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WO2004094316A2 - Traitement par biorestauration d'une pollution par du petrole faisant appel a de l'acide urique non hydrosoluble comme source d'azote - Google Patents

Traitement par biorestauration d'une pollution par du petrole faisant appel a de l'acide urique non hydrosoluble comme source d'azote Download PDF

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WO2004094316A2
WO2004094316A2 PCT/IL2004/000351 IL2004000351W WO2004094316A2 WO 2004094316 A2 WO2004094316 A2 WO 2004094316A2 IL 2004000351 W IL2004000351 W IL 2004000351W WO 2004094316 A2 WO2004094316 A2 WO 2004094316A2
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petroleum
uric acid
bacteria
degrading
crude oil
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WO2004094316A3 (fr
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Eugene Rosenberg
Eliora Ron
Omry Koren
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Ramot at Tel Aviv University Ltd
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Ramot at Tel Aviv University Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/002—Reclamation of contaminated soil involving in-situ ground water treatment
    • 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
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/68—Treatment of water, waste water, or sewage by addition of specified substances, e.g. trace elements, for ameliorating potable water
    • C02F1/681—Treatment of water, waste water, or sewage by addition of specified substances, e.g. trace elements, for ameliorating potable water by addition of solid materials for removing an oily layer on water
    • 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
    • C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
    • 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
    • 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
    • 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
    • C12N1/26—Processes using, or culture media containing, hydrocarbons
    • 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

Definitions

  • the present invention relates to a method of degrading petroleum using uric acid as a nitrogen source for petroleum-degrading bacteria, and more particularly, to the identification and characterization of petroleum-degrading bacteria which can be used to degrade petroleum pollutions.
  • Petroleum is a major source . of energy for industry and daily life, and its transport across the world is frequent. As a result, petroleum spill has become a significant, world-wide environmental hazard, particularly to shorelines and seawater.
  • Crude oil includes saturate, aromatic, resin and asphaltene components.
  • aromatic components with four or more aromatic rings and asphaltene or resin fractions which contain higher molecular weight compounds are less accessible for biodegradation.
  • Inipol EAP 22 contains large amounts of oleic acid (which serves as an alternative carbon source) its use increases the carbon-to-nitrogen ratio in the environment, which limits the nitrogen availability.
  • Inipol EAP 22 is an emulsifier and its use in seawater is likely to harm the environment.
  • the interaction of the fertilizer with water results in the release of urea (i.e., the nitrogen source) from the emulsion into the water phase, making it un- accessible to the petroleum-degrading microorganisms.
  • a method of degrading petroleum or petroleum product(s) comprising applying to the petroleum or petroleum product(s): (a) bacteria capable of using petroleum as a source of carbon; and (b) uric acid, at an amount effective for providing a nitrogen source to the bacteria to thereby degrade the petroleum or petroleum product(s).
  • a bacterial strain capable of using petroleum or petroleum product(s) as a source of carbon and uric acid as a nitrogen source.
  • a method of degrading petroleum or petroleum product(s) comprising applying to the petroleum or petroleum product(s) uric acid, at an amount effective for providing a nitrogen source to petroleum-degrading bacteria to thereby degrade the petroleum or petroleum product(s).
  • a method of isolating a bacterial strain capable of degrading petroleum or petroleum product(s) comprising: (a) culturing a bacteria-containing sample in a culture medium containing petroleum or petroleum product(s) and uric acid as a nitrogen source; and (b) isolating a bacterial strain from the bacterial-containing sample exhibiting a petroleum or petroleum product degrading activity to thereby obtain a bacterial strain capable of degrading petroleum or petroleum product(s).
  • the petroleum or petroleum product(s) form a part of an oil spill in a water body.
  • the bacteria is selected from the group consisting of Acinetobacter sp. OKI, Alcanivorax sp. OK2, Acinetobacter calcoaceticus RAG-1, and Pseudomonas fluorescens.
  • the uric acid is provided at a concentration ratio of 1 part of uric acid to 5-50 parts of petroleum or petroleum product(s).
  • the uric acid forms a part of a fertilizer composition.
  • the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Acinetobacter having the NCIMB designation No. 41212.
  • the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Alcanivorax having the NCIMB designation No. 41213.
  • an environment of the petroleum or petroleum product(s) includes petroleum-degrading bacteria.
  • the bacteria-containing sample is beach tar, seawater and/or pigeon manure.
  • the culturing is effected by subjecting the bacteria-containing sample to a repeated inoculation/culturing cycle in the culture medium.
