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WO2014067023A1 - Production of sulfhydric acid from sulphur by means of a microbial consortium - Google Patents

Production of sulfhydric acid from sulphur by means of a microbial consortium Download PDF

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Publication number
WO2014067023A1
WO2014067023A1 PCT/CL2013/000070 CL2013000070W WO2014067023A1 WO 2014067023 A1 WO2014067023 A1 WO 2014067023A1 CL 2013000070 W CL2013000070 W CL 2013000070W WO 2014067023 A1 WO2014067023 A1 WO 2014067023A1
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Prior art keywords
microbial consortium
bioreactor
halophilic
sulfur
hydrogen sulfide
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Spanish (es)
French (fr)
Inventor
Davor COTORAS TADIC
Cristian Patricio MARTÍNEZ LUCO
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Universidad de Chile
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Universidad de Chile
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Priority to AU2013337536A priority Critical patent/AU2013337536B2/en
Priority to CA2879839A priority patent/CA2879839A1/en
Publication of WO2014067023A1 publication Critical patent/WO2014067023A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the present invention relates to a biological process for the production of hydrogen sulfide from sulfur, to be used for the industrial production of sodium sulfhydrate (NaSH), for its use in water treatment, in mining, cellulose and in the industry of tanneries, or directly as a precipitating agent of metals in mining or industrial effluents.
  • NaSH sodium sulfhydrate
  • PAQUES BV® is a company that develops technologies for water treatment, among which the biogenic production of H 2 S stands out with the patented THIOTEQ TM technology, which generates H 2 S from different sulfur sources (S °, sulfuric acid or other sources of sulfur) with an electron donor such as ethanol, acetic acid, hydrogen gas and others, at room temperature and pressure (US Patent 6,852,305; Buisman et al. 2006. Biologically produced sulphide for purification of process streams , effluent treatment and recovery of metais in the metal and mining industry. Hydrometallurgy. 83: 106-113).
  • the sulphide produced is introduced into a contact tank with the tributary, dissolving and precipitating the dissolved metals.
  • the metal sulphides are separated by sedimentation and dehydrated, generating an effluent treated with low levels of dissolved metals.
  • Another process for the production of biogenic H 2 S is described in Chilean Patent Application No. 200700022, which discloses a process of bacterial reduction of elemental sulfur, using sulfate reducing bacteria in the presence of sulfate and carbon dioxide .
  • This process uses hydrogen gas as an electron donor for the metabolism of sulfur reducing bacteria.
  • this process becomes restrictive for an application in the treatment of wastewater with high metal contents.
  • the alternatives currently available have different disadvantages.
  • the main difficulty is that sulfur-reducing bacteria use only low molecular weight organic molecules as electron donors, such as pyruvate, lactate, ethanol, mixtures of alcohols or hydrogen.
  • This invention solves the problems of the state of the art using a halophilic sulfur reducing microbial consortium, which is capable of using complex organic substrates, such as agroindustrial products or wastes, thus reducing the operating costs of the system.
  • Another feature of the invention is that the high pH values and high salt concentrations, at which the microbial consortium of this invention is capable of growing and producing hydrogen sulfide, generates a restrictive environment for the growth of other competing microorganisms (such as methanogenic archaea) or other contaminating microorganisms. This gives the process the additional advantage of greater stability.
  • the main object of the present invention is a biological method of producing sulfuric acid from sulfur by means of a microbial consortium, which comprises at least the steps of:
  • a culture medium that contains at least one suspension of one or more complex carbon organic substrates, such as donors of electrons and powdered or finely ground sulfur.
  • the bioreactor support material is the same complexed particulate carbon organic compound.
  • the halophilic sulfur reducing microbial consortium is enriched from an environmental sample.
  • the environmental sample is the anaerobic mud of a saline lagoon or a salt flat.
  • the halophilic sulfur reducing microbial consortium is composed of at least hydrolytic, fermentative, acetogenic and sulfur reducing microorganisms (see Figure 1).
  • the halophilic sulfur reducing microbial consortium is composed of bacteria and archaea.
  • the bacteria belong, at least, to the phylogenetic groups of Proteobacteria ⁇ , ⁇ , and ⁇ and bacteria of the Citofaga-Flavobacterium group.
  • the complex organic substrate (s) are naturally occurring products rich in polymeric organic compounds.
  • products of natural origin rich in polymeric organic compounds are selected from the group of cellulose, lignocellulosic plant products or residues, starch, starch-rich plant products or residues, seaweed, microalgae and cyanobacteria.
  • the support materials are selected from the group of ceramics, silicon stone, glass, diatomaceous earth extrudate and plastic.
  • the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 5.0 and 10.
  • the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 6.5 and 10. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 8.5 and 9.5.
  • the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 0 and 100 g / L. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 20 and 80 g / L.
  • the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 25 and 70 g / L.
  • the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 30 and 60 g / L.
  • the hydrogen sulfide acid produced is used as a metal precipitating agent in mining or industrial effluents.
  • the hydrogen sulphide acid produced is used for the industrial production of sodium hydrochloride (NaSH).
  • Microbial consortium in this invention, the concept of microbial consortium is understood as a group of different microorganisms that act together.
  • microorganisms with different metabolic capacities can be found.
  • sulfur reducing microbial consortium it is composed, for example, of hydrolytic, fermentative, acetogenic and sulfur reducing microorganisms.
  • hydrolytic proteolytic microorganisms capable of degrading proteins
  • sucroitic microorganisms capable of degrading several sugars
  • Lipolytic microorganisms capable of digesting lipids or fats
  • cellulite microorganisms capable of degrading cellulose or plant matter.
  • This figure shows a general outline of the stages and metabolic products in anaerobic microbial digestion of complex organic matter, using sulfur as an electron acceptor (Modified by Muyzer G., Stams A. 2008. The ecology and biotechnology of sulphate-reducing bacteria. Nature Reviews Microbiology. 6: 441-454).
  • BRS ° sulfur reducing bacteria.
  • FIGURE 2 is a diagrammatic representation of FIGURE 1
  • This figure shows the change in appearance that occurs in culture medium with starch, cellulose and spirulina as a substrate, before and after cultivating the microbial sulfur reducing consortium.
  • the black color is produced by the precipitation of iron sulfide generated by the interaction of the H 2 S produced with and the ferrous ion present in the culture medium.
  • FIGURE 3 is a diagrammatic representation of FIGURE 3
  • FIGURE 4 shows production of H 2 S by the microbial consortium, grown in media with cellulose (t3 ⁇ 43 ⁇ 43 ⁇ 4), starch (BH) and spirulina (IB) as electron donors, respectively. These cultures were grown for 14, 10 and 8 days, respectively. The values represent the means ⁇ standard deviation. The means with common letter (a, b) do not present statistically significant differences, according to Duncan (p ⁇ 0.05).
  • FIGURE 4 shows production of H 2 S by the microbial consortium, grown in media with cellulose (t3 ⁇ 43 ⁇ 43 ⁇ 4), starch (BH) and spirulina (IB) as electron donors, respectively. These cultures were grown for 14, 10 and 8 days, respectively. The values represent the means ⁇ standard deviation. The means with common letter (a, b) do not present statistically significant differences, according to Duncan (p ⁇ 0.05).
  • This figure shows the in situ hybridization of the microbial consortium grown in a starch culture medium.
  • the percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.
  • FIGURE 5
  • This figure shows the in situ hybridization of the microbial consortium grown in a cellulose culture medium.
  • the percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.
  • FIGURE 6 is a diagrammatic representation of FIGURE 6
  • This figure shows the in situ hybridization of the microbial consortium grown in a culture medium with spirulina.
  • the percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.
  • FIGURE 7 is a diagrammatic representation of FIGURE 7
  • This figure shows the effect of pH on the production of H 2 S by the starch-grown microbial consortium as a substrate for 21 days of culture.
  • the concentration of H 2 S is shown on days 5 (O), 8 (O), 14 ( ⁇ ) and 21
  • FIGURE 8
  • This figure shows the effect of pH on the production of H 2 S by the microbial consortium grown with spirulina as a substrate for 21 days of culture.
  • the concentration of H 2 S during days 5 ( ⁇ ), 8 (O), 14 () and 21 () of culture is shown.
  • the values represent the means ⁇ standard deviation.
  • the average values with the same letter are not statistically different, except the averages of the 21st day of cultivation (Duncan, p ⁇ 0.05).
  • FIGURE 9 This figure represents a simplified diagram of the fixed bed bioreactor used in the tests.
  • the culture medium containing the sulfur powder and one or more complex organic substrates is fed through the feed duct (1) with a pump (2) and entered through an inlet duct (3) to the bioreactor (5), filled with a support material (4), which can also be the same complex organic substrate in solid or particulate form).
  • the culture medium is recirculated through the recirculation duct (6) with a recirculation pump (7).
  • the effluent containing hydrogen sulfide is removed from the bioreactor through the outlet duct (8).
  • FIGURE 10 is a diagrammatic representation of FIGURE 10
  • This figure shows the performance profile of the cellulose-fed bioreactor and Celite R-635 as support material, during stage one of the bioreactor start-up.
  • the concentration values of H 2 S represent the means ⁇ standard deviation.
  • the concentration of hydrogen sulfide acid in effluent () is shown on the left axis and on the right axis the fed pH (+++) and the effluent pH () during operation.
  • the vertical segmented line ( ⁇ ) and the upper box in each figure represent the change in the mode of operation and the percentage of feeding with respect to the total volume of the bioreactor.
  • FIGURE 11 is a diagrammatic representation of FIGURE 11
  • This figure shows the performance profile of the cellulose-fed bioreactor and Celite R-635 as support material, during stage two of the bioreactor commissioning.
  • the concentration values of H 2 S represent the means ⁇ standard deviation.
  • FIGURE 12 is a diagrammatic representation of FIGURE 12
  • This figure shows the performance profile of the bioreactor without support and fed with spirulina as an organic substrate, during stage one of the bioreactor commissioning.
  • the concentration values of H 2 S represent the averages ⁇ standard deviation.
  • the concentration of hydrogen sulfide acid in effluent () is shown on the left axis and on the right axis the fed pH (+++) and the effluent pH () during operation.
  • the vertical segmented line (!) And the top box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor.
  • FIGURE 13 is a diagrammatic representation of FIGURE 13
  • This figure shows the performance profile of the bioreactor without support and fed with spirulina as an organic substrate, during stage two of commissioning the bioreactor.
  • the H2S concentration values represent the means ⁇ standard deviation.
  • the vertical segmented line (j) and the upper box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor.
  • the interval (//) represents a recirculated batch operation time in which there was no parameter determination.
  • FIGURE 14 is a diagrammatic representation of FIGURE 14
  • This figure shows the performance profile of the bioreactor fed with spirulina and Celite R-635 as a support material, during the bioreactor commissioning stage.
  • the concentration values of H 2 S represent the means ⁇ standard deviation.
  • ) and the top box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor.
  • the interval (//) represents a recirculated batch operation time in which there was no parameter determination.
  • Example 1 Cultivation of the sulfur reducing microbial consortium with complex organic substrates
  • the growth of the microbial consortium was evaluated with the three organic substrates studied in triplicate, determining the production of sulphides by the appearance of black precipitate. of iron sulfide (Postgate, 1979) and by the spectrophotometric method of production of methylene blue (Conagua. 1982. Water analysis - Determination of sulphides. Mexican Standard NMX-AA-084-1982. Mexico).
  • the method of production of methylene blue is based on the formation of blue color produced by the reaction of H 2 S with N, N-dimethyl-1,4-phenylenediamine oxalate and iron (III) chloride.
  • the quantification of hydrogen sulfide is carried out by taking 5 mL of sample, to which 500 ⁇ of a solution of N, N-dimethyl-1,4-phenylenediamino oxalate (6.75 g L "1 ) and 150 are added. ⁇ of ferric chloride (2500 g L '1 ), which is allowed to react for about 5 minutes, subsequently adding diamonium diphosphate (500 gL "1 ) to eliminate excess ferric chloride interference. After 5 minutes the absorbency of the sample was measured at 664 nm, which is proportional to the concentration of dissolved sulphides.
  • Table 1 Composition of the modified Postage C culture medium with the different substrates studied
  • Figure 2 shows the development of sulfur reducing bacteria due to the appearance of black precipitate in the culture media with starch, cellulose and spirulina as carbon sources, respectively.
  • Figure 3 the generation of H 2 S by the microbial consortium grown at pH 7.1 is shown in the three substrates studied by the methylene blue method. A greater production of sulfides is observed in the media grown with spirulina and starch, while with the cellulose substrate there is a significantly lower production of sulphides, according to Duncan's test (p ⁇ 0.05).
  • Example 2 In situ hybridization with fluorescent probe of the sulfur reducing microbial consortium.
  • the characterization of the microbial consortium grown on the different substrates was performed using the fluorescent in situ hybridization technique. Cultures used for hybridization were performed using the methodology used in Example 1. The probes used and their characteristics are shown in Table 2.
  • Table 2 Probes used in the study of microorganism characterization of the microbial consortium, its sequence, position in the rRNA and specificity in in situ hybridization (Amann et al. 1995. Phylogenetic Identification and In Situ Detection of Individual microbial cell without cultivation. icrobiol Rev. 59: 143-169).
  • ALF1b a-Proteobacteria C ⁇ 6S, 19-35) CGTTCGYTCTGAGCCAG
  • the slides with the samples were incubated for ninety minutes at 45 ° C, which after this time were washed with their respective wash solution for 30 minutes at 45 ° C (Table 4). Once washed, the slides with the fixed samples were allowed to dry at room temperature and then stained with 20 ⁇ of DAPI (4 ', 6-Diamidino-2-phenylindole) at 50 ⁇ g ⁇ mL "1 for 10 minutes, then rinsed with distilled water to remove excess DAPI The samples were finally observed in an epifluorescence microscope, with Zeiss filter No. 20 for probe labeled with CY3 and with Zeiss filter No. 09 to see the microorganisms labeled with DAPI.
  • DAPI 4- ', 6-Diamidino-2-phenylindole
  • Table 3 Composition of the hybridization solution used for the study of in situ hybridization for the different probes analyzed.
  • Table 4 Composition of the wash solution used for the study of in situ hybridization for the different probes analyzed.
  • the sulfur reducing microbial consortium is composed of bacteria and archaea. Also this consortium has in its microbial structure bacteria of the subclass Proteobacteria ⁇ , ⁇ and ⁇ , in addition to bacteria of the Citofaga-Flavobacterium group, depending on their composition of the type of complex carbon organic substrate used as for their cultivation.
  • Example 2 Effect of pH on the production of hydrogen sulfide by the sulfur reducing microbial consortium.
  • culture media were prepared as in Example 1, with the different substrates studied, with the exception of pH and the absence of ferric chloride.
  • the pH of the media was adjusted before autoclaving from 5.5 to 10.0 with an amplitude of 0.5 units. However, these levels changed after sterilization.
  • the actual pH with which one worked was 5.2; 5.6; 6.1; 6.6; 7.1; 7.5; 8.0; 8.5; 8.9 and 9.4.
  • In these media sulfide measurements were made by the methylene blue method.
  • Figure 7 shows that the production of H 2 S by the microbial starch consortium, as an electron donor, there was a significantly higher production at pH 8.9 and 9.4, compared to the other pH analyzed for all the days studied (Duncan test, p ⁇ 0.05), those that remained relatively stable within the range of 3 - 30 ppm of H 2 S.
  • Example 3 Development of the microbial sulfur reducing consortium in a reactor with support and with cellulose as an organic substrate.
  • This bioreactor consisted of a main column with two upper and lower inlets, to which it was filled with support material, culture medium (similar to that used in Example 1, except for the absence of ferric chloride and the concentration of NaCI, which was 40 g L "1 ) and cellulose as an organic substrate at a concentration of 1 gL " which was subsequently autoclaved at 110 ° C for 30 minutes.
  • the culture medium containing the sulfur powder and cellulose is fed through the feed line (1) with a pump (2) and the bioreactor (5), filled with Celite R-635 (4), is entered through an inlet duct (3).
  • the recirculating culture medium through the recirculation duct (6) with a recirculation pump (7).
  • the effluent containing hydrogen sulfide is removed from the bioreactor through the outlet duct (8).
  • this bioreactor was operated in batch, and then passed to a semi-continuous upward recirculated batch stage to achieve a 24-hour recirculation hydraulic retention time (HRT).
  • HRT 24-hour recirculation hydraulic retention time
  • a fresh inlet and substrate were fed through a lower inlet in a g COD / g S ° ratio of «1, 00 with a feed flow of 1.73 ⁇ 0.31 mL min " through a peristaltic pump, in volumes equivalent to the total volume of the bioreactors, while the effluent to be evaluated was removed from the upper part.
  • H 2 S accordinging to Example 1
  • pH of the effluents obtained to obtain the greatest sulfurogenic activity were estimated daily
  • the chemical demand for oxygen or COD was also determined (Kit Hanna Hl 93754A-25 LR, based on Official Standard Methods procedure, Standard Methods for
  • the bioreactor operation was temporarily separated into two stages; a first stage in which the parameters and optimal start-up times and the period of immobilization of the microorganisms were established; and a second stage in which the bioreactors were operated under a semi-continuous regime, which sought to increase the volumetric productivity of H 2 S, modifying parameters such as hydraulic retention times and the volume and pH of the fed medium.
  • Figures 10 and 11 show the performance profile of the bioreactor fed with cellulose and filled with Celite R-635 as a support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S, reaching a maximum of 1.94 mol / m 3 d in this bioreactor with cellulose as organic substrate.
  • Example 4 Development of the sulfur reducing microbial consortium in an unsupported reactor and with spirulina as an organic substrate.
  • Example 3 A Teflon reactor of the same characteristics used in Example 3 and with the same methodology was used, with the exception of the substrate supplied, the absence of support material and the concentration of NaCI, which was 30 gL "1. This bioreactor was fed with spirulina at a concentration of 1 gL "1 as an organic nutrient.
  • Figures 12 and 13 show the performance profile of the bioreactor fed with spirulina and without support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S was observed, reaching a maximum of 2.75 mol / m 3 d in this bioreactor with spirulina as organic substrate.
  • Example 5 Development of the microbial sulfur reducing consortium in a reactor with support and with spirulina as organic substrate.
  • the methodology of operation and maintenance of this bioreactor was similar to that used in Example 3, with the exception of the substrate supplied and the concentration of NaCI, which was 30 gL "1.
  • This bioreactor was fed with 1 gL " 1 of spirulina as a nutrient organic.
  • the production of H 2 S (according to Example 1) and the pH of the effluents obtained to obtain the greatest sulfurogenic activity were estimated daily.
  • the chemical oxygen demand or COD (as in Example 3) of some important points of the operation was also determined.
  • Figure 14 shows the performance profile of the bioreactor fed with spirulina and without support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S was observed, reaching a maximum of 2.94 mol / m 3 d in this bioreactor with spirulina as organic substrate and Celite R-635 as support material.

