WO2004094316A2 - Bioremediation of petroleum pollution using water-insoluble uric acid as the nitrogen source - Google Patents

Abstract

A method of degrading petroleum or petroleum product(s) and bacterial strains suitable for use in such a method are provided. The method 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.

Description

BIOREMEDIATION OF PETROLEUM POLLUTION USING WATER- INSOLUBLE URIC ACID AS THE NITROGEN SOURCE

FIELD AND BACKGROUND OF THE INVENTION 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.

The fate of petroleum in a marine environment has been extensively studied

(Harayama S., et al, J. Mol. Microbiol. Biotechnol. 1999, 1: 63-70, Van Hamme SO, et al., Microbiol. Mol. Biol. Rev. 2003, 67: 503-49; Cohen Y., Int. Microbiol. 2002, 5: 189-93; Prince RC, Crit. Rev. Microbiol. 1993, 19: 217-42) and biodegradation has been suggested as effective, environmentally benign treatment for oil spill - contaminated shorelines.

When crude oil is dispersed in seawater, the interaction of the fine mineral particles in the water with the oil reduces the adhesion of the oil to solid surfaces (e.g., to sediments or bedrock), and increases the formation of stable, micron-sized, oil droplets. These oil droplets have increased surface area and as such are more accessible for biodegradation (Owens and Lee, 2003. Mar Pollut Bull. 47: 397-405).

Crude oil includes saturate, aromatic, resin and asphaltene components.

Following its discharge into the sea, the saturate and aromatic components with one, two or three aromatic rings are readily biodegraded in the marine environment.

However, aromatic components with four or more aromatic rings and asphaltene or resin fractions which contain higher molecular weight compounds are less accessible for biodegradation.

Generally, the conversion of 1 kg hydrocarbon to cell material consumes approximately 150 g of nitrogen. Since petroleum contains only traces of nitrogen, microbial degradation of petroleum in open systems such as lakes, oceans and wastelands, requires the availability of a utilizable source of nitrogen (Atlas, R.M.

1991. J. Chem. Technol. Biotechnol. 52: 149-156; Atlas and Bartha, 1972. Biotechnol. Bioeng. 14: 309-317; Rosenberg, E.,S. et al., 1998. Rate-limiting steps in the microbial degradation of petroleum hydrocarbons, p. 159-171. In H. Rubin, N. Narkis, and J. Carberry (Ed.). Soil and aquifer pollution. Springer- Verlag, Berlin Heidelberg; Rosenberg, E., and Ron, E.Z. 1996. Bioremediation of petroleum contamination, p.100- 124. In R.L. Crawford, and D.G. Crawford. (Eds.), Bioremediation: principles and applications. Cambridge University Press, Cambridge).

Under laboratory conditions, the nitrogen requirement for optimum growth of hydrocarbon oxidizers can be readily satisfied with urea or salts that contain ammonium or nitrate ions. However, the high water solubility of urea and nitrogen containing salts makes it less effective in open systems due to rapid dilution.

To overcome the nitrogen-limitation for petroleum degradation in open systems, Atlas and Bartha (Environ. Sci. Technol. 1973, 7: 538-541) have suggested the use of several oleophilic nitrogen compounds with low carbon-nitrogen ratios. Such oleophilic nutrients have high affinity towards the oil phase, and as such may enhance the selective growth of petroleum-degrading microorganisms. Thus, the Inipol oleic acid, urea, lauryl phosphate (Inipol EAP 22) oleophilic fertilizer was used in the bioremediation of polluted shorelines following the Exxon Valdez spill [Atlas, 1991 (Supra); Lindstrom, J.E., et al., 1991. Appl. Environ. Microbiol. 57: 2514-2522]. However, since 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. In addition, Inipol EAP 22 is an emulsifier and its use in seawater is likely to harm the environment. Most importantly, 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.

Another water-insoluble polymer that adheres to oil is based on the urea- formaldehyde formulation (Rosenberg, E., et al., 1992. Biodegradation 3: 337-350; Rosenberg, E., et al., 1996. J. Biotechnol. 51: 273-278). This polymer was successfully used to bioremediate a heavily oil-contaminated sandy beach, but is inefficient in seawater since it causes the oil to sink.

There is thus a widely recognized need for, and it would be highly advantageous to have, a method of degrading petroleum and petroleum products present in seawater devoid of the above limitations. SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided 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).

According to another aspect of the present invention there is provided a bacterial strain capable of using petroleum or petroleum product(s) as a source of carbon and uric acid as a nitrogen source. According to yet another aspect of the present invention there is provided 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). According to still another aspect of the present invention there is provided a method of isolating a bacterial strain capable of degrading petroleum or petroleum product(s), the method 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).

According to further features in preferred embodiments of the invention described below, the petroleum or petroleum product(s) form a part of an oil spill in a water body. According to still further features in the described preferred embodiments the bacteria is selected from the group consisting of Acinetobacter sp. OKI, Alcanivorax sp. OK2, Acinetobacter calcoaceticus RAG-1, and Pseudomonas fluorescens.

According to still further features in the described preferred embodiments 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).

According to still further features in the described preferred embodiments the uric acid forms a part of a fertilizer composition. According to still further features in the described preferred embodiments the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Acinetobacter having the NCIMB designation No. 41212.

According to still further features in the described preferred embodiments the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Alcanivorax having the NCIMB designation No. 41213.

According to still further features in the described preferred embodiments an environment of the petroleum or petroleum product(s) includes petroleum-degrading bacteria. According to still further features in the described preferred embodiments the bacteria-containing sample is beach tar, seawater and/or pigeon manure.

According to still further features in the described preferred embodiments the culturing is effected by subjecting the bacteria-containing sample to a repeated inoculation/culturing cycle in the culture medium. According to still further features in the described preferred embodiments each of the repeated inoculation/culturing cycle is effected for at least 3 days.

According to still further features in the described preferred embodiments the inoculation/culturing cycle is repeated at least 8 times.

According to still further features in the described preferred embodiments the culturing is effected at 30 °C using aeration.

According to still further features in the described preferred embodiments the petroleum is crude oil.

According to still further features in the described preferred embodiments the crude oil is provided at a concentration of 5 mg/ml. According to still further features in the described preferred embodiments the uric acid is provided at a concentration range of 0.1-5 mg/ml.

According to still further features in the described preferred embodiments the culture medium includes 15 mg/L MgSO₄7H₂O, 15 mg/L FeSO₄7H₂O, 5 mg/L CaCl₂ and 500 mg/L NaCl in 1 mM potassium phosphate buffer at pH 6.5. According to still further features in the described preferred embodiments the culture medium is 1 mM potassium phosphate buffer at pH 8.0 in sterile seawater. The present invention successfully addresses the shortcomings of the presently known configurations by providing a method of degrading petroleum using uric acid as a sole nitrogen source and petroleum-degrading bacteria.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIG. 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. Bacteria were inoculated into E. salts containing crude oil (5 mg/ml) and uric acid (0.5 mg/ml) and were incubated at 30 °C with shaking; cm = colony forming unit; h = hour.

FIG. 2 is a scanning electron photomicrograph depicting the structure of the Acinetobacter sp. OKI. Note the presence of extracellular appendages. FIG. 3 is an electron photomicrograph illustrating the adhesion of uric acid to crude oil. Note the uric acid crystals adhering on the left and upper sides of the oil droplet; UA = uric acid. FIG. 4 is a graph illustrating the affinity of Acinetobacter sp. OKI to hexadecane. Bacterial suspensions (1 ml, A₅₆o = 1.0) were mixed with varying amounts of hexadecane. Results are expressed as percentage of the initial absorbance (A₅₆₀) of the aqueous suspension as a function of hexadecane volume. 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, A₅₆₀: 1.0) were mixed with varying amounts of hexadecane. Results are expressed as percentage of the initial absorbance (A₅₆₀) 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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

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.

Specifically, 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. Before explaining at least one embodiment of the invention in detail, it is to be understood that 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. Also, it is to be understood that 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.

Under laboratory conditions, the nitrogen requirement for optimum growth of hydrocarbon oxidizers can be readily satisfied with urea or salts that contain ammonium or nitrate ions. However, since these nitrogen sources have high water solubility they are not effective in supporting bacterial degradation of petroleum due to rapid dilution thereof in water.

Several nitrogen-containing nutrients have been suggested for use in petroleum degradation in open systems. These include oleophilic nitrogen compounds such as Inipol EAP 22 (Atlas, 1991 (Supra); Lindstrom, J.E., et al., 1991. Appl. Environ. Microbiol. 57: 2514-2522) and urea-formaldehyde formulation (Rosenberg, E., et al, 1992. Biodegradation 3: 337-350; Rosenberg, E., et al., 1996. J. Biotechnol. 51: 273-278). However, while the oleophilic compounds are limited by the release of urea (i.e., the nitrogen source) from the emulsion into the water phase, 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).

In sharp contrast to its prior use, 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. Indeed, as is shown in Examples 1-3 of the Examples section which follows, 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. In addition, as is shown in Figure 3, 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.

Thus, according to one aspect of the present invention there is provided a method of degrading petroleum or petroleum product(s).

As used herein the phrase "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.

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. For example, 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.

Preferably, 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). As used herein, the phrase "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.

According to preferred embodiments of the present invention 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). According to this aspect of the present invention, 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).

According to preferred embodiments of the present invention the uric acid is part of a composition such as a Guano fertilizer.

It will be appreciated that 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.

Preferably, 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).

It will be appreciated that the 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.

While reducing the present invention to practice the present inventors have uncovered two independent bacterial strains (strains OKI and OK2 mentioned hereinabove) which are capable of using petroleum or petroleum product(s) as a source of carbon and uric acid as a source of nitrogen.

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. In addition, as is shown in Figure 2, microscopic evaluation of 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. Appl. Microbiol. Biotechnol. 44: 660- 667). Moreover, as is further shown in Example 1 of the Examples section which follows, the nucleotide sequence of the variable region of the 16S rDNA sequence of strain OKI (755 bp in length; GenBank accession No. AY260854, SEQ ID NO:5) had the closest similarity to A. baumannii (DSM 3008), with an identity of 99 %. Thus, it can be concluded that strain OKI of the present invention (NCIMB designation number 41212) is an isolate of petroleum-degrading bacterial strain of the genus Acinetobacter.

As is further shown in Example 3 of the Examples section which follows, the second bacterial strain (strain OK2, NCIMB designation number 41213) 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. As is further shown in Example 3 of the Examples section which follows, 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 %.

Thus, it can be concluded that strain OK2 of the present invention is an isolate of a petroleum degrading bacterial strain of the genus Alcanivorax. In order to facilitate use, 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.

It will be appreciated that in cases where the polluted environment already includes the petroleum degrading bacteria, the method described above can be practiced without the step of including the petroleum degrading bacteria.

For example, in cases where degradation of petroleum pollution of water is desired, addition of uric acid alone can_^also lead to petroleum degradation since it has been shown that seawater and fresh water include bacteria which can degrade petroleum (Rosenberg E, Prokaryotes II, pp. 446-460, Springer- Verlag, New York,

1992).

Thus, according to another aspect of the present invention there is provided a method of degrading petroleum or petroleum product(s). The method 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).

According to preferred embodiments of the present invention such an environment can be, for example, fresh water or sea water (Rosenberg E, 1992 (supra);

Hara A, et al., 2003, Environ Microbiol. 5: 746-53) and/or soil (Bogan BW et al.,

2003, Int. J. Syst. Evol. Microbiol. 53: 1389-95; Margesin R et al., 2003, Appl.

Environ. Microbiol. 69: 3085-92). Thus, 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.

It will be appreciated that the 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. Thus, according to another aspect of the present invention there is provided 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).

As used herein, the phrase "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).

According to presently preferred embodiments, the bacteria-containing sample is derived from beach tar, seawater or pigeon manure, such samples can be collected using simple laboratory tools.

In order to isolate petroleum-degrading bacteria according to the method of the present invention 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.

As used herein 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

(e.g., PU medium, E salts) and can be cultured in the presence or absence of air to enable the growth of aerobic or anaerobic bacteria, respectively.

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.

For example, as is shown in Figure 1, a significant growth rate from 5xl0⁴ o

CFU/ml to 5x10 CFU/ml was achieved within 35 hours of culturing of strain OKI at 30 °C using aeration.

Thus, according to presently preferred configurations, 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.,

3-4 days) and diluted in a fresh preparation of the same culture medium (e.g., containing crude oil and uric acid) and be subjected to an additional culturing period.

For example, as is shown in Examples 1 and 3 of the Examples section which follows, after an initial culturing period of four days in a culture medium containing crude oil and uric acid, the bacteria-containing sample was diluted in a fresh culture medium containing the same concentrations of crude oil and uric acid and was cultured for additional three days. This inoculation/culturing cycle was repeated 8 times. Thus, according to preferred embodiments of the present invention culturing is effected by subjecting the bacteria-containing sample to a repeated inoculation/culturing cycle in the culture medium. According to presently preferred configurations, the repeated inoculation/culturing cycle is effected for at least 3 days. In addition, to efficiently select petroleum-degrading bacteria according to the method of the present invention, 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. Preferably, 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.

It will be appreciated that following the repeated inoculation/culturing cycles, the bacterial culture may include more than one type of bacteria. To separate the various types of bacteria from each other the bacterial culture is preferably streaked on a solid medium, such as on 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.

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 MgSO₄7H₂O, 15 mg/L FeSO₄7H₂O, 5 mg/L CaCl₂ 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.

Once obtained, 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.

For example, 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).

The metabolic activity of the isolated petroleum-degrading bacteria (i.e., functional characterization) can be evaluated using the carbon compound utilization test on the Biolog GN Microplate (Biolog Inc., Haywood, CA) according to manufacturer's instructions. Briefly, fresh colonies on LB agar are transferred to a Biolog inoculation medium to reach a turbidity of A_{5 0} = 0.13-0.14. The inoculated medium is then distributed into the microwells containing different carbon sources and incubated at 30 °C for 48 hours. Wells that turn the tetrazolium violet indicator purple are considered positive for metabolic activity. For a more precise identification of a bacterial strain the genomic sequence of the variable region of the 16S rDNA is determined. 16S rDNA sequence-based bacterial identification is the most advanced and accurate method of identifying bacterial strains since it is based on highly conserved sequence stretches.

Briefly, 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. To evaluate the bacterial adhesion to hydrocarbon molecules (e.g., hexadecane) a bacterial adhesion test (BATH) can be performed (Rosenberg, M., and

Rosenberg, E., 1985. Bacterial adherence at the hydrocarbon- water interface. Oil

Petrochem. Pollut. 2: 155-162). Briefly, cells are harvested during stationary phase, centrifuged and washed twice with PUM buffer (22.2 g/L K₂HPO₄3H₂O, 7.26 g/L

KH₂PO₄, 1.8 g/L urea, 0.2 g/L MgSO₄7H₂O at pH 7.1). 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.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes 1-111 Cellis, J. E., Ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (Eds.), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., Ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., Ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); Doetsch, R. N. 1981. Determinative methods of light microscopy, p. 21-33. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C.; all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. EXAMPLE 1 IDENTIFICATION AND CHARACTERIZATION OF ACINETOBACTER SP. OKI

To test the hypothesis that crude oil can be biodegraded using uric acid as a sole nitrogen source, crude oil and uric acid in E salt buffer were inoculated with pigeon manure and pure bacteria isolated were identified, as follows. Materials and Experimental Methods

Isolation of Acinetobacter sp. OKI - An enrichment culture medium containing 5 mg/ml crude oil (weathered Alaska North Slope ANS) and 5 mg/ml uric acid in E salts (15 mg MgSO₄7H₂O, 15 mg FeSO₄7H₂O, 5 mg CaCl₂ and 500 mg NaCl per liter 1 mM potassium phosphate buffer, pH 6.5) was inoculated with 1 teaspoon of pigeon manure and incubated at 30 °C with aeration. After 4 days, a 10 μl sample was transferred to a fresh enrichment culture medium and incubation continued for 3 days. The latter procedure was repeated eight times yielding a mixed culture (by phase microscopy) that visibly emulsified and degraded the petroleum.

To obtain pure cultures, the last enrichment culture was streaked onto Luria-

Bertuni (LB) agar. Several isolated colonies were re-streaked on LB agar to obtain pure isolates. One of the isolates, referred to as strain OKI, which was able to grow as a pure culture on the petroleum/uric acid enrichment medium, was chosen for further study.

Cell viability and phenotypic characterization of Acinetobacter sp. OKI - Cell morphology was determined by phase-contrast microscopy and scanning electron microscopy (Jeol 840A, JEOL Inc. Peabody, MA, USA) after negative staining with 1 % uranyl acetate. The metabolic activity of the isolated bacteria was evaluated using the carbon compound utilization test on the Biolog GN Microplate (Biolog Inc., Haywood, CA). Fresh colonies on LB agar were transferred to Biolog inoculation medium to reach a turbidity of A₅₉o = 0.13-0.14. The inoculated medium was then distributed into the microwells containing different carbon sources and incubated at 30 °C for 48 hours. Wells that turned the tetrazolium violet indicator purple were marked as positive for metabolic activity. Gram-negative rods were identified using the API-20 system (bioMerieux, France) according to manufacturer's instructions. Sequencing analysis of the 16S rDNA from isolated OKI strain — Genomic DNA was extracted from 2 ml of strain OKI overnight culture using the Wizard DNA genomic purification kit (Promega Corp., Madison, WT) and was used as a template for a PCR reaction using the forward [5'-GAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:l)] and reverse [5'-AGAAAGGAGGTGATCCAGCC-3' (SEQ ID NO:2)] PCR primers specific to the 16S rDNA. PCR reaction (50 μl) included 5 μl 10 x buffer, 1 μl 2.5 mM total dNTP mixture, 10 ng template DNA and 2.5 units Ex Taq DNA polymerase (Takara Shuzo) and 5 μM 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). Experimental Results Isolation of bacterial strains utilizing crude oil and uric acid- To isolate bacterial strains capable of degrading crude oil, an enrichment culture medium (E salts) containing crude oil, as a carbon source, and uric acid, as a nitrogen source, was inoculated with pigeon manure. Following 28 days in culture and several transfers the crude oil was emulsified and partially degraded. When the culture was plated on LB agar five types of colonies were observed. Examination of the five isolated colonies indicated that, as pure cultures, two were unable to grow, two grew poorly and one grew well on the enrichment culture medium. The strain that grew well, referred to as OKI, was chosen for further analysis.

A comparison of the growth of strain OKI and the mixed culture on the crude oil/uric acid emichment medium revealed that although the mixed culture exhibited a greater growth rate within the first 10 hours in culture, the overall growth of the isolated OKI strain was in the same order of magnitude as the mixed culture following 33 hours in culture (Figure 1).

Phenotypic properties of the isolated OKI strain - The isolated strain (strain

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

[Grimont, P.A.D., and P.J.M. Bouvet. 1991. Taxonomy of Acinetobacter sp., p. 28-35.

In K.J. Towner, E. Bergogne-Berezin, and CA. Fewson (Ed.). The biology of

Acinetobacter. Plenum Press, New York; Juni, E. 1978. Genetics and physiology of

Acinetobacter. Annu. Rev. Microbiol. 32: 349-371]. Microscopic evaluation of the isolated OKI strain revealed the presence of extracellular appendages (Figure 2) which are similar to 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. Appl. Microbiol. Biotechnol. 44: 660-667).

Metabolic characterization of the isolated OKI strain - The Biolog and API- 20 identification kits indicated that strain OKI was an Acinetobacter, most closely related to A. baumannii (ID = 76 %). Like most Acinetobacter strains, 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.

Sequence analysis of the isolated Acinetobacter sp. OKI strain - The nucleotide sequence of the variable region of strain OKI (755 bp in length; SEQ ID NO:5, GenBank accession No. AY260854) had the closest similarity to A. baumannii (DSM 3008), with an identity of 813/821 nucleotides (99 %).

Thus, the phentotypic, metabolic and genotypic tests performed demonstrate that strain OKI is a new Acinetobacter species, most closely related to A. baumannii. EXAMPLE 2

ADHESION OF URIC ACID AND ACINETOBACTER SP. OKI TO CRUDE OIL

AND BACTERIAL HYDROCARBON AND NITROGEN SUBSTRATE

SPECIFICITY Materials and Experimental Methods

Growth on crude oil and uric acid after removal of water solubles - One- liter flasks containing 200 ml E salts, 5 mg/ml crude oil and varying concentrations of uric acid or ammonium sulfate were mixed for 1 hour at 30 °C. Following one hour, the flasks were allowed to stand for 5 min to enable the separation of the aqueous phase from the insoluble phase (containing crude oil bound to uric acid). Once separated, the aqueous phase was carefully replaced with 200 ml sterile water. The procedure was repeated two additional times. After the third removal of water- soluble compounds, 200 ml E salts were added and the flasks were inoculated with 0.01 ml of a 3-day culture of Acinetobacter sp. OKI in crude oil/uric acid enrichment culture medium. The flasks were then incubated at 30 °C for 96 h with shaking. Viable cell number was determined on Luria-Bertuni (LB) agar. Petroleum degradation was determined by extracting the cultures with equal volumes of dichloromethane, transferring the organic phase to tarred beakers and drying to constant weight at room temperature. The percent degradation was determined by comparison with a sterile control treated in exactly the same manner. Approximately 90 % of the input crude oil was recovered in the sterile control.

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 K₂HPO₄3H₂O, 7.26 g KH₂PO₄, 1.8 g urea, 0.2 g MgSO₄7H₂O 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. Following 10 min preincubation at 30 °C, the mixtures were agitated uniformly for 2 min. After allowing 15 min for the hexadecane phase to rise, the aqueous phase was carefully removed using a Pasteur pipette and transferred to a 1 ml cuvette. Turbidity was determined at 560 nm. Experimental Results

Adhesion of uric acid and strain OKI to crude oil - Scanning electron microscopy revealed that uric acid crystals adhered to droplets of crude oil in the culture medium (Figure 3). Using low ratios of uric acid to crude oil (1:20, w/w) the crude oil/uric acid complex had a density less than 1.0 and floated to the surface of the medium, whereas at higher ratios (1:2), the complexes sedimented. These data also demonstrated the interaction between uric acid and crude oil. Using the BATH test it was shown that stationary phase cells of strain OKI adhere to hexadecane (Figure 4). Exponentially growing cells adhered poorly (data not shown). Growth of strain OKI on crude oil and uric acid following removal of water soluble nutrients - To test the hypothesis that uric acid would bind to crude oil and would not be diluted in the surrounding water in a simulated open system, 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). As is shown in Table 1, hereinbelow, the high cell yields (2-6x10⁸ 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 (9x10⁸ cells per ml) and petroleum degradation value (67 %). As is further shown in 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 5x10⁶ cells per ml and 2 % petroleum degradation.

These results demonstrate the effectiveness of uric acid as a sole nitrogen source for petroleum degradation in simulated open systems.

Table 1 Growth of Acinetobacter sp. OKI on crude oil and uric acid following removal of water soluble nutrients"

N-source Water Cell yield Petroleum exchange (CFU/ml) degradation (%)

NH4SO4 (1 mg/ml) None 8xl0⁸ 62

NH4SO4 (1 mg ml) 3 x ^b 5xlO^fi 2

Uric acid (1 mg/ml) None 9x10^s 67

Uric acid (1 mg/ml) 3 x ^b 4x10^s 50

Uric acid (0.5 mg/ml) 3 x ^b 6xl0⁸ 49

Uric acid (0.25 mg/ml) 3 x ^b 2x10^s 48 ^(a) Bacteria were grown in E salts containing 5 mg/ml crude oil and the different nitrogen sources at 30 °C with shaking for 96 h; ^b) After mixing the media, the clear aqueous phase was removed and replaced with E salts. The procedure was repeated three times to remove water soluble compounds.

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.

Growth yield as a function of crude oil and uric acid concentrations - To determine growth yields of Acinetobacter sp. OKI as a function of petroleum and uric acid concentration, 0.01 ml of an overnight culture on LB medium was inoculated into 200 ml of E salts containing varying concentrations of crude oil and uric acid in 1 -liter flasks. After incubation with shaking for 96 hours at 30 °C, cell yields were determined by spreading appropriate dilutions on LB agar. The growth of strain OKI was proportional to crude oil concentration from 0-5 mg/ml (Figure 5a) and uric acid concentration from 0-0.5 mg/ml (Figure 5b), reaching 9x10⁸ cells per ml. The minimum doubling time during exponential phase on the crude oil/uric acid medium at 30 °C was ca. 50 minutes.

Hydrocarbon and nitrogen substrate specificity - The ability of strain OKI to utilize various aliphatic and aromatic hydrocarbons as carbon sources was examined in 125 shake flask experiments containing 20 ml E salts, 0.5 mg/ml uric acid and 2 mg/ml of the test hydrocarbon. After inoculation with 0.01 ml from a 4-day culture in crude oil/uric acid enrichment culture medium, the flasks were incubated for 96 hours at 30 °C with shaking. With volatile toxic hydrocarbons (benzene, toluene, xylene, pentane, heptane, octane, nonane and decane) growth experiments were also performed by adding the hydrocarbon to side-arm flaks, such that the inoculated medium was exposed to the hydrocarbon vapors, rather than the liquid hydrocarbon. The growth tests revealed that strain OKI grows only on straight and branched-chain (phytane and pristane) aliphatic hydrocarbons containing 12 or more carbons. Strain OKI failed to grow on shorter alkanes or the 16 aromatic hydrocarbons tested. The Agha Jari crude oil used in the above washout experiment contained only 14 % aromatics (Gutnick, D.L., and E. Rosenberg. 1977. Oil tankers and pollution: a microbiological approach. Ann. Rev. Microbiol. 31:379-396). Compounds that could serve as nitrogen sources (Oxonic acid, Murexide, guanine, xanthine, allantoin, allantoate, urea and ammonia) were examined as described above, using 5 mg/ml crude oil as the carbon source and 0.5 mg/ml of the test nitrogen source. In all cases growth was determined visibly and by viable count on LB agar. Uric acid is catabolized by bacteria sequentially to allantoin, allantoate, ureidoglycolate, urea and ammonia (Shultz, A.C., et al., 2001. Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J. Bacteriol. 185: 3293-3302). All of these compounds (except ureidoglycolate which was not available for testing) served as nitrogen sources for strain OKI .

Altogether, these results demonstrate that uric acid can serve as a nitrogen source for hydrocarbon-degrading bacteria and can bind to crude oil. In addition, the identification of the 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/cm³), 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. Theoretically, a uric acid/crude oil complex of 1:10 (w/w) would have a density of approximately 1.0 (gram/cm³). Thus, it was not surprising that when 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. Clearly, in 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.

The ability of microorganisms to degrade uric acid is not an unusual trait. In 1929, Den Dooren de Jorg described Bacillus fastidiosus, a bacterium which grew only on uric acid and allantoin (Den Dooren de Jong, L.E. 1929. Ueber Bacillus fastidiosus. Zantralbl. Bacteriol. Parasitenkd. Infektionskr. Hyg. Abt. II 79:344-358). Since then, numerous uric acid-degrading bacteria and yeasts have been described and various routes of catabolism studied (Christiansen, L.C., S. Schou, P. Nygaard, and H.H. Saxild. 1997. J. Bacteriol. 179: 2540-2550; DeMoll, E., and Auffenberg, T. 1993. J. Bacteriol. 175: 5754-5761; Diallinas, G., and Scazzocchio, C. 1989. Genetics 122: 341-350; Marin, M., et al., 1996. Appl. Microbiol. Biotechnol. 44: 660-667; Mayser, P., et al., 1998. Arch. Dermatol. Res. 290: 277-282; Rosenberg, M., and Rosenberg, E. 1985. Oil Petrochem. Pollut. 2:155-162; Vogels, G.D., and van der Drift, C. 1976. Bacteriol. Rev. 40: 402-469). Some of these bacteria utilize uric acid as both a carbon and nitrogen source; others use it only as a nitrogen source. Strain OKI fits into the latter group. Thus, uric acid can serve as a general water- insoluble nitrogen source for hydrocarbon oxidizers.

EXAMPLE 3 BIODEGRADATION OF PETROLEUM IN SEAWATER BY ALCANIVORAX SP. OK2 USING WATER-INSOLUBLE URIC ACID AS THE NITROGEN SOURCE

Uric acid was used as a sole nitrogen source for the bioremediation of petroleum in seawater, as follows.

Materials and Experimental Methods

Isolation of Alcanivorax sp. OK2 - An enrichment culture medium containing 5 mg/ml crude oil, 0.5 mg/ml uric acid (PU medium) in 1 mM potassium phosphate buffer, pH 8.0 in autoclaved Mediterranean seawater was inoculated with beach tar and incubated at 30 °C with aeration. After 4 days, a sample was transferred to a fresh enrichment culture medium and incubation continued for 7 days. The latter procedure was repeated several times yielding a mixed culture (by phase microscopy) that visibly emulsified and degraded the petroleum. To obtain pure cultures, the last enrichment culture was diluted 10^"6 and streaked onto Marine agar (Difco, Detroit, Mich.). To obtain pure isolates, several isolated colonies were restreaked on Marine agar. One of the isolates, referred to as 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. PCR amplification, sequencing and phylogenetic analysis of the 16S rDNA sequence of strain OK2 - The preparation of genomic DNA, PCR amplification, sequencing and BLAST analysis of the 16S rDNA of strain OK2 were performed essentially as described for strain OKI in Example 1 hereinabove, using the following primers: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO:3) and 5'- TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO:4).

Growth yield as a function of crude oil and uric acid concentrations - To determine growth yields of Alcanivorax sp. OK2 as a function of petroleum concentration, 0.01 ml of an exponentially growing culture on PU medium was inoculated into 20 ml of seawater containing 0.5 mg/ml uric acid, 1 mM potassium phosphate buffer, pH 8.0, and varying concentrations of crude oil. After incubation with shaking for 1 week at 30 °C, cell yields were determined by spreading appropriate dilutions on BHI agar (BD Diagnostic System, Sparks, MD, USA). Growth yield as a function of uric acid concentration was determined as described above, using 5 mg/ml petroleum and varying concentrations of uric acid.

Growth on crude oil and uric acid after removal of water solubles - Flasks containing 25 mg crude oil and varying amounts of uric acid or ammonium sulfate in 20 ml sterile artificial seawater (SAS) were mixed for 1 hour at 30 °C. After allowing the flask to stand for 5 minute, the clear aqueous phase was removed, without disturbing the insoluble crude oil and bound uric acid, and then replaced with 20 ml SAS. The procedure was repeated two additional times. After the third removal of water-soluble compounds, 20 ml SAS containing lmM potassium phosphate buffer, pH 8, were added and the flasks inoculated with 0.01 ml of a 3-day culture of Alcanivorax sp. OK2 in PU medium. The flasks were then incubated at 30 °C with shaking. Viable cell number was determined after 2, 7, 15 and 20 days as described above. Petroleum degradation was determined after 20 days by extracting the cultures with equal volumes of dichloromethane, transferring the organic phase to tarred beakers and drying to constant weight at room temperature. The percent degradation was determined by comparison with a no nitrogen supplementation control treated in exactly the same manner.

BATH test - was performed as described for strain OKI in Example 2 hereinabove. Experimental Results

Isolation and characterization of Alcanivorax sp. OK2 - Using crude oil as the carbon source and uric acid as the nitrogen source in a seawater culture medium, a mixed culture was obtained after several transfers that emulsified and partially degraded the crude oil. Plating on Marine agar yielded six colony types. Examination of the six isolated colonies indicated that, as pure cultures, only one grew well on the enrichment culture medium. That strain, referred to as OK2, was chosen for further study.

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 μm) are connected by strings of extracellular material, giving rise to multicellular sheets. The Biolog and API-20 identification kits failed to classify strain OK2. Noteworthy, Alcanivorax strains are not included in these data banks. Like other Alcanivorax strains, 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 %.

Altogether, these results demonstrate that 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).

Adhesion of Alcanivorax sp. OK2 to hydrocarbons - During the growth of strain OK2 on crude oil, it was observed that most of the cells were attached to oil droplets. Using the BATH test it was shown that most of the stationary phase cells of strain OK2 adhered to hexadecane (Figure 7). The population appeared to be heterogenous with regard to cell surface hydrophobicity. Approximately 45 % of the cells bound avidly to low concentrations of hexadecane, whereas 35 % of the cells required higher amounts of hexadecane and 20 % failed to adhere. Exponentially growing cells adhered poorly (data not shown).

Growth yield as a function of crude oil and uric acid concentrations - Cell growth yield of Alcanivorax sp. OK2 was directly proportional to petroleum concentration from 0-5 mg/ml (Figure 8a) and to uric acid concentration from 0-2 mg/ml (Figure 8b). No growth was observed in the absence of either crude oil or uric acid.

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. With 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. These tests revealed that strain OK2 grew on linear and branched long chain aliphatic hydrocarbons and one aromatic hydrocarbon, fluoranthene (Table 2, hereinbelow). Highest growth yields were obtained on crude oil and hexadecane. The ability to use branch chain alkanes (e.g., pristine and phytane) has recently been shown to give Alcanivorax an advantage over Acinetobacter in oil contaminated water (Hara et al. 2003. Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation. Environ. Microbiol. 5: 746-753).

Table 2 Hydrocarbon substrate specificity of Alcanivorax sp. OK2 ilkane (0.2 %) Growth (cells/ml) Aromatics (0.2 %) Growth (cells/ml)

None < 10⁶. Benzene < 10⁶

Pentane < 10⁶ Xylene < 10⁶

Cyclohexane < 10⁶ Naphthalene < 10⁶

Heptane < 10⁶ 2-methylnaρhthalene < 10⁶

Octane < 10⁶ Phenanthroline < 10^δ

Nonane < 10⁶ Fluorene < 10⁶

Decane 6 x l0⁷ Anthracene < 10⁶

Phytane 3 x l0⁷ Phenanthrene < 10⁶

Pristane 8 x l0⁷ Chrysene < 10⁶

Tetradecane 1 x 10^s Perylene < 10^δ

Hexadecane 2 x l0⁹ Benzopyrene < 10⁶

Crude oil 2 x l0⁹ Hexaphenylbenzene < 10^δ

Fluoranthene 2 x l0⁷ 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.

Growth of Alcanivorax sp. OK2 on crude oil and uric acid following removal of water soluble nutrients - To test the effectiveness of uric acid as a nitrogen source for petroleum degradation in simulated open system using seawater, the growth of strain OK2 was measured on crude oil-uric acid media after the media was washed three times with seawater (a simulated open system). As is shown in Table 3 hereinbelow, the growth yields of the Alcanivorax sp. OK2 bacteria were 6.9x10 and 8.0x10 CFU per ml using initial concentrations of 1 mg and 4 mg per ml uric acid, respectively. In addition, under these conditions (i.e., 1-4 mg/ml uric acid in a simulated seawater open system), 17-31 % of the petroleum was degraded (Table 3, hereinbelow). On the other hand, the water-soluble ammonium sulfate control did not support any significant petroleum degradation following the washout procedure.

Table 3 Growth of Alcanivorax sp. OK2 on crude oil and uric acid after removal of water soluble contents⁰

Nitrogen supplement Water wash Cellyield^b Petroleum

(CFU/ml ±S.E) degradation⁰ (% ±S.E)

0.5 mg/ml (NH₄)₂S0₄ None 1.3 ± 0.2xl0⁸ 13 ± 2.2

0.5 mg/ml (NH₄)₂S0₄ 3X 5.0 ± 1.5xl0⁶ 1 ± 2.0

0.5 mg/ml uric acid None 4.9 ± l.lxl0⁸ 20 + 1.1

1 mg/ml uric acid 3X 6.9 ± 0.5x10⁷ 17 + 1.2

4 mg/ml uric acid 3X 8.0 ± 2.5xl0⁸ 31 + 1.5

^(a> After mixing 5 mg/ml crude oil and the nitrogen supplement in sterile artificial seawater

(SAS), the 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.

^ Maximum cell yield was obtained at 7 days.

^(o) Petroleum degradation was measured after 20 days.

Altogether, these results demonstrate that Alcanivorax sp. OK2 can grow on crude oil/uric acid seawater medium and suggest the use of uric acid as potentially useful nitrogen source for the bioremediation of petroleum pollution in open systems. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. 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 said bacteria to thereby degrade the petroleum or petroleum product(s).

2. The method of claim 1 , wherein said petroleum or petroleum product(s) form a part of an oil spill in a water body.

3. The method of claim 1, wherein said bacteria is selected from the group consisting of Acinetobacter sp. OKI, Alcanivorax sp. OK2, Acinetobacter calcoaceticus RAG-1, and Pseudomonas fluorescens.

4. The method of claim 1, wherein said uric acid is provided at a concentration ratio of 1 part of uric acid to 5-50 parts of petroleum or petroleum product(s).

5. The method of claim 1, wherein said uric acid forms a part of a fertilizer composition.

6. A bacterial strain capable of using petroleum or petroleum product(s) as a source of carbon and uric acid as a nitrogen source.

7. The bacterial strain of claim 6, wherein the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Acinetobacter having the NCIMB . designation No. 41212.

8. The bacterial strain of claim 6, wherein the bacteria is an isolate of a petroleum degrading bacterial strain of the genus Alcanivorax having the NCIMB designation No. 41213.

9. 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).

10. The method of claim 9, wherein an environment of the petroleum or petroleum product(s) includes petroleum-degrading bacteria.

11. The method of claim 9, wherein said uric acid is provided at a concentration ratio of 1 part of uric acid to 5-50 parts of petroleum or petroleum product(s).

12. The method of claim 9, wherein said uric acid forms a part of a fertilizer composition.

13. A method of isolating a bacterial strain capable of degrading petroleum or petroleum product(s), the method 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 said 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).

14. The method of claim 13, wherein said bacteria-containing sample is beach tar, seawater and/or pigeon manure.

15. The method of claim 13, wherein said culturing is effected by subjecting said bacteria-containing sample to a repeated inoculation/culturing cycle in said culture medium.

16. The method of claim 15, wherein each of said repeated inoculation/culturing cycle is effected for at least 3 days.

17. The method of claim 15, wherein said inoculation/culturing cycle is repeated at least 8 times.

18. The method of claim 13, wherein said culturing is effected at 30 °C using aeration.

19. The method of claim 13, wherein said petroleum is crude oil.

20. The method of claim 19, wherein said crude oil is provided at a concentration of 5 mg/ml.

21. The method of claim 13, wherein said uric acid is provided at a concentration range of 0.1-5 mg/ml.

22. The method of claim 13, wherein said culture medium includes 15 mg/L MgSO₄7H₂O, 15 mg/L FeSO₄7H₂O, 5 mg/L CaCl₂ and 500 mg/L NaCl in 1 mM potassium phosphate buffer at pH 6.5.

23. The method of claim 13, wherein said culture medium is 1 mM potassium phosphate buffer at pH 8.0 in sterile seawater.