WO2007093993A2

WO2007093993A2 - Microbial compositions useful for the degradation of hydrocarbons

Info

Publication number: WO2007093993A2
Application number: PCT/IL2007/000203
Authority: WO
Inventors: Eugene Rosenberg; Eliora Ron
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2006-02-14
Filing date: 2007-02-14
Publication date: 2007-08-23
Also published as: WO2007093993A3

Abstract

The present invention relates in general to the field of hydrocarbon degradation, and more particularly, to environmentally safe bacterial compositions useful for cleaning and treating hydrocarbon-contaminated water and surfaces. The compositions have utility in degrading hydrocarbons, including petroleum, in seawater and on surfaces.

Description

MICROBIAL COMPOSITIONS USEFUL FOR THE DEGRADATION OF HYDROCARBONS

FIELD OF THE INVENTION The present invention relates in general to the field of degradation of hydrocarbons, and more particularly, to environmentally safe compositions useful for cleaning and treating hydrocarbon-contaminated water and surfaces. The compositions have utility in degrading soluble and insoluble hydrocarbons, including petroleum, in bilge, ballast, drainage water and in holding and storage compartments, including on-shore tanks, oil pipelines, and cargo, bilge and slop tanks of maritime vessels.

BACKGROUND OF THE INVENTION

Hydrocarbon based Pollution

Petroleum fuels and potable water are two of the world's most important resources, with the demand for both continually increasing. One of the most economical methods of transporting liquid petroleum fuels, such as crude oil, fuel oil, heavy diesel oil and lubricating oil, is by maritime carriers and, unfortunately, this has led to considerable pollution problems on the high seas and on waterways. This pollution problem has been intensified by the use of carriers with enormous capacity, including the Very Large Crude Carrier (VLCC) of 200,000 to 299,999 dead weight ton (dwt) and the Ultra Large Crude

Carrier (ULCC) of 300,000 to 550,000 dwt.

While water pollution occurs through accidental oil spills, an equally serious source of pollution results from petroleum fuel that is intentionally discharged by carriers. According to International maritime law, the shipping industry is responsible for discharging seawater that is hydrocarbon free. Three of the major sources of this polluted water are bilge water, ballast water and drainage water. Ballast and bilge water originate from maritime vessels while drainage water originates from the crude oil that is stored in tanks on shore.

Others sources of hydrocarbon contaminated water, include inter alia water discharged from a marine vessel's bilge, wastewater collected from cleaning on-shore oil storage tanks, the water by-product from the oil refining process, water used to raise oil and gas from oil wells, and the wash-water from a wide range of machine repair shops. In some ships residual oil can mix with ballast water to create an oily water mixture that requires treatment prior to discharge into the ocean.

International maritime conventions delineate the levels of hydrocarbons allowed in water to be discharged ("effluent"). Within territorial waters, domestic legislation and regulation govern the maximum permitted levels of hydrocarbon content for off-shore maritime discharge of this polluted water. For example, regulations of the International Convention for Prevention of Pollution of the Seas by Oil (1954, and amendments thereto) specify (1) that the instantaneous rate of oil discharge should not exceed 10 liters per nautical mile, (2) that the total quantity discharged should not exceed 1/15,000 of the total cargo-carrying capacity of the discharging vessel, and (3) that the tanker should not be less than 50 miles from the nearest land at the time of discharge. International law requires that water released from a tanker contain less than 15-ppm oil. Hydrocarbon-contaminated water is highly contaminated with soluble hydrocarbons, which are not detectable by currently used methods. In particular, the soluble hydrocarbons benzene, toluene and xylenes, are the most toxic components of the hydrocarbon mixes typical of crude oil, and are commonly present in concentrations many times higher than the concentrations permitted by regulation and convention for discharge of the non-soluble, far less toxic, suspended hydrocarbon droplets.