  • each of the repeated inoculation/culturing cycle is effected for at least 3 days.
  • the inoculation/culturing cycle is repeated at least 8 times.
  • the culturing is effected at 30 °C using aeration.
  • the petroleum is crude oil.
  • the crude oil is provided at a concentration of 5 mg/ml.
  • the uric acid is provided at a concentration range of 0.1-5 mg/ml.
  • the culture medium includes 15 mg/L MgSO 4 7H 2 O, 15 mg/L FeSO 4 7H 2 O, 5 mg/L CaCl 2 and 500 mg/L NaCl in 1 mM potassium phosphate buffer at pH 6.5.
  • the culture medium is 1 mM potassium phosphate buffer at pH 8.0 in sterile seawater.
  • 1 is a graph illustrating the growth of a mixed enrichment culture (open circles) and strain OKI (closed circles) on crude oil and uric acid.
  • FIG. 2 is a scanning electron photomicrograph depicting the structure of the Acinetobacter sp. OKI. Note the presence of extracellular appendages.
  • FIGs. 5a-b are graphs illustrating the growth of Acinetobacter sp. OKI as a function of increasing concentrations of petroleum or uric acid. Strain OKI was grown in E. salts (further described in Example 1) containing 0.5 mg/ml uric acid and varying concentrations of crude oil ( Figure 5a) or in E. salts containing 5 mg/ml crude oil and varying concentrations of uric acid ( Figure 5b). Cell concentrations were determined after incubation at 30 °C for 4 days.
  • FIGs. 6a-b are electron photomicrographs depicting the structure of strain Alcanivorax OK2.
  • FIG. 7 is a graph illustrating the affinity of Alcanivorax sp. OK2 to hexadecane.
  • Bacterial suspensions of stationary phase cells (1 ml, A 560 : 1.0) were mixed with varying amounts of hexadecane. Results are expressed as percentage of the initial absorbance (A 560 ) of the aqueous suspension as a function of hexadecane volume.
  • FIGs 8a-b are graphs illustrating the growth of Alcanivorax sp. OK2 as a function of petroleum concentration ( Figure 8a) or uric acid concentration ( Figure 8b).
  • Alcanivorax sp. OK2 was grown in seawater containing 0.5 mg/ml uric acid and varying concentrations of crude oil ( Figure 8a) or in seawater containing 5 mg/ml crude oil and varying concentrations of uric acid ( Figure 8b). Cell concentrations were determined after incubation at 30°C for 7 days.
  • the present invention is of a method of degrading petroleum or petroleum product(s) using uric acid as a nitrogen source for petroleum-degrading bacteria.
  • the present invention is particularly suitable for degrading petroleum pollution of seawater and fresh water.
  • the principles and operation of the method of degrading petroleum according to the present invention may be better understood with reference to the drawings and accompanying descriptions.
  • the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples.
  • the invention is capable of other embodiments or of being practiced or carried out in various ways.
  • the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
  • Petroleum spill is a world-wide environmental hazard, particularly to shorelines, lakes, and seawater. Petroleum can be degraded using several bacteria, which utilize petroleum as a hydrocarbon source. However, since the conversion of one kg of hydrocarbon to cell material requires the utilization of 150 grams of nitrogen, and since petroleum contains only traces of nitrogen, the rate-limiting step in bacterial petroleum degradation is the availability of nitrogen.
  • urea i.e., the nitrogen source
  • the urea-formaldehyde formulations cause the oil to sink, thus making it ineffective for use in open systems such as seawater.
  • Uric acid is the major nitrogen waste product of birds, terrestrial reptiles and many insects and was used for decades as a main soil fertilizer (e.g. the commercially inexpensive guano fertilizer). Many different species of bacteria are known to degrade uric acid [Vogels and van der Drift (1976). Bacteriol. Rev. 40: 402-469; Christiansen, L.C., et al., (1997). J. Bacteriol. 179: 2540-2550; Shultz, A.C., et al, 2001. J. Bacteriol. 185: 3293-3302) and one species, Bacillus fastidiosus, was reported to grow only on uric acid and allantoin (Bongaerts and Vogels. (1976). Uric acid degradation by Bacillus fastidiosus strains. J. Bacteriol. 125: 689-697).
  • uric acid which has low-water solubility
  • the present inventors have predicted that uric acid, which has low-water solubility, can be used as a sole nitrogen source for petroleum-degrading bacteria.
  • the present inventors have uncovered two petroleum-degrading bacteria, Acinetobacter sp. OKI and Alcanivorax sp. OK2, which can utilize uric acid as a sole nitrogen source.
  • the present inventors have uncovered that uric acid can adhere to crude oil droplets, and as such would be highly accessible to petroleum-degrading bacteria utilized to degrade petroleum present in water.
  • petroleum or petroleum product(s) refers to crude or refined petroleum, oil, fuel oil, diesel oil, gasoline, hydraulic oil, and/or kerosene.
  • Benzene, toluene, ethylbenzene and xylenes are the most volatile constituents of gasoline and Trimethylbenzenes, and other polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, anthracene, acenaphthene, acenaphthylene, benzo (a) anthracene, benzo (a) pyrene, benzo (b) fluoranthene, benzo (g,h,i) perylene, benzo (k) fluoranthene and pyrene which are common constituents of fuel oils and heavier petroleum compounds are also part of the petroleum or petroleum produces) of the present invention.
  • PAHs polycyclic aromatic hydrocarbons
  • the petroleum or petroleum product(s) of the present invention can be present as a gas, solid or liquid which is dispersed in or on solid or liquid surfaces.
  • the petroleum or petroleum product(s) can be deposited in a liquid form on solid surfaces such as soil, land, metal surfaces and the like, in a gas form on a liquid body, and/or in a liquid form on a water body, such as an ocean, lake, and seawater.
  • the petroleum or petroleum product(s) of the present invention form a part of an oil spill in a water body.
  • the water body of the present invention can be a sea, an ocean, a lake, a river or the like.
  • Such an oil spill can result from oil transportation via the sea or ocean and/or from industrial discharges of oil into a river, lake, sea and/or ocean water.
  • the method of the present invention is effected by applying to the petroleum or petroleum product(s) bacteria capable of using petroleum as a source of carbon, and uric acid, at an amount effective for providing a nitrogen source to the bacteria to thereby degrade the petroleum or petroleum product(s).
  • bacteria capable of using petroleum as a source of carbon refers to one or more strains of bacteria capable of utilizing petroleum or petroleum products as a sole source of carbon.
  • the bacterial strain is Acinetobacter sp. OKI (NCIMB No. 41212), Alcanivorax sp. OK2 (NCIMB No. 41213) both of which are further described hereinbelow, Acinetobacter calcoaceticus RAG-1 (ATCC 31012) which is described in detail in Sar and Rosenberg (Current Microbiology, 1983, 9: 309-314) and/or Pseudomonas fluorescens which is described in Barathi and Vasudevan (J. Environ. Sci. Health Part. A Tox. Hazard. Subst. Environ. Eng. 2003, 38: 1857-66).
  • the petroleum-degrading bacteria of the present invention is applied on the petroleum or petroleum product(s) concomitantly and/or sequentially to the application of uric acid.
  • the uric acid of the present invention can be provided to the petroleum or petroleum product(s) of the present invention as is (i.e., in a solid purified form), as part of a water suspension, or as part of a composition such as a fertilizer (e.g., Guano).
  • a fertilizer e.g., Guano
  • the uric acid is part of a composition such as a Guano fertilizer.
  • the amount of uric acid effective for petroleum or petroleum product(s) degradation depends on the type of bacteria and the amounts of petroleum or petroleum product(s). For example, as is shown in the examples section which follows, when the Acinetobacter sp. OKI strain was used in a culture medium containing E salts and 5 mg/ml crude oil, 0.25-1 mg/ml of uric acid were sufficient for the degradation of 48-50 % of crude oil. On the other hand, when the Alcanivorax sp. OK2 strain was used in a culture medium containing seawater and 5 mg/ml crude oil, 0.5-2 mg/ml of uric acid were needed for the degradation of 17-31 % of cmde oil.
  • uric acid is provided to the petroleum-degrading bacteria at a concentration ratio of 1 part uric acid to 5-100 parts petroleum or petroleum product(s), more preferably, at a concentration ratio of 1 part uric acid to 5-50 parts petroleum or petroleum product(s), more preferably, at a concentration ratio of 1 part uric acid to 10-20 parts petroleum or petroleum product(s).
  • teachings of the method of the present invention can be used to degrade petroleum pollutant in seawater, ocean water and shorelines, and as such can reduce the world-environmental hazard associated with the use of petroleum.
  • strains OKI and OK2 are capable of using petroleum or petroleum product(s) as a source of carbon and uric acid as a source of nitrogen.
  • strain OKI As is shown in Example 1 of the Examples section which follows, the first isolated bacterial strain (strain OKI, NCIMB designation number 41212) has phenotypic properties which are typical to the genus Acinetobacter, i.e., a Gram- negative, strictly aerobic, non motile, oxidase-negative, short rod.
  • strain OKI exhibited extracellular appendages, such as those described for the hydrocarbon-utilizing bacterium Acinetobacter calcoaceticus MM5 (Marin, M., et al., 1996. Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5.
  • strain OKI of the present invention (NCIMB designation number 41212) is an isolate of petroleum-degrading bacterial strain of the genus Acinetobacter.
  • the second bacterial strain isolated by the present study has phenotypic properties which are typical of the genus Alcanivorax, i.e., a Gram-negative, strictly aerobic, non motile, oxidase-positive, short rod.
  • the nucleotide sequence of the 16S rDNA sequence of strain OK2 (1459 bp in length; GenBank accession No. AY307381, SEQ ID NO:6) had the closest similarity to Alcanivorax TE9 (Accession No. AB055207) with an identity of 99 %.
  • strain OK2 of the present invention is an isolate of a petroleum degrading bacterial strain of the genus Alcanivorax.
  • any of the petroleum degrading bacterial strains described herein can be packaged in a kit along with appropriate instructions for use and labels indicating EPA approval for use in petroleum or petroleum product(s) degradation.
  • the kit for petroleum or petroleum product(s) degradation can include, for example, a container including petroleum-degrading bacteria provided with a suitable buffer, culture medium etc, and an additional container which includes an effective amount of uric acid as described above.
  • Such a kit can be used to degrade petroleum or petroleum product(s) in a variety of oil spills in oceans, seas, lakes and the like which exhibit a world-wide environmental hazard.
  • a method of degrading petroleum or petroleum product(s) is effected by applying to the petroleum or petroleum product(s) uric acid, at an amount effective for providing a nitrogen source to petroleum-degrading bacteria to thereby degrade the petroleum or petroleum product(s).
  • such an environment can be, for example, fresh water or sea water (Rosenberg E, 1992 (supra);
  • the method of the present invention can be used to degrade petroleum pollutant in seawater, fresh water, shorelines and soil by adding uric acid in any form as described above, including part of a composition such as a Guano fertilizer.
  • uric acid used according to this aspect of the present invention can be packaged in a kit along with appropriate instructions for use and labels indicating EPA approval for use in petroleum or petroleum product(s) degradation.
  • the teachings of the present invention can be used to identify more petroleum-degrading bacteria which utilize uric acid as a sole nitrogen source.
  • a method of isolating petroleum-degrading bacterial strains which preferably utilize uric acid as a source of nitrogen.
  • the method according to this aspect of the present invention is effected by culturing a bacteria-containing sample in a culture medium containing petroleum or petroleum product(s) and uric acid as a nitrogen source and isolating a bacterial strain from the bacterial-containing sample exhibiting a petroleum or petroleum product degrading activity to thereby obtain a bacterial strain capable of degrading petroleum or petroleum product(s).
  • bacteria-containing sample refers to any biological sample containing bacteria, including, but not limited to, bacteria- containing samples which are found in water bodies (e.g., in seawater, lake water, and the like), on the beach (e.g., beach tar) or on any place on a land or a soil material (e.g., birds manure).
  • the bacteria-containing sample is derived from beach tar, seawater or pigeon manure, such samples can be collected using simple laboratory tools.
  • the bacteria-containing sample is subjected to inoculation in a culture medium containing petroleum or petroleum product(s) and uric acid as a nitrogen source.
  • the phrase "inoculation” refers to placing a bacteria-containing sample in a culture medium. Inoculation is usually followed by culturing the bacteria in the culture medium under culturing conditions suitable for bacterial growth. Methods of inoculating and culturing bacteria-containing samples are known to anyone with skills in the arts. For example, the bacteria-containing sample can be placed in a container (e.g., flask) in the presence of the appropriate culture medium
  • PU medium e.g., PU medium, E salts
  • E salts e.g., PU medium, E salts
  • the culturing temperature may vary between 20-45 °C, depending on the bacterial strain used, and can be optimized using methods known in the arts.
  • CFU/ml to 5x10 CFU/ml was achieved within 35 hours of culturing of strain OKI at 30 °C using aeration.
  • culturing is effected at 30 °C using aeration.
  • the culturing period of a bacteria-containing sample depends on the bacterial strain and culture medium used.
  • the selection of a particular bacterial strain from a bacteria-containing sample may require subjecting the bacteria-containing sample to prolonged incubation periods under the selective culture conditions (e.g., a culture medium containing crude oil and uric acid).
  • Such incubation time is preferably in the range of 1-50 days, more preferably, 3-40 days, more preferably, 10-35 days, more- preferably 25-35 days. It will be appreciated that in order to improve the selection efficacy, an aliquot of the cultured bacteria can be obtained following a few days in culture (e.g.,
  • culturing is effected by subjecting the bacteria-containing sample to a repeated inoculation/culturing cycle in the culture medium.
  • the repeated inoculation/culturing cycle is effected for at least 3 days.
  • the inoculation/culturing cycle is repeated at least 8 times.
  • the petroleum or petroleum product(s) contained in the culture medium used by the present invention can be any kind of crude or refined petroleum, oil, and the like, as discussed hereinabove.
  • the petroleum used by the present invention is crude oil.
  • the concentration of crude oil in the culture medium is preferably in the range of 0.5-10 mg/ml, more preferably, in the range of 1-8 mg/ml, more preferably, in the range of 3-6 mg/ml, most preferably 5 mg/ml.
  • the concentration of uric acid contained in the culture medium of the present invention can be in the range of 0.1-10 mg/ml, more preferably, in the range of 0.5-8 mg/ml, most preferably, in the range of 0.5-5 mg/ml. It will be appreciated that the concentration of uric acid in the culture medium can be determined according to the concentration of crude oil used, such as that 1 part of uric acid is provided to 5-50 parts of crude oil.
  • the bacterial culture may include more than one type of bacteria.
  • the bacterial culture is preferably streaked on a solid medium, such as on culture plates (e.g., agar plates).
  • culture plates e.g., agar plates.
  • the various bacterial types can be distinguished based on the shape, size and color of the isolated colonies. Methods of streaking and identifying bacterial types on culture plates are known in the arts. Following their growth on a culture plate, single colonies can be picked using a bacteriological loop and re-cultured on the same culture medium (e.g., containing crude oil and uric acid) to obtain pure isolates of petroleum-degrading bacteria.
  • a bacteriological loop e.g., containing crude oil and uric acid
  • the culture medium used by the present invention can be any bacterial culture medium suitable for growing petroleum-degrading bacteria.
  • Such culture media are described in Ridgway, HF., et al., (Appl. Environ. Microbiol. 1990, 56: 3565-3575; Sutherland, TD., et al., (Applied and Environmental Microbiology, 2000, 66: 2822- 2828; Rosenberg E, Prokaryotes II, pp. 446-460, Springer- Verlag, New York, 1992).
  • Non-limiting examples of suitable culture medium include the E salts (15 mg/L MgSO 4 7H 2 O, 15 mg/L FeSO 4 7H 2 O, 5 mg/L CaCl 2 and 500 mg/L NaCl in 1 mM potassium phosphate buffer at pH 6.5) and a medium containing 1 mM potassium phosphate buffer at pH 8.0 in sterile seawater.
  • the pure isolates of petroleum-degrading bacteria can be subjected to a variety of phenotypic and functional characterization tests, including, but not limited to, morphological assessments following morphological stainings, metabolic activity staining, adherence to hydrocarbons, and sequence analysis of genomic DNA.
  • isolated bacteria can be subjected to Gram staining in order to classify them into Gram-positive of Gram-negative strains.
  • Gram-negative rods are identified using the API-20 system (bioMerieux, France) according to manufacturer's instructions.
  • the cell morphology can be determined using phase-contrast microscopy and scanning electron microscopy (Jeol 840A, JEOL Inc. Peabody, MA, USA) after negative staining with 1 % uranyl acetate (Amano K, Fukushi K., 1984, Microbiol Immunol. 28: 149-59).
  • PCR primers which are designed according to the 16S rDNA sequence of Escherichia coli axe used in a PCR reaction using, as a template, a sample of genomic DNA which is prepared from the petroleum-degrading bacteria.
  • hydrocarbon molecules e.g., hexadecane
  • BATH bacterial adhesion test
  • Hexadecane (0.05-0.2 ml) is added to round-bottom test tubes containing 1.2 ml of washed cells. Following 10 minutes of preincubation at 30 °C, the mixtures are agitated uniformly for 2 minutes, following which the hexadecane phase is allowed to rise. Following 15 minutes of phase separation, the aqueous phase is carefully removed using a Pasteur pipette and transferred to a 1-ml cuvette for determination of turbidity at 560 nm.
  • LB Bertuni
  • strain OKI which was able to grow as a pure culture on the petroleum/uric acid enrichment medium, was chosen for further study.
  • PCR reaction (50 ⁇ l) included 5 ⁇ l 10 x buffer, 1 ⁇ l 2.5 mM total dNTP mixture, 10 ng template DNA and 2.5 units Ex Taq DNA polymerase (Takara Shuzo) and 5 ⁇ M of each of the PCR primers.
  • PCR conditions included an initial denaturation at 95 °C for 3 min followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min and a final extension step at 72 °C for 10 min.
  • the size and purity of the reaction products were evaluated on 1 % agarose gel and the amplified DNA fragments were recovered using a QIAquick PCR purification kit (Qiagen Inc., CA USA) according to manufacturer's instructions.
  • Dye-Terminator DNA sequencing was performed using the ABI Prism automatic sequencer (model 377, version 2.1.1, PE, Applied Biosystems, HITACHI) using the PCR primers (i.e., SEQ ID Nos. 1 and 2). To determine the most likely identity of the isolated bacteria strain the obtained sequence was subjected to the BLAST basic local alignment search tool using default parameters (www.ncbi.nlm.nih.gov/blast).
  • OKI was found to be a Gram-negative, strictly aerobic, non motile, oxidase-negative, short rod. These phenotypic properties are typical of the genus Acinetobacter
  • strain OKI can use a wide variety of organic compounds as carbon sources (39 out of the 95 of the compounds in Biolog test), including the sugars D-glucose and L-arabinose and amino acids A, N, D, E, L, P, H and hydroxyl-L-proline.
  • strain OKI is a new Acinetobacter species, most closely related to A. baumannii.
  • BATH test Bacterial adhesion to hydrocarbon (BATH) test was performed by a modification of the standard method (Rosenberg, M., and Rosenberg, E., 1985. Bacterial adherence at the hydrocarbon- water interface. Oil Petrochem. Pollut. 2: 155-162). Cells were harvested during stationary phase, centrifuged and washed twice with PUM buffer (22.2 g K 2 HPO 4 3H 2 O, 7.26 g KH 2 PO 4 , 1.8 g urea, 0.2 g MgSO 4 7H 2 O and distilled water in 1000 ml, pH 7.1). Hexadecane (0.05-0.2 ml) was added to round-bottom test tubes containing 1.2 ml of washed cells.
  • strain OKI Growth of strain OKI on crude oil and uric acid following removal of water soluble nutrients -
  • the growth of strain OKI was measured on crude oil/uric acid and crude oil/ammonium sulfate media after the media were mixed and the aqueous phase removed and replaced with water three times (simulated open system).
  • Table 1 hereinbelow, the high cell yields (2-6x10 8 cells per ml) and petroleum degradation (48-50 %) occurred with 0.25-1.0 mg/ml initial concentrations of uric acid.
  • the unwashed control containing 1 mg/ml uric acid yielded a slightly higher cell yield (9x10 8 cells per ml) and petroleum degradation value (67 %).
  • Table 1 hereinbelow, when ammonium sulfate was provided without the washout procedure, 62 % of the petroleum was degraded. However, in the simulated open system the ammonium sulfate yielded only 5x10 6 cells per ml and 2 % petroleum degradation.
  • strain OKI As is many Acinetobacter strains (Sar and Rosenberg, 1983. Curr. Microbiol. 9: 309-314), the growth of strain OKI on crude oil was accompanied by emulsification of the oil. Preliminary data indicate the extracellular emulsifier is a glucosamine-containing polysaccharide.
  • uric acid can serve as a nitrogen source for hydrocarbon-degrading bacteria and can bind to crude oil.
  • Acinetobacter sp. OKI which can grow on crude oil and uric acid further suggest its use in oil bioremediation in open water systems. Analysis and discussion - Because uric acid has a density of 1.89 (gram/cm 3 ), it rapidly sediments in aqueous medium. However, when the aqueous medium contained crude oil, the added uric acid did not settle, but remained on the surface bound to the oil. The interaction of the uric acid and crude oil was observed by phase and electron microscopy.
  • a uric acid/crude oil complex of 1:10 would have a density of approximately 1.0 (gram/cm 3 ).
  • ratios of uric acid to crude oil exceeded 1:10, the complex sedimented.
  • the simulated open system experiment demonstrates that when a complex containing 1 g crude oil and 50-200 mg bound uric acid in 200 ml aqueous medium is mixed for 1 hour and then the water replaced, and the procedure repeated three times, enough uric acid remains with the crude oil to stimulate maximum growth of strain OKI and petroleum degradation.
  • a true open system such as a lake, the complex would be exposed to a much larger body of water and the uric acid would have to remain bound to the oil for at least a few days.
  • uric acid can serve as a general water- insoluble nitrogen source for hydrocarbon oxidizers.
  • Uric acid was used as a sole nitrogen source for the bioremediation of petroleum in seawater, as follows.
  • strain OK2 which was able to grow as a pure culture on the petroleum/uric acid enrichment medium, was chosen for further study. Phenotypic characterization of Alcanivorax sp. OK2 - Performed as described for strain OKI in Example 1 hereinabove.
  • Strain OK2 was found to be a Gram-negative, strictly aerobic, non motile, oxidase-positive, short rod. These properties are typical of the genus Alcanivorax (Yakimov MM et al., 1998. Alcanivorax borkumensis gen. nov., sp. nov., a new hydrocarbon-degrading and surfactant-producing marine bacterium. Int. J. Syst. Bact. 48: 339-348). As is shown in Figures 6a-b, the cells (ca. 0.9x0.4 ⁇ m) are connected by strings of extracellular material, giving rise to multicellular sheets. The Biolog and API-20 identification kits failed to classify strain OK2.
  • strain OK2 can only use a narrow range of organic compounds as carbon sources. For example, strain OK2 failed to grow on any of the 15 amino acids and peptides in the kit and grew on only one carbohydrate (D-mannose) of the 28 tested.
  • the nucleotide sequence of the variable region of strain OK2 (1459 bp in length; SEQ ID NO:6, GenBank accession No. AY307381) had the closest similarity to Alcanivorax TE9 (Accession No. AB055207) with an identity of 99 %.
  • strain OK2 is a new Alcanivorax species, most closely related to strain TE9 isolated from the sea of Japan (Syutsubo et al. 2001, Environ. Microbiol. 3: 371-379).
  • Hydrocarbon and nitrogen substrate specificity The ability of Alcanivorax sp OK2 to utilize various aliphatic and aromatic hydrocarbons as carbon sources was examined in PU medium in which the crude oil was replaced with 2 mg/ml of the test hydrocarbon.
  • volatile toxic hydrocarbons benzene, toluene, xylene, pentane, heptane, octane, nonane and decane growth experiments were also performed by adding the hydrocarbon to side-arm flasks, such that the inoculated medium was exposed to the hydrocarbon vapors, rather than the liquid hydrocarbon.
  • Fluoranthene 2 x l0 7 Compounds that could serve as nitrogen sources were examined as described in Example 2, hereinabove. All of these compounds (except ureidoglycolate which was not available for testing) served as nitrogen sources for strain OK2.
  • SAS clear aqueous phase was removed and replaced with fresh SAS. The procedure was repeated three times to remove water soluble components. The medium was then supplemented with lmM potassium phosphate buffer and inoculated with a 0.01 ml of an OK2 culture grown in PU medium.

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Abstract

L'invention concerne un procédé de dégradation du pétrole ou d'un ou de plusieurs produits du pétrole, ainsi que des souches bactériennes pouvant être utilisées dans un tel procédé. Ledit procédé consiste à appliquer au pétrole ou au(x) produit(s) du pétrole des bactéries pouvant utiliser le pétrole comme source de carbone et de l'acide urique en une quantité pouvant constituer une source d'azote pour lesdites bactéries.
PCT/IL2004/000351 2003-04-24 2004-04-25 Traitement par biorestauration d'une pollution par du petrole faisant appel a de l'acide urique non hydrosoluble comme source d'azote Ceased WO2004094316A2 (fr)

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WO2007093993A3 (fr) * 2006-02-14 2007-11-15 Univ Ramot Préparations microbiennes pouvant être employées dans la dégradation d'hydrocarbures
WO2011153364A1 (fr) * 2010-06-02 2011-12-08 Eudes De Crecy Evolution de microorganismes sur des hydrocarbures complexes
CN102321659A (zh) * 2011-08-30 2012-01-18 浙江工业大学 氯代脂肪烃降解性质粒pRC11、工程菌及其应用
CN102583845A (zh) * 2012-01-16 2012-07-18 上海市自来水市北有限公司 一种应对饮用水源地突发性油类污染的方法
WO2012137220A2 (fr) 2011-04-04 2012-10-11 Indian Oil Corporation Ltd. Composition de bioaugmentation et son utilisation pour améliorer l'efficacité d'un traitement d'effluent dans une installation de traitement d'hydrocarbures
DE102013207197A1 (de) * 2013-04-22 2014-10-23 Helmholtz-Zentrum Dresden - Rossendorf E.V. Verfahren und System zur Abtrennung von Schwermetallen
WO2016001928A1 (fr) 2014-07-02 2016-01-07 Mekorot Water Company, Ltd Procédé de bioremédiation d'eau contaminée
CN108042968A (zh) * 2017-11-23 2018-05-18 浙江海洋大学 一种针对多环芳烃特异性降解的试剂的制备方法
CN111778187A (zh) * 2020-07-07 2020-10-16 内蒙古恒盛环保科技工程有限公司 一种微生物修复菌剂及其制备方法
RU2808248C1 (ru) * 2023-03-17 2023-11-28 федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский горный университет императрицы Екатерины II" Состав для биоремедиации почвы, загрязненной нефтью и нефтепродуктами
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US3871957A (en) * 1973-05-09 1975-03-18 Exxon Research Engineering Co Use of microorganisms to disperse and degrade oil spills
US4045582A (en) * 1973-08-15 1977-08-30 The Gates Rubber Company Beneficiation of uric acid from natural products
US3959127A (en) * 1973-12-05 1976-05-25 The United States Of America As Represented By The Secretary Of The Navy Biodegradation of oil on water surfaces
CA2085432A1 (fr) * 1991-12-24 1993-06-25 Eugene Rosenberg Compositions non polluantes servant a decomposer les hydrocarbures, et micro-organismes destines a leur utilisation
US5656486A (en) * 1996-06-10 1997-08-12 Daniels; Thomas Brent Method of treating and conditioning poultry manure to form a composition for biodegrading petroleum compounds

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WO2007093993A3 (fr) * 2006-02-14 2007-11-15 Univ Ramot Préparations microbiennes pouvant être employées dans la dégradation d'hydrocarbures
WO2011153364A1 (fr) * 2010-06-02 2011-12-08 Eudes De Crecy Evolution de microorganismes sur des hydrocarbures complexes
WO2012137220A2 (fr) 2011-04-04 2012-10-11 Indian Oil Corporation Ltd. Composition de bioaugmentation et son utilisation pour améliorer l'efficacité d'un traitement d'effluent dans une installation de traitement d'hydrocarbures
US9272933B2 (en) 2011-04-04 2016-03-01 Indian Oil Corporation Ltd. Bio-augmentation composition and use thereof for improving efficiency of effluent treatment in hydrocarbon processing plant
CN102321659A (zh) * 2011-08-30 2012-01-18 浙江工业大学 氯代脂肪烃降解性质粒pRC11、工程菌及其应用
CN102583845A (zh) * 2012-01-16 2012-07-18 上海市自来水市北有限公司 一种应对饮用水源地突发性油类污染的方法
DE102013207197B4 (de) 2013-04-22 2019-05-02 Helmholtz-Zentrum Dresden - Rossendorf E.V. Verfahren zur Abtrennung von Schwermetallen, Verwendung des Verfahrens und Verwendung eines Systems zur Aufreinigung schwermetallbelasteter Gewässer oder zum Nachweis von Schwermetallen in Gewässern
DE102013207197A1 (de) * 2013-04-22 2014-10-23 Helmholtz-Zentrum Dresden - Rossendorf E.V. Verfahren und System zur Abtrennung von Schwermetallen
WO2016001928A1 (fr) 2014-07-02 2016-01-07 Mekorot Water Company, Ltd Procédé de bioremédiation d'eau contaminée
US20180201531A1 (en) * 2014-07-02 2018-07-19 Mekorot Water Company, Ltd A method for bioremediation of contaminated water
US10647601B2 (en) 2014-07-02 2020-05-12 Mekorot Water Company, Ltd Method for bioremediation of contaminated water
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CN111778187A (zh) * 2020-07-07 2020-10-16 内蒙古恒盛环保科技工程有限公司 一种微生物修复菌剂及其制备方法
RU2808248C1 (ru) * 2023-03-17 2023-11-28 федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский горный университет императрицы Екатерины II" Состав для биоремедиации почвы, загрязненной нефтью и нефтепродуктами
CN118726223A (zh) * 2024-08-22 2024-10-01 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) 一株基因工程菌及其构建方法和应用

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