WO1995001311A1 - PROCESS FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND/OR NITRITES USING STRAINS OF $i(KLEBSIELLA OXYTOCA) - Google Patents

PROCESS FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND/OR NITRITES USING STRAINS OF $i(KLEBSIELLA OXYTOCA) Download PDF

Info

Publication number
WO1995001311A1
WO1995001311A1 PCT/ES1994/000069 ES9400069W WO9501311A1 WO 1995001311 A1 WO1995001311 A1 WO 1995001311A1 ES 9400069 W ES9400069 W ES 9400069W WO 9501311 A1 WO9501311 A1 WO 9501311A1
Authority
WO
WIPO (PCT)
Prior art keywords
nitrates
nitrites
effluent
clone
microorganisms
Prior art date
Application number
PCT/ES1994/000069
Other languages
Spanish (es)
French (fr)
Inventor
José Luis RAMOS MARTIN
Guadalupe PIÑAR LARRUBIA
Estrella Duque Martin De Oliva
Ali Haidour
Original Assignee
Union Española De Explosivos S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from ES9301496A external-priority patent/ES2064287B1/en
Application filed by Union Española De Explosivos S.A. filed Critical Union Española De Explosivos S.A.
Priority to AU70742/94A priority Critical patent/AU7074294A/en
Publication of WO1995001311A1 publication Critical patent/WO1995001311A1/en

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/22—Klebsiella

Definitions

  • the invention relates to new strains of Klebsiella oxytoca suitable for use in a process for the biological elimination of nitrates and / or nitrites.
  • the invention provides a process for the elimination of nitrates and / or nitrites contained in industrial or urban effluents and in soils, by the use of Klebsiella ox ⁇ toca strains capable of using said compounds as a nitrogen source, thus avoiding the environmental pollution produced by said compounds.
  • Effluents from explosive factories especially those producing 2,4,6-trinitrotoluene (TNT), nitroethylene glycol and nitroglycerin, have low levels of the aforementioned nitroorganics and high levels of nitrates, in addition to an acidic pH (values between 1 and two).
  • Current effluent treatment systems from explosives manufacturing plants mainly include neutralization of water and infinite dilution of water with them. This treatment entails a problem of continuous addition of pollutants to rivers, groundwater and soils, so it is not adequate.
  • an object of this invention is the isolation and characterization of microorganisms capable of eliminating nitrates and / or nitrites.
  • microorganisms belonging to two strains of Klebsiella oxytoca. they constitute an additional object of this invention.
  • Another additional object of this invention is a process for the biological elimination of nitrates and / or nitrites contained in industrial effluents, urban wastewater or soils, by using the microorganisms provided by this invention.
  • the invention provides new strains of Klebsiella oxytoca capable of using nitrates and / or nitrites as a source of nitrogen, as well as a process for the biological elimination of nitrates and / or nitrites contained in industrial effluents, urban wastewater and soils. , by using the strains of Klebsiella oxytoca provided by this invention.
  • the invention comprises, on the one hand, the isolation and characterization of microorganisms capable of using nitrates and / or nitrites as a source of N, and on the other, the use of isolated microorganisms for the elimination of nitrates and / or nitrites present in effluents from industrial plants, in urban wastewater and in soils contaminated with these compounds.
  • a sugar preferably fructose or glucose, or a suitable organic acid, preferably acid, can be used acetic or succinic, or alcohols of the type of glycerin and ethylene glycol.
  • the resulting suspension is incubated at temperatures between 15 ° C and 42 ° C, preferably between 25 ° C and 30 ° C, with optional stirring, and, after one week of incubation, appropriate dilutions are plated on selective solid medium plates.
  • M8 noble agar
  • potassium nitrate (20-100 mM
  • Examples 1 describe the isolation of bacteria capable of using nitrate as a source of N, in particular, of the two strains of Klebsiella oxytoca of this invention.
  • Isolated bacteria have a bacillary form, and do not show pigmentation when grown on plates of minimal medium or medium rich in LB (Maniatis et al. Molecular Cloning, A Labor atory Manual, Cold Spring Harbor Labor atory, NY, 1982).
  • One of the strains isolated, specifically, the so-called Klebsiella oxytoca clone-15, in a minimal medium type M8 can use fructose, glucose, acetic acid, succinic acid and glycerol as a source of C and ammonia, nitrate and nitrite as a source of N aerobic or anaerobic conditions, while the other isolated strain, the so-called Klebsiella oxytoca clone AH, can use nitrate and nitrite as a source of N and ethylene glycol as a source of C in aerobic conditions.
  • the common taxonomic characteristics of the Klebsiella oxytoca clone-15 and Klebsiella oxytoca clone strains are the following:
  • oxytoca clone AHI only uses ethylene glycol as a source of C in aerobiosis ⁇ -galactosidase: Positive Arginine dehydrolase: Negative Lysine decarboxylase: Positive Ornithine decarboxylase: Negative Citrate use: Positive Production of H 2 S: Negative Ureasa: Positive Tryptophan : Negative Production of indole: Positive Gelatinase: Negative Fermentation / oxidation of sugars: Glucose: Positive Mannitol: Positive Inositol: Positive
  • Sorbitol Positive Ramnosa: Positive Sucrose: Positive Melibiosa: Positive Tonsil: Positive
  • the Klebsiella oxytoca clone AH strain is capable of growing in the presence of high nitrate loads, at concentrations of the order of 225 mM in watertight systems, making it very suitable for the elimination of nitrates and / or nitrites present in effluents, sewage and soil.
  • colonies of bacteria capable of using nitrates and / or nitrites as a source of N are isolated and purified, they can be used to eliminate these compounds present in effluents from industrial plants, in urban wastewater and in soils.
  • a first application of the bacteria of this invention is its use in a process of Biological elimination of nitrates and / or nitrites contained in an industrial effluent. Said procedure basically comprises the following steps:
  • Effluents capable of being purified by this procedure can be effluents from any industrial process in which nitrates and / or nitrites are used or produced, such as effluents from explosive manufacturing plants. Likewise, any type of water contaminated with nitrates and / or nitrites can be treated.
  • wastewater from explosive manufacturing plants is acidic water that can be neutralized by adding any alkali, preferably a hydroxide, carbonate or bicarbonate of an alkali metal or alkaline earth metal, in order that these waters reach a pH comprised between 6.0 and 10.5 after neutralization.
  • alkali preferably a hydroxide, carbonate or bicarbonate of an alkali metal or alkaline earth metal
  • This pH range is adequate when the strain used is the so-called Klebsiella oxytoca clone-15, while if the Klebsiella oxytoca clone strain is used THERE, it is more convenient neutralize the effluent and maintain its pH in a range between 6.0 and 8.0, since that is its range of action.
  • the necessary nutrients such as phosphate or any other nutrient or micronutrient that are necessary for the optimal functioning of the process are added.
  • suitable amounts of the A9 micronutrient solution may be added, the composition of which is described in April et al. cited ⁇ upra, together with appropriate amounts of Fe, Mg, Co and Mo (of the order of microraolar).
  • the choice and amount of nutrients and micronutrients to be added will depend on the composition of the effluent to be treated and the microbiological demand.
  • a source of C of those mentioned above is added, appropriate for the strain to be used in the process.
  • the neutralized and duly supplemented effluents are then inoculated with a culture of the bacteria provided by this invention (Klebsiella oxytoca clone-15 or Klebsiella oxytoca clone AH).
  • the bacterial load may be between 0.01 and 0.1 absorbance units at 660 n per culture, preferably, about 0.03 absorbance units at 660 nm per ml of culture.
  • the incubation should be carried out at a temperature between 20 ° and 30 ° C with a contribution of up to 2 liters of air per liter of culture, if the procedure is performed using Klebsiella oxytoca clone AH, while air may not be supplied or up to 2 liters of air per liter of culture if Klebsiella ox ⁇ toca clone-15 is used in the procedure.
  • the course of the incubation is continued periodically measuring the amount of nitrate and nitrite present in the effluent or water to be purified, by means of a specific electrode of nitrate (for example, a Crison type nitrate electrode) and by an extremely sensitive chemical test for nitrite detection (Snell and Snell, Colorimetric Methods of Analysis, Vol. 3, p. 804-805, Van Nostrand Co. Inc ., New York, 1949) respectively, ending this stage once nitrates and / or nitrites and source of C. cease to be detected.