An important consideration in the washing of oil cargo tanks following oil removal relates to the risk of serious explosions and fires. Despite the efforts made at the oil-terminal ports to completely unload the oil from the marine cargo tanks, residual oil on the tank walls and floor poses an explosion hazard, and requires that the oil cargo tanks be filled with inert gas until the tank is either refilled with crude or cleaned prior to entry into dry-dock. The washing techniques in very large crude carriers involve the use of light oil for an initial wash, delivered through high velocity rotating jets, and followed by a similarly delivered stream of cold, clean, unrecirculated seawater, usually at flow rates of approximately 180 tons per hour at 140 psig. The disintegration of the water jet on the tank walls has been shown to give rise to a cloud of charged water droplets, which are believed to be responsible for the ignition of the explosive atmosphere. Earlier investigations had indicated that forced ventilation of the cargo tanks before and during the cleaning would reduce the danger of explosions. However, explosions have occurred in very large crude carriers even though the oxygen levels in the atmosphere in the crude oil cargo tanks was kept below the lower explosive limit. It would be highly desirable to eliminate these electrostatic hazards.

Crude oil is an impure product containing insoluble solids and sludge, and the heavy deposits formed on the tank surfaces necessitate stringent cleaning methods. One solution is the manual cleaning of the post-wash residuals that remain adhering to the container surfaces and crevices; another solution is use of very low pressure water containing chemical detergents. However, the toxicity of the chemical detergents on marine life can cause at least as much damage as the oil itself. Current thinking is that the use of chemical detergents would add to the pollution problem, unless the cleaning operation was carried out at a shore facility having regulated disposal procedures.

Microbial Hydrocarbon Degradation The use of microorganisms for degrading hydrocarbons has been described in the art.

US Patent No. 3,769,164 teaches a process for the microbial degradation of petroleum using specific Nocardia bacterial species, and certain yeast species and filamentous fungi.

US Patent No. 3,941,692 to some of the inventors of the present invention teaches a process for removing oil from sea water in ship compartments comprising the use of a species of Arthrobacter species in combination with nitrogen and phosphorous sources.

Rosenberg (App. Microb., 24(3):363, 1972) discloses factors affecting the microbial degradation of crude oil in seawater.

US Patent No. 5,780,290 to some of the inventors of the present invention teaches hydrocarbon degrading bacterial strains that utilize urea-formaldehyde resins as a nitrogen source.

International Patent application WO 2004/094316 to some of the inventors of the present invention relates to a petroleum-degrading bacteria using uric acid as a nitrogen source. The bacteria belong to the Alcanivorax and Acinetobacter genera. US Patent application publication no. 2005/0106702 relates to methods for the microbial degradation of petroleum pollutants including polyaromatic hydrocarbons (PAHs) using bacterial isolates from an oil refinery field. Certain of those isolates have the ability to produce useful biosurfactants, which contribute to the remediation properties of the bacteria. Acinetobacter (sp El l) was shown to use long chain aliphatic hydrocarbons as a substrate, while aromatic and cyclic hydrocarbons inhibited growth (Razak, et al., Acta biotechnol., 19(2):213, 1999). Hedlund et al., disclose the isolation of a novel genus and species of marine bacterium that degrade polycyclic aromatic hydrocarbons, Neptunamonas naphthovorans (Appl. Environ. Microb., 65(1):251, 1999). There remains an unmet need for safe and economical methods of cleaning hydrocarbon polluted surfaces and water, in particular bilge water, ballast water and drainage water. Novel bacterial isolates capable of degrading both the soluble and insoluble hydrocarbons are desired.

SUMMARY OF THE INVENTION

The present invention provides compositions useful for degrading hydrocarbons in water and on surfaces. In particular, novel bacterial isolates are provided that are capable of degrading both the soluble and insoluble components of crude oil under aerobic conditions, thus facilitating the decontamination of bilge water, ballast water and drainage water prior to release into the sea and the cleaning of marine vessel compartments and tanks, coastal oil and water-oil storage and holding tanks, oil pipelines, on-shore oil storage tanks, and the like.

According to one aspect the present invention provides novel bacterial strains useful for the degradation of hydrocarbons. In one embodiment the present invention provides a novel bacterial isolate belonging to the Idiomarina genus (strain no. ASHl), having a 16S rRNA sequence set forth in SEQ ID NO:1; a novel bacterial isolate belonging to the Rhodobacteriacea genus (strain no. ASH2), having a 16S rRNA sequence set forth in SEQ ID NO:2; a novel bacterial isolate belonging to the Alter omonas genus (strain no. OKI), having a 16S rRNA sequence set forth in SEQ ID NO:3; and a novel bacterial isolate belonging to the Alcanivorax genus (strain no. ASH4), having a 16S rRNA sequence set forth in SEQ ID NO:4. Each of the isolates was found to be effective in degrading hydrocarbons in seawater.

Surprisingly the bacteria are able to degrade soluble and insoluble hydrocarbons in the presence of other organic and inorganic chemicals, which may be considered detrimental to bacteria and other life forms.

In one embodiment the present invention provides a variant bacterial strain having a 16S rRNA polynucleotide sequence at least 99.5% homologous to the 16S rRNA sequence set forth in any one of SEQ ID NO: 1-SEQ ID NO:4. In another aspect, there is provided an inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, and mixtures thereof. In one embodiment the bacterial inoculum comprises a single bacterial isolate. In other embodiments the bacterial inoculum comprises plurality of isolates selected from SEQ ID NOS: 1-4. In one particular embodiment the bacterial inoculum comprises a mixture of the four isolated bacterial strains according to the present invention. In one embodiment the bacterial inoculum is provided as a liquid. In other embodiments the bacterial inoculum is provided as a powder.

Another aspect of the present invention provides a method for the degradation of hydrocarbons in hydrocarbon-contaminated seawater comprising the steps of a. providing a bacterial inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, and mixtures thereof; and b. contacting said hydrocarbon-contaminated water with said bacterial inoculum under conditions suitable to degrade said hydrocarbon. In one embodiment the method further comprises the step of providing a source of nitrogen and a source of phosphorous. In some embodiments the method further comprises providing an oxygen source. In another embodiment the bacterial inoculum comprises bacterial isolates having a 16S rRNA sequence SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4.

In some embodiments the hydrocarbons refer to petroleum. In other embodiments petroleum refers to crude petroleum or refined petroleum.

In some embodiments of the present invention the hydrocarbon-contaminated water is selected from bilge water, ballast water and drainage water. In preferred embodiments the present invention provides a method for cleaning hydrocarbon contaminated ballast water, bilge water or drainage water to a final hydrocarbon concentration of less than about 50 ppm hydrocarbons. In one embodiment the final hydrocarbon concentration is less than about 20 ppm, preferably less than a concentration of less than 10 ppm.

In one embodiment the method for the degradation of hydrocarbons is carried out at a temperature in the range of about 1O⁰C to about 35°C. In some embodiments the temperature is about 15⁰C to about 3O⁰C. In other embodiments the temperature is about 20⁰C to about 28°C.

In another aspect the present invention provides a method for the degradation of hydrocarbons on hydrocarbon-contaminated surfaces. In one embodiment the method of cleaning a hydrocarbon contaminated surface comprise the steps of a. exposing the hydrocarbon contaminated surface to salt water; and b. contacting said hydrocarbon-surface with a bacterial inoculum, the bacterial inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4 under conditions suitable to degrade said hydrocarbons. In one embodiment the hydrocarbon-contaminated surface is exposed to salt water and the bacteria are brought into contact with the surface. In other embodiments the bacteria are diluted in salt water and the surface is exposed to the bacteria-salt water mixture.

In one embodiment the hydrocarbon-contaminated surface is selected from the surfaces of a cargo tank, slop tank, a coastal tank or an oil-pipeline. In one embodiment the method further comprised the step of providing a source of nitrogen and a source of phosphorous. In some embodiments the method further comprises providing an oxygen source.

In some embodiments the bacteria come into contact with the hydrocarbons by circulating through the tank. In some embodiments the bacteria-treated saltwater circulates through the tank. In one embodiment the saltwater containing the bacteria circulates by means of a spraying mechanism. In another embodiment the spraying mechanism is a rotary sprayer. In some embodiments the circulating means is placed in the tank. In another embodiment the container is filled with water and the water agitated.

In one embodiment the tank is filled to about 100%, in other embodiments the tank is filled to about 50% volume capacity. In yet other embodiments the tank is filled to about 20% volume capacity, preferably the tank is filled to about 5% volume capacity.

These and other embodiments of the invention will be apparent upon reference to the following description.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, it has been discovered that soluble and insoluble hydrocarbons present in bilge water, ballast water and drainage water can be converted wholly or in part to non-oily, non-toxic products by means of microbial action, enabling the subsequent discharge of treated water that meets maritime regulatory and local jurisdiction environmental standards. Furthermore, hydrocarbon holding compartments, including cargo compartments and on-shore holding tanks can be cleaned efficiently and in a manner obviating the risk of explosions and fires. It is a feature of the invention that bacterial cultures are used to convert both the soluble and the insoluble hydrocarbons to non-toxic products. This action can take place in any site but preferably takes place in a maritime vessel compartment, in a coastal hydrocarbon-holding tank or a contaminated water treatment tank. Preferred maritime vessel compartments include ballast tanks, bilge tanks, slop tanks, cargo tanks, oil/water separation tanks, on-board specialized decontamination tanks, and the like.

Furthermore the bacterial cultures are able to degrade soluble and insoluble hydrocarbons in the presence of other organic and inorganic chemicals that would be considered detrimental to bacterial survival. Although the use of microbial cultures to degrade hydrocarbons is known, the degradation of hydrocarbons by marine microorganisms has been investigated primarily in relationship to their effect on oil slicks that pollute the seas. It has been known that the degradation of oil slicks occurs naturally; however, the process is very slow and is rarely of any immediate value in preventing danger to marine life or pollution of the coastal areas. Once hydrocarbons have been discharged into the sea, and an oil slick formed, there is little that can be done except to attempt to confine and remove the oil. Methods of treating spilled oil are costly and rarely successful. It is one of the objects of this invention to provide a simple, economical and environmentally safe procedure for preventing hydrocarbon based water pollution by treating bilge, ballast and drainage water prior to its release into bodies of water, including rivers, seas and oceans.

The bacterial strains of the present invention are novel isolates of marine bacteria that have been include a novel bacterial isolate belonging to the Idiomarina genus, having a 16S rRNA sequence set forth in SEQ ID NO:1; a novel bacterial isolate belonging to the Rhodobacteriacea genus, having a 16S rRNA sequence set forth in SEQ ID NO:2; a novel bacterial isolate belonging to the Alteromonas genus, having a 16S rRNA sequence set forth in SEQ ID NO:3; and a novel bacterial isolate belonging to the Alcanivorax genus, having a 16S rRNA sequence set forth in SEQ ID NO:4. Each of the isolates was found to be effective in degrading soluble and or insoluble hydrocarbons in seawater. These strains were deposited with the National Collections of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen, Scotland, pursuant to the provisions of the Budapest treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.

The bacterial isolate belonging to the Idiomarina genus was deposited on January 12, 2006 and was given accession number NCIMB 41371; the isolate belonging to the Rhodobacteriaceae was deposited on January 12, 2006 and was deposited on January 12, 2006 and was given accession number NCIMB 41372; the isolate belonging to the Alteromonas genus was deposited on January 4, 2006 and was given accession number NCIMB 41370; the Alcanivorax isolate was deposited on January 12, 2005 was deposited on January 12, 2006 and was given accession number NCIMB 41373.

Definitions

For convenience, certain terms employed in the specification, examples and claims are described herein.

The term "hydrocarbons" is meant to include chemicals comprising hydrogen and carbon as well as hydrocarbon derivatives. Water-soluble and water-insoluble hydrocarbons are included. Hydrocarbons include petroleum (crude and refined), oils including fuel oil, diesel oil, gasoline, hydraulic oil, and/or kerosene. The products benzene, toluene, ethylbenzene and xylenes 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 hydrocarbons of the present invention. Hydrocarbon derivatives include alcohols, aldehydes, ethers, carboxylic acids, and halocarbons.

The physical properties and exact chemical composition ("fingerprint") of crude oil (petroleum) varies from one venue to another. Therefore the origin of petroleum can be identified by its hydrocarbon and hydrocarbon derivative content. Petroleum comprises, for example aliphatics including straight chain, branched and cyclic alkanes (cycloalkanes) and cycloalkanes having multiple rings and or alkyl side chains; aromatics such as benzene and structural derivatives thereof and polyaromatic hydrocarbons (PAH); resins; and asphaltenes.

The term "ppm" as used in this document is a measure of the level of hydrocarbon pollution in parts per million, using acceptable methods and measuring devices. It is to be noted that methods and devices utilized by the various regulatory bodies controlling the permitted levels of hydrocarbon pollutants release into the environment are not able to measure the soluble fraction of hydrocarbons in the hydrocarbon-contaminated water.

"Total hydrocarbons" includes both soluble and insoluble fractions of hydrocarbons. Typical contaminating soluble hydrocarbons include benzene, xylenes and toluene (also known as "BXT").

Often hydrocarbon-contaminated water, in particular the drainage water, is highly contaminated with soluble hydrocarbons. Presently, the methods and equipment used to measure hydrocarbons in the discharge water does not measure these toxic contaminants, which are freely released into the environment. The term "bacterial inoculum" refers to a composition comprising one or more bacterial strains of the present invention. The bacterial inoculum can be liquid or dry. For example, the bacteria can be provided in a nutrient broth or alternatively provided as a dehydrated or lyophilized powder. After the inoculum is added to water, the mixture of inoculum and water is referred to as "bacteria-treated water". The bacterial species of the present invention are aerobic bacteria. Preferably the oxygen content in the water is at least about 5% oxygen (0.4 mg per liter). Methods and devices for the aeration of water are known in the art.

The term "salt water" refers to water having a salt (sodium chloride) concentration of about 0.5% to about 4%. Salt water is meant to encompass both natural salt water and artificially produced salt water. On average, sea water in the world's oceans and seas has a salinity of about 3.5%. Sodium chloride makes up about 98% of the salt in the oceans and seas. The bacterial isolates of the present invention can tolerate a salt concentration from about 0.5% to about 4%, and function equally well in artificially prepared salt water, natural sea water and dilute sea water. Bacteria belonging to a "bacterial strain" refer to bacteria having identical or essentially identical 16S rRNA polynucleotide sequences. Essentially identical refers to a bacterial variant having an 16S rRNA sequence deviating by less than 0.5%, preferably less than 0.3% and more preferably less than 0.1% from the 16S rRNA sequences set forth in SEQ ID NO:1-SEQ ID NO:4, and retaining hydrocarbon degradation activity. A bacterial variant can have one or more base substitutions, deletions and or insertions compared to the sequences set forth in SEQ ID NO: 1 -SEQ ID NO:4. The bacteria are non-pathogenic and non-opportunistic.

The term "bilge water" refers to water in the bilge tank of a maritime vessel. The bilge water typically includes crude oil, gasoline, motor oil and/or diesel fuel, which can create hazardous conditions on board.

The term "ballast water" refers to the water used by ships to maintain stability. Large ships often carry millions of gallons of ballast water in ballast tanks. This water is characteristically contaminated with oil and sludge. The term "drainage water" refers to water that has been separated via gravitation from the crude hold held in a maritime or on-shore hydrocarbon holding compartment.

Other sources of hydrocarbon contaminated water include oil-well water, post- refinery water, oil-water mixes from machinery repair facilities and oil-water mix from cleaning fuel tanks and fuel lines. Bilge waster, ballast water and drainage water are contaminated with soluble and insoluble hydrocarbons and must be decontaminated before discharge to meet international and national maritime and other regulatory demands.

It is to be understood that methods of the present invention are not limited to maritime industries and are meant to encompass methods for the degradation of hydrocarbons originating in different industries including the railroad, airline, and land transport industries, as well as the oil storage, transportation, refining and fuel-dispensing industries. Embodiments of the Invention

The present invention provides bacterial isolates and compositions comprising at least one said isolate and methods of use thereof for the degradation of hydrocarbons in contaminated water and compartments. International laws delineate the levels of hydrocarbons allowed in discharge water.

For example, regulations of the International Convention for Prevention of Pollution of the Seas by Oil (1954, and amendments thereto) specify (1) that the instantaneous rate of oil discharge should not exceed 10 liters per mile, (2) that the total quantity discharged should not exceed 1/15,000 of the total cargo-carrying capacity, and (3) that the tanker should not be less than 50 miles from the nearest land at the time of discharge. International law requires that water released from a tanker contain less than 15ppm hydrocarbons.

Treatment of Hydrocarbon Contaminated Water

In some embodiments the present invention provides a method for cleaning hydrocarbon contaminated ballast water, bilge water or drainage water, the method comprising the steps of providing an inoculum of bacteria at least one bacterial strain according to the present invention and exposing the hydrocarbon contaminated ballast water, bilge water and drainage water to the bacteria for a sufficient time to degrade the hydrocarbon.

Ballast water, bilge water or drainage water can be collected in a maritime vessel compartment, an off-shore compartment or an on-shore compartment. Bacteria are added to the hydrocarbon-contaminated water and are allowed to degrade the hydrocarbons until a desired level of hydrocarbon remains. In some embodiments a source of nitrogen and a source of phosphorous are added to the water. In other embodiments the water temperature can be adjusted to about 10°C to about 35°C, preferably about 15⁰C to about 3O⁰C and more preferably about 20⁰C to about 28⁰C.

In some embodiments a continuous flow system is desired and in other embodiments a batch system of cleaning is preferred. For example, a continuous flow system may be preferred for the treatment of drainage water, wherein hydrocarbon contaminated water flows into a compartment comprising an inoculum of hydrocarbon degrading bacteria, and treated water flows out.

Since the bacteria are harmless to people and the environment they may be released into the environment with the treated water. In some embodiments the bacteria will be collected. In other embodiments it may be desired to destroy the bacteria following the treatment process.

Treatment of Hydrocarbon Contaminated Compartments

The compartments in which hydrocarbons or hydrocarbon-contaminated water are carried or held can also be cleaned according to the methods of the present invention. In one embodiment the method of cleaning a surface in a hydrocarbon contaminated tank comprise the steps of a) exposing the hydrocarbon contaminated surface to salt water; and b) contacting said hydrocarbon-surface with a bacterial inoculum, the bacterial inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO: 1 ; SEQ ID NO:2;

SEQ ID NO:3 and SEQ ID NO:4 under conditions suitable to degrade said hydrocarbons.

In one embodiment of the invention, seawater is added to the compartment tank, after it has been emptied of its hydrocarbons or hydrocarbon-contaminated water. The seawater is added primarily as a vehicle for the subsequent microbial fermentation process and no attempt is made at this point to wash the compartment. The water is preferably added under low pressure without jet nozzles in order to avoid any impingement on the tank walls and resultant buildup of electrostatic charges. Seawater typically contains about 3% salts with relatively low nitrogen and phosphorus levels. Additional nitrogen and phