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    • 专利摘要:
    Htf degrading bacteria. The present invention relates to the bacterium pseudomonas oleovorans hbd2 deposited in the spanish collection of type cultures with the access number 8969, and to the use thereof for the degradation of the htf heat carrier fluid, either in liquid media or in contaminated soils. Htf or its chemical components. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2641967A1
    申请号:ES201630622
    申请日:2016-05-13
    公开日:2017-11-14
    发明作者:Lara Paloma SÁEZ MELERO；Rafael BLANCO MORENO； Víctor Manuel LUQUE ALMAGRO；Conrado MORENO VIVIÁN；María Dolores ROLDÁN RUIZ
    申请人:Universidad de Cordoba；
    IPC主号:
    专利说明:

    HTF DEGRADING BACTERIA
    Field of the Invention
    The present invention falls within the general field of bioremediation and in particular refers to Pseudomonas oleovorans HBD2, deposited in the Spanish Type Culture Collection (CECT) for identification and deposit with the access code CECT 8969.
    State of the art
    Some solar thermal power plants use the Dowtherm A heat carrier fluid (HTF) to transfer heat from the parabolas to the turbines that generate electricity. This fluid is composed of a eutectic mixture of biphenyl (26.5%) and biphenyl oxide (73.5%); It is a highly toxic, viscous, colorless-yellowish liquid that has a boiling point of 257 OC.
    Sometimes the pipes of the plants lose their tightness as a result of changes in temperature during the operation of the plant, causing leaks in their elbows, contaminating the floor of the plants.
    Numerous bacterial genera are known that have the ability to degrade biphenyl (Fugihara et aL, 2015; Draft genome sequence of Pseudomonas aeruginosa KF702 (NBRC11 0665), a polychlorinated biphenyl-degrading bacterium isolated from biphenylcontaminated soil. Genome Announc 3: e00517-15 ), while the number of microorganisms that can use diphenyl oxide is reduced (Wang et aL, 2015 Characterization of the molecular degradation mechanism of diphenyl ethers by Cupriavidus sp. WS. Enviran Sci Pollut Res, DOI10.1007 / s11356-015 -4854-3). As for the HTF fluid, so far no microorganism has been described that can simultaneously degrade the two organic compounds present in this eutectic mixture.
    Despite the progress that has been made in this field in recent years, there is still a need to provide a method for the elimination of contaminating waste in and around solar thermal power plants.
    Brief Description of the Invention
    The present invention solves the problems described in the state of the art since it refers to the use of a microorganism that degrades HTF in such a way that it provides a bioremediation method of soils contaminated with HTF.
    Thus, in a first aspect the present invention relates to a strain of Pseudomonas oJeovorans HBD2 isolated and deposited in the Spanish Type Culture Collection with the accession number CECT 8969 (hereinafter strain of the present invention).
    In another aspect, the present invention relates to a pure bacterial culture, and / or extract and / or bioactive compound comprising the strain of the present invention.
    In the present invention, bioactive products refers to products obtained from the strain of the present invention, both its cellular and intracellular components such as DNA, peptides, etc. which confer to the strain of the present invention, the degrading and / or tolerant activity to HTF.
    In another aspect, the present invention relates to a composition comprising the strain of the present invention, or a pure culture, or an extract or a bioactive compound of the strain of the present invention (hereinafter composition of the present invention ).
    In a particular aspect, the composition of the present invention is a biological composition. In another aspect in particular, the biological composition comprises biologically acceptable carriers and / or excipients.
    In the present invention by biological composition refers to those compositions that incorporate the strain of the present invention, and / or the extract, culture and / or bioactive compounds thereof with at least one biologically acceptable carrier and / or excipient. The biologically acceptable carriers and / or excipients to be used in the present invention are known in the state of the art to one skilled in the art.
    In a particular aspect, the composition of the present invention is a chemical composition. In another aspect in particular, the chemical composition comprises biologically acceptable carriers and / or excipients.
    In the present invention by chemical composition refers to those compositions that incorporate the strain of the present invention, and / or the extract, culture and / or bioactive compounds thereof with at least one chemically acceptable carrier and / or excipient. The chemically acceptable vehicles and / or excipients to be used in the present invention are known in the state of the art to one skilled in the art.
    In another aspect, the present invention relates to the use of the strain of Pseudomonas oJeovorans HBD2 deposited in the Spanish Type Culture Collection with the accession number CECT 8969 and / or to the use of the extract, culture and / or bioactive compounds of the strain of the present invention and / or the use of the composition of the present invention for the degradation of the heat-carrying fluid. In a particular embodiment, it is for the degradation of heat-carrying fluid and / or biphenyl. In another particular embodiment, it is for the degradation of heat-carrying fluid and / or diphenyl ether.
    In another aspect, the present invention relates to the use of the strain of Pseudomonas oJeovorans HBD2 deposited in the Spanish Type Culture Collection with the accession number CECT 8969 and / or to the use of the extract, culture and / or bioactive compounds of the strain of the present invention and / or the use of the composition of the present invention for the treatment of bioremediation of contaminated soils.
    Description of the figures
    Figure 1 shows the consumption of biphenyl (BP), diphenyl ether (DE) and heat transfer fluid (HTF). The strain of Pseudomonas oleovorans of the present invention and Cupriavidus necator strain H850 deposited in the CECT with the number 4424 were cultured aerobically with 10 mM ammonium as a source of nitrogen and BP (2 g L-1), DE (2 mL L-1 ) or HTF (2 mL L-1) as the only carbon source. The concentration of BP (empty bars) and DE (filled bars) in the medium was determined by GC / MS after 1 or 5 days of culture (left and right respectively for each strain). The data, which correspond to the average of three experiments with their standard deviation, are expressed as% with respect to the initial concentration of each compound.
    Figure 2 shows the tolerance to heat-carrying fluid (HTF) by bacteria. The Pseudomonas oleovorans HBD2 bacterial strains of the present invention and the Cupriavidus necator H850 biphenyl degrading bacteria were cultured aerobically with 10 mM ammonium as a source of nitrogen. HTF at different concentrations was used as the only carbon source: 50,000 ppm (50 mL L-1; squares), 100,000 ppm (100 mL L-1; triangles) 0150000 ppm (150 mL L-1; circulars). At the indicated times the bacterial growth (A600, filled symbols) and the concentration of ammonium (empty symbols) were determined. The data correspond to the average of three experiments with their standard deviation.
    Detailed description of the invention
    The inventors of the present invention have managed to identify a new bacterial strain, Pseudomonas oleovorans HBD2, deposited in the Spanish Type Culture Collection with the access number CECT 8969 that has novel biological activities with respect to other bacteria since to date it has not been has described no microorganism with degrading activity of HTF, that is to say that it simultaneously degrades the two organic compounds present in this eutectic mixture. This degrade activity of HTF has been analyzed by comparison with Cupriavidus necator H850 deposited in the CECT with the number 4424, diphenyl degrading bacteria and Sphingomonas sp.SS3, diphenyl ether degrading strain. In addition, the tolerance of Pseudomonas oleovorans HBD2 CECT 8969 to HTF was checked.
    From the plant rhizosphere of the "La Africana" solar thermal power plant (Córdoba, Spain), through enrichment techniques, the Pseudomonas or / eovorans HBD2 lineage, deposited in the Spanish Type Culture Collection with the code of CECT access 8969.
    The cells were inoculated in 50 mL capacity flasks, with 10 mL of M9 medium (Sambrook and Russel, 2001 Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbar, NY: Cold Spring Harbor Laboratory Press; 2001), a pH 7, and incubated in aerobiosis on a thermostated orbital shaker at 300C and 220 rpm. The nitrogen source used was 10 mM ammonium chloride and biphenyl, biphenyl ether, or HTF (2000 ppm) was used as the only carbon source. The flasks were sealed with paraffin and incubated at 302C on shaker at 220 rpm.
    For bacterial enrichment, once a week for a month, aliquots of the culture were taken and transferred to flasks containing 9 ml of fresh medium with the same sources of carbon and nitrogen and incubated under the same conditions as the initial culture.
    Finally, aliquots of the turbids were taken with turbidity (which indicated the bacterial growth), then they were transferred to LB medium plates with HTF to obtain the individual colonies. Bacterial line was maintained by taking 750 111 of a pure culture in liquid LB medium and adding 250 111 of 80% glycerol (v / v), and subsequently stored at -80 2C.
    Determination of bacterial growth
    The Pseudomonas o / eovorans HBD2 bacteria, deposited in the Spanish Type Culture Collection with accession number CECT 8969, Cupriavidus necator H850 and Sphingomonas sp.SS3, were grown at 302C in LB medium or M9 medium containing ammonium chloride (10 mM) as a source of nitrogen and BF, DE or HTF (2,000 ppm) as the sole source of carbon.
    To measure the tolerance to HTF, 50, 100 or 150 ml of HTF heat carrier fluid (50,000, 100,000 0150000 ppm respectively) were used. Bacterial growth was determined by different methods including monitoring the absorbance of cultures at 600 nm (A600), by counting colony forming units (CFU) and by determining the concentration of proteins in whole cells by the method of Lorry modified (Shakir et aL, 1994. A rapid protein determination by modification of the Lowry procedure. Anal. Biochem. 216: 232-233).
    Chemical and analytical determinations
    To quantify the consumption of BF, DE and HTF its concentration in the culture media was determined. As control, identical media were used in composition that had not been inoculated with the bacteria. In each case, a liquid-liquid extraction of the culture supernatant with 1/10 chloroform was performed and analyzed by Gas Chromatography coupled to Mass Spectrometry coupled to a VarianSarturno 2200 Ion Trap. The experimental conditions were as follows: injection at 2752C, with helium as a carrier gas at a constant flow of 1 ml / min, initially a temperature gradient from 65 pcs was maintained (maintained for 0.5 minutes), increasing 8 pcs per minute to reach 180 pcs (maintained 0.1 min) Subsequently, 300 'C was reached by applying a gradient of 25 pcs per minute and 300' C were maintained for 2 min. The volume of the injected sample was 1111. The detection by Mass Spectrometry analysis was carried out by ion Full Sean between 50 and 275 (0.5 s / scan) and 70 eVo
    To detect the presence of the enzyme 2,3-dihydroxybiphenyl dioxygenase (BphC), the solid medium plates containing the colonies of bacteria were sprayed with a solution of 0.1% dihydroxybiphenyl (w / v) and 10% acetone ( v / v), as described in Wagner-Dóbler et al., 1998 Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments. Appl Environ Microbiol 1998; 64: 3014-22. The appearance of the yellow color after several minutes indicated the formation of 2-hydroxy-6-oxo-6hexaphenyl -2,4-dienic acid (HOPDA).
    The reaction products (biphenyl, diphenyl ether and others, were purchased from Sigma-Aldrich (SI. Louis, MO). The HTF was obtained from the company MAGTEL, S.L, which is the company that manages the thermosolar plant.
    Under the previously described experimental conditions, it was shown that Pseudomonas oleovorans HBD2 degraded the eutectic mixture, with an almost 85% consumption of HTF after 5 days of culture (Fig. 2). This bacterium also independently degrades the chemical compounds BP and DE, although more efficiently the BP than the DE when grown with each of these compounds as the sole source of carbon, leaving only 5% of PB but more 50% DE after 5 days of culture (Fig. l).
    5 On the contrary, the bacterial strain Cupriavidus necator H850, which could degrade 95% of SP and 40% of ED after 5 days, in the HTF mixture only degrades 45% DE and 40% SP at 5 days of cultivation Although, it was also demonstrated that strain H850 was tolerant of high concentrations of HTF (Fig. 2)
    权利要求:
    Claims (9)
    [1]
    1. Pseudomonas oleovorans HBD2 strain isolated and deposited in the Spanish Type Crops Collection with accession number CECT 8969.
    [2]
    2. Pure bacterial culture, extract or bioactive compound comprising the strain of Pseudomonas oleovorans HBD2 of claim 1.
    [3]
    3. Composition comprising the strain of Pseudomonas oleovorans HBD2 according to claim 1, or a pure culture, extract or bioactive compound according to claim 2.
    [4]
    Four. Composition according to claim 3, wherein said composition is a biological composition.
    Composition according to claim 4, which comprises at least one vehicle and / or a biologically acceptable excipient.
    [6]
    6. Composition according to claim 3, wherein said composition is a chemical composition.
    [7]
    7. Composition according to claim 6, comprising at least one vehicle and / or an acceptable chemical excipienle.
    [8]
    8. Use of the strain of Pseudomonas oJeovorans HBD2 according to claim 1, or of a pure bacterial culture, extract or bioactive compound according to claim 2 or of a composition according to any of claims 3-7, for the degradation of the heat-carrying fluid.
    Use according to claim 8, for the degradation of biphenyl.
    [10]
    10. Use according to any of claims 8-9 for the degradation of diphenyl ether.
    [11 ]
    eleven . Use of the strain of Pseudomonas oleovorans HBD2 according to claim 1, or of a pure bacterial culture, extract or bioactive compound according to claim 2 or of a composition according to any of claims 3-7 for the treatment of
    25 bioremediation of contaminated soils.
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    同族专利:
    公开号 | 公开日
    ES2641967B2|2018-05-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4664805A|1985-07-23|1987-05-12|Regents Of The University Of California|Analog enrichment decontamination process|
    US20030148501A1|1999-03-31|2003-08-07|Picardal Flynn W.|Compositions and methods useful in bioremediation of polychlorinated biphenyls|ES2841973A1|2020-01-07|2021-07-12|Acciona Energia Sa|BACTERIAL STRAINS AND THEIR USE FOR THE BIOREMEDIATION OF SOILS CONTAMINATED WITH BIPHENYL AND DIPHENYL OXIDE|
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