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Abstract

The invention relates to a biological method for producing sulfhydric acid from sulphur by means of a microbial consortium, wherein said method comprises at least the steps of: (a) growing a halophilic sulfur-reducing microbial consortium, (b) recirculating the culture medium of the bioreactor until a biofilm of the sulfur-reducing consortium forms over the supporting material of the bioreactor, (c) adding to the bioreactor, continuously or semi-continuously, a culture medium containing at least one suspension of at least one complex carbonaceous organic substrate, and (d) removing the effluent containing the sulfhydric acid from the bioreactor.

Description

PRODUCCIÓN DE ÁCIDO SULFHÍDRICO PARTIR DE AZUFRE MEDIANTE UN SULFHYDIC ACID PRODUCTION FROM SULFUR BY MEANS OF

CONSORCIO MICROBIANO. MICROBIAN CONSORTIUM

CAMPO DE APLICACIÓN DE LA INVENCIÓN FIELD OF APPLICATION OF THE INVENTION

La presente invención se refiere a un proceso biológico de producción de ácido sulfhídrico a partir de azufre, para ser utilizado para la producción industrial de sulfhidrato de sodio (NaSH), para su uso tratamiento de aguas, en minería, celulosa y en la industria de las curtiembres, o directamente como agente precipitante de metales en efluentes mineros o industriales. The present invention relates to a biological process for the production of hydrogen sulfide from sulfur, to be used for the industrial production of sodium sulfhydrate (NaSH), for its use in water treatment, in mining, cellulose and in the industry of tanneries, or directly as a precipitating agent of metals in mining or industrial effluents.

DESCRIPCIÓN DE LO CONOCIDO EN LA MATERIA DESCRIPTION OF THE KNOWN IN THE MATTER

Se ha descrito numerosos diseños de biorreactores activos para la reducción biológica de sulfato por la acción de las bacterias reductoras de sulfato (Kaksonen A., Puhakka J. 2007. Sulfate Reduction Based Bioprocesses for the Treatment of Acid Mine Drainage and the Recovery of Metals. Engineering in Life Sciences. 7(6): 541- 564.), lo que les permite operar a altas cargas volumétricas y mantener altas eficiencias de tratamiento. Sin embargo, la mayoría de las aplicaciones de los biorreactores basados en bacterias reductoras de sulfato consideran el H2S como un subproducto de la remediación aguas residuales con alto contenido de sulfatos y drenajes ácidos de mina, dejando en segundo plano la remoción y/o recuperación de metales. Numerous designs of active bioreactors for the biological reduction of sulfate by the action of sulfate reducing bacteria have been described (Kaksonen A., Puhakka J. 2007. Sulfate Reduction Based Bioprocesses for the Treatment of Acid Mine Drainage and the Recovery of Metals. Engineering in Life Sciences. 7 (6): 541-564.), which allows them to operate at high volumetric loads and maintain high treatment efficiencies. However, most applications of bioreactors based on sulfate reducing bacteria consider H 2 S as a byproduct of wastewater remediation with high sulfate content and acid mine drains, leaving in the background the removal and / or metal recovery.