  • a specific electrode of nitrate for example, a Crison type nitrate electrode
  • Snell and Snell Colorimetric Methods of Analysis, Vol. 3, p. 804-805, Van Nostrand Co. Inc ., New York, 1949
  • the bacteria are removed by flocculation or by any other method that allows, if desired, their reuse in a new process and the purified water that can be freely discharged is removed.
  • This procedure can be carried out continuously or in airtight, as well as in large rafts or in fermenters.
  • the flow and composition of the inlet and outlet effluent must be adequately regulated, as well as the concentration of bacterial culture and the necessary nutrient and micronutrient intake.
  • a second application of the bacteria of this invention is their use in a process for the biological elimination of nitrates and / or nitrites in surface soils contaminated with said compounds. Said procedure comprises the following steps:
  • microorganisms The injection of microorganisms is done to reach high cell densities in the soil, of the order of 10 5 bacteria per cm 2 of soil, which facilitate the removal of the contaminant. If necessary, it would add phosphate and a carbon source to the soil that facilitates its survival.
  • a third application of the bacteria of this invention is their use in the elimination of nitrates and / or nitrites contained in wastewater following a procedure similar to that described above for the elimination of nitrates and / or nitrites contained in industrial effluents.
  • the resulting suspensions I to III were incubated at a temperature between 25 ° C and 30 ° C, with optional stirring, for one week. Subsequently, appropriate dilutions of said suspensions were seeded on plates of selective solid medium consisting of M8 medium, 1.5% noble agar (w / v) and
  • the isolated colonies were purified and their phenotype was tested by culturing them in the above-indicated media.
  • the isolated bacteria were classified as Klebsiella oxytoca.
  • the two most efficient strains were the so-called Klebsiella oxytoca clone-15 and Klebsiella oxytoca clone AH, which have the characteristics previously mentioned in the description. Both cultures of these strains were deposited on June 15, 1993 and June 10, 1994 in the CECT, with accession numbers B CECT 4460 and CECT 4500 respectively.
  • EXAMPLE 2 Use of nitrate as a source of N under aerobic conditions 100 ml of M8 minimum medium is supplemented with: 250 ⁇ l of A9 micronutrient solution; - 6 ⁇ g of iron citrate;
  • the culture is started by adding an amount of bacteria (Klebsiella oxytoca clone-15) such that an initial turbidity is reached at 660 nm of 0.03 units and incubated at 30 ° C with shaking (150 rpm in an orbital incubator) and aerobiosis. After 24 hours of incubation the turbidity of the culture at 660 nm is between 1 and 2 units and the concentration of nitrate in the culture medium is undetectable by selective analysis with a supernatant nitrate electrode. The concentration of C source is equally undetectable.
  • Example 2 The procedure described in Example 2 was repeated, but using potassium nitrate in a concentration of 100 mM. The nitrate disappeared completely within 48 hours.
  • EXAMPLE 4 Use of nitrate as a source of N in aerobic conditions
  • Example 2 The procedure described in Example 2 was repeated, but using water from a factory so that the final concentration in HN0 3 was 25-30 mM. The nitrate disappeared completely within 24 hours.
  • EXAMPLE 5 Use of nitrate as a source of N in aerobic conditions The procedure described in Example 2 was repeated, but using water from a factory so that the concentration in HN0 3 was 75-100 mM. The nitrate disappeared completely within 48 hours.
  • Example 2 The procedure described in Example 2 was repeated, but using a continuous nitrate addition system.
  • the fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 3 units at 660 mm. Water from a diluted factory to supply 25 to 30 mM of HN0 3 was added at a flow of 1-20 ml / h. The air volume was 0.5 volumes / min. The nitrate in the overflowing medium was undetectable.
  • Example 4 The procedure described in Example 4 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter.
  • the operating conditions were as follows: pH: 6.5-8.0
  • Example 2 The procedure described in Example 2 was repeated, but using anaerobic conditions. The nitrate disappeared completely within 24 hours.
  • Example 8 The procedure described in Example 8 was repeated, but using potassium nitrate at a concentration of 100 mM. The nitrate disappeared completely within 48 hours.
  • Example 8 The procedure described in Example 8 was repeated, but using water from a factory until reaching a concentration in HN0 3 of 25-30 mM. The nitrate disappeared completely within 24 hours.
  • EXAMPLE 11 Use of nitrate as a source of N under anaerobic conditions The procedure described in Example 8 was repeated, but using water from a factory until reaching a concentration in HN0 3 of 75-100 mM. The nitrate disappeared completely within 48 hours.
  • EXAMPLE 12 Use of nitrate as a source of N under anaerobic conditions, continuously The procedure described in Example 6 was repeated except that no air was bubbled.
  • the fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 1 unit at 66? mm Diluted water from a factory containing a concentration of 25 to 30 mM in HN0 3 was added at a flow of 1-20 ml / h. The nitrate in the medium that overflowing was undetectable.
  • Example 4 The procedure described in Example 4 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter.
  • the operating conditions were as follows: pH: 6.5-8.0
  • Nitrates 200 mg / ml Sulfates: 1000 mg / ml - pH: 2.0
  • a volume of 1 liter of said effluent was neutralized with NaOH until a pH of 7.0 was reached. 100 ml of this effluent was taken up to 1 liter with distilled water and the nutrients and micronutrients of the M8 medium were added, using an appropriate source of C such as those mentioned in Example 1.
  • the evolution of the incubation was continued by periodically measuring the nitrate content by means of a selective electrode, checking that after 24 hours the nitrate had completely disappeared.
  • 100 ml of M8 sterile minimum medium is supplemented with: 250 ⁇ l of A9 micronutrient solution; 6 ⁇ g of iron citrate; 100 ⁇ l of Mg S0 4 1M solution; 2 mM NaN0 2
  • the culture is started by adding a quantity of bacteria (Klebsiella oxytoca clone-15) such that an initial turbidity is reached at 660 nm of 0.03 units and it is incubated at 30 ° C with agitation (150 rpm in an orbital incubator) and aerobiosis. After 24 h of incubation the turbidity of the culture at 660 nm is between 0.5 and 1 unit and the concentration of nitrite in the culture medium is undetectable by chemical analysis of supernatants. It is also undetectable the concentration of source of
  • EXAMPLE 16 Use of nitrite as a source of N under aerobic conditions The procedure described in Example 15 was repeated, but using a 10 mM concetration of sodium nitrite. The nitrite disappeared completely within 48 hours.
  • Example 15 The procedure described in Example 15 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter.
  • the operating conditions were as follows: pH: 6. 5-8. 0
  • Example 15 The procedure described in Example 15 was repeated, but using anaerobic conditions. The nitrite disappeared completely within 24 hours.
  • Example 18 The procedure described in Example 18 was repeated, but using a concentration of 10 mM in sodium nitrite. The nitrite disappeared completely within 48 hours.
  • EXAMPLE 20 Use of nitrate as a source of N under aerobic conditions 100 ml of M8 minimum medium is supplemented with: 250 ⁇ l of A9 micronutrient solution; 6 ⁇ g of iron citrate;
  • the culture is started by adding a quantity of bacteria (Klebsiella oxytoca clone AH) such that an initial turbidity is reached at 660 nm of 0.03 units and it is incubated at 30 ° C with agitation (150 rpm in an orbital incubator) and aerobiosis. After 48 hours of incubation the turbidity of the culture at 660 nm is between 5 and 15 units and the concentration of nitrate in the culture medium is undetectable by selective analysis with a supernatant nitrate electrode. The concentration of C source is equally undetectable.
  • Example 20 The procedure described in Example 20 was repeated, but using potassium nitrate at a concentration of 225 mM. The nitrate disappeared completely within 48 hours.
  • Example 20 The procedure described in Example 20 was repeated, but using a continuous nitrate addition system.
  • the fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 3 units at 660 mm.
  • Undiluted water from a factory supplied 200 to 400 roM of HN0 3 and was added at a flow of 1-5 ml / h.
  • the air volume was 0.5 volumes / min.
  • the nitrate in the overflowing medium was undetectable.

Abstract

Two new strains of Klebsiella oxytoca have been isolated, identified as K. oxytoca clone-15 and clone AH1, which are capable of using nitrates and/or nitrites as nitrogen source and, consequently may be used for the biological elimination of nitrates and/or nitrites contained in industrial effluents, waters and grounds contaminated by said substances, through a process which comprises the inoculation of cultures of said strains into said effluents, waters and grounds contaminated of cultures of said strains. The strain K. oxytoca clone-15 is capable of using, both in aerobic and anaerobic conditions, nitrates and/or nitrites present in a concentration up to 100 mM, whereas the strain K. oxytoca clone AH1 is capable of using, in aerobic conditions, nitrates present in a concentration up to 400 mM. The process applies to the elimination of nitrates and/or nitrites contained in industrial effluents, waters and grounds which have been contaminated.

Description

PROCEDIMIENTO PARA LA ELIMINACIÓN BIOLÓGICA DE NITRATOS Y/O NITRITOS UTILIZANDO CEPAS DE KT.KRHTTCT.T OXYTOCAPROCEDURE FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND / OR NITRITES USING KP.KRHTTCT.T OXYTOCA
CAMPO DE LA INVENCIÓN La invención se refiere a nuevas cepas de Klebsiella oxytoca adecuadas para su empleo en un procedimiento para la eliminación biológica de nitratos y/o nitritos. En particular, la invención proporciona un procedimiento para la eliminación de nitratos y/o nitritos contenidos en efluentes industriales o urbanos y en suelos, mediante el empleo de cepas de Klebsiella oxγtoca capaces de utilizar dichos compuestos como fuente de nitrógeno, evitando de esta manera la contaminación medioambiental producida por dichos compuestos.FIELD OF THE INVENTION The invention relates to new strains of Klebsiella oxytoca suitable for use in a process for the biological elimination of nitrates and / or nitrites. In particular, the invention provides a process for the elimination of nitrates and / or nitrites contained in industrial or urban effluents and in soils, by the use of Klebsiella oxγtoca strains capable of using said compounds as a nitrogen source, thus avoiding the environmental pollution produced by said compounds.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
La industria en general, y en particular, la industria de explosivos fabrica grandes cantidades de diversos compuestos sustituidos con grupos nitro que se utilizan como explosivos y propelentes. Los efluentes de lavado de plantas de nitración de diversos compuestos contienen altas cantidades de nitratos y niveles detectables de nitritos, que si no se tratan de manera adecuada, constituyen una fuente de contaminación medioambiental por lo que deben ser eliminados ya que los primeros, a concentraciones moderadas, son tóxicos y los segundos, agentes mutagénicos, y, por tanto, agentes sospechosos de inducir cáncer.The industry in general, and in particular, the explosives industry manufactures large quantities of various compounds substituted with nitro groups that are used as explosives and propellants. The washing effluents of nitration plants of various compounds contain high amounts of nitrates and detectable levels of nitrites, which, if not treated properly, constitute a source of environmental contamination, so they must be eliminated since the former, at concentrations Moderate, they are toxic and the latter are mutagenic agents, and therefore agents suspected of inducing cancer.
Los efluentes de fábricas de explosivos, especialmente de aquéllas productoras de 2,4,6- trinitrotolueno (TNT) , nitroetilenglicol y nitroglicerina, presentan bajos niveles de los nitroorgánicos mencionados y altos niveles de nitratos, amén de un pH ácido (valores entre 1 y 2). Los sistemas actuales de tratamiento de los efluentes procedentes de plantas de fabricación de explosivos incluyen fundamentalmente la neutralización de las aguas y la dilución al infinito de los mismos con agua. Este tratamiento conlleva un problema de adición continuada de contaminantes a ríos, aguas subterráneas y suelos, por lo que no resulta adecuado.Effluents from explosive factories, especially those producing 2,4,6-trinitrotoluene (TNT), nitroethylene glycol and nitroglycerin, have low levels of the aforementioned nitroorganics and high levels of nitrates, in addition to an acidic pH (values between 1 and two). Current effluent treatment systems from explosives manufacturing plants mainly include neutralization of water and infinite dilution of water with them. This treatment entails a problem of continuous addition of pollutants to rivers, groundwater and soils, so it is not adequate.
La microbiología en general y la biotecnología en particular, pueden ofrecer una alternativa biológica a la eliminación de nitratos y nitritos mediante la utilización de los mismos como fuente de N por microorganismos adecuados. Así, en varios estudios se recoge la posibilidad de tratar biológicamente efluentes que contienen nitratos. Entre estos trabajos cabe destacar los siguientes:Microbiology in general and biotechnology in particular, can offer a biological alternative to the elimination of nitrates and nitrites by using them as a source of N by suitable microorganisms. Thus, several studies reflect the possibility of treating biologically effluents that contain nitrates. These works include the following:
Kaplan et al. (International Biodeterioration. 23 : 233-248 , 1987 ) utilizaron microorganismos no definidos lo cual genera problemas de reproducibilidad de tratamiento; - Hensen Christensen y Harremoes {Prog. Wat . Tech . 8: 509-555, 1977 ) revisan el estado de la técnica en plantas de tratamientos con cargas bajas de nitrato; du Toit y Davies (Water Res . 7 : 489-500 , 1973 ) estudian la eliminación de nitratos en flujo continuo con residuos domésticos con baja carga de nitrato;Kaplan et al. (International Biodeterioration. 23: 233-248, 1987) used undefined microorganisms which causes reproducibility problems of treatment; - Hensen Christensen and Harremoes {Prog. Wat. Tech 8: 509-555, 1977) review the state of the art in treatment plants with low nitrate loads; du Toit and Davies (Water Res. 7: 489-500, 1973) study the elimination of nitrates in continuous flow with household waste with low nitrate load;
Hochstein y Tomlinson (Annu . Rev . Microbiol . 42: 231- 261 ) revisan el proceso de desnitrificación desde distintos puntos de vista; yHochstein and Tomlinson (Annu. Rev. Microbiol. 42: 231-261) review the denitrification process from different points of view; Y
Ye et al. (Applied Environmental Microbiology 59 : 250-254 , 1993 ) muestran la versatilidad de enzimas implicados en los procesos de desnitrificación.Ye et al. (Applied Environmental Microbiology 59: 250-254, 1993) show the versatility of enzymes involved in denitrification processes.
Sería conveniente, por tanto, disponer de microorganismos capaces de utilizar los nitratos y/o nitritos presentes en los efluentes de las plantas de fabricación de explosivos, como fuente de N, al objeto de efectuar una eliminación biológica de los mismos, superando los inconvenientes anteriormente citados y evitando de este modo los problemas medioambientales asociados con el vertido incontrolado de dichos efluentes o con su tratamiento inadecuado.It would be convenient, therefore, to have microorganisms capable of using the nitrates and / or nitrites present in the effluents of the plants of manufacture of explosives, as a source of N, in order to carry out a biological elimination thereof, overcoming the aforementioned inconveniences and thus avoiding the environmental problems associated with the uncontrolled discharge of said effluents or with their inadequate treatment.
Por consiguiente, un objeto de esta invención lo constituye el aislamiento y caracterización de microorganismos capaces de eliminar nitratos y/o nitritos. Tales microorganismos, pertenecientes a dos cepas de Klebsiella oxytoca. constituyen un objeto adicional de esta invención.Therefore, an object of this invention is the isolation and characterization of microorganisms capable of eliminating nitrates and / or nitrites. Such microorganisms, belonging to two strains of Klebsiella oxytoca. they constitute an additional object of this invention.
Otro objeto adicional de esta invención lo constituye un procedimiento para la eliminación biológica de nitratos y/o nitritos contenidos en efluentes industriales, aguas residuales urbanas o suelos, mediante el empleo de los microorganismos proporcionados por esta invención.Another additional object of this invention is a process for the biological elimination of nitrates and / or nitrites contained in industrial effluents, urban wastewater or soils, by using the microorganisms provided by this invention.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN La invención proporciona nuevas cepas de Klebsiella oxytoca capaces de utilizar nitratos y/o nitritos como fuente de nitrógeno, así como un procedimiento para la eliminación biológica de nitratos y/o nitritos contenidos en efluentes industriales, aguas residuales urbanas y suelos, mediante el empleo de las cepas de Klebsiella oxytoca proporcionadas por esta invención.DETAILED DESCRIPTION OF THE INVENTION The invention provides new strains of Klebsiella oxytoca capable of using nitrates and / or nitrites as a source of nitrogen, as well as a process for the biological elimination of nitrates and / or nitrites contained in industrial effluents, urban wastewater and soils. , by using the strains of Klebsiella oxytoca provided by this invention.
A modo de resumen, la invención comprende, por una parte, el aislamiento y caracterización de microorganismos capaces de utilizar nitratos y/o nitritos como fuente de N, y por otra, el empleo de los microorganismos aislados para la eliminación de nitratos y/o nitritos presentes en efluentes de plantas industriales, en aguas residuales urbanas y en suelos contaminados con dichos compuestos.By way of summary, the invention comprises, on the one hand, the isolation and characterization of microorganisms capable of using nitrates and / or nitrites as a source of N, and on the other, the use of isolated microorganisms for the elimination of nitrates and / or nitrites present in effluents from industrial plants, in urban wastewater and in soils contaminated with these compounds.
1. Aislamiento y caracterización de microorganismos capaces de utilizar nitratos y/o nitritos. 1.1 Aislamiento1. Isolation and characterization of microorganisms able to use nitrates and / or nitrites. 1.1 Isolation
Para el aislamiento de microorganismos capaces de utilizar nitrato como fuente de N se procedió del siguiente modo. Una muestra de un suelo próximo a una planta generadora de efluentes cargados con nitratos se suspendió en un medio de cultivo mínimo del tipo M8 [el medio M8 es idéntico al medio M9 excepto en que se omite la fuente de nitrógeno (NH4C1)]. La composición del medio M9 se describe en Abril et al . , Journal of Bacteriology , Vol . 171 , No. 12, págε . 6782-6790, ( 1989 ) . A dicha suspensión se añadió un volumen del agua efluente de la fábrica, rica en nitratos, y una cantidad adecuada de una fuente de C. Como fuente de C puede utilizarse un azúcar, preferentemente fructosa o glucosa, o un ácido orgánico adecuado, preferentemente ácido acético o succínico, o alcoholes del tipo de la glicerina y el etilenglicol. Posteriormente se incuba la suspensión resultante a temperaturas comprendidas entre 15°C y 42°C, preferentemente entre 25°C y 30°C, con agitación opcional, y, tras una semana de incubación, se siembran diluciones apropiadas en placas de medio sólido selectivo tal como el constituido por M8, agar noble, nitrato potásico (20-100 mM) y una fuente de C. Tras un periodo de incubación comprendido entre 24 h y una semana las colonias aisladas se purifican y su capacidad para crecer a expensas de nitratos y/o nitritos se comprueba en medio liquido. En el Ejemplos 1 se describe el aislamiento de bacterias capaces de utilizar nitrato como fuente de N, en particular, de las dos cepas de Klebsiella oxytoca de esta invención.For the isolation of microorganisms capable of using nitrate as a source of N, the following procedure was followed. A sample of a soil near a nitrate-charged effluent generating plant was suspended in a minimum culture medium of type M8 [the M8 medium is identical to the M9 medium except where the nitrogen source is omitted (NH 4 C1)] . The composition of the M9 medium is described in April et al. , Journal of Bacteriology, Vol. 171, No. 12, p. 6782-6790, (1989). To this suspension was added a volume of effluent water from the factory, rich in nitrates, and an adequate amount of a source of C. As a source of C a sugar, preferably fructose or glucose, or a suitable organic acid, preferably acid, can be used acetic or succinic, or alcohols of the type of glycerin and ethylene glycol. Subsequently, the resulting suspension is incubated at temperatures between 15 ° C and 42 ° C, preferably between 25 ° C and 30 ° C, with optional stirring, and, after one week of incubation, appropriate dilutions are plated on selective solid medium plates. such as that constituted by M8, noble agar, potassium nitrate (20-100 mM) and a source of C. After an incubation period between 24 h and one week the isolated colonies are purified and their ability to grow at the expense of nitrates and / or nitrites is checked in liquid medium. Examples 1 describe the isolation of bacteria capable of using nitrate as a source of N, in particular, of the two strains of Klebsiella oxytoca of this invention.
1.2 Caracterización de los microorganismos aislados1.2 Characterization of isolated microorganisms
Siguiendo análisis tipo [Test API 20E, de Biomerieux,Following type analysis [Biomerieux API Test 20E,
Francia, nB de referencia del catálogo 20.100] se ha podido verificar que los microorganismos aislados capaces de utilizar nitratos y/o nitritos como fuente de N, eran bacterias pertenecientes al género Klebsiella. Dos de las cepas aisladas más eficientes fueron las denominadas Klebsiella oxytoca clón-15 y Klebsiella oxytoca clon AHÍ, que han sido depositadas en la Colección Española de Cultivos Tipo (CECT), Valencia, España, los días 15 de Junio de 1993 y 10 de Junio de 1994, correspondiéndoles los números de accesión CECT 4460 y CECT 4500, respectivamente. Las bacterias aisladas presentan forma bacilar, y no presentan pigmentación cuando se cultivan en placas de medio mínimo o medio rico en LB ( Maniatis et al . Molecular Cloning, A Labor atory Manual , Cold Spring Harbor Labor atory , NY , 1982) . Una de las cepas aisladas, concretamente, la denominada Klebsiella oxytoca clón-15, en medio mínimo tipo M8 puede utilizar fructosa, glucosa, ácido acético, ácido succínico y glicerol como fuente de C y amoníaco, nitrato y nitrito como fuente de N, en condiciones aeróbicas o anaeróbicas, mientras que la otra cepa aislada, la denominada Klebsiella oxytoca clon AHÍ, puede utilizar nitrato y nitrito como fuente de N y etilenglicol como fuente de C en condiciones aeróbicas.France, reference number B of the catalog 20,100] it has been verified that the isolated microorganisms capable of using nitrates and / or nitrites as a source of N, they were bacteria belonging to the genus Klebsiella. Two of the most efficient isolated strains were the so-called Klebsiella oxytoca clón-15 and Klebsiella oxytoca clón AHÍ, which have been deposited in the Spanish Type Culture Collection (CECT), Valencia, Spain, on June 15, 1993 and 10 June 1994, corresponding accession numbers CECT 4460 and CECT 4500, respectively. Isolated bacteria have a bacillary form, and do not show pigmentation when grown on plates of minimal medium or medium rich in LB (Maniatis et al. Molecular Cloning, A Labor atory Manual, Cold Spring Harbor Labor atory, NY, 1982). One of the strains isolated, specifically, the so-called Klebsiella oxytoca clone-15, in a minimal medium type M8 can use fructose, glucose, acetic acid, succinic acid and glycerol as a source of C and ammonia, nitrate and nitrite as a source of N aerobic or anaerobic conditions, while the other isolated strain, the so-called Klebsiella oxytoca clone AH, can use nitrate and nitrite as a source of N and ethylene glycol as a source of C in aerobic conditions.
Las características taxonómicas comunes de las cepas Klebsiella oxytoca clón-15 y Klebsiella oxytoca clon AHÍ son las siguientes:The common taxonomic characteristics of the Klebsiella oxytoca clone-15 and Klebsiella oxytoca clone strains are the following:
1. Características morfológicas Forma: Bacilar Movilidad: No móvil Esporas: No forman1. Morphological characteristics Form: Bacilar Mobility: Non-mobile Spores: They do not form
Tinción Gram: NegativaGram stain: Negative
2. Características de cultivo2. Cultivation characteristics
Cultivo en medio Mac Conkey: Crecimiento Siembra en placas de agar nutritivo: Crecimiento Siembra en tubo inclinado de agar nutritivo: CrecimientoCultivation in Mac Conkey medium: Growth Sowing on nutrient agar plates: Growth Sowing in an inclined nutrient agar tube: Growth
3. Características fisiológicas pH: 6.0-10.5 (para K. oxytoca clón-15) 6.0-8.0 (para K. oxytoca clon AHÍ) Temperatura: 15eC-42sC Comportamiento frente al 02: ambas son anaeróbicas facultativas, pero la cepa K. oxytoca clon AHÍ sólo utiliza etilenglicol como fuente de C en aerobiosis β-galactosidasa: Positiva Arginina dehidrolasa: Negativa Lisina descarboxilasa: Positiva Ornitina descarboxilasa: Negativa Utilización de citrato: Positiva Producción de H2S: Negativa Ureasa: Positiva Triptófano desaminasa: Negativa Producción de indol: Positiva Gelatinasa: Negativa Fermentación/oxidación de azúcares: Glucosa: Positiva Manitol: Positiva Inositol: Positiva3. Physiological characteristics pH: 6.0-10.5 (for K. oxytoca clone-15) 6.0-8.0 (for K. oxytoca clone AH) Temperature: 15 e C-42 s C Behavior vs. 0 2 : both are optional anaerobic, but the strain K. oxytoca clone AHI only uses ethylene glycol as a source of C in aerobiosis β-galactosidase: Positive Arginine dehydrolase: Negative Lysine decarboxylase: Positive Ornithine decarboxylase: Negative Citrate use: Positive Production of H 2 S: Negative Ureasa: Positive Tryptophan : Negative Production of indole: Positive Gelatinase: Negative Fermentation / oxidation of sugars: Glucose: Positive Mannitol: Positive Inositol: Positive
Sorbitol: Positiva Ramnosa: Positiva Sacarosa: Positiva Melibiosa: Positiva Amigdalina: PositivaSorbitol: Positive Ramnosa: Positive Sucrose: Positive Melibiosa: Positive Tonsil: Positive
Arabinosa: Positiva Citocromo oxidasa: Negativa Producción de N02: Positiva Reducción a gas N2: Positiva La cepa Klebsiella oxytoca clón-15 es capaz de crecer en presencia de altas y bajas cargas de nitratos y/o nitritos. Una característica de esta cepa es el hecho de que es capaz de crecer en presencia de relativamente altas conci traciones de nitratos (hasta 100 mM) , lo que le da un carácter muy particular y constituye una ventaja fundamental para su aplicación en la eliminación de nitratos y/o nitritos presentes en efluentes, aguas residuales y suelos. Por otra parte, la cepa Klebsiella oxytoca clon AHÍ es capaz de crecer en presencia de altas cargas de nitratos, a concentraciones del orden de 225 mM en sistemas estancos, por lo que es muy adecuada para la eliminación de nitratos y/o nitritos presentes en efluentes, aguas residuales y suelos.Arabinose: Positive Cytochrome oxidase: Negative Production of N0 2 : Positive Gas reduction N 2 : Positive The Klebsiella oxytoca clone-15 strain is capable of growing in the presence of high and low nitrate and / or nitrite loads. A characteristic of this strain is the fact that it is capable of growing in the presence of relatively high nitrate concentrations (up to 100 mM), which gives it a very particular character and constitutes a fundamental advantage for its application in nitrate removal and / or nitrites present in effluents, wastewater and soils. On the other hand, the Klebsiella oxytoca clone AH strain is capable of growing in the presence of high nitrate loads, at concentrations of the order of 225 mM in watertight systems, making it very suitable for the elimination of nitrates and / or nitrites present in effluents, sewage and soil.
Dado que la vía de asimilación del nitrato incluye, entre otras reacciones, la reducción del nitrato a nitrito y amoniaco, se estudió la posibilidad de que las cepas aisladas fueran capaces de asimilar nitritos, observándose que dichas cepas son capaces de utilizar nitritos como fuente de N, en condiciones aeróbicas o anaeróbicas rKlebsiella oxytoca clón-15], tal como se describe en los Ejemplos 2 a 19, como en condiciones aeróbicas [Klebsiella oxytoca clon AHÍ], como se describe en los Ejemplos 20 a 22.Since the route of assimilation of nitrate includes, among other reactions, the reduction of nitrate to nitrite and ammonia, the possibility that isolated strains were able to assimilate nitrites was studied, observing that these strains are capable of using nitrites as a source of N, in aerobic or anaerobic conditions rKlebsiella oxytoca clone-15], as described in Examples 2 to 19, as in aerobic conditions [Klebsiella oxytoca clone AH], as described in Examples 20 to 22.
Una vez aisladas y purificadas las colonias de bacterias capaces de utilizar nitratos y/o nitritos como fuente de N, éstas pueden utilizarse para eliminar dichos compuestos presentes en efluentes de plantas industriales, en aguas residuales urbanas y en suelos.Once the colonies of bacteria capable of using nitrates and / or nitrites as a source of N are isolated and purified, they can be used to eliminate these compounds present in effluents from industrial plants, in urban wastewater and in soils.
2. Procedimiento de eliminación biológica de nitratos y/o nitritos.2. Procedure for biological elimination of nitrates and / or nitrites.
Una primera aplicación de las bacterias de esta invención, lo constituye su empleo en un procedimiento de eliminación biológica de nitratos y/o nitritos contenidos en un efluente industrial. Dicho procedimiento comprende básicamente las siguientes etapas:A first application of the bacteria of this invention is its use in a process of Biological elimination of nitrates and / or nitrites contained in an industrial effluent. Said procedure basically comprises the following steps:
a) neutralización del efluente a tratar; b) suplemento de los nutrientes necesarios para el funcionamiento óptimo del procedimiento; c) inoculación del efluente a tratar con cultivos de microorganismos capaces de utilizar nitratos y/o nitritos hasta la desaparición de los mismos; d) retirada de los microorganismos que pueden ser reutilizados en un nuevo ciclo de tratamiento de efluentes, si se desea; y e) vertido de las aguas depuradas.a) neutralization of the effluent to be treated; b) supplement of the nutrients necessary for the optimal functioning of the procedure; c) inoculation of the effluent to be treated with cultures of microorganisms capable of using nitrates and / or nitrites until their disappearance; d) removal of microorganisms that can be reused in a new effluent treatment cycle, if desired; and e) discharge of purified water.
Como puede apreciarse, el procedimiento comienza con una etapa de neutralización de los efluentes. Los efluentes susceptibles de ser depurados mediante este procedimiento pueden ser efluentes de cualquier proceso industrial en el que se utilicen o produzcan nitratos y/o nitritos, tales como efluentes procedentes de plantas de fabricación de explosivos. Asimismo también pueden tratarse cualquier tipo de aguas contaminadas con nitratos y/o nitritos.As can be seen, the process begins with a stage of neutralization of effluents. Effluents capable of being purified by this procedure can be effluents from any industrial process in which nitrates and / or nitrites are used or produced, such as effluents from explosive manufacturing plants. Likewise, any type of water contaminated with nitrates and / or nitrites can be treated.
En general, las aguas de desecho procedentes de las plantas de fabricación de explosivos son aguas acidas que se pueden neutralizar añadiendo cualquier álcali, preferentemente un hidróxido, carbonato o bicarbonato de un metal alcalino o alcalinotérreo, al objeto de qué dichas aguas alcancen un pH comprendido entre 6.0 y 10.5 tras la neutralización. Este intervalo de pH es adecuado cuando la cepa que se utiliza es la denominada Klebsiella oxytoca clón-15, mientras que si se utiliza la cepa Klebsiella oxytoca clon AHÍ, resulta más conveniente neutralizar el efluente y mantener el pH del mismo en un intervalo comprendido entre 6.0 y 8.0, ya que ese es su intervalo de actuación.In general, wastewater from explosive manufacturing plants is acidic water that can be neutralized by adding any alkali, preferably a hydroxide, carbonate or bicarbonate of an alkali metal or alkaline earth metal, in order that these waters reach a pH comprised between 6.0 and 10.5 after neutralization. This pH range is adequate when the strain used is the so-called Klebsiella oxytoca clone-15, while if the Klebsiella oxytoca clone strain is used THERE, it is more convenient neutralize the effluent and maintain its pH in a range between 6.0 and 8.0, since that is its range of action.
Posteriormente, y tras análisis del efluente neutralizado, se añaden los nutrientes necesarios tales como fosfato o cualquier otro nutriente o micronutriente que sea necesario para el óptimo funcionamiento del proceso. A modo de ejemplo, pueden añadirse cantidades adecuadas de la solución de micronutrientes A9, cuya composición se describe en Abril et al . citado εupra , junto con cantidades apropiadas de Fe, Mg, Co y Mo (del orden de microraolar). En cualquier caso, la elección y cantidad de nutrientes y micronutrientes a añadir será función de la composición del efluente a tratar y de la demanda microbiológica. Adicionalmente se añade una fuente de C de las mencionadas anteriormente, apropiada para la cepa que se va a utilizar en el procedimiento.Subsequently, and after analysis of the neutralized effluent, the necessary nutrients such as phosphate or any other nutrient or micronutrient that are necessary for the optimal functioning of the process are added. By way of example, suitable amounts of the A9 micronutrient solution may be added, the composition of which is described in April et al. cited εupra, together with appropriate amounts of Fe, Mg, Co and Mo (of the order of microraolar). In any case, the choice and amount of nutrients and micronutrients to be added will depend on the composition of the effluent to be treated and the microbiological demand. Additionally, a source of C of those mentioned above is added, appropriate for the strain to be used in the process.
A continuación se inoculan los efluentes neutralizados y debidamente suplementados con un cultivo de las bacterias proporcionadas por esta invención (Klebsiella oxytoca clón-15 o Klebsiella oxytoca clon AHÍ). La carga bacteriana puede estar comprendida entre 0.01 y 0.1 unidades de absorbancia a 660 n por i de cultivo, preferentemente, alrededor de 0.03 unidades de absorbancia a 660 nm por mi de cultivo.The neutralized and duly supplemented effluents are then inoculated with a culture of the bacteria provided by this invention (Klebsiella oxytoca clone-15 or Klebsiella oxytoca clone AH). The bacterial load may be between 0.01 and 0.1 absorbance units at 660 n per culture, preferably, about 0.03 absorbance units at 660 nm per ml of culture.
La incubación debe realizarse a una temperatura comprendida entre 20° y 30°C con un aporte de hasta 2 litros de aire por litro de cultivo, si el procedimiento se realiza utilizando Klebsiella oxytoca clon AHÍ, mientras que puede no aportarse aire o pueden aportarse hasta 2 litros de aire por litro de cultivo si en el procedimiento se utiliza Klebsiella oxγtoca clón-15. El curso de la incubación se sigue midiendo periódicamente la cantidad de nitrato y nitrito presente en el efluente o agua a depurar, mediante un electrodo especifico de nitrato (por ejemplo, un electrodo de nitrato del tipo Crison) y mediante un ensayo químico extremadamente sensible para la detección de nitrito (Snell y Snell , Colorimetric Methods of Analysis, Vol . 3 , págε . 804-805, Van Nostrand Co . Inc. , New York, 1949 ) respectivamente, dándose por finalizada esta etapa una vez dejan de detectarse nitratos y/o nitritos y fuente de C.The incubation should be carried out at a temperature between 20 ° and 30 ° C with a contribution of up to 2 liters of air per liter of culture, if the procedure is performed using Klebsiella oxytoca clone AH, while air may not be supplied or up to 2 liters of air per liter of culture if Klebsiella oxγtoca clone-15 is used in the procedure. The course of the incubation is continued periodically measuring the amount of nitrate and nitrite present in the effluent or water to be purified, by means of a specific electrode of nitrate (for example, a Crison type nitrate electrode) and by an extremely sensitive chemical test for nitrite detection (Snell and Snell, Colorimetric Methods of Analysis, Vol. 3, p. 804-805, Van Nostrand Co. Inc ., New York, 1949) respectively, ending this stage once nitrates and / or nitrites and source of C. cease to be detected.
Posteriormente se retiran las bacterias por floculación o por cualquier otro método que permita, si se desea, su reutilización en un nuevo proceso y se retiran las aguas depuradas que pueden ser libremente vertidas.Subsequently, the bacteria are removed by flocculation or by any other method that allows, if desired, their reuse in a new process and the purified water that can be freely discharged is removed.
Este procedimiento puede realizarse en continuo o en estanco, así como en balsas de grandes dimensiones o en fermentadores. Cuando se realiza en continuo debe regularse adecuadamente el caudal y la composición del efluente de entrada y del de salida, asi como la concentración de cultivo bacteriano y el aporte de nutrientes y micronutrientes necesarios.This procedure can be carried out continuously or in airtight, as well as in large rafts or in fermenters. When it is carried out continuously, the flow and composition of the inlet and outlet effluent must be adequately regulated, as well as the concentration of bacterial culture and the necessary nutrient and micronutrient intake.
Una segunda aplicación de las bacterias de esta invención lo constituye su empleo en un procedimiento para la eliminación biológica de nitratos y/o nitritos en suelos superficiales contaminados con dichos compuestos. Dicho procedimiento comprende las siguientes etapas:A second application of the bacteria of this invention is their use in a process for the biological elimination of nitrates and / or nitrites in surface soils contaminated with said compounds. Said procedure comprises the following steps:
a) inyección primaria de un cultivo de microorganismos capaces de utilizar nitratos y/o nitritos; y b) inyecciones sucesivas de cultivos de los microorganismos antes citados hasta que el suelo quede libre de nitratos y/o nitritos.a) primary injection of a culture of microorganisms capable of using nitrates and / or nitrites; and b) successive injections of cultures of the aforementioned microorganisms until the soil is free of nitrates and / or nitrites.
La inyección de microorganismos se realiza para alcanzar altas densidades celulares en los suelos, del orden de 105 bacterias por cm2 de suelo, que faciliten la eliminación del contaminante. Si fuese necesario se añadiría a los suelos fosfatos y una fuente de carbono que facilite su supervivencia.The injection of microorganisms is done to reach high cell densities in the soil, of the order of 10 5 bacteria per cm 2 of soil, which facilitate the removal of the contaminant. If necessary, it would add phosphate and a carbon source to the soil that facilitates its survival.
Finalmente, una tercera aplicación de las bacterias de esta invención lo constituye su empleo en la eliminación de nitratos y/o nitritos contenidos en aguas residuales siguiendo un procedimiento similar al descrito anteriormente para la eliminación de nitratos y/o nitritos contenidos en efluentes industriales.Finally, a third application of the bacteria of this invention is their use in the elimination of nitrates and / or nitrites contained in wastewater following a procedure similar to that described above for the elimination of nitrates and / or nitrites contained in industrial effluents.
Las ventajas derivadas de los procedimientos de eliminación biológica de los nitratos y/o nitritos de esta invención, pueden resumirse en:The advantages derived from the biological elimination procedures of the nitrates and / or nitrites of this invention can be summarized in:
1) presentan una alta especificidad en la eliminación de nitratos y nitritos;1) have a high specificity in the elimination of nitrates and nitrites;
2) funcionan en un amplio intervalo de concentraciones de nitrato, nitrito o sus mezclas, en particular, funciona a concentraciones de:2) they work in a wide range of concentrations of nitrate, nitrite or mixtures thereof, in particular, it works at concentrations of:
0.01 a 100 mM de nitrato y de 0.01 a 50 mM de nitrito o sus mezclas, cuando el procedimiento se efectúa utilizando bacterias de la cepa Klebsiella oxytoca clón-15, o de0.01 to 100 mM of nitrate and 0.01 to 50 mM of nitrite or mixtures thereof, when the procedure is carried out using bacteria from the Klebsiella oxytoca clone-15 strain, or from
0.01 a 225 mM de nitrato en estanco y con suplemento de hasta 400 mM de nitrato en continuo, controlando la velocidad de suministro, cuando el procedimiento se efectúa utilizando bacterias de la cepa Klebsiella oxytoca clon AHÍ; y0.01 to 225 mM of nitrate in airtight and with a supplement of up to 400 mM of nitrate in continuous, controlling the speed of supply, when the procedure is carried out using bacteria of the strain Klebsiella oxytoca clone THERE; Y
3) presentan una alta versatilidad, ya que pueden ser utilizados ¿n situ para eliminar nitratos y/o nitritos contenidos en efluentes de plantas industriales, aguas residuales y suelos.3) they have high versatility, since they can be used in situ to eliminate nitrates and / or nitrites contained in effluents from industrial plants, wastewater and soils.
A continuación se describen unos ejemplos que sirven para ilustrar realizaciones particulares de la presente invención sin que deban ser tomados en sentido limitativo del alcance de la misma.Some examples are described below which serve to illustrate particular embodiments of the present. invention without having to be taken in a limiting sense of its scope.
EJEMPLO 1 Aislamiento de las bacterias \Klebsiella oxytoca clón-15 y Klebsiella oxytoca clon AHÍ]EXAMPLE 1 Isolation of bacteria \ Klebsiella oxytoca clone-15 and Klebsiella oxytoca clone AH]
Una muestra de 10 g de un suelo de Páramo de Masa (Burgos) se suspendió en un medio de cultivo mínimo del tipo M8, en una relación 1:10 (p/v). Posteriormente se añadió KN03 al medio hasta alcanzar una concentración de 20 mM o de 100 mM, y de la suspensión se tomaron 3 alícuotas idénticas a las que se añadieron:A sample of 10 g of a soil of Páramo de Masa (Burgos) was suspended in a minimum culture medium of type M8, in a ratio 1:10 (w / v). Subsequently KN0 3 was added to the medium until a concentration of 20 mM or 100 mM was reached, and from the suspension 3 identical aliquots were taken to which were added:
MUESTRA GLICEROL ETILENGLICOL FRUCTOSA (g/1) (g/1) (g/1) I 5SAMPLE GLYCEROL ETHYLENE GLYCOLOL FRUCTOSE (g / 1) (g / 1) (g / 1) I 5
II 5II 5
III 5III 5
Las suspensiones I a III resultantes se incubaron a una temperatura comprendida entre 25°C y 30°C, con agitación opcional, durante una semana. Posteriormente se sembraron diluciones apropiadas de dichas suspensiones en placas de medio sólido selectivo constituido por medio M8, agar noble 1.5% (p/v) yThe resulting suspensions I to III were incubated at a temperature between 25 ° C and 30 ° C, with optional stirring, for one week. Subsequently, appropriate dilutions of said suspensions were seeded on plates of selective solid medium consisting of M8 medium, 1.5% noble agar (w / v) and
20 mM KN03 + 5 g/1 de glicerol (muestra I) 100 mM KNO3 + 10 g/1 de glicerol (muestra I)20 mM KN0 3 + 5 g / 1 glycerol (sample I) 100 mM KNO3 + 10 g / 1 glycerol (sample I)
20 mM KNO3 + 5 g/1 de etilenglicol (muestra II) 100 mM KNO3 + 5 g/1 de etilenglicol (muestra II) 20 mM KNO3 + 5 g/1 de fructosa (muestra III) 100 mM KNO3 + 5 g/1 de fructosa (muestra III)20 mM KNO 3 + 5 g / 1 ethylene glycol (sample II) 100 mM KNO 3 + 5 g / 1 ethylene glycol (sample II) 20 mM KNO 3 + 5 g / 1 fructose (sample III) 100 mM KNO 3 + 5 g / 1 fructose (sample III)
Tras incubar entre 24 horas y una semana, se purificaron las colonias aisladas y se comprobó su fenotipo cultivándolas en los medios arriba indicados. Siguiendo los análisis del Kit API 20E (Biomerieux) , las bacterias aisladas fueron clasificadas como Klebsiella oxytoca. Las dos cepas más eficientes fueron las denominadas Klebsiella oxytoca clón-15 y Klebsiella oxytoca clon AHÍ, que presentan las características mencionadas previamente en la descripción. Sendos cultivos de esas cepas, se depositaron los días 15 de Junio de 1993 y 10 de Junio de 1994 en la CECT correspondiéndoles los nB de accesión CECT 4460 y CECT 4500 respectivamente.After incubating between 24 hours and one week, the isolated colonies were purified and their phenotype was tested by culturing them in the above-indicated media. Following the analysis of the API 20E Kit (Biomerieux), the isolated bacteria were classified as Klebsiella oxytoca. The two most efficient strains were the so-called Klebsiella oxytoca clone-15 and Klebsiella oxytoca clone AH, which have the characteristics previously mentioned in the description. Both cultures of these strains were deposited on June 15, 1993 and June 10, 1994 in the CECT, with accession numbers B CECT 4460 and CECT 4500 respectively.
EJEMPLO 2 Utilización de nitrato como fuente de N en condiciones aeróbicas 100 mi de medio mínimo M8 se suplementan con: 250 μl de solución de micronutrientes A9; - 6 μg de citrato de hierro;EXAMPLE 2 Use of nitrate as a source of N under aerobic conditions 100 ml of M8 minimum medium is supplemented with: 250 μl of A9 micronutrient solution; - 6 μg of iron citrate;
100 μl de solución de Mg S04 1M; 20 mM KN03; y 1 g de glicerol. El cultivo se inicia añadiendo una cantidad de bacteria (Klebsiella oxytoca clón-15) tal que se alcanza una turbidez inicial a 660 nm de 0.03 unidades y se incuba a 30°C con agitación (150 rpm en un incubador orbital) y aerobiosis. Tras 24 horas de incubación la turbidez del cultivo a 660 nm está comprendida entre 1 y 2 unidades y la concentración de nitrato en el medio de cultivo es indetectable mediante análisis selectivo con un electrodo de nitrato de sobrenadante. Es igualmente indetectable la concentración de fuente de C.100 μl of Mg S0 4 1M solution; 20 mM KN0 3 ; and 1 g of glycerol. The culture is started by adding an amount of bacteria (Klebsiella oxytoca clone-15) such that an initial turbidity is reached at 660 nm of 0.03 units and incubated at 30 ° C with shaking (150 rpm in an orbital incubator) and aerobiosis. After 24 hours of incubation the turbidity of the culture at 660 nm is between 1 and 2 units and the concentration of nitrate in the culture medium is undetectable by selective analysis with a supernatant nitrate electrode. The concentration of C source is equally undetectable.
EJEMPLO 3 Utilización de nitrato como fuente de N en condiciones aeróbicasEXAMPLE 3 Use of nitrate as a source of N in aerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 2, pero utilizando nitrato potásico en una concentración de 100 mM. El nitrato desapareció completamente en un plazo de 48 horas. EJEMPLO 4 Utilización de nitrato como fuente de N en condiciones aeróbicasThe procedure described in Example 2 was repeated, but using potassium nitrate in a concentration of 100 mM. The nitrate disappeared completely within 48 hours. EXAMPLE 4 Use of nitrate as a source of N in aerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 2, pero utilizando agua de una factoría de manera que la concentración final en HN03 fuera de 25-30 mM. El nitrato desapareció completamente en un plazo de 24 horas.The procedure described in Example 2 was repeated, but using water from a factory so that the final concentration in HN0 3 was 25-30 mM. The nitrate disappeared completely within 24 hours.
EJEMPLO 5 Utilización de nitrato como fuente de N en condiciones aeróbicas Se repitió el procedimiento descrito en el Ejemplo 2, pero utilizando agua de una factoría de manera que la concentración en HN03 fuera de 75-100 mM. El nitrato desapareció completamente en un plazo de 48 h.EXAMPLE 5 Use of nitrate as a source of N in aerobic conditions The procedure described in Example 2 was repeated, but using water from a factory so that the concentration in HN0 3 was 75-100 mM. The nitrate disappeared completely within 48 hours.
EJEMPLO 6 Utilización de nitrato como fuente de N en condiciones aeróbicas, en continuoEXAMPLE 6 Use of nitrate as a source of N in aerobic conditions, in continuous
Se repitió el procedimiento descrito en el Ejemplo 2, pero utilizando un sistema de adición en continuo de nitrato. El fermentador contenía 110 mi de medio y la turbidez del cultivo era del orden de 3 unidades a 660 mm. Agua de una factoría diluida para suministrar de 25 a 30 mM de HN03 se añadía a un flujo de 1-20 ml/h. El volumen de aire era de 0.5 volúmenes/min. El nitrato en el medio que rebosaba era indetectable.The procedure described in Example 2 was repeated, but using a continuous nitrate addition system. The fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 3 units at 660 mm. Water from a diluted factory to supply 25 to 30 mM of HN0 3 was added at a flow of 1-20 ml / h. The air volume was 0.5 volumes / min. The nitrate in the overflowing medium was undetectable.
EJEMPLO 7 Utilización de nitrato como fuente de NEXAMPLE 7 Use of nitrate as a source of N
Se repitió el procedimiento descrito en el Ejemplo 4, pero se utilizaron 2 litros de medio de cultivo y la incubación se realizó en un fermentador. Las condiciones de operación fueron las siguientes: pH: 6.5-8.0The procedure described in Example 4 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter. The operating conditions were as follows: pH: 6.5-8.0
Temperatura: 25°C-35°C. Agitación: 400-800 rpm Aire: 0.5 volúmenes/minuto. El nitrato desapareció tras 24-30 h de cultivo. EJEMPLO 8 Utilización de nitrato como fuente de N en anaerobiosisTemperature: 25 ° C-35 ° C. Agitation: 400-800 rpm Air: 0.5 volumes / minute. The nitrate disappeared after 24-30 h of culture. EXAMPLE 8 Use of nitrate as a source of N in anaerobiosis
Se repitió el procedimiento descrito en el Ejemplo 2, pero utilizando condiciones anaeróbicas. El nitrato desapareció completamente en un plazo de 24 horas.The procedure described in Example 2 was repeated, but using anaerobic conditions. The nitrate disappeared completely within 24 hours.
EJEMPLO 9 Utilización de nitrato como fuente de N en condiciones anaeróbicasEXAMPLE 9 Use of nitrate as a source of N under anaerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 8, pero utilizando nitrato potásico a una concentración de 100 mM. El nitrato desapareció completamente en un plazo de 48 horas.The procedure described in Example 8 was repeated, but using potassium nitrate at a concentration of 100 mM. The nitrate disappeared completely within 48 hours.
EJEMPLO 10 Utilización de nitrato como fuente de N en condiciones anaeróbicasEXAMPLE 10 Use of nitrate as a source of N under anaerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 8, pero utilizando agua de una factoría hasta alcanzar una concentración en HN03 de 25-30 mM. El nitrato desapareció completamente en un plazo de 24 horas.The procedure described in Example 8 was repeated, but using water from a factory until reaching a concentration in HN0 3 of 25-30 mM. The nitrate disappeared completely within 24 hours.
EJEMPLO 11 Utilización de nitrato como fuente de N en condiciones anaeróbicas Se repitió el procedimiento descrito en el Ejemplo 8, pero utilizando agua de una factoría hasta alcanzar una concentración en HN03 de 75-100 mM. El nitrato desapareció completamente en un plazo de 48 horas.EXAMPLE 11 Use of nitrate as a source of N under anaerobic conditions The procedure described in Example 8 was repeated, but using water from a factory until reaching a concentration in HN0 3 of 75-100 mM. The nitrate disappeared completely within 48 hours.
EJEMPLO 12 Utilización de nitrato como fuente de N en condiciones anaeróbicas, en continuo Se repitió el procedimiento descrito en el Ejemplo 6 excepto que no se burbujeaba aire. El fermentador contenia 110 mi de medio y la turbidez del cultivo era del orden de 1 unidad a 66? mm. Agua diluida de una factoría que contenía una concentración de 25 a 30 mM en HN03 se añadía a un flujo de 1-20 ml/h. El nitrato en el medio que rebosaba era indetectable.EXAMPLE 12 Use of nitrate as a source of N under anaerobic conditions, continuously The procedure described in Example 6 was repeated except that no air was bubbled. The fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 1 unit at 66? mm Diluted water from a factory containing a concentration of 25 to 30 mM in HN0 3 was added at a flow of 1-20 ml / h. The nitrate in the medium that overflowing was undetectable.
EJEMPLO 13 Utilización de nitrato como fuente de NEXAMPLE 13 Use of nitrate as a source of N
Se repitió el procedimiento descrito en el Ejemplo 4, pero se utilizaron 2 litros de medio de cultivo y la incubación se realizó en un fermentador. Las condiciones de operación fueron las siguientes: pH: 6.5-8.0The procedure described in Example 4 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter. The operating conditions were as follows: pH: 6.5-8.0
Temperatura : 25°C-35°C . Agitación : 400-800 rpmTemperature: 25 ° C-35 ° C. Agitation: 400-800 rpm
Sin Aire .Without air .
El nitrato desapareció tras 24-30 horas de cultivo.The nitrate disappeared after 24-30 hours of culture.
EJEMPLO 14 Tratamiento de un efluente real Un efluente procedente de una fábrica de explosivos presentaba las siguientes características:EXAMPLE 14 Treatment of a real effluent An effluent from an explosives factory had the following characteristics:
- Composición:- Composition:
Nitratos: 200 mg/ml Sulfatos: 1000 mg/ml - pH: 2.0Nitrates: 200 mg / ml Sulfates: 1000 mg / ml - pH: 2.0
Un volumen de 1 litro de dicho efluente se neutralizó con NaOH hasta alcanzar un pH de 7.0. 100 mi de este efluente se llevaron hasta 1 litro con agua destilada y se añadieron los nutrientes y micronutrientes del medio M8, utilizándose una fuente apropiada de C tal como las mencionadas en el Ejemplo 1.A volume of 1 liter of said effluent was neutralized with NaOH until a pH of 7.0 was reached. 100 ml of this effluent was taken up to 1 liter with distilled water and the nutrients and micronutrients of the M8 medium were added, using an appropriate source of C such as those mentioned in Example 1.
Posteriormente se inoculó un cultivo de las bacterias de la cepa Klebsiella oxytoca clón-15, con una turbidez inicial de 0.05 a 660 nm. Las condiciones de la incubación fueron las siguientes:Subsequently, a culture of the bacteria of the Klebsiella oxytoca clone-15 strain was inoculated, with an initial turbidity of 0.05 at 660 nm. The conditions of the incubation were the following:
- agitación: 600 rpm en un incubador orbital- agitation: 600 rpm in an orbital incubator
- temperatura: 25°C- temperature: 25 ° C
- tiempo: 24 h- time: 24 h
La evolución de la incubación se siguió midiendo periódicamente el contenido de nitrato mediante un electrodo selectivo, comprobándose que al cabo de 24 horas había desaparecido completamente el nitrato.The evolution of the incubation was continued by periodically measuring the nitrate content by means of a selective electrode, checking that after 24 hours the nitrate had completely disappeared.
EJEMPLO 15 Utilización de nitrito como fuente de N en condiciones aeróbicasEXAMPLE 15 Use of nitrite as a source of N in aerobic conditions
100 mi de medio mínimo M8 estéril se suplementan con: 250 μl de solución de micronutrientes A9; 6 μg de citrato de hierro; 100 μl de solución de Mg S04 1M; 2 mM NaN02 100 ml of M8 sterile minimum medium is supplemented with: 250 μl of A9 micronutrient solution; 6 μg of iron citrate; 100 μl of Mg S0 4 1M solution; 2 mM NaN0 2
1 g de glicerol1 g of glycerol
El cultivo se inicia añadiendo una cantidad de bacteria (Klebsiella oxytoca clón-15) tal que se alcanza una turbidez inicial a 660 nm de 0.03 unidades y se incuba a 30°C con agitación (150 rpm en un incubador orbital) y aerobiosis. Tras 24 h de incubación la turbidez del cultivo a 660 nm está comprendida entre 0.5 y 1 unidad y la concentración de nitrito en el medio de cultivo es indetectable mediante análisis químico de sobrenadantes. Es igualmente indetectable la concentración de fuente deThe culture is started by adding a quantity of bacteria (Klebsiella oxytoca clone-15) such that an initial turbidity is reached at 660 nm of 0.03 units and it is incubated at 30 ° C with agitation (150 rpm in an orbital incubator) and aerobiosis. After 24 h of incubation the turbidity of the culture at 660 nm is between 0.5 and 1 unit and the concentration of nitrite in the culture medium is undetectable by chemical analysis of supernatants. It is also undetectable the concentration of source of
Carbono.Carbon.
EJEMPLO 16 Utilización de nitrito como fuente de N en condiciones aeróbicas Se repitió el procedimiento descrito en el Ejemplo 15, pero utilizando una concetración de 10 mM de nitrito sódico. El nitrito desapareció completamente en un plazo de 48 horas.EXAMPLE 16 Use of nitrite as a source of N under aerobic conditions The procedure described in Example 15 was repeated, but using a 10 mM concetration of sodium nitrite. The nitrite disappeared completely within 48 hours.
EJEMPLO 17 Utilización de nitrito como fuente de N en condiciones aeróbicasEXAMPLE 17 Use of nitrite as a source of N in aerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 15, pero se utilizaron 2 litros de medio de cultivo y la incubación se realizó en un fermentador. Las condiciones de operación fueron las siguientes: pH : 6 . 5-8 . 0The procedure described in Example 15 was repeated, but 2 liters of culture medium was used and the incubation was carried out in a fermenter. The operating conditions were as follows: pH: 6. 5-8. 0
Temperatura: 25°C-35°C. Agitación: 400-800 rpm Aire: 0.5 volúmenes/minuto. El nitrito desapareció tras 24-30 horas de cultivo.Temperature: 25 ° C-35 ° C. Agitation: 400-800 rpm Air: 0.5 volumes / minute. The nitrite disappeared after 24-30 hours of cultivation.
EJEMPLO 18 Utilización de nitrito como fuente de N en anaerobiosisEXAMPLE 18 Use of nitrite as a source of N in anaerobiosis
Se repitió el procedimiento descrito en el Ejemplo 15, pero utilizando condiciones anaeróbicas. El nitrito desapareció completamente en un plazo de 24 horas.The procedure described in Example 15 was repeated, but using anaerobic conditions. The nitrite disappeared completely within 24 hours.
EJEMPLO 19 Utilización de nitrito como fuente de N en condiciones anaeróbicasEXAMPLE 19 Use of nitrite as a source of N under anaerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 18, pero utilizando una concentración de 10 mM en nitrito sódico. El nitrito desapareció completamente en un plazo de 48 horas.The procedure described in Example 18 was repeated, but using a concentration of 10 mM in sodium nitrite. The nitrite disappeared completely within 48 hours.
EJEMPLO 20 Utilización de nitrato como fuente de N en condiciones aeróbicas 100 mi de medio mínimo M8 se suplementan con: 250 μl de solución de micronutrientes A9; 6 μg de citrato de hierro;EXAMPLE 20 Use of nitrate as a source of N under aerobic conditions 100 ml of M8 minimum medium is supplemented with: 250 μl of A9 micronutrient solution; 6 μg of iron citrate;
100 μl de solución de Mg S04 1M; 20 mM KN03; y 1.0 g de etilenglicol. El cultivo se inicia añadiendo una cantidad de bacteria (Klebsiella oxytoca clon AHÍ) tal que se alcanza una turbidez inicial a 660 nm de 0.03 unidades y se incuba a 30°C con agitación (150 rpm en un incubador orbital) y aerobiosis. Tras 48 horas de incubación la turbidez del cultivo a 660 nm está comprendida entre 5 y 15 unidades y la concentración de nitrato en el medio de cultivo es indetectable mediante análisis selectivo con un electrodo de nitrato de sobrenadante. Es igualmente indetectable la concentración de fuente de C.100 μl of Mg S0 4 1M solution; 20 mM KN0 3 ; and 1.0 g of ethylene glycol. The culture is started by adding a quantity of bacteria (Klebsiella oxytoca clone AH) such that an initial turbidity is reached at 660 nm of 0.03 units and it is incubated at 30 ° C with agitation (150 rpm in an orbital incubator) and aerobiosis. After 48 hours of incubation the turbidity of the culture at 660 nm is between 5 and 15 units and the concentration of nitrate in the culture medium is undetectable by selective analysis with a supernatant nitrate electrode. The concentration of C source is equally undetectable.
EJEMPLO 21 Utilización de nitrato como fuente de N en condiciones aeróbicasEXAMPLE 21 Use of nitrate as a source of N in aerobic conditions
Se repitió el procedimiento descrito en el Ejemplo 20, pero utilizando nitrato potásico en una concentración de 225 mM. El nitrato desapareció completamente en un plazo de 48 horas.The procedure described in Example 20 was repeated, but using potassium nitrate at a concentration of 225 mM. The nitrate disappeared completely within 48 hours.
EJEMPLO 22 Utilización de nitrato como fuente de N en condiciones aeróbicas, en continuoEXAMPLE 22 Use of nitrate as a source of N in aerobic conditions, in continuous
Se repitió el procedimiento descrito en el Ejemplo 20, pero utilizando un sistema de adición en continuo de nitrato. El fermentador contenía 110 mi de medio y la turbidez del cultivo era del orden de 3 unidades a 660 mm. Agua sin diluir de una factoría suministraba de 200 a 400 roM de HN03 y se añadía a un flujo de 1-5 ml/h. El volumen de aire era de 0.5 volúmenes/min. El nitrato en el medio que rebosaba era indetectable.The procedure described in Example 20 was repeated, but using a continuous nitrate addition system. The fermenter contained 110 ml of medium and the turbidity of the culture was of the order of 3 units at 660 mm. Undiluted water from a factory supplied 200 to 400 roM of HN0 3 and was added at a flow of 1-5 ml / h. The air volume was 0.5 volumes / min. The nitrate in the overflowing medium was undetectable.
Descrito el objetivo de la presente invención se declara que lo que constituye la esencialidad de la misma es lo que se declara en las siguientes: Once the objective of the present invention has been described, it is stated that what constitutes its essentiality is what is stated in the following:

Claims

REIVINDICACIONES
1. Klebsiella oxytoca clón-15, depositada en la CECT con el nE de accesión CECT 4460, capaz de utilizar nitratos y/o nitritos como fuente de nitrógeno bajo condiciones aeróbicas o anaeróbicas.1. Klebsiella oxytoca-clone 15, deposited in the CECT with CECT Accession E n 4460, capable of using nitrate and / or nitrite as nitrogen source under aerobic or anaerobic conditions.
2. Klebsiella oxytoca clon AHÍ, depositada en la CECT con el nB de accesión CECT 4500, capaz de utilizar nitratos y/o nitritos como fuente de nitrógeno y etilenglicol como fuente de carbono bajo condiciones aeróbicas.2. Klebsiella oxytoca clone AH, deposited in the CECT with accession number B CECT 4500, capable of using nitrates and / or nitrites as a source of nitrogen and ethylene glycol as a source of carbon under aerobic conditions.
3. Procedimiento para la eliminación biológica de nitratos y/o nitritos contenidos en efluentes industriales o en aguas contaminadas con dichos compuestos, caracterizado porque comprende las etapas de: a) neutralizar el efluente o el agua contaminada a tratar; b) añadir los nutrientes necesarios para el funcionamiento óptimo del procedimiento; c) inocular el efluente o el agua contaminada a tratar con un cultivo de alguno de los microorganismos citados en las reivindicaciones3. Procedure for the biological elimination of nitrates and / or nitrites contained in industrial effluents or in waters contaminated with said compounds, characterized in that it comprises the steps of: a) neutralizing the effluent or the contaminated water to be treated; b) add the necessary nutrients for the optimal functioning of the procedure; c) inoculate the effluent or contaminated water to be treated with a culture of any of the microorganisms cited in the claims
1 ó 2 hasta la desaparición de los nitratos y/o nitritos contenidos en dichos efluente o agua contaminada; y d) retirar dichos microorganismos y verter los efluentes y las aguas depuradas.1 or 2 until the disappearance of nitrates and / or nitrites contained in said effluent or contaminated water; and d) remove said microorganisms and pour effluents and purified water.
4. Procedimiento según la reivindicación 3, caracterizado porque el efluente a tratar es un efluente procedente de una fábrica productora de explosivos o de una fábrica que utilice o produzca nitratos y/o nitritos.4. Method according to claim 3, characterized in that the effluent to be treated is an effluent from an explosive producing factory or from a factory that uses or produces nitrates and / or nitrites.
5. Procedimiento según la reivindicación 3, caracterizado porque el efluente o el agua a tratar se neutraliza por adición de un álcali hasta alcanzar un pH comprendido entre 6.0 y 10.5, cuando se utilizan bacterias de la cepa Klebsiella oxytoca clón-15.5. Method according to claim 3, characterized in that the effluent or the water to be treated is neutralized by the addition of an alkali until a pH between 6.0 and 10.5 is reached, when bacteria of the Klebsiella oxytoca clone-15 strain are used.
6. Procedimiento según la reivindicación 3, caracterizado porque el efluente o el agua a tratar se neutraliza por adición de un álcali hasta alcanzar un pH comprendido entre 6.0 y 8.0, cuando se utilizan bacterias de la cepa Klebsiella oxytoca clon AHÍ.Method according to claim 3, characterized in that the effluent or the water to be treated is neutralized by the addition of an alkali until a pH between 6.0 and 8.0 is reached, when bacteria of the Klebsiella oxytoca clone strain AH are used.
Procedimiento según la reivindicación 3, caracterizado porque los microorganismos retirados, pueden ser reutilizados en un nuevo ciclo de tratamiento de efluentes o de aguas contaminadas.Method according to claim 3, characterized in that the microorganisms removed can be reused in a new cycle of effluent or contaminated water treatment.
8. Procedimiento para la eliminación biológica de nitratos y/o nitritos contenidos en suelos superficiales contaminados con dichos compuestos, caracterizado porque comprende las etapas de: a) efectuar una inyección primaria con un cultivo de los microorganismos de las reivindicaciones8. Procedure for the biological elimination of nitrates and / or nitrites contained in surface soils contaminated with said compounds, characterized in that it comprises the steps of: a) performing a primary injection with a culture of the microorganisms of the claims
1 ó 2 en el suelo a tratar; y b) efectuar inyecciones sucesivas de dichos microorganismos hasta que el suelo quede libre de nitratos y/o nitritos.1 or 2 in the soil to be treated; and b) make successive injections of said microorganisms until the soil is free of nitrates and / or nitrites.
9. Procedimiento según la reivindicación 8, caracterizado porque el suelo a tratar se inyecta con dichos microorganismos hasta alcanzar una densidad del orden de 105 microorganismos por cm2 de suelo. 9. Method according to claim 8, characterized in that the soil to be treated is injected with said microorganisms until reaching a density of the order of 10 5 microorganisms per cm 2 of soil.
PCT/ES1994/000069 1993-07-02 1994-07-01 PROCESS FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND/OR NITRITES USING STRAINS OF $i(KLEBSIELLA OXYTOCA) WO1995001311A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU70742/94A AU7074294A (en) 1993-07-02 1994-07-01 Process for the biological elimination of nitrates and/or nitrites using strains of (klebsiella oxytoca)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
ESP9301496 1993-07-02
ES9301496A ES2064287B1 (en) 1993-07-02 1993-07-02 PROCEDURE FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND / OR NITRITES USING KLEBSIELLA OXYTOCA CLON-15.
ESP9401427 1994-06-30
ES9401427A ES2083327B1 (en) 1993-07-02 1994-06-30 IMPROVEMENTS INTRODUCED IN THE PURPOSE OF THE SPANISH PATENT APPLICATION N-P 9301496 FOR "PROCEDURE FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND / OR NITRITES USING KLEBSIELLA OXYTOCA CLONE-15-A.

Publications (1)

Publication Number Publication Date
WO1995001311A1 true WO1995001311A1 (en) 1995-01-12

Family

ID=26154733

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES1994/000069 WO1995001311A1 (en) 1993-07-02 1994-07-01 PROCESS FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND/OR NITRITES USING STRAINS OF $i(KLEBSIELLA OXYTOCA)

Country Status (2)

Country Link
AU (1) AU7074294A (en)
WO (1) WO1995001311A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028475A2 (en) * 1994-04-18 1995-10-26 Idaho Research Foundation, Inc. Biological isolates for degrading nitroaromatics and nitramines in water and soils
US5616162A (en) * 1990-04-11 1997-04-01 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils
US6066772A (en) * 1997-08-29 2000-05-23 Waste Management, Inc. Treatment of TNT-contaminated soil
US6481929B1 (en) 1998-04-27 2002-11-19 Arcadis Geraghty & Miller Aerobic bioreduction of municipal solid waste landfill mass
US6644200B1 (en) 1995-11-17 2003-11-11 The Ensign-Bickford Company Method for bioremediating undetonated explosive device
US6668725B2 (en) 1995-11-17 2003-12-30 The Ensign-Brickford Company Methods, apparatus, and systems for accelerated bioremediation of explosives

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1423585A1 (en) * 1986-03-05 1988-09-15 Макеевский Инженерно-Строительный Институт Strain of pseudomonas fluorescens bacteria used for biological purification of waste water of aromatic nitrocompounds
WO1991015440A1 (en) * 1990-04-11 1991-10-17 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1423585A1 (en) * 1986-03-05 1988-09-15 Макеевский Инженерно-Строительный Институт Strain of pseudomonas fluorescens bacteria used for biological purification of waste water of aromatic nitrocompounds
WO1991015440A1 (en) * 1990-04-11 1991-10-17 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 110, no. 7, 13 February 1989, Columbus, Ohio, US; abstract no. 54294g, WONG,S.H.: "effects of carbon sources on nitrate assimilation of kebsiella species" page 374; *
DATABASE WPI Section Ch Week 8912, Derwent World Patents Index; Class D04, AN 89-091696 *
MICROBIOS LETT., vol. 38, no. 150, 1988, pages 55 - 60 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5616162A (en) * 1990-04-11 1997-04-01 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils
US6334954B1 (en) 1990-04-11 2002-01-01 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils
US6348639B1 (en) 1990-04-11 2002-02-19 Idaho Research Foundation, Inc. Biological system for degrading nitroaromatics in water and soils
WO1995028475A3 (en) * 1994-04-18 1995-11-16 Idaho Res Found Biological isolates for degrading nitroaromatics and nitramines in water and soils
WO1995028475A2 (en) * 1994-04-18 1995-10-26 Idaho Research Foundation, Inc. Biological isolates for degrading nitroaromatics and nitramines in water and soils
US7077044B2 (en) 1995-11-17 2006-07-18 Dyno Nobel Inc. Method for bioremediating undetonated explosive device
US7240618B2 (en) 1995-11-17 2007-07-10 Dyno Nobel Inc. Explosive device with accelerated bioremediation capacity
US6644200B1 (en) 1995-11-17 2003-11-11 The Ensign-Bickford Company Method for bioremediating undetonated explosive device
US6660112B1 (en) 1995-11-17 2003-12-09 The Ensign-Bickford Company Method for manufacturing explosive device having self-remediating capacity
US6668725B2 (en) 1995-11-17 2003-12-30 The Ensign-Brickford Company Methods, apparatus, and systems for accelerated bioremediation of explosives
US6066772A (en) * 1997-08-29 2000-05-23 Waste Management, Inc. Treatment of TNT-contaminated soil
US6916136B2 (en) 1998-04-27 2005-07-12 Waste Management Holdings, Inc. Aerobic bioreduction of municipal solid waste landfill mass
US6481929B1 (en) 1998-04-27 2002-11-19 Arcadis Geraghty & Miller Aerobic bioreduction of municipal solid waste landfill mass

Also Published As

Publication number Publication date
AU7074294A (en) 1995-01-24

Similar Documents

Publication Publication Date Title
Wallace et al. Perchlorate reduction by a mixed culture in an up-flow anaerobic fixed bed reactor
Shradha et al. Isolation and characterization of phenol degrading bacteria from oil contaminated soil
CN101955885B (en) High-efficiency denitrification mixed bacterial agent and application thereof
US10364415B2 (en) 1,4-dioxane-degrading bacteria culture method, medium, and 1,4-dioxane treatment method using 1,4-dioxane-degrading bacteria
Abd El–Rahim et al. Microflora involved in textile dye waste removal
Hsu et al. Successful enrichment of rarely found Candidatus Anammoxoglobus propionicus from leachate sludge
WO1995001311A1 (en) PROCESS FOR THE BIOLOGICAL ELIMINATION OF NITRATES AND/OR NITRITES USING STRAINS OF $i(KLEBSIELLA OXYTOCA)
KR20210105849A (en) Yellow soil fermentation strain composition for water purification and water purification method using the same
CN113414232A (en) Method for treating high-concentration cadmium pollution through calcium-enhanced microbial mineralization and combined phytoremediation
JP3432214B2 (en) Algae treatment
EP0589818A2 (en) Process for the biological removal of nitrated derivatives
CN115386520B (en) Rhodococcus pyridine-philic RL-GZ01 strain and application thereof
CN109370931B (en) Complex microbial inoculant for efficiently degrading polycyclic aromatic hydrocarbon and application thereof
Okubo et al. Distribution and capacity for utilization of lower fatty acids of phototrophic purple nonsulfur bacteria in wastewater environments
Jun et al. Aerobic denitrification by a novel Pseudomonas sp. JN5 in different bioreactor systems
CN112522158B (en) Marine bacterium and application thereof
TW201631145A (en) Anaerobic bacteria strain for degrading chlorinated organic contaminant and use thereof
CN110468066B (en) Aerobic denitrifying strain and application thereof
KR20140114564A (en) Complex strain for the waste water treatment and nitrogen treatment process using the same
Eltarahony et al. Isolation, characterization and identification of nitrate reductase producing bacteria
Yoo et al. Eco-friendly and efficient in situ restoration of the constructed sea stream by bioaugmentation of a microbial consortium
KR101222602B1 (en) Castellaniella sp. and mixotrophic denitrification process using castellaniella sp
RU2114174C1 (en) Consortium of yeast candida maltosa for biodegradation of petroleum pollution
CN105861375B (en) A kind of microphenomenon of degradation of aniline and application thereof
Zhu et al. Characteristics of an aerobic denitrifier isolated from unconfined aquifer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CN CZ DE DK FI GB HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA