) and 20 minutes (Fig. 1 c).
La Figura 2 muestra un registro fotográfico del desempeño típico de las probetas con tratamiento Ph1 , durante la realización de los ensayos en túnel de viento a diferentes tiempos: 0 minutos (Fig. 2a), 10 minutos (Fig. 2b), 20 minutos (Fig. 2c), 60 minutos (Fig. 2d), 120 minutos (Fig. 2e) y 180 minutos (Fig. 2f).  Figure 2 shows a photographic record of the typical performance of the Ph1 treatment specimens, during the wind tunnel tests at different times: 0 minutes (Fig. 2a), 10 minutes (Fig. 2b), 20 minutes ( Fig. 2c), 60 minutes (Fig. 2d), 120 minutes (Fig. 2e) and 180 minutes (Fig. 2f).
La Figura 3 muestra un registro fotográfico del desempeño típico de las probetas tratadas con la mezcla 1 de cianobacterias, durante la realización de los ensayos en túnel de viento a diferentes tiempos: 0 minutos (Fig. 3a), 10 minutos (Fig. 3b), 20 minutos (Fig. 3c), 60 minutos (Fig. 3d), 180 minutos (Fig. 3e) y 360 minutos (Fig. 3f). La Figura 4 muestra los pesos (Fig. 4A) y porcentajes de pérdida de relave (Fig. 4B) por efecto eólico, registrados en los ensayos de túnel de viento para las muestras de relave sin tratamiento. Las barras negras indican el peso seco inicial; las barras blancas indican el peso seco final. Figure 3 shows a photographic record of the typical performance of the specimens treated with the cyanobacterial mixture 1, during the wind tunnel tests at different times: 0 minutes (Fig. 3a), 10 minutes (Fig. 3b) , 20 minutes (Fig. 3c), 60 minutes (Fig. 3d), 180 minutes (Fig. 3e) and 360 minutes (Fig. 3f). Figure 4 shows the weights (Fig. 4A) and percentages of tailings loss (Fig. 4B) by wind effect, recorded in wind tunnel tests for tailings samples without treatment. The black bars indicate the initial dry weight; the white bars indicate the final dry weight.
La Figura 5 muestra los pesos (Fig. 5A) y porcentajes de pérdida de relave Figure 5 shows the weights (Fig. 5A) and percentages of tailings loss
(Fig. 5B) por efecto eólico, registrados en los ensayos de túnel de viento para las muestras de relave con tratamiento Ph1 . Las barras negras indican el peso seco inicial; las barras blancas indican el peso seco final. (Fig. 5B) by wind effect, recorded in wind tunnel tests for tailings samples with Ph1 treatment. The black bars indicate the initial dry weight; the white bars indicate the final dry weight.
La Figura 6 muestra los pesos (Fig. 6A) y porcentajes de pérdida de relave (Fig. 6B) por efecto eólico, registrados en los ensayos de túnel de viento para las muestras de relave tratadas con la mezcla 1 de cianobacterias. Las barras negras indican el peso seco inicial; las barras blancas indican el peso seco final.  Figure 6 shows the weights (Fig. 6A) and percentages of tailings loss (Fig. 6B) by wind effect, recorded in wind tunnel tests for tailings samples treated with cyanobacterial mixture 1. The black bars indicate the initial dry weight; the white bars indicate the final dry weight.
La Figura 7 muestra los pesos (Fig. 6A) y porcentajes de pérdida de relave (Fig. 6B) por efecto eólico, registrados en los ensayos de túnel de viento para las muestras de relave tratadas con la mezcla 2 de cianobacterias y microalgas. Las barras negras indican el peso seco inicial; las barras blancas indican el peso seco final. Figure 7 shows the weights (Fig. 6A) and percentages of tailings loss (Fig. 6B) by wind effect, recorded in wind tunnel tests for tailings samples treated with the mixture 2 of cyanobacteria and microalgae. The black bars indicate the initial dry weight; the white bars indicate the final dry weight.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención se refiere a un método y una composición para suprimir el polvo en suspensión proveniente de material particulado de relaves, donde dicho método comprende las etapas de:  The present invention relates to a method and a composition for suppressing suspended dust from particulate tailings material, wherein said method comprises the steps of:
a. obtener composiciones biológicas en medio de cultivo líquido adecuado, compuesta por mezcla de microorganismos, obtenidos de los depósitos KCTC13158BP y KCTC13159BP;  to. obtain biological compositions in suitable liquid culture medium, composed of a mixture of microorganisms, obtained from the KCTC13158BP and KCTC13159BP tanks;
b. Aplicar mediante riego por aspersión una vez, entre 100 a 200 (cm3) de la composición biológica por cada 500 a 1 .000 (cm2) de superficie de relave o sustrato a tratar, con una tasa riego entre 2 y 2,5 (L/m2); b. Apply by sprinkler irrigation once, between 100 to 200 (cm 3 ) of the biological composition for every 500 to 1, 000 (cm 2 ) of tailings or substrate surface to be treated, with an irrigation rate between 2 and 2.5 (L / m 2 );
c. Estabilizar el material particulado de manera de evitar la erosión eólica o la dispersión de dicho material particulado desde la superficie del sustrato.  C. Stabilize the particulate material so as to avoid wind erosion or dispersion of said particulate material from the substrate surface.
La obtención de las composiciones biológicas se realizó cultivando muestras de costras de suelo en medio líquido estéril adecuado. El protocolo completo comprende: The biological compositions were obtained by cultivating samples of soil scabs in suitable sterile liquid medium. The complete protocol includes:
incubar los tubos con las muestras aplicando fotoperiodos de 12 h de luz y 12 h oscuridad, a una temperatura de 28°C; sembrar y cultivar en medio estéril sólido adecuado e incubar aplicando fotoperiodos de 12 h de luz y 12 h oscuridad, a una temperatura de 28°C y obtener colonias aisladas; secuenciar las colonias aisladas obtenidas; generar cultivos líquidos de las colonias aisladas caracterizadas; criopreservar centrifugando 2 mL de cultivo de cada colonia aislada y caracterizada de cianobacterias y microalgas a 4.000 rpm durante 10 minutos; descartar el sobrenadante y resuspender el precipitado en medio líquido adecuado; enfriar la suspensión durante 5 minutos a 3°C, luego durante 30 minutos a -20°C y sucesivamente congelar a -86°C; descongelar las muestras criopreservadas realizando una inoculación al 10% v/v desde el cultivo madre en medio nutritivo adecuado incubando durante 14 días a 3.000 lux con ciclo lumínico de 14/12 horas y agitación orbital con velocidad constante a 120 rpm. Los medios adecuados para los cultivos son seleccionados entre los siguientes: incubate the tubes with the samples by applying photoperiods of 12 h light and 12 h darkness, at a temperature of 28 ° C; sow and cultivate in suitable solid sterile medium and incubate by applying photoperiods of 12 h of light and 12 h darkness, at a temperature of 28 ° C and obtain isolated colonies; sequence the isolated colonies obtained; generate liquid cultures of the isolated isolated colonies; cryopreserve centrifuging 2 mL of culture from each isolated colony and characterized by cyanobacteria and microalgae at 4,000 rpm for 10 minutes; discard the supernatant and resuspend the precipitate in suitable liquid medium; cool the suspension for 5 minutes at 3 ° C, then for 30 minutes at -20 ° C and then freeze at -86 ° C; Thaw cryopreserved samples by inoculating at 10% v / v from the mother culture in a suitable nutrient medium by incubating for 14 days at 3,000 lux with a light cycle of 14/12 hours and orbital agitation with constant speed at 120 rpm. Suitable media for the crops are selected from the following:
Cultivo de muestras de costras de suelo: BG1 1 o MDM;  Cultivation of soil crust samples: BG1 1 or MDM;
Medio estéril sólido: BG1 1 ;  Solid sterile medium: BG1 1;
Medios para criopreservar las muestras: medio BG1 1 con dimetilsulfóxido Means for cryopreservation of samples: BG1 1 medium with dimethylsulfoxide
3% v/v y metanol 5% v/v o en medio BG1 1 con glicerol 10% v/v; 3% v / v and 5% v / v methanol or in BG1 1 medium with 10% v / v glycerol;
Medio para descongelar los viales: medio nutritivo conteniendo N:P:K y elementos traza Ca, Fe, Mg, Mn y S.  Means for defrosting the vials: nutritive medium containing N: P: K and trace elements Ca, Fe, Mg, Mn and S.
Además, en la presente invención se reivindican composiciones biológicas que comprenden: In addition, biological compositions are claimed in the present invention comprising:
• mezclas de microorganismos obtenidas de los depósitos KCTC13158BP y KCTC13159BP en una concentración entre 106 a 108 células por mL; y• mixtures of microorganisms obtained from the KCTC13158BP and KCTC13159BP deposits at a concentration between 10 6 to 10 8 cells per mL; Y
• medio de cultivo. • culture medium.
Lo microorganismos de la composición biológica pueden ser seleccionadas, pero no se limitan a los géneros de las cianobacterias Leptoiyngbya y Trichocoleus. Estas pueden ser seleccionadas, pero no se limitan a las especies Leptoiyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptoiyngbya boryana, Leptoiyngbya sp. Las microalgas pueden ser seleccionadas, pero no se limitan a especies del género Chlorella. Además, es posible emplear combinaciones de dichas cianobacterias y microalgas. The microorganisms of the biological composition can be selected, but they are not limited to the genera of the Leptoiyngbya and Trichocoleus cyanobacteria. These can be selected, but are not limited to the species Leptoiyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptoiyngbya boryana, Leptoiyngbya sp. Microalgae can be selected, but are not limited to species of the genus Chlorella. In addition, it is possible to use combinations of said cyanobacteria and microalgae.
Es conocido que cianobacterias y microalgas tienen la capacidad de crecer en suelos de granulometrías que abarcan un rango entre 1 a 125 pm, formando biocostras que facilitan la recuperación de suelos empobrecidos, pues estas favorecen el crecimiento de plantas y vegetales sobre suelos de granulometría fina. Sin embargo, el método y composición biológica descritos en la presente invención, están dirigidos a estabilizar el polvo en suspensión proveniente del matenal particulado de relaves, lo que minimiza la infiltración de agua y la erosión causada por el viento en relaves mineros. No existen antecedentes de que las cianobacterias y microalgas sean utilizadas con el propósito descrito en la presente invención. It is known that cyanobacteria and microalgae have the ability to grow in granulometry soils that range from 1 to 125 pm, forming biocostras that facilitate the recovery of impoverished soils, as these favor the growth of plants and vegetables on fine granulometry soils. However, the method and biological composition described in the present invention are aimed at stabilizing the suspended dust from the particulate tailings malt, which minimizes water infiltration and erosion caused by wind in mining tailings. There is no history that cyanobacteria and microalgae are used for the purpose described in the present invention.
EJEMPLOS EXAMPLES
Ejemplo N° 1 : obtención de los cultivos usados en los ensayos de estabilización Example No. 1: obtaining the crops used in the stabilization tests
1 . Las muestras iniciales fueron obtenidas desde suelo de la IV Región de Coquimbo ubicada en la zona sem ¡-árida del oeste de Sudamérica, al sur del gran desierto de Atacama (29°00'S; 32°10'S). En 6 sectores distintos dentro de la Cuarta Región de Coquimbo fueron extraídas 34 muestras de suelo. Los sitios de muestreo fueron escogidos en base al color y textura aparente. Para la extracción de muestras, se removió con cuidado la primera capa de suelo (1 cm de profundidad aprox.) recolectando entre 8 a 15 g de suelo, que se almacenó en tubos Falcon estériles. Una vez colectadas todas las muestras estas fueron conservadas a 4°C.  one . The initial samples were obtained from the soil of the IV Region of Coquimbo located in the semi-arid zone of western South America, south of the great Atacama Desert (29 ° 00'S; 32 ° 10'S). In 6 different sectors within the Fourth Region of Coquimbo, 34 soil samples were extracted. Sampling sites were chosen based on color and apparent texture. For the extraction of samples, the first layer of soil (approximately 1 cm deep) was carefully removed, collecting 8 to 15 g of soil, which was stored in sterile Falcon tubes. Once all samples were collected, they were stored at 4 ° C.
2. Paralelamente, para el aislamiento de los microorganismos, se tomó 1 gramo aproximado de cada muestra de suelo y se le agregaron 10 mL de medio de cultivo estéril (BG1 1 o MDM) en tubos Falcon de 15 mL estériles, o con cantidades menores respetando la proporción 1/10, esto es, por cada gramo de suelo se adicionaron 10 mL de medio. Posteriormente se agitó en un agitador orbital por 30 min a 34°C, se esperó a que decantara el material particulado y se sacó una alícuota de 100 μί, la cual se sembró en ambiente estéril con rastrillo en medio BG1 1 sólido cubriendo toda la placa, se etiquetó cada placa con la información de la muestra, el volumen de inoculo y la fecha. Los tubos con medio líquido con proporción 1/10 y las placas sembradas se incubaron en una cámara de cultivo, la cual disponía de un fotoperiodo de 12 h luz-12 h oscuridad, a una temperatura de 28°C. Los cultivos líquidos se dejaron en la cámara de cultivo hasta que se observaron filamentos verdes oscuros o manchas de color verde claro. Se realizó un análisis por microscopía óptica, en un microscopio Zeiss Axiostarplus® a 100X con un aumento del ocular del 10X, para confirmar la presencia de microorganismos y se traspasó una porción a medio BG1 1 líquido estéril. Para el caso de las placas en medio sólido con evidente crecimiento de colonias pigmentadas, se realizó un análisis por microscopía óptica y se sembró en placas de BG1 1 sólido para la separación de colonias, como también se inocularon en medio líquido para la obtención de biomasa. 2. In parallel, for the isolation of microorganisms, approximately 1 gram of each soil sample was taken and 10 mL of sterile culture medium (BG1 1 or MDM) was added in sterile 15 mL Falcon tubes, or with smaller amounts respecting the 1/10 ratio, that is, for each gram of soil, 10 mL of medium was added. Subsequently, it was stirred on an orbital shaker for 30 min at 34 ° C, the particulate was expected to decant and a 100 μί aliquot was removed, which was seeded in a sterile environment with a rake in solid BG1 1 medium covering the entire plate , each plate was labeled with the sample information, the inoculum volume and the date. The tubes with liquid medium with a ratio of 1/10 and the seeded plates were incubated in a culture chamber, which had a photoperiod of 12 h light-12 h darkness, at a temperature of 28 ° C. Liquid cultures were left in the culture chamber until dark green filaments or light green spots were observed. An analysis was performed by Optical microscopy, in a Zeiss Axiostarplus® 100X microscope with a 10X magnification of the eyepiece, to confirm the presence of microorganisms and a portion was transferred to sterile liquid BG1 1 medium. In the case of the plates in solid medium with evident growth of pigmented colonies, an analysis by optical microscopy was carried out and seeded in plates of solid BG1 for the separation of colonies, as well as inoculated in liquid medium to obtain biomass .
3. Las muestras se identificaron mediante obtención del ADN genómico para lo cual se tomaron 200 μί del cultivo, cuidando que se obtuvieran filamentos en la recolección; para el caso de los microorganismos más compactos se sacaron del medio de cultivo y se obtuvo una porción cuidadosamente removida con un bisturí de forma estéril. Para cultivos en medio sólido, se cortaron secciones con un bisturí de forma estéril. Todas las muestras se introdujeron en los tubos de recolección respectivos y de ser necesario, se enrasaron hasta 200 μί con agua estéril. Para la extracción de DNA genómico se utilizó el kit FavorPrep, Soil DNA isolation minikit de Favorgen Biotech corp®. Se cuantificó ADN y se realizó amplificación de fragmentos de ADN para determinar la presencia de microorganismos en las muestras. 25 μί de productos de PCR de la amplificación de cada muestra, fueron enviados a la empresa Macrogen para su secuenciación. Una vez obtenidos los resultados de la secuenciación se procesó cada secuencia con el software Geneious en relación a la presencia de los primers y la calidad de la secuenciación entregados por los electroferogramas de la secuenciación realizada. Luego se realizó un alineamiento de cada secuencia con la herramienta BLASTn dirigido a cada género de microorganismo. Posteriormente se analizó la lista de resultados significativos obtenidos y los resultados del árbol de distancia entregados por la misma herramienta, identificando el género más probable al que podría pertenecer el microorganismo. Estos análisis indicaron que los microorganismos que se encontraban presentes en las muestras extraídas de costras de suelo correspondían a cianobacterias, específicamente a las especies cianobacteriales Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp. Además, se identificó la presencia del género de microalga Chlorella. 4. Para obtener los cultivos que se usaron en el análisis de estabilidad, se realizó una inoculación al 10% v/v desde del cultivo criopreservado de las especie de cianobaterias y microalgas aisladas, y las muestras fueron crecidas durante dos semanas en medio nutritivo conteniendo una proporción de N:P:K y elementos traza: Ca, Fe, Mg, Mn y S. Las condiciones de crecimiento correspondieron a 3.000 lux proveniente de luz fluorescente (40,5 pmol m-2 s-1 ), agitación orbital con velocidad constante a 120 rpm, ciclo lumínico de 14/12 horas, humedad entre 40% y 60%. 3. The samples were identified by obtaining genomic DNA for which 200 μί of the culture was taken, taking care that filaments were obtained in the collection; in the case of the most compact microorganisms, they were removed from the culture medium and a carefully removed portion was obtained with a sterile scalpel. For cultures in solid medium, sections were cut with a sterile scalpel. All samples were introduced into the respective collection tubes and, if necessary, flush up to 200 μί with sterile water. For the extraction of genomic DNA, the FavorPrep kit, Soil DNA isolation minikit from Favorgen Biotech corp® was used. DNA was quantified and amplification of DNA fragments was performed to determine the presence of microorganisms in the samples. 25 μί of PCR products amplifying each sample were sent to the company Macrogen for sequencing. Once the results of the sequencing were obtained, each sequence was processed with the Geneious software in relation to the presence of the primers and the quality of the sequencing delivered by the electropherograms of the sequencing performed. An alignment of each sequence was then performed with the BLASTn tool aimed at each genre of microorganism. Subsequently, the list of significant results obtained and the results of the distance tree delivered by the same tool were analyzed, identifying the most probable genus to which the microorganism could belong. These analyzes indicated that the microorganisms that were present in the samples extracted from soil scabs corresponded to cyanobacteria, specifically the cyanobacterial species Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp. In addition, the presence of the Chlorella microalgae genus was identified. 4. To obtain the cultures that were used in the stability analysis, a 10% v / v inoculation was made from the cryopreserved culture of the isolated cyanobacterial and microalgae species, and the samples were grown for two weeks in nutrient medium containing a proportion of N: P: K and trace elements: Ca, Fe, Mg, Mn and S. The growth conditions corresponded to 3,000 lux from fluorescent light (40.5 pmol m-2 s-1), orbital agitation with constant speed at 120 rpm, light cycle of 14/12 hours, humidity between 40% and 60%.
Ejemplo N° 2: Criopreservación de las muestras Example No. 2: Cryopreservation of samples
2 mi de cultivo de cada aislado fue centrifugado a 4.000 rpm a temperatura ambiente durante 10 minutos. A continuación, se descartó el sobrenadante y se resuspendió el precipitado en medio de criopreservación. Dos medios de criopreservación fueron utilizados para preservar a -86°C: a) Medio A: BG1 1 (ATCC Médium 616), dimetilsulfóxido (DMSO en concentración al 3% v/v y metanol en concentración al 5% v/v). b) Medio B: BG1 1 y glicerol en concentración al 10% v/v. 2 ml of culture of each isolate was centrifuged at 4,000 rpm at room temperature for 10 minutes. Then, the supernatant was discarded and the precipitate was resuspended in cryopreservation medium. Two cryopreservation media were used to preserve at -86 ° C: a) Medium A: BG1 1 (ATCC Medium 616), dimethylsulfoxide (DMSO in 3% v / v concentration and methanol in 5% v / v concentration). b) Medium B: BG1 1 and glycerol in concentration at 10% v / v.
Una vez resuspendida la biomasa de cianobacterias y microalgas, se enfrió la nueva suspensión durante 5 minutos a 4°C, 30 minutos a -20°C y finalmente se llevó el cultivo a -86°C. Once the cyanobacterial and microalgae biomass was resuspended, the new suspension was cooled for 5 minutes at 4 ° C, 30 minutes at -20 ° C and finally the culture was brought to -86 ° C.
Las muestras obtenidas desde costras del suelo han sido depositadas bajo las especificaciones del tratado de Budapest en la autoridad internacional de depósitos "Korean Collection for Type Cultures", bajo los números KCTC 13158BP y KCTC 13159BP. Dichas muestras depositadas contienen las especies cianobacteriales Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp. y microalgas del género Chlorella. Samples obtained from soil crusts have been deposited under the specifications of the Budapest Treaty at the international deposit authority "Korean Collection for Type Cultures", under the numbers KCTC 13158BP and KCTC 13159BP. Said deposited samples contain the cyanobacterial species Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp. and microalgae of the genus Chlorella.
Ejemplo N° 3: Evaluación de viabilidad celular de las muestras criopreservadas Example No. 3: Evaluation of cell viability of cryopreserved samples
Previo a la evaluación de viabilidad se descongelaron los cultivos mantenidos a -86°C, manteniendo cada muestra a 4°C, tras lo cual fueron centrifugadas a 4.000 rpm durante 5 minutos a 4°C. Posterior a esto se eliminó el sobrenadante y se inoculó toda la biomasa precipitada, sobre medio líquido o sólido (BG1 1 ) a una concentración de 10% v/v. Para evaluar la viabilidad se inocularon los aislados criopreservados sobre medio de cultivo nutritivo sólido (BG1 1 con 1 % agar) y se mantuvieron durante 7 días a 3.000 lux proveniente de luz fluorescente (40,5 pmol m"2 s"1), ciclo lumínico de 14/12 horas, humedad entre 40% y 60%. En este caso la viabilidad se verificó por crecimiento en placa. Prior to the feasibility assessment, the cultures maintained at -86 ° C were thawed, keeping each sample at 4 ° C, after which they were centrifuged at 4,000 rpm for 5 minutes at 4 ° C. After this, the supernatant was removed and all the precipitated biomass was inoculated on liquid or solid medium (BG1 1) at a concentration of 10% v / v. To evaluate the viability, the cryopreserved isolates were inoculated on solid nutrient culture medium (BG1 1 with 1% agar) and kept for 7 days at 3,000 lux from fluorescent light (40.5 pmol m "2 s " 1 ), cycle light of 12/14 hours, humidity between 40% and 60%. In this case the viability was verified by plaque growth.
Para evaluar la viabilidad en medio líquido, se inoculó la biomasa de cianobacterias y microalgas criopreservadas sobre medio de cultivo nutritivo (BG1 1 ) y se mantuvo el cultivo durante 30 días bajo las siguientes condiciones de crecimiento: 3.000 lux proveniente de luz fluorescente (40,5 pmol m"2 s"1), ciclo lumínico de 14/12 horas, humedad entre 40% y 60%, y agitación orbital a velocidad constante de 120 rpm. En este caso la viabilidad se verifica por crecimiento, a través de la observación de la biomasa crecida luego de una y dos semanas. Ejemplo N° 4: Caracterización geotécnica del material de relave To assess the viability in liquid medium, the cryopreserved cyanobacteria and microalgae biomass was inoculated on nutrient culture medium (BG1 1) and the culture was maintained for 30 days under the following growth conditions: 3,000 lux from fluorescent light (40, 5 pmol m "2 s " 1 ), light cycle of 14/12 hours, humidity between 40% and 60%, and orbital agitation at a constant speed of 120 rpm. In this case, the viability is verified by growth, through the observation of the biomass grown after one and two weeks. Example No. 4: Geotechnical characterization of tailings material
Se realizaron ensayos de laboratorio para evaluar la composición geotécnica del material de relave midiendo los parámetros de granulometría por método mecánico (Tabla N° 1 ) y granulometría por método del hidrómetro bajo la norma de ASTM (Tabla N° 2). Tabla N° 1 : Granulometría por método mecánico
Figure imgf000015_0001
Laboratory tests were carried out to evaluate the geotechnical composition of the tailings material by measuring the parameters of granulometry by mechanical method (Table No. 1) and granulometry by hydrometer method under the ASTM standard (Table No. 2). Table N ° 1: Granulometry by mechanical method
Figure imgf000015_0001
Tabla N° 2: Granulometría por método del hidrómetro (norma ASTM)
Figure imgf000015_0002
Además, se midieron los parámetros de gravedad específica (Tabla N° 3), densidad máxima compactada (Tabla N° 4) y densidad aparente suelta (Tabla N° 5) a las muestras de relave. Table N ° 2: Granulometry by hydrometer method (ASTM standard)
Figure imgf000015_0002
In addition, the parameters of specific gravity (Table No. 3), maximum compacted density (Table No. 4) and bulk bulk density (Table No. 5) were measured to the tailings samples.
Tabla N° 3: Gravedad específica
Figure imgf000016_0001
Table No. 3: Specific Gravity
Figure imgf000016_0001
Tabla N° 4: Densidad máxima compactada
Figure imgf000016_0002
Table N ° 4: Maximum compacted density
Figure imgf000016_0002
Tabla N° 5: Densidad aparente suelta
Figure imgf000016_0003
Ejemplo N° 5: Ensayos en túnel de viento Table 5: Loose bulk density
Figure imgf000016_0003
Example No. 5: Wind tunnel tests
Los ensayos en túnel de viento fueron realizados bajo la norma chilena de depósitos de relaves NCh 3266 - 2012, simulando condiciones de velocidades y regímenes de viento que se registran en sitios de emplazamiento de faenas mineras afectadas por erosión eólica, de manera que los resultados son extrapolables a las condiciones de terreno.  Wind tunnel tests were carried out under the Chilean tailings deposit standard NCh 3266 - 2012, simulating wind speed and speed conditions that are registered at sites of mining sites affected by wind erosion, so that the results are extrapolated to ground conditions.
a) Preparación de las probetas: La preparación de las probetas contempló el llenado de los moldes metálicos con relave a densidad aparente suelta. Para esto se utilizó un embudo que permite la caída libre y constante del relave hasta completar su capacidad, finalmente enrasando la superficie para dejarla uniforme. El volumen promedio de las probetas es de 3.804 cm3 La probeta preparada se deja reposar por aproximadamente 12 horas, antes de someterla al ensayo en el túnel de viento. Las muestras tratadas con el agente mitigador (Ph1 ) y con los microorganismos fueron aplicados mediante riego superficial, por lo que para una superficie de 726 cm2 se utilizaron los volúmenes y las tasas de riego especificadas en la tabla N° 6. A partir de las muestras criopreservadas se prepararon 2 mezclas de microorganismos: la mezcla 1 contiene Leptolyngbya badia, Trichocoleus sociatus, y la mezcla 2 contiene Trichocoleus desertorum, Leptolyngbya boryana y Chlorella.sp. a) Preparation of the specimens: The preparation of the specimens contemplated the filling of the metal molds with tailings at loose bulk density. For this, a funnel was used that allows the free and constant fall of the tailings until its capacity is completed, finally flush the surface to leave it uniform. The average volume of the specimens is 3,804 cm 3 The prepared specimen is allowed to stand for approximately 12 hours, before being tested in the wind tunnel. The samples treated with the mitigating agent (Ph1) and with the microorganisms were applied by surface irrigation, so that for an area of 726 cm 2 the volumes and irrigation rates specified in Table No. 6 were used. From The cryopreserved samples were prepared 2 mixtures of microorganisms: mixture 1 contains Leptolyngbya badia, Trichocoleus sociatus, and mixture 2 contains Trichocoleus desertorum, Leptolyngbya boryana and Chlorella.sp.
Tabla N° 6: Tasa de riego y volumen aplicado a las muestras Table N ° 6: Irrigation rate and volume applied to the samples
Figure imgf000017_0001
b) Instalación de la probeta y ejecución del ensayo: La probeta se instaló en el soporte de plano regulable del túnel de viento; la inclinación para esta serie de pruebas fue entre 9o y 10° con respecto a la horizontal. Una vez instalada la probeta en el soporte, se inició el ensayo aplicando viento a una velocidad de 12 m/s, considerando un tiempo de exposición de seis horas continuas. Para cada ensayo se tomó el peso y humedad inicial de cada probeta, y después de finalizado el ensayo se pesó el material remanente en el molde y se midió la humedad final. Cada uno de los ensayos realizados fue registrado fotográficamente. En el caso de las probetas sin tratamiento se realizó una toma cada 5 minutos. En el caso de las probetas tratadas, se realizaron tomas cada 5 minutos hasta completar 20 minutos, luego a los 60 de exposición y posteriormente a cada hora hasta completar las seis horas de ensayo. Las figuras 1 -3 muestran un registro fotográfico del desempeño típico de las probetas sin tratamiento (Figura 1 ), tratamiento con el agente mitigador Ph1 (Figura 2), y tratamiento con la mezcla 1 (Figura 3), en diferentes tiempos durante la realización de los ensayos sometidos al efecto eólico en el túnel de viento. Para el ensayo con la mezcla 2, no proporcionamos fotografías. Resultados obtenidos de ensayos en túnel de viento: Se realizaron ensayos a doce probetas, de acuerdo a la norma chilena de depósitos de relaves NCh 3266 - 2012, de las cuales tres no fueron tratadas, tres fueron tratadas con Ph1 , tres fueron tratadas con la mezcla 1 y otras tres fueron tratadas con la mezcla 2. Los pesos y porcentajes de pérdida de relave por efecto eólico registrados en los ensayos realizados, se presentan en la tabla N° 7 y en las figuras 4 (Sin tratamiento), 5 (Tratamiento con Ph1 ), 6 (Tratamiento con la mezcla 1 ) y 7 (Tratamiento con la mezcla 2). Es importante destacar que en el caso de las probetas no tratadas, la pérdida de material se alcanzó a los 20 minutos, tal como se observa en las figuras 1 y 4. En el caso de las probetas tratadas con Ph1 , la pérdida de material se registró luego de tres y seis horas de exposición de la probeta a la acción del viento, como se puede observar en las figuras 2 y 5. En las probetas tratadas con la mezcla 1 , no se registró pérdida de material particulado, como se puede apreciar en las figuras 3 y 6. Este resultado también se observó al tratar el material particulado proveniente de relave con la mezcla 2, según se puede apreciar en la figura 7.
Figure imgf000017_0001
b) Installation of the specimen and execution of the test: The specimen was installed on the adjustable plane support of the wind tunnel; the inclination for this series of tests was between 9 ° and 10 ° with respect to the horizontal. Once the specimen was installed in the support, the test was started by applying wind at a speed of 12 m / s, considering an exposure time of six continuous hours. For each test, the initial weight and humidity of each specimen was taken, and after the end of the test, the remaining material in the mold was weighed and the final humidity was measured. Each of the tests performed was registered. photographically. In the case of untreated specimens, one shot was taken every 5 minutes. In the case of the treated specimens, shots were taken every 5 minutes to complete 20 minutes, then at 60 exposure and then every hour until six hours of testing were completed. Figures 1 -3 show a photographic record of the typical performance of the specimens without treatment (Figure 1), treatment with the mitigating agent Ph1 (Figure 2), and treatment with the mixture 1 (Figure 3), at different times during the realization of the tests subjected to the wind effect in the wind tunnel. For the test with mixture 2, we do not provide photographs. Results obtained from wind tunnel tests: Twelve test pieces were performed, according to the Chilean norm of tailings deposits NCh 3266 - 2012, of which three were not treated, three were treated with Ph1, three were treated with the mixture 1 and three others were treated with mixture 2. The weights and percentages of tailings loss due to wind effect recorded in the tests performed, are presented in table No. 7 and in figures 4 (Without treatment), 5 (Treatment with Ph1), 6 (Treatment with mixture 1) and 7 (Treatment with mixture 2). It is important to note that in the case of untreated specimens, the loss of material was reached after 20 minutes, as shown in Figures 1 and 4. In the case of specimens treated with Ph1, the loss of material is recorded after three and six hours of exposure of the specimen to the action of the wind, as can be seen in figures 2 and 5. In the specimens treated with mixture 1, no loss of particulate material was recorded, as can be appreciate in figures 3 and 6. This result was also observed when treating the particulate material coming from tailings with the mixture 2, as can be seen in figure 7.
Tabla N° 7: Resultados de pesos y porcentajes de pérdida de relave Table N ° 7: Results of weights and percentages of tailings loss
Figure imgf000019_0001
Figure imgf000019_0001
s.t.: Ensayos sin tratamiento  s.t .: Untreated trials
Mientras esta invención ha sido descrita bajo las modalidades señaladas anteriormente, podría parecer evidente que otras alternativas, modificaciones o variaciones entregarían los mismos resultados, sin embrago, hemos podido establecer que las proporciones en las que se encuentran presentes las cianobacterias y las algas son fundamentales para el éxito de la metodología. Consecuentemente, las modalidades de la invención pretenden ser ilustrativas, no limitantes. Varios cambios pueden ser realizados sin alejarse del espíritu y alcance de la invención como se define en las siguientes reivindicaciones. While this invention has been described under the modalities indicated above, it might seem obvious that other alternatives, modifications or variations would deliver the same results, however, we have been able to establish that the proportions in which cyanobacteria and algae are present are fundamental for The success of the methodology. Consequently, the embodiments of the invention are intended to be illustrative, not limiting Various changes can be made without departing from the spirit and scope of the invention as defined in the following claims.
Todas las patentes, solicitudes de patentes, artículos científicos y otros documentos públicos que en conocimiento del solicitante constituyen el estado del arte, han sido adecuadamente citados en la presente solicitud. All patents, patent applications, scientific articles and other public documents that, in the applicant's knowledge, constitute the state of the art, have been properly cited in the present application.

Claims

REIVINDICACIONES
1 . Un método para suprimir el polvo en suspensión proveniente de material particulado de relaves, CARACTERIZADA porque comprende: one . A method for suppressing suspended dust from particulate tailings material, CHARACTERIZED because it comprises:
a. obtener composiciones biológicas en medio de cultivo líquido adecuado, compuestas por mezclas de microorganismos obtenidos de los depósitos to. obtain biological compositions in suitable liquid culture medium, composed of mixtures of microorganisms obtained from the deposits
KCTC13158BP y KCTC13159BP; KCTC13158BP and KCTC13159BP;
b. Aplicar una vez entre 100 a 200 cm3 una composición biológica por cada 500 a 1 .000 cm2 de superficie de relave o sustrato a tratar, con una tasa riego entre 2 y 2,5 L/m2; b. Apply once between 100 to 200 cm3 biological composition 500 to 1 000 cm 2 of surface tailings or substrate to be treated with an irrigation rate between 2 and 2.5 L / m 2;
c. Estabilizar el material particulado.  C. Stabilize the particulate material.
2. El método de la reivindicación 1 , CARACTERIZADO porque la aplicación de la composición biológica se realiza mediante riego por aspersión. 2. The method of claim 1, CHARACTERIZED in that the application of the biological composition is carried out by sprinkler irrigation.
3. El método según la reivindicación 1 , CARACTERIZADO porque las composiciones biológicas se obtienen al cultivar muestras de costras de suelo en medio líquido estéril adecuado; incubar los tubos con las muestras aplicando fotoperiodos de 12 h de luz y 12 h oscuridad, a una temperatura de 28°C; cultivar en medio estéril sólido adecuado e incubar aplicando fotoperiodos de 12 h de luz y 12 h oscuridad, a una temperatura de 28°C y obtener colonias aisladas; secuenciar las colonias aisladas obtenidas; criopreservar centrifugando 2 ml_ de cultivo de cada muestra aislada y caracterizada a 4.000 rpm durante 10 minutos; descartar el sobrenadante y resuspender el precipitado en medio líquido adecuado; enfriar la suspensión durante 5 minutos a 3°C, luego 3 durante 30 minutos a -20°C y congelar a -86°C; descongelar las muestras criopreservadas realizando una inoculación al 10% v/v desde el cultivo madre en medio nutritivo adecuado incubando durante 14 días a 3.000 lux con ciclo lumínico de 14/12 horas y agitación orbital con velocidad constante a 120 rpm; mezclar las especies de cianobacterias y/o microalgas; cultivar hasta obtener los volúmenes necesanos y densidad celular de 106 a 108 células por ml_. 3. The method according to claim 1, CHARACTERIZED in that the biological compositions are obtained by growing samples of soil scabs in suitable sterile liquid medium; incubate the tubes with the samples by applying photoperiods of 12 h light and 12 h darkness, at a temperature of 28 ° C; cultivate in a suitable solid sterile medium and incubate by applying photoperiods of 12 h of light and 12 h darkness, at a temperature of 28 ° C and obtain isolated colonies; sequence the isolated colonies obtained; cryopreserve centrifuging 2 ml_ of culture of each isolated and characterized sample at 4,000 rpm for 10 minutes; discard the supernatant and resuspend the precipitate in suitable liquid medium; cool the suspension for 5 minutes at 3 ° C, then 3 for 30 minutes at -20 ° C and freeze at -86 ° C; thaw cryopreserved samples by inoculating at 10% v / v from the mother culture in suitable nutrient medium incubating for 14 days at 3,000 lux with a light cycle of 14/12 hours and orbital agitation with constant speed at 120 rpm; mix the species of cyanobacteria and / or microalgae; Cultivate to obtain the necessary volumes and cell density of 10 6 to 10 8 cells per ml.
4. El método según las reivindicaciones 1 a 3, CARACTERIZADO porque el medio líquido estéril adecuado para cultivar las muestras de costras de suelo puede ser seleccionado entre medio BG1 1 o MDM. 4. The method according to claims 1 to 3, CHARACTERIZED in that the sterile liquid medium suitable for cultivating the soil crust samples can be selected from BG1 1 or MDM medium.
5. El método según las reivindicaciones 1 a 3, CARACTERIZADO porque el medio estéril sólido adecuado es medio sólido BG1 1 . 5. The method according to claims 1 to 3, CHARACTERIZED in that the suitable solid sterile medium is BG1 1 solid medium.
6. El método según las reivindicaciones 1 a 3, CARACTERIZADO porque el medio adecuado para criopreservar es seleccionado entre medio BG1 1 con dimetiisulfoxido 3% v/v y metanol 5% v/v o en medio BG1 1 con glicerol 10% v/v. 6. The method according to claims 1 to 3, CHARACTERIZED in that the suitable medium for cryopreservation is selected from BG1 1 medium with 3% v / v dimethisulfoxide and 5% v / v methanol or BG1 1 medium with 10% v / v glycerol.
7. El método según las reivindicaciones 1 a 3, CARACTERIZADO porque el medio adecuado para descongelar los viales de las muestras es medio nutritivo conteniendo N:P:K y elementos traza Ca, Fe, Mg, Mn y S. 7. The method according to claims 1 to 3, CHARACTERIZED in that the suitable means for thawing the sample vials is nutritive medium containing N: P: K and trace elements Ca, Fe, Mg, Mn and S.
8. Una composición biológica como supresor de polvo proveniente de material particulado de relaves, CARACTERIZADA porque comprende: 8. A biological composition as a dust suppressor from tailings particulate material, CHARACTERIZED because it comprises:
• Una mezcla de microorganismos obtenida de los depósitos KCTC13158BP y KCTC13159BP, en una concentración entre 106 a 108 células por ml_; y• A mixture of microorganisms obtained from the KCTC13158BP and KCTC13159BP deposits, at a concentration between 10 6 to 10 8 cells per ml_; Y
• medio de cultivo. • culture medium.
9. La composición biológica de la reivindicación 8, CARACTERIZADA porque los microorganismos son seleccionados de cianobacterias, más específicamente son seleccionadas entre las especies Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp., o combinaciones de ellas. 9. The biological composition of claim 8, CHARACTERIZED in that the microorganisms are selected from cyanobacteria, more specifically are selected from the species Leptolyngbya badia, Trichocoleus sociatus, Trichocoleus desertorum Leptolyngbya boryana, Leptolyngbya sp., Or combinations thereof.
10. La composición biológica de la reivindicación 8, CARACTERIZADA porque los microorganismos son seleccionados de microalgas del género Chlorella sp. 10. The biological composition of claim 8, CHARACTERIZED in that the microorganisms are selected from microalgae of the genus Chlorella sp.
1 1 . La composición biológica de la reivindicación 8, CARACTERIZADA porque el medio de cultivo es seleccionado entre medio líquido estéril BG1 1 , MDM, o medio nutritivo conteniendo N:P:K y elementos traza Ca, Fe, Mg, Mn y S eleven . The biological composition of claim 8, CHARACTERIZED in that the culture medium is selected from sterile liquid medium BG1 1, MDM, or nutrient medium containing N: P: K and trace elements Ca, Fe, Mg, Mn and S
12. El uso de la composición biológica, CARACTERIZADO porque se emplea para supnmir el polvo en suspensión proveniente de material particulado de relaves de minería en general, producido por actividades industriales, de transporte, y fuentes naturales como la erosión eólica 12. The use of the biological composition, CHARACTERIZED because it is used to suppress suspended dust from particulate material from mining tailings in general, produced by industrial activities, transportation, and natural sources such as wind erosion
3 3
PCT/CL2017/050092 2016-12-30 2017-12-28 Method for eliminating suspended dust originating from particulate tailings generated by means of wind erosion, comprising obtaining a biological composition, applying the biological composition, and stabilising the particulate matter, as well as the resulting biological composition and the application thereof WO2018119541A1 (en)

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BR112019013552-9A BR112019013552A2 (en) 2016-12-30 2017-12-28 METHOD FOR SUPPRESSING DUST IN SUSPENSION OF PARTICULATED WASTE MATERIAL, BIOLOGICAL COMPOSITION AND USE OF BIOLOGICAL COMPOSITION

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