Actualmente existen pocas publicaciones de la producción de H2S mediante la utilización de bacterias reductoras de azufre. PAQUES B.V® es una compañía que desarrolla tecnologías para el tratamiento de aguas, entre las cuales destaca la producción biogénica de H2S con la tecnología patentada THIOTEQ™, la cual genera H2S desde distintas fuentes de azufre (S°, ácido sulfúrico u otras fuentes de azufre) con un donador de electrones como el etanol, ácido acético, gas hidrógeno y otros, a temperatura y presión ambiente (Patente norteamericana US 6.852.305; Buisman et al. 2006. Biologically produced sulphide for purification of process streams, effluent treatment and recovery of metáis in the metal and mining industry. Hydrometallurgy. 83: 106- 113). En este proceso, el sulfuro producido se introduce a un tanque contactador con el afluente, disolviéndose y precipitando los metales disueltos. Los sulfuras metálicos se separan por sedimentación y se deshidratan, generando un efluente tratado con bajos niveles de metales disueltos. Otro proceso para la producción de biogénica de H2S está descrito en la Solicitud de patente chilena N° 200700022, la cual da a conocer un proceso de reducción bacteriana de azufre elemental, utilizando bacterias reductoras de sulfato en presencia de sulfato y dióxido de carbono. Este proceso emplea hidrógeno gaseoso como dador de electrones para el metabolismo de las bacterias reductoras de azufre. Sin embargo, y debido al alto costo del hidrógeno molecular, este proceso se torna restrictivo para una aplicación en el tratamiento de aguas residuales con altos contenidos de metales. There are currently few publications on the production of H 2 S through the use of sulfur reducing bacteria. PAQUES BV® is a company that develops technologies for water treatment, among which the biogenic production of H 2 S stands out with the patented THIOTEQ ™ technology, which generates H 2 S from different sulfur sources (S °, sulfuric acid or other sources of sulfur) with an electron donor such as ethanol, acetic acid, hydrogen gas and others, at room temperature and pressure (US Patent 6,852,305; Buisman et al. 2006. Biologically produced sulphide for purification of process streams , effluent treatment and recovery of metais in the metal and mining industry. Hydrometallurgy. 83: 106-113). In this process, the sulphide produced is introduced into a contact tank with the tributary, dissolving and precipitating the dissolved metals. The metal sulphides are separated by sedimentation and dehydrated, generating an effluent treated with low levels of dissolved metals. Another process for the production of biogenic H 2 S is described in Chilean Patent Application No. 200700022, which discloses a process of bacterial reduction of elemental sulfur, using sulfate reducing bacteria in the presence of sulfate and carbon dioxide . This process uses hydrogen gas as an electron donor for the metabolism of sulfur reducing bacteria. However, and due to the high cost of molecular hydrogen, this process becomes restrictive for an application in the treatment of wastewater with high metal contents.

Las alternativas disponibles en la actualidad presentan diferentes desventajas. La principal dificultad es que las bacterias reductoras de azufre utilizan como los donadores de electrones solo moléculas orgánicas de bajo peso molecular, tales como piruvato, lactato, etanol, mezclas de alcoholes o hidrógeno. Esto representa una desventaja desde el punto de vista económico ya que el alto costo actual de estos sustratos encarece y hace poco viable la utilización de este proceso a escalas industriales. Esta invención resuelve los problemas del estado de la técnica utilizando un consorcio microbiano reductor de azufre halófilo, que es capaz de emplear sustratos orgánicos complejos, tales como productos o desechos agroindustriales, disminuyendo, de esta forma, los costos de operación del sistema. Otra característica de la invención es que los altos valores de pH y altas concentraciones de sal, a los cuales el consorcio microbiano de esta invención es capaz de crecer y producir ácido sulfhídrico, genera un ambiente restrictivo para el crecimiento de otros microrganismos competidores (tales como las arqueas metanogénicas) u otros microorganismos contaminantes. Esto le confiere al proceso la ventaja adicional de una mayor estabilidad. The alternatives currently available have different disadvantages. The main difficulty is that sulfur-reducing bacteria use only low molecular weight organic molecules as electron donors, such as pyruvate, lactate, ethanol, mixtures of alcohols or hydrogen. This represents a disadvantage from the economic point of view since the current high cost of these substrates makes the use of this process at industrial scales more expensive and unfeasible. This invention solves the problems of the state of the art using a halophilic sulfur reducing microbial consortium, which is capable of using complex organic substrates, such as agroindustrial products or wastes, thus reducing the operating costs of the system. Another feature of the invention is that the high pH values and high salt concentrations, at which the microbial consortium of this invention is capable of growing and producing hydrogen sulfide, generates a restrictive environment for the growth of other competing microorganisms (such as methanogenic archaea) or other contaminating microorganisms. This gives the process the additional advantage of greater stability.

DEFINICIÓN DE LA INVENCIÓN DEFINITION OF THE INVENTION

El principal objeto de la presente invención es un método biológico de producción de ácido sulfhídrico a partir de azufre mediante un consorcio microbiano, que comprende al menos los pasos de: The main object of the present invention is a biological method of producing sulfuric acid from sulfur by means of a microbial consortium, which comprises at least the steps of:

a) crecer un consorcio microbiano reductor de azufre halófilo, capaz de utilizar sustratos orgánicos carbonados complejos como donadores de electrones en un biorreactor anaerobio de lecho fijo que contiene el material de soporte del biorreactor y un medio de cultivo constituido, al menos, por uno o más sustratos orgánicos carbonados complejos, como donadores de electrones, y azufre, b) recircular el medio de cultivo del biorreactor hasta que se forme la biopelícula del consorcio reductor de azufre sobre el material de soporte del biorreactor, a) grow a halophilic sulfur reducing microbial consortium, capable of using complex carbon-based organic substrates as electron donors in a fixed-bed anaerobic bioreactor containing the bioreactor support material and a culture medium consisting of at least one or more complex carbon organic substrates, such as electron donors, and sulfur, b) recirculating the bioreactor culture medium until the biofilm of the sulfur reducing consortium is formed on the bioreactor support material,

c) agregar al biorreactor, en forma continua o semicontinua, un medio de cultivo que contiene al menos una suspensión de uno o más sustratos orgánicos carbonados complejos, como donadores de electrones y azufre en polvo o finamente molido. c) add to the bioreactor, continuously or semi-continuously, a culture medium that contains at least one suspension of one or more complex carbon organic substrates, such as donors of electrons and powdered or finely ground sulfur.

d) retirar del biorreactor el efluente que contiene el ácido sulfhídrico. d) remove the effluent containing hydrogen sulfide from the bioreactor.

En una realización de la invención, el material de soporte del biorreactor es el mismo compuesto orgánicos carbonado complejo en forma particulada.  In one embodiment of the invention, the bioreactor support material is the same complexed particulate carbon organic compound.

En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo se enriquece a partir de una muestra ambiental. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium is enriched from an environmental sample.

En otra realización de la invención, la muestra ambiental es el lodo anaerobio de una laguna salina o un salar. En una realización de la invención, el consorcio microbiano reductor de azufre halófilo está compuesto al menos por microorganismos hidrolíticos, fermentativos, acetogénicos y reductores de azufre (ver Figura 1 ). In another embodiment of the invention, the environmental sample is the anaerobic mud of a saline lagoon or a salt flat. In one embodiment of the invention, the halophilic sulfur reducing microbial consortium is composed of at least hydrolytic, fermentative, acetogenic and sulfur reducing microorganisms (see Figure 1).

En otra realización de la invención el consorcio microbiano reductor de azufre halófilo está compuesto por bacterias y arqueas. In another embodiment of the invention the halophilic sulfur reducing microbial consortium is composed of bacteria and archaea.

En otra realización de la invención, las bacterias pertenecen, a lo menos, a los grupos filogenéticos de las Proteobacterias α, β, y δ y bacterias del grupo Citofaga- Flavobacterium. In another embodiment of the invention, the bacteria belong, at least, to the phylogenetic groups of Proteobacteria α, β, and δ and bacteria of the Citofaga-Flavobacterium group.

En una realización de la invención, el o los sustratos orgánicos complejos son productos de origen natural ricos en compuestos orgánicos poliméricos. In one embodiment of the invention, the complex organic substrate (s) are naturally occurring products rich in polymeric organic compounds.

En otra realización de la invención, los productos de origen natural ricos en compuestos orgánicos poliméricos se seleccionan del grupo de la celulosa, los productos o residuos vegetales lignocelulósicos, el almidón, los productos o residuos vegetales ricos en almidón, las algas marinas, las microalgas y las cianobacterias. In another embodiment of the invention, products of natural origin rich in polymeric organic compounds are selected from the group of cellulose, lignocellulosic plant products or residues, starch, starch-rich plant products or residues, seaweed, microalgae and cyanobacteria.

En una realización de la invención, los materiales de soporte se seleccionan del grupo de la cerámica, la piedra silícica, el vidrio, extruido de tierra de diatomeas y el plástico. En una realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 5,0 y 10. In one embodiment of the invention, the support materials are selected from the group of ceramics, silicon stone, glass, diatomaceous earth extrudate and plastic. In one embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 5.0 and 10.

En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 6,5 y 10. En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 8,5 y 9,5. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 6.5 and 10. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 8.5 and 9.5.

En una realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 0 y 100 g/L. En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 20 y 80 g/L. In one embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 0 and 100 g / L. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 20 and 80 g / L.

En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 25 y 70 g/L. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 25 and 70 g / L.

En otra realización de la invención, el consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 30 y 60 g/L. In another embodiment of the invention, the halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 30 and 60 g / L.

En otra realización de la invención, el ácido sulfhídrico producido se emplea como agente precipitante de metales en efluentes mineros o industriales In another embodiment of the invention, the hydrogen sulfide acid produced is used as a metal precipitating agent in mining or industrial effluents.

En otra realización de la invención, el ácido sulfhídrico producido se emplea para la producción industrial de sulfhidrato de sodio (NaSH). In another embodiment of the invention, the hydrogen sulphide acid produced is used for the industrial production of sodium hydrochloride (NaSH).

Definiciones: Definitions:

Consorcio microbiano: en ésta invención, el concepto de consorcio microbiano se entiende como un grupo de diferentes microorganismos que actúan en conjunto. En un consorcio microbiano se puede encontrar microorganismos con diferentes capacidades metabólicas. En el caso particular del consorcio microbiano reductor de azufre, éste está compuesto, por ejemplo, por microorganismos hidrolíticos, fermentativos, acetogénicos y reductores de azufre. Entre los microrganismos hidrolíticos se podría encontrar microorganismos proteolíticos (capaces de degradar proteínas); microorganismos sacaroiíticos (capaces de degradar varios azúcares); microorganismos lipolíticos (capaces de digerir los lípidos o grasas), o microorganismos celulíticos (capaz para degradar la celulosa o la materia vegetal), Estas capacidades metabólicas diferentes permiten que el consorcio sea capaz de degradar una variedad de residuos orgánicos complejos. Microbial consortium: in this invention, the concept of microbial consortium is understood as a group of different microorganisms that act together. In a microbial consortium, microorganisms with different metabolic capacities can be found. In the particular case of the sulfur reducing microbial consortium, it is composed, for example, of hydrolytic, fermentative, acetogenic and sulfur reducing microorganisms. Among the microorganisms hydrolytic proteolytic microorganisms (capable of degrading proteins) could be found; sucroitic microorganisms (capable of degrading several sugars); Lipolytic microorganisms (capable of digesting lipids or fats), or cellulite microorganisms (capable of degrading cellulose or plant matter). These different metabolic capacities allow the consortium to be able to degrade a variety of complex organic waste.

Descripción de las Figuras FIGURA 1 : Description of the Figures FIGURE 1:

Esta figura muestra un esquema general de las etapas y productos metabólicos en la digestión microbiana anaerobia de materia orgánica compleja, utilizando azufre como aceptor de electrones (Modificada de Muyzer G., Stams A. 2008. The ecology and biotechnology of sulphate- reducing bacteria. Nature Reviews Microbiology. 6: 441- 454). BRS° = bacterias reductoras de azufre.  This figure shows a general outline of the stages and metabolic products in anaerobic microbial digestion of complex organic matter, using sulfur as an electron acceptor (Modified by Muyzer G., Stams A. 2008. The ecology and biotechnology of sulphate-reducing bacteria. Nature Reviews Microbiology. 6: 441-454). BRS ° = sulfur reducing bacteria.

FIGURA 2: FIGURE 2:

Esta figura muestra el cambio de aspecto que se produce en medio de cultivo con almidón, celulosa y espirulina como sustrato, antes y después de cultivar el consorcio microbiano reductor de azufre. El color negro se produce por la precipitación de sulfuro de hierro generado por la interacción del H2S producido con y el ion ferroso presente en el medio de cultivo. This figure shows the change in appearance that occurs in culture medium with starch, cellulose and spirulina as a substrate, before and after cultivating the microbial sulfur reducing consortium. The black color is produced by the precipitation of iron sulfide generated by the interaction of the H 2 S produced with and the ferrous ion present in the culture medium.

FIGURA 3: FIGURE 3:

Esta figura muestra producción de H2S por el consorcio microbiano, crecidos en medios con celulosa (t¾¾¾), almidón (BH) y espirulina (IB) como donadores de electrones, respectivamente. Estos cultivos fueron crecidos durante 14, 10 y 8 días, respectivamente. Los valores representan los promedios ± desviación estándar. Las medias con letra común (a,b) no presentan diferencias estadísticamente significativas, según Duncan (p<0,05). FIGURA 4: This figure shows production of H 2 S by the microbial consortium, grown in media with cellulose (t¾¾¾), starch (BH) and spirulina (IB) as electron donors, respectively. These cultures were grown for 14, 10 and 8 days, respectively. The values represent the means ± standard deviation. The means with common letter (a, b) do not present statistically significant differences, according to Duncan (p <0.05). FIGURE 4:

Esta figura muestra la hibridación in situ del consorcio microbiano cultivado en un medio de cultivo con almidón. Se muestra los porcentajes de cada uno de los grupos, marcados con las sondas específicas, respecto a los microorganismos totales marcados con DAPI. Las barras de error corresponden a la desviación estándar entre los porcentajes de microorganismos marcados con la sonda. This figure shows the in situ hybridization of the microbial consortium grown in a starch culture medium. The percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.

FIGURA 5: FIGURE 5:

Esta figura muestra la hibridación in situ del consorcio microbiano cultivado en un medio de cultivo con celulosa. Se muestra los porcentajes de cada uno de los grupos, marcados con las sondas específicas, respecto a los microorganismos totales marcados con DAPI. Las barras de error corresponden a la desviación estándar entre los porcentajes de microorganismos marcados con la sonda. This figure shows the in situ hybridization of the microbial consortium grown in a cellulose culture medium. The percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.

FIGURA 6: FIGURE 6:

Esta figura muestra la hibridación in situ del consorcio microbiano cultivado en un medio de cultivo con espirulina. Se muestra los porcentajes de cada uno de los grupos, marcados con las sondas específicas, respecto a los microorganismos totales marcados con DAPI. Las barras de error corresponden a la desviación estándar entre los porcentajes de microorganismos marcados con la sonda.  This figure shows the in situ hybridization of the microbial consortium grown in a culture medium with spirulina. The percentages of each of the groups, marked with the specific probes, are shown with respect to the total microorganisms marked with DAPI. Error bars correspond to the standard deviation between the percentages of microorganisms marked with the probe.

FIGURA 7: FIGURE 7:

Esta figura muestra el efecto del pH en la producción de H2S por el consorcio microbiano crecido con almidón como sustrato durante 21 días de cultivo. Se muestra la concentración de H2S durante los días 5 ( O ), 8 ( O ), 14 ( Δ ) y 21This figure shows the effect of pH on the production of H 2 S by the starch-grown microbial consortium as a substrate for 21 days of culture. The concentration of H 2 S is shown on days 5 (O), 8 (O), 14 (Δ) and 21

( ) de cultivo. Los valores representan los promedios ± desviación estándar. (*) Los promedios de los datos son significativamente diferentes para todos los días de cultivo, según Duncan (p<0,05). () of cultivation. The values represent the means ± standard deviation. (*) The data averages are significantly different for every day of cultivation, according to Duncan (p <0.05).

FIGURA 8: FIGURE 8:

Esta figura muestra el efecto del pH en la producción de H2S por el consorcio microbiano crecido con espirulina como sustrato durante 21 días de cultivo. Se muestra la concentración de H2S durante los días 5 ( Π ), 8 ( O ), 14 ( ) y 21 ( ) de cultivo. Los valores representan los promedios ± desviación estándar. (a,b) Los valores promedio con misma letra no son diferentes estadísticamente, excepto las medias del día 21 de cultivo (Duncan, p<0,05). This figure shows the effect of pH on the production of H 2 S by the microbial consortium grown with spirulina as a substrate for 21 days of culture. The concentration of H 2 S during days 5 (Π), 8 (O), 14 () and 21 () of culture is shown. The values represent the means ± standard deviation. (a, b) The average values with the same letter are not statistically different, except the averages of the 21st day of cultivation (Duncan, p <0.05).

FIGURA 9: Esta figura representa un diagrama simplificado del biorreactor de lecho fijo usado en los ensayos. En este proceso, el medio de cultivo que contiene el azufre en polvo y uno a más sustratos orgánicos complejos se alimenta mediante el conducto de alimentación (1 ) con una bomba (2) y se ingresa mediante un conducto de entrada (3) al biorreactor (5), relleno de un material de soporte (4), que también puede ser el mismo sustrato orgánico complejo en forma sólida o particulada). El medio de cultivo se recircula mediante el conducto de recirculación (6) con una bomba de recirculación (7). El efluente que contiene el ácido sulfhídrico se retirar del biorreactor mediante el conducto de salida (8). FIGURE 9: This figure represents a simplified diagram of the fixed bed bioreactor used in the tests. In this process, the culture medium containing the sulfur powder and one or more complex organic substrates is fed through the feed duct (1) with a pump (2) and entered through an inlet duct (3) to the bioreactor (5), filled with a support material (4), which can also be the same complex organic substrate in solid or particulate form). The culture medium is recirculated through the recirculation duct (6) with a recirculation pump (7). The effluent containing hydrogen sulfide is removed from the bioreactor through the outlet duct (8).

FIGURA 10: FIGURE 10:

Esta figura muestra el perfil de desempeño del biorreactor alimentado con celulosa y Celite R-635 como material de soporte, durante la etapa uno de puesta en marcha del biorreactor. Los valores de concentración de H2S representan los promedios ± desviación estándar. En el eje izquierdo se muestra la concentración de ácido sulfhídrico en efluente ( ) y en el eje derecho el pH alimentado (+++) y el pH del efluente ( ) durante la operación. La línea segmentada vertical (¡) y el recuadro superior en cada figura representa el cambio en el modo de operación y el porcentaje de alimentación respecto al volumen total del biorreactor. This figure shows the performance profile of the cellulose-fed bioreactor and Celite R-635 as support material, during stage one of the bioreactor start-up. The concentration values of H 2 S represent the means ± standard deviation. The concentration of hydrogen sulfide acid in effluent () is shown on the left axis and on the right axis the fed pH (+++) and the effluent pH () during operation. The vertical segmented line (¡) and the upper box in each figure represent the change in the mode of operation and the percentage of feeding with respect to the total volume of the bioreactor.

FIGURA 11 : FIGURE 11:

Esta figura muestra el perfil de desempeño del biorreactor alimentado con celulosa y Celite R-635 como material de soporte, durante la etapa dos de puesta en marcha del biorreactor. Los valores de concentración de H2S representan los promedios ± desviación estándar. A) Eje izquierdo, concentración de ácido sulfhídrico en efluenteThis figure shows the performance profile of the cellulose-fed bioreactor and Celite R-635 as support material, during stage two of the bioreactor commissioning. The concentration values of H 2 S represent the means ± standard deviation. A) Left axis, concentration of hydrogen sulfide in effluent

( ); eje derecho 1 , tiempo de retención hidráulico ( ); eje derecho 2, demanda química de oxígeno (·); B) pH alimentado ( ) y pH del efluente ( ) durante la operación. La línea segmentada vertical (¡) y el recuadro superior en cada figura representa el cambio en el modo de operación y el porcentaje de alimentación respecto al volumen total del biorreactor. El intervalo (//) representa un tiempo de operación en lote recirculado en el cual no hubo determinación de parámetros. (); right axis 1, hydraulic retention time (); right axis 2, chemical oxygen demand (·); B) fed pH () and effluent pH () during operation. The vertical segmented line (¡) and the upper box in each figure represent the change in the mode of operation and the percentage of feeding with respect to the total volume of the bioreactor. The interval (//) represents a recirculated batch operation time in which there was no parameter determination.

FIGURA 12: FIGURE 12:

Esta figura muestra el perfil de desempeño del biorreactor sin soporte y alimentado con espirulina como sustrato orgánico, durante la etapa uno de puesta en marcha del biorreactor. Los valores de concentración de H2S representan los promedios ± desviación estándar. En el eje izquierdo se muestra la concentración de ácido sulfhídrico en efluente ( ) y en el eje derecho el pH alimentado (+++) y el pH del efluente ( ) durante la operación. La línea segmentada vertical (!) y el recuadro superior en cada figura representa el cambio en el modo de operación y el porcentaje de alimentación respecto al volumen total del biorreactor. This figure shows the performance profile of the bioreactor without support and fed with spirulina as an organic substrate, during stage one of the bioreactor commissioning. The concentration values of H 2 S represent the averages ± standard deviation. The concentration of hydrogen sulfide acid in effluent () is shown on the left axis and on the right axis the fed pH (+++) and the effluent pH () during operation. The vertical segmented line (!) And the top box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor.

FIGURA 13: FIGURE 13:

Esta figura muestra el perfil de desempeño del biorreactor sin soporte y alimentado con espirulina como sustrato orgánico, durante la etapa dos de puesta en marcha del biorreactor. Los valores de concentración de H2S representan los promedios ± desviación estándar. A) Eje izquierdo, concentración de ácido sulfhídrico en efluente This figure shows the performance profile of the bioreactor without support and fed with spirulina as an organic substrate, during stage two of commissioning the bioreactor. The H2S concentration values represent the means ± standard deviation. A) Left axis, concentration of hydrogen sulfide in effluent

( ); eje derecho 1 , tiempo de retención hidráulico ( ); eje derecho 2, demanda química de oxígeno (·); B) pH alimentado ( ) y pH del efluente ( ) durante la operación. La línea segmentada vertical (j) y el recuadro superior en cada figura representa el cambio en el modo de operación y el porcentaje de alimentación respecto al volumen total del biorreactor. El intervalo (//) representa un tiempo de operación en lote recirculado en el cual no hubo determinación de parámetros. (); right axis 1, hydraulic retention time (); right axis 2, chemical oxygen demand (·); B) fed pH () and effluent pH () during operation. The vertical segmented line (j) and the upper box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor. The interval (//) represents a recirculated batch operation time in which there was no parameter determination.

FIGURA 14: FIGURE 14:

Esta figura muestra el perfil de desempeño del biorreactor alimentado con espirulina y Celite R-635 como material de soporte, durante la etapa de puesta en marcha del biorreactor. Los valores de concentración de H2S representan los promedios ± desviación estándar. A) Eje izquierdo, concentración de ácido sulfhídrico en efluente ( ); eje derecho 1 , tiempo de retención hidráulico ( ); eje derecho 2, demanda química de oxígeno (·); B) pH alimentado ( ) y pH del efluente ( ) durante la operación. La línea segmentada vertical (|) y el recuadro superior en cada figura representa el cambio en el modo de operación y el porcentaje de alimentación respecto al volumen total del biorreactor. El intervalo (//) representa un tiempo de operación en lote recirculado en el cual no hubo determinación de parámetros. This figure shows the performance profile of the bioreactor fed with spirulina and Celite R-635 as a support material, during the bioreactor commissioning stage. The concentration values of H 2 S represent the means ± standard deviation. A) Left axis, concentration of hydrogen sulfide in effluent (); right axis 1, hydraulic retention time (); right axis 2, chemical oxygen demand (·); B) fed pH () and effluent pH () during operation. The vertical segmented line (|) and the top box in each figure represent the change in the mode of operation and the percentage of feed with respect to the total volume of the bioreactor. The interval (//) represents a recirculated batch operation time in which there was no parameter determination.

Los siguientes ejemplos ilustran algunas aplicaciones concretas de la invención, pero no pretenden limitar el marco ni los alcances de la presente invención. EJEMPLOS The following examples illustrate some specific applications of the invention, but are not intended to limit the scope or scope of the present invention. EXAMPLES

Ejemplo 1 : Cultivo del consorcio microbiano reductor de azufre con sustratos orgánicos complejos Example 1: Cultivation of the sulfur reducing microbial consortium with complex organic substrates

El cultivo del consorcio se realizó en tubos de ensayo de 20 mL, a los cuales se les agregó 10 mL de medio Postgate C modificado (Tabla 1) (Postgate J. 1979. The Sulphate-Reducing Bacteria. Cambridge University Press. Chapter 3: Cultivation and growth. 24 - 27) utilizando distintas fuentes de carbono y una alta concentración de cloruro de sodio. A estos medios se les ajustó el pH a 7,5 usando hidróxido de potasio (KOH) 3M para alcalinizar y ácido fosfórico (H3P04) 1M para acidificar, para posteriormente autoclavar a 110°C por 30 minutos. Para generar condiciones anaeróbicas a los medios se cubrieron con una capa de aceite de parafina estéril luego de ser autoclavados. Los medios de cultivo se inocularon con aproximadamente 200 μί de cultivos previos y se mantuvieron en una incubadora a 28°C Se evaluó el crecimiento del consorcio microbiano con los tres sustratos orgánicos estudiados por triplicado, determinando la producción de sulfuras por la aparición de precipitado negro de sulfuro de hierro (Postgate, 1979) y por el método espectrofotométrico de producción de azul de metileno (Conagua. 1982. Análisis de agua - Determinación de sulfuras. Norma Mexicana NMX-AA-084-1982. México). El método de producción de azul de metileno se basa en la formación de color azul producido por la reacción del H2S con el N,N-dimetil-1 ,4-fenilendiamino oxalato y cloruro de hierro (III). La cuantificación de ácido sulfhídrico se lleva a cabo tomando 5 mL de muestra, a los cuales se les adiciona 500 μΐ de una solución de N,N-dimetil- 1 ,4-fenilendiamino oxalato (6,75 g L"1) y 150 μΐ de cloruro férrico (2500 g L'1), el cual se deja reaccionar por alrededor de 5 minutos, agregando posteriormente difosfato de diamonio (500 g-L"1) para eliminar la interferencia del cloruro férrico en exceso. Luego de 5 minutos se midió la absorbencia de la muestra a 664 nm, la que es proporcional a la concentración de sulfuras disueltos. Consortium culture was performed in 20 mL test tubes, to which 10 mL of modified Postgate C medium (Table 1) was added (Postgate J. 1979. The Sulphate-Reducing Bacteria. Cambridge University Press. Chapter 3: Cultivation and growth 24-27) using different carbon sources and a high concentration of sodium chloride. To these media the pH was adjusted to 7.5 using 3M potassium hydroxide (KOH) to alkalize and 1M phosphoric acid (H 3 P0 4 ) to acidify, then autoclave at 110 ° C for 30 minutes. To generate anaerobic conditions the media were covered with a layer of sterile paraffin oil after being autoclaved. The culture media were inoculated with approximately 200 μί of previous cultures and kept in an incubator at 28 ° C. The growth of the microbial consortium was evaluated with the three organic substrates studied in triplicate, determining the production of sulphides by the appearance of black precipitate. of iron sulfide (Postgate, 1979) and by the spectrophotometric method of production of methylene blue (Conagua. 1982. Water analysis - Determination of sulphides. Mexican Standard NMX-AA-084-1982. Mexico). The method of production of methylene blue is based on the formation of blue color produced by the reaction of H 2 S with N, N-dimethyl-1,4-phenylenediamine oxalate and iron (III) chloride. The quantification of hydrogen sulfide is carried out by taking 5 mL of sample, to which 500 μΐ of a solution of N, N-dimethyl-1,4-phenylenediamino oxalate (6.75 g L "1 ) and 150 are added. μΐ of ferric chloride (2500 g L '1 ), which is allowed to react for about 5 minutes, subsequently adding diamonium diphosphate (500 gL "1 ) to eliminate excess ferric chloride interference. After 5 minutes the absorbency of the sample was measured at 664 nm, which is proportional to the concentration of dissolved sulphides.

Tabla 1 : Composición del medio de cultivo Postage C modificado con los diferentes sustratos estudiados Table 1: Composition of the modified Postage C culture medium with the different substrates studied

Medio de cultivo  Culture medium

Compuesto [g-L 1] Compound [gL 1 ]

Almidón Celulosa Espirulina  Spirulina Cellulose Starch

K2HP04 0,5 0,5 0,5 NH4CI 1 ,0 1 ,0 1 ,0 K 2 HP0 4 0.5 0.5 0.5 NH 4 CI 1, 0 1, 0 1, 0

Azufre (S°) 1 ,0 1 ,0 1 ,0  Sulfur (S °) 1, 0 1, 0 1, 0

CaCI2 · 6H20 0,1 0,1 0,1 CaCI 2 · 6H 2 0 0.1 0.1 0.1

MgCI2 6H20 2,0 2,0 2,0 MgCI 2 6H 2 0 2.0 2.0 2.0

NaCI 60,0 60,0 60,0  NaCI 60.0 60.0 60.0

Extracto de levadura 0,5 0,5 0,5  Yeast Extract 0.5 0.5 0.5

FeCI3 · 6H20 0,5 0,5 0,5 FeCI 3 · 6H 2 0 0.5 0.5 0.5

Ácido tioglicólico 0,1 0,1 0,1  Thioglycolic acid 0.1 0.1 0.1

Almidón 20 — —  Starch 20 - -

Celulosa — 40 —  Cellulose - 40 -

Espirulina — — 3  Spirulina - - 3

En la Figura 2 se observa el desarrollo de las bacterias reductoras de azufre por la aparición de precipitado negro en los medios de cultivo con almidón, celulosa y espirulina como fuentes de carbono, respectivamente. En la Figura 3, se muestra la generación de H2S por el consorcio microbiano crecido a pH 7,1 en los tres sustratos estudiados por método azul de metileno. Se observa una mayor producción de sulfuros en los medios crecidos con espirulina y almidón, mientras que con el sustrato celulosa existe una producción de sulfuros significativamente menor, según prueba de Duncan (p<0,05). Ejemplo 2: Hibridación in situ con sonda fluorescente del consorcio microbiano reductor de azufre. Figure 2 shows the development of sulfur reducing bacteria due to the appearance of black precipitate in the culture media with starch, cellulose and spirulina as carbon sources, respectively. In Figure 3, the generation of H 2 S by the microbial consortium grown at pH 7.1 is shown in the three substrates studied by the methylene blue method. A greater production of sulfides is observed in the media grown with spirulina and starch, while with the cellulose substrate there is a significantly lower production of sulphides, according to Duncan's test (p <0.05). Example 2: In situ hybridization with fluorescent probe of the sulfur reducing microbial consortium.

La caracterización del consorcio microbiano crecido en los distintos sustratos se realizó utilizando la técnica de hibridación fluorescente in situ. Los cultivos utilizados para la hibridación se realizaron con la metodología empleada en el Ejemplo 1. Las sondas utilizadas y sus características se muestran en la Tabla 2.  The characterization of the microbial consortium grown on the different substrates was performed using the fluorescent in situ hybridization technique. Cultures used for hybridization were performed using the methodology used in Example 1. The probes used and their characteristics are shown in Table 2.

Tabla 2: Sondas utilizadas en el estudio de caracterización de microorganismos del consorcio microbiano, su secuencia, posición en el rRNA y especificidad en la hibridación in situ (Amann et al. 1995. Phylogenetic Identification and In Situ Detection of Individual microbial cell without cultivation. icrobiol. Rev. 59: 143- 169).  Table 2: Probes used in the study of microorganism characterization of the microbial consortium, its sequence, position in the rRNA and specificity in in situ hybridization (Amann et al. 1995. Phylogenetic Identification and In Situ Detection of Individual microbial cell without cultivation. icrobiol Rev. 59: 143-169).

Sondas Especificidad (rRNA, posición)(a) Secuencia Probes Specificity (rRNA, position) (a) Sequence

EUB338 Bacteria (16S, 338-355) GCTGCCTCCCGTAGGAGT Archaea Archara ( 16S , 915- 934) GTGCTCCCCCGCCAATTCCTEUB338 Bacteria (16S, 338-355) GCTGCCTCCCGTAGGAGT Archaea Archara (16S, 915-934) GTGCTCCCCCGCCAATTCCT

ALF1b a-Proteobacteria C\6S, 19-35) CGTTCGYTCTGAGCCAGALF1b a-Proteobacteria C \ 6S, 19-35) CGTTCGYTCTGAGCCAG

BET42a β-Proteobacteria (23S, 1027-1043) GCCTTCCCACTTCGTTTBET42a β-Proteobacteria (23S, 1027-1043) GCCTTCCCACTTCGTTT

SRB385 δ-Proteobacteria (16S, 385-402) CGGCGTCGCTGCGTCAGGSRB385 δ-Proteobacteria (16S, 385-402) CGGCGTCGCTGCGTCAGG

CF319a Cytophaga-Flavobacterium (16S, 319- TGGTCCGTGTCTCAGTAC CF319a Cytophaga-Flavobacterium (16S, 319- TGGTCCGTGTCTCAGTAC

336)  336)

(a) Posición de unión de sonda de acuerdo al rRNA 16S de E. coli. (a) Probe binding position according to E. coli 16S rRNA.

Para realizar la hibridación se tomó una muestra de 100 μΙ_ de cada cultivo crecido durante 7 días, la que se mezcló con 900 μΙ_ de PBS y se centrifugó durante 5 minutos a 13.400 x g. Una vez centrifugado, se descartó el sobrenadante para luego resuspender el pellet en 900 μΙ_ de PBS, centrifugando durante 3 minutos a 2061 x g. Se tomó 50 μΙ_ de sobrenadante, lo que se depositó en un portaobjeto para fijar la muestra con calor. Una vez fijada, se agregó 20 μΙ_ de formaldehído 37% sobre cada muestra durante 20 minutos. Luego de esto, se agregó 50 pL de solución de hibridación a cada muestra (según Tabla 3), la que contenía 20 ng de sonda marcada con CY3. Los portaobjetos con las muestras se incubaron durante noventa minutos a 45°C, que luego de este tiempo fueron lavadas con su solución de lavado respectiva durante 30 minutos a 45°C (Tabla 4). Una vez lavados, los portaobjetos con las muestras fijadas se dejaron secar a temperatura ambiente para luego teñir con 20 μί de DAPI (4',6-Diamidino-2-fenilindol) a 50 μg■mL"1 durante 10 minutos, posteriormente se enjuagó con agua destilada para retirar el exceso de DAPI. Finalmente se observó las muestras en un microscopio de epifluorescencia, con filtro Zeiss N°20 para sonda marcada con CY3 y con filtro Zeiss N°09 para ver los microorganismos marcados con DAPI. Se fotografió las muestras utilizando una cámara Canon PowerShot sx110 IS y se capturó con el software Remote Capture v.3.0.1.8. Las imágenes se analizaron utilizando el software ImageJ (Abramoff ef al. 2004. Image Processing with ImageJ. Biophotonics International. 11(7): 36- 42), para realizar un conteo de microorganismos marcados con la sonda respectiva versus microorganismos totales marcados con DAPI. To perform the hybridization, a sample of 100 μΙ_ of each culture grown for 7 days was taken, which was mixed with 900 μΙ_ of PBS and centrifuged for 5 minutes at 13,400 x g. Once centrifuged, the supernatant was discarded and then resuspended in 900 μΙ_ of PBS, centrifuged for 3 minutes at 2061 x g. 50 μΙ_ of supernatant was taken, which was deposited on a slide to fix the sample with heat. Once fixed, 20 μΙ_ of 37% formaldehyde was added on each sample for 20 minutes. After this, 50 pL of hybridization solution was added to each sample (according to Table 3), which contained 20 ng of CY3 labeled probe. The slides with the samples were incubated for ninety minutes at 45 ° C, which after this time were washed with their respective wash solution for 30 minutes at 45 ° C (Table 4). Once washed, the slides with the fixed samples were allowed to dry at room temperature and then stained with 20 μί of DAPI (4 ', 6-Diamidino-2-phenylindole) at 50 μg ■ mL "1 for 10 minutes, then rinsed with distilled water to remove excess DAPI The samples were finally observed in an epifluorescence microscope, with Zeiss filter No. 20 for probe labeled with CY3 and with Zeiss filter No. 09 to see the microorganisms labeled with DAPI. samples using a Canon PowerShot sx110 IS camera and captured with Remote Capture software v.3.0.1.8. Images were analyzed using ImageJ software (Abramoff ef al. 2004. Image Processing with ImageJ. Biophotonics International. 11 (7): 36-42), to perform a count of microorganisms labeled with the respective probe versus total microorganisms labeled with DAPI.

Tabla 3: Composición de la solución de hibridación utilizada para el estudio de la hibridación in situ para las distintas sondas analizadas. Table 3: Composition of the hybridization solution used for the study of in situ hybridization for the different probes analyzed.

Formamida NaCI [M] Tris/ HCI (pH 7,2) SDS Formamide NaCI [M] Tris / HCI (pH 7.2) SDS

Sondas Probes

[%] [mM] [%] ALF1 b/ EUB338/ Archaea 20 0,9 20 0,01[%] [mM] [%] ALF1 b / EUB338 / Archaea 20 0.9 20 0.01

BET42a/ CF319a / SRB385 35 0,9 20 0,01BET42a / CF319a / SRB385 35 0.9 20 0.01

Tabla 4: Composición de la solución de lavado utilizada para el estudio de la hibridación in situ para las distintas sondas analizadas. Table 4: Composition of the wash solution used for the study of in situ hybridization for the different probes analyzed.

Tris/HCI (pH 7,2) SDS NaCI EDTA Tris / HCI (pH 7.2) SDS NaCI EDTA

Sondas Probes

[mM] [%] [M] [mM] [mM] [%] [M] [mM]

ALF1 b/ EUB338/ Archaea 20 0,010 180 5 ALF1 b / EUB338 / Archaea 20 0.010 180 5

BET42a/ CF319a / SRB385 20 0,021 40 5  BET42a / CF319a / SRB385 20 0.021 40 5

Cuando se utiliza almidón como sustrato orgánico carbonado complejo para el desarrollo del consorcio microbiano (Figura 4), los resultados muestran que el consorcio está compuesto por todos los microorganismos estudiados. Un 44,5% de los microorganismos corresponde a Bacterias, mientras que un 10,7% corresponde a Arqueas. Se puede observar que el grupo más representativo corresponde a las Proteobacterias δ con un 16,6%, seguido por la subclase α con un 14,8%, el grupo Citofaga-Flavobacterium con un 12,8% y la subclase β con un 3,4%. When starch is used as a complex carbon organic substrate for the development of the microbial consortium (Figure 4), the results show that the consortium is composed of all the microorganisms studied. 44.5% of microorganisms correspond to bacteria, while 10.7% correspond to archaea. It can be seen that the most representative group corresponds to δ Proteobacteria with 16.6%, followed by the α subclass with 14.8%, the Citofaga-Flavobacterium group with 12.8% and the β subclass with a 3 ,4%.

Como se muestra en la Figura 5, cuando el consorcio microbiano es mantenido con medio con celulosa como sustrato orgánico carbonado complejo, el consorcio está compuesto por todos los microorganismos estudiados. Así, un 47,8% de los microorganismos corresponde a Bacterias, mientras que un 3,8% de ellos corresponde a Arqueas. Por otro lado, las Proteobacterias δ corresponde a la subclase más representativa con un 15,7%. También se muestra la presencia de las Proteobacterias α y β y microorganismos del grupo Citofaga-Flavobacterium, cada uno con una representación menor al 10% del total de microorganismos. As shown in Figure 5, when the microbial consortium is maintained with cellulose medium as a complex carbon organic substrate, the consortium is composed of all the microorganisms studied. Thus, 47.8% of the microorganisms correspond to Bacteria, while 3.8% of them correspond to Archaea. On the other hand, δ Proteobacteria corresponds to the most representative subclass with 15.7%. The presence of the α and β Proteobacteria and microorganisms of the Citofaga-Flavobacterium group is also shown, each with a representation of less than 10% of the total microorganisms.

Cuando se utiliza espirulina como sustrato orgánico carbonado complejo para el desarrollo del consorcio microbiano (Figura 6), se observa que este está compuesto por todos los microorganismos estudiados. Un 59,0% de los microorganismos corresponde a Bacterias, mientras que un 4,3% corresponde a Arqueas. Se puede observar que el grupo más representativo corresponde a las Proteobacterias δ con un 17,8%, seguido por el grupo Citofaga-Flavobacterium con un 10,6%, y finalmente las subclases α y β con un 8,4 y un 8,2%, respectivamente. When spirulina is used as a complex carbon organic substrate for the development of the microbial consortium (Figure 6), it is observed that it is composed of all the microorganisms studied. 59.0% of the microorganisms correspond to Bacteria, while 4.3% corresponds to Arqueas. It can be observed that the most representative group corresponds to the δ Proteobacteria with 17.8%, followed by the Citofaga-Flavobacterium group with 10.6%, and finally the α and β subclasses with 8.4 and 8, 2%, respectively.

Es así que el consorcio microbiano reductor de azufre está compuesto por bacterias y arqueas. También este consorcio posee en su estructura microbiana bacterias de la subclase Proteobacterias α, β y δ, además de bacterias del grupo Citofaga- Flavobacterium, dependiendo su composición del tipo del sustrato orgánico carbonado complejo utilizado como para su cultivo. Ejemplo 2: Efecto del pH en la producción de ácido sulfhídrico por el consorcio microbiano reductor de azufre. Thus, the sulfur reducing microbial consortium is composed of bacteria and archaea. Also this consortium has in its microbial structure bacteria of the subclass Proteobacteria α, β and δ, in addition to bacteria of the Citofaga-Flavobacterium group, depending on their composition of the type of complex carbon organic substrate used as for their cultivation. Example 2: Effect of pH on the production of hydrogen sulfide by the sulfur reducing microbial consortium.

Para observar el efecto del pH sobre la producción de H2S por el consorcio microbiano se preparó medios de cultivo como en el Ejemplo 1 , con los distintos sustratos estudiados, a excepción del pH y la ausencia del cloruro férrico. El pH de los medios se ajustó antes de autoclavado desde 5,5 a 10,0 con una amplitud de 0,5 unidades. Sin embargo, estos niveles cambiaron luego de la esterilización. Los pH reales con los que se trabajó fueron 5,2; 5,6; 6,1 ; 6,6; 7,1 ; 7,5; 8,0; 8,5; 8,9 y 9,4. En estos medios se realizaron las mediciones de sulfuras por el método de azul de metileno. To observe the effect of pH on the production of H 2 S by the microbial consortium, culture media were prepared as in Example 1, with the different substrates studied, with the exception of pH and the absence of ferric chloride. The pH of the media was adjusted before autoclaving from 5.5 to 10.0 with an amplitude of 0.5 units. However, these levels changed after sterilization. The actual pH with which one worked was 5.2; 5.6; 6.1; 6.6; 7.1; 7.5; 8.0; 8.5; 8.9 and 9.4. In these media sulfide measurements were made by the methylene blue method.

En la Figura 7 se observa que la producción de H2S por el consorcio microbiano con almidón, como donador de electrones, existió una producción significativamente mayor en los pH 8,9 y 9,4, en comparación a los otros pH analizados para todos los días estudiados (prueba de Duncan, p<0,05), los que se mantuvieron relativamente estables dentro del rango de 3 - 30 ppm de H2S. Figure 7 shows that the production of H 2 S by the microbial starch consortium, as an electron donor, there was a significantly higher production at pH 8.9 and 9.4, compared to the other pH analyzed for all the days studied (Duncan test, p <0.05), those that remained relatively stable within the range of 3 - 30 ppm of H 2 S.

En la Figura 8 se muestra la producción de H2S en medios con espirulina como donador de electrones. Se observa una mayor producción -a pH alcalinos, observándose una diferencia significativa en los niveles de sulfuras entre los medios con pH 9,4 con respecto a los otros pH para todos los días estudiados (según prueba de Duncan, p<0,05). The production of H 2 S in spirulina media as an electron donor is shown in Figure 8. A higher production is observed - at alkaline pH, with a significant difference in sulphide levels between the media with pH 9.4 with respect to the other pH for all days studied (according to Duncan test, p <0.05) .

Ejemplo 3: Desarrollo del consorcio microbiano reductor de azufre en un reactor con soporte y con celulosa como sustrato orgánico. Example 3: Development of the microbial sulfur reducing consortium in a reactor with support and with cellulose as an organic substrate.

Se cargó un reactor de teflón con un volumen útil 410 cm3 (dimensiones 49 cm de alto y 3,3 de diámetro), el que se rellenó con 200 g de Celite R-635 (extruido de tierra de diatomeas producido por World Minerals, Inc., Lompoc, CA, EEUU). Este biorreactor consistió en una columna principal con dos entradas superiores e inferiores, a las que se rellenó con material de soporte, medio de cultivo (similar al utilizado en el Ejemplo 1 , excepto por la ausencia de cloruro férrico y por la concentración de NaCI, que fue de 40 g L"1) y celulosa como sustrato orgánico en una concentración 1 g-L"\ la que posteriormente se autoclavó a 110°C por 30 minutos. Una vez frió, se inoculó con 100 ml_ de un cultivo en lote en fase de crecimiento, sellando el biorreactor para evitar la oxigenación. El biorreactor se mantuvo a una temperatura de 28°C y con flujo de recirculación ascendente, como se esquematiza en la Figura 9. En este proceso, el medio de cultivo que contiene el azufre en polvo y la celulosa se alimenta mediante el conducto de alimentación (1) con una bomba (2) y se ingresa mediante un conducto de entrada (3) al biorreactor (5), relleno con Celite R-635 (4). El medio de cultivo de recircula mediante el conducto de recirculación (6) con una bomba de recirculación (7). El efluente que contiene el ácido sulfhídrico se retirar del biorreactor mediante el conducto de salida (8). En una primera etapa, este biorreactor se operó en lote, para luego pasar a una etapa de lote recirculado ascendente en forma semicontinua para lograr un tiempo de retención hidráulico (TRH) de recirculación de 24 horas. Durante las etapas posteriores, por una entrada inferior se alimentó con medio fresco y sustrato en una relación g DQO/g S° de «1 ,00 con un flujo de alimentación de 1 ,73±0,31 mL min" a través de una bomba peristáltica, en volúmenes porcentualmente equivalentes al volumen total de los biorreactores, mientras que por la parte superior se removió el efluente a evaluar. Luego de la alimentación del biorreactor y extracción del efluente, esta se mantuvo siempre con recirculación semicontinua para mantener un TRH de 24 horas. Se estimó diariamente la producción de H2S (según Ejemplo 1 ) y el pH de los efluentes obtenidos para obtener la mayor actividad sulfurogénica. También se determinó la demanda química de oxígeno o DQO (Kit Hanna Hl 93754A-25 LR, basado en procedimiento Oficial de Métodos Estándar, Standard Methods for the Examination of Water & Wastewater. 1997. Chemical Oxygen Demand (COD). N° 5220D, Part 5000. 20th Edition) de algunos puntos importantes de la operación. A Teflon reactor with a useful volume of 410 cm3 (dimensions 49 cm high and 3.3 in diameter) was loaded, which was filled with 200 g of Celite R-635 (diatomaceous earth extrudate produced by World Minerals, Inc ., Lompoc, CA, USA). This bioreactor consisted of a main column with two upper and lower inlets, to which it was filled with support material, culture medium (similar to that used in Example 1, except for the absence of ferric chloride and the concentration of NaCI, which was 40 g L "1 ) and cellulose as an organic substrate at a concentration of 1 gL " which was subsequently autoclaved at 110 ° C for 30 minutes. Once cold, he was inoculated with 100 ml_ of a growing batch culture, sealing the bioreactor to prevent oxygenation. The bioreactor was maintained at a temperature of 28 ° C and with upward recirculation flow, as outlined in Figure 9. In this process, the culture medium containing the sulfur powder and cellulose is fed through the feed line (1) with a pump (2) and the bioreactor (5), filled with Celite R-635 (4), is entered through an inlet duct (3). The recirculating culture medium through the recirculation duct (6) with a recirculation pump (7). The effluent containing hydrogen sulfide is removed from the bioreactor through the outlet duct (8). In a first stage, this bioreactor was operated in batch, and then passed to a semi-continuous upward recirculated batch stage to achieve a 24-hour recirculation hydraulic retention time (HRT). During the subsequent stages, a fresh inlet and substrate were fed through a lower inlet in a g COD / g S ° ratio of «1, 00 with a feed flow of 1.73 ± 0.31 mL min " through a peristaltic pump, in volumes equivalent to the total volume of the bioreactors, while the effluent to be evaluated was removed from the upper part.After the bioreactor feed and effluent extraction, it was always maintained with semi-continuous recirculation to maintain a HRT of 24 hours, the production of H 2 S (according to Example 1) and the pH of the effluents obtained to obtain the greatest sulfurogenic activity were estimated daily The chemical demand for oxygen or COD was also determined (Kit Hanna Hl 93754A-25 LR, based on Official Standard Methods procedure, Standard Methods for the Examination of Water & Wastewater, 1997. Chemical Oxygen Demand (COD) No. 5220D, Part 5000. 20th Edition) of some important points of the operation.

La operación de los biorreactores se separó temporalmente en dos etapas; una primera etapa en que se estableció los parámetros y tiempos óptimos de puesta en marcha y el periodo de inmovilización de los microorganismos; y una segunda etapa en que se operó los biorreactores bajo un régimen semicontinuo, que buscó aumentar la productividad volumétrica de H2S, modificando parámetros como los tiempos de retención hidráulica y el volumen y el pH del medio alimentado. The bioreactor operation was temporarily separated into two stages; a first stage in which the parameters and optimal start-up times and the period of immobilization of the microorganisms were established; and a second stage in which the bioreactors were operated under a semi-continuous regime, which sought to increase the volumetric productivity of H 2 S, modifying parameters such as hydraulic retention times and the volume and pH of the fed medium.

En las Figuras 10 y 11 se muestra el perfil de desempeño del biorreactor alimentado con celulosa y rellenado con Celite R-635 como material de soporte durante las dos etapas estudiadas. Se observó un aumento gradual de la productividad volumétrica de H2S, alcanzando un máximo de 1 ,94 mol/m3 d en este biorreactor con celulosa como sustrato orgánico. Figures 10 and 11 show the performance profile of the bioreactor fed with cellulose and filled with Celite R-635 as a support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S, reaching a maximum of 1.94 mol / m 3 d in this bioreactor with cellulose as organic substrate.

Ejemplo 4: Desarrollo del consorcio microbiano reductor de azufre en un reactor sin soporte y con espirulina como sustrato orgánico. Example 4: Development of the sulfur reducing microbial consortium in an unsupported reactor and with spirulina as an organic substrate.

Se utilizó un reactor de teflón de iguales características utilizado en el Ejemplo 3 y con la misma metodología, a excepción del sustrato suministrado, la ausencia de material de soporte y la concentración de NaCI, que fue de 30 g-L"1. Este biorreactor se alimentó con espirulina a una concentración de 1 g-L"1 como nutriente orgánico. A Teflon reactor of the same characteristics used in Example 3 and with the same methodology was used, with the exception of the substrate supplied, the absence of support material and the concentration of NaCI, which was 30 gL "1. This bioreactor was fed with spirulina at a concentration of 1 gL "1 as an organic nutrient.

Se estimó diariamente la producción de H2S (según Ejemplo 1) y el pH de los efluentes obtenidos para obtener la mayor actividad sulfurogénica. También se determinó la demanda química de oxígeno o DQO (como en Ejemplo 3) de algunos puntos importantes de la operación. The production of H 2 S (according to Example 1) and the pH of the effluents obtained to obtain the greatest sulfurogenic activity were estimated daily. The chemical oxygen demand or COD (as in Example 3) of some important points of the operation was also determined.

En las Figuras 12 y 13 se muestra el perfil de desempeño del biorreactor alimentado con espirulina y sin material de soporte durante las dos etapas estudiadas. Se observó un aumento gradual de la productividad volumétrica de H2S, alcanzando un máximo de 2,75 mol/m3 d en este biorreactor con espirulina como sustrato orgánico. Figures 12 and 13 show the performance profile of the bioreactor fed with spirulina and without support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S was observed, reaching a maximum of 2.75 mol / m 3 d in this bioreactor with spirulina as organic substrate.

Ejemplo 5: Desarrollo del consorcio microbiano reductor de azufre en un reactor con soporte y con espirulina como sustrato orgánico. Example 5: Development of the microbial sulfur reducing consortium in a reactor with support and with spirulina as organic substrate.

Se cargó un reactor de vidrio con un volumen útil 500 cm3 (dimensiones 49 cm de alto y 3,6 de diámetro), el que se rellenó con 200 g de Celite R-635. La metodología de operación y mantenimiento de este biorreactor fue similar al utilizado en el Ejemplo 3, a excepción del sustrato suministrado y la concentración de NaCI, que fue de 30 g-L"1. Este biorreactor se alimentó con 1 g-L"1 de espirulina como nutriente orgánico. Se estimó diariamente la producción de H2S (según Ejemplo 1 ) y el pH de los efluentes obtenidos para obtener la mayor actividad sulfurogénica. También se determinó la demanda química de oxígeno o DQO (como en Ejemplo 3) de algunos puntos importantes de la operación. A glass reactor with a useful volume 500 cm 3 (dimensions 49 cm high and 3.6 in diameter) was loaded, which was filled with 200 g of Celite R-635. The methodology of operation and maintenance of this bioreactor was similar to that used in Example 3, with the exception of the substrate supplied and the concentration of NaCI, which was 30 gL "1. This bioreactor was fed with 1 gL " 1 of spirulina as a nutrient organic. The production of H 2 S (according to Example 1) and the pH of the effluents obtained to obtain the greatest sulfurogenic activity were estimated daily. The chemical oxygen demand or COD (as in Example 3) of some important points of the operation was also determined.

En la Figura 14 se muestra el perfil de desempeño del biorreactor alimentado con espirulina y sin material de soporte durante las dos etapas estudiadas. Se observó un aumento gradual de la productividad volumétrica de H2S, alcanzando un máximo de 2,94 mol/m3 d en este biorreactor con espirulina como sustrato orgánico y Celite R-635 como material de soporte. Figure 14 shows the performance profile of the bioreactor fed with spirulina and without support material during the two stages studied. A gradual increase in volumetric productivity of H 2 S was observed, reaching a maximum of 2.94 mol / m 3 d in this bioreactor with spirulina as organic substrate and Celite R-635 as support material.

Claims

REIVINDICACIONES 1. Un método biológico de producción de ácido sulfhídrico a partir de azufre mediante un consorcio microbiano, CARACTERIZADO porque comprende al menos los pasos de: 1. A biological method of producing sulfuric acid from sulfur by means of a microbial consortium, CHARACTERIZED because it comprises at least the steps of: a) crecer un consorcio microbiano reductor de azufre halófilo, capaz de utilizar sustratos orgánicos carbonados complejos como donadores de electrones en un biorreactor anaerobio de lecho fijo que contiene el material de soporte del biorreactor y un medio de cultivo constituido, al menos, por uno o más sustratos orgánicos carbonados complejos, como donadores de electrones, y azufre,  a) grow a halophilic sulfur reducing microbial consortium, capable of using complex carbon-based organic substrates as electron donors in a fixed-bed anaerobic bioreactor containing the bioreactor support material and a culture medium consisting of at least one or more complex carbon organic substrates, such as electron donors, and sulfur, b) recircular el medio de cultivo del biorreactor hasta que se forme la biopelícula del consorcio reductor de azufre sobre el material de soporte del biorreactor,  b) recirculating the bioreactor culture medium until the biofilm of the sulfur reducing consortium is formed on the bioreactor support material, c) agregar al biorreactor, en forma continua o semicontinua, un medio de cultivo que contiene al menos una suspensión de uno o más sustratos orgánicos carbonados complejos, como donadores de electrones y azufre en polvo o finamente molido.  c) add to the bioreactor, continuously or semi-continuously, a culture medium that contains at least one suspension of one or more complex carbon organic substrates, such as donors of electrons and powdered or finely ground sulfur. d) retirar del biorreactor el efluente que contiene el ácido sulfhídrico.  d) remove the effluent containing hydrogen sulfide from the bioreactor. 2. Un método de acuerdo a la reivindicación 1 , CARACTERIZADO porque dicho material de soporte del biorreactor es el mismo compuesto orgánicos carbonado complejo en forma particulada. 2. A method according to claim 1, CHARACTERIZED in that said bioreactor support material is the same particulate complex carbon organic compound. 3. Un método de acuerdo a las reivindicaciones 1 o 2, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo se enriquece a partir de una muestra ambiental. 3. A method according to claims 1 or 2, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium is enriched from an environmental sample. 4. Un método de acuerdo a la reivindicación 3, CARACTERIZADO porque dicha muestra ambiental es el lodo anaerobio de una laguna salina o un salar. 4. A method according to claim 3, CHARACTERIZED in that said environmental sample is the anaerobic sludge of a saline lagoon or a salt flat. 5. Un método de acuerdo a las reivindicaciones 1 - 4, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo está compuesto al menos por microorganismos hidrolíticos, fermentativos, acetogénicos y reductores de azufre. 5. A method according to claims 1-4, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium is composed of at least hydrolytic, fermentative, acetogenic and sulfur reducing microorganisms. 6. Un método de acuerdo a las reivindicaciones 1 - 5, CARACTERIZADO porque dicho consorcio microbiano reductor de sulfato halófilo está compuesto por bacterias y arqueas. 6. A method according to claims 1-5, CHARACTERIZED in that said halophilic sulfate reducing microbial consortium is composed of bacteria and archaea. 7. Un método de acuerdo a las reivindicaciones 5 o 6, CARACTERIZADO porque dichas bacterias pertenecen, a lo menos, a los grupos filogenéticos de las Proteobacterias α, β, y δ y bacterias del grupo Citofaga-Flavobacterium. 7. A method according to claims 5 or 6, CHARACTERIZED in that said bacteria belong, at least, to the phylogenetic groups of Proteobacteria α, β, and δ and bacteria of the Citofaga-Flavobacterium group. 8. Un método de acuerdo a las reivindicaciones 1 - 7, CARACTERIZADO porque dichos uno o más sustratos orgánicos complejos son productos de origen natural ricos en compuestos orgánicos poliméricos. 8. A method according to claims 1-7, CHARACTERIZED in that said one or more complex organic substrates are naturally occurring products rich in polymeric organic compounds. 9. Un método de acuerdo a la reivindicación 8, CARACTERIZADO porque dichos productos de origen natural ricos en compuestos orgánicos poliméricos se seleccionan del grupo de la celulosa, los productos o residuos vegetales lignocelulósicos, el almidón, los productos o residuos vegetales ricos en almidón, las algas marinas, las microalgas y las cianobacterias. 9. A method according to claim 8, CHARACTERIZED in that said naturally occurring products rich in polymeric organic compounds are selected from the group of cellulose, lignocellulosic plant products or residues, starch, starch-rich plant products or residues, seaweed, microalgae and cyanobacteria. 10. Un método de acuerdo a las reivindicaciones 1 - 9, CARACTERIZADO porque dichos materiales de soporte se seleccionan del grupo de la cerámica, la piedra silícica, el vidrio, el extruido de tierra de diatomeas y el plástico. 10. A method according to claims 1-9, CHARACTERIZED in that said support materials are selected from the group of ceramics, silicon stone, glass, diatomaceous earth extrudate and plastic. 1 1. Un método de acuerdo a las reivindicaciones 1 - 10, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 5,0 y 10. 1 1. A method according to claims 1-10, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 5.0 and 10. 12. Un método de acuerdo a las reivindicaciones 1 - 10, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 6,5 y 10. 12. A method according to claims 1-10, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 6.5 and 10. 13. Un método de acuerdo a las reivindicaciones 1 - 10, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a un pH 8,5 y 9,5. 014/067023 PCT/CL2013/000070 13. A method according to claims 1-10, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at pH 8.5 and 9.5. 014/067023 PCT / CL2013 / 000070 14. Un método de acuerdo a las reivindicaciones 1 - 13, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 0 y 100 g/L. 14. A method according to claims 1-13, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 0 and 100 g / L. 15. Un método de acuerdo a las reivindicaciones 1 - 13, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 20 y 80 g/L. 15. A method according to claims 1-13, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 20 and 80 g / L. 16. Un método de acuerdo a las reivindicaciones 1 - 13, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 25 y 70 g/L. 16. A method according to claims 1-13, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 25 and 70 g / L. 17. Un método de acuerdo a las reivindicaciones 1 - 13, CARACTERIZADO porque dicho consorcio microbiano reductor de azufre halófilo presenta la capacidad de crecer y producir ácido sulfhídrico a concentraciones de cloruro de sodio entre 30 y 60 g/L. 17. A method according to claims 1-13, CHARACTERIZED in that said halophilic sulfur reducing microbial consortium has the ability to grow and produce hydrogen sulfide at sodium chloride concentrations between 30 and 60 g / L. 18. Uso del método de acuerdo a las reivindicaciones 1 - 17, CARACTERIZADO porque dicho ácido sulfhídrico producido se emplea como agente precipitante de metales en efluentes mineros o industriales 18. Use of the method according to claims 1-17, CHARACTERIZED in that said produced hydrogen sulfide is used as a metal precipitating agent in mining or industrial effluents 19. Uso del método de acuerdo a las reivindicaciones 1 - 17, CARACTERIZADO porque dicho ácido sulfhídrico producido se emplea para la producción industrial de sulfhidrato de sodio (NaSH). 19. Use of the method according to claims 1-17, CHARACTERIZED in that said hydrogen sulphide acid produced is used for the industrial production of sodium hydrochloride (NaSH).
PCT/CL2013/000070 2012-10-31 2013-10-04 Production of sulfhydric acid from sulphur by means of a microbial consortium Ceased WO2014067023A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587079A (en) * 1995-04-21 1996-12-24 Rowley; Michael V. Process for treating solutions containing sulfate and metal ions.
US20040115120A1 (en) * 1998-11-16 2004-06-17 Paques Bio System B.V. Process for the production of hydrogen sulphide from elemental sulphur and use thereof in heavy metal recovery
WO2009100537A1 (en) * 2008-02-12 2009-08-20 Bioteq Environmental Technologies Inc. Processes for producing h2s using sulphur-reducing bacteria

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587079A (en) * 1995-04-21 1996-12-24 Rowley; Michael V. Process for treating solutions containing sulfate and metal ions.
US20040115120A1 (en) * 1998-11-16 2004-06-17 Paques Bio System B.V. Process for the production of hydrogen sulphide from elemental sulphur and use thereof in heavy metal recovery
WO2009100537A1 (en) * 2008-02-12 2009-08-20 Bioteq Environmental Technologies Inc. Processes for producing h2s using sulphur-reducing bacteria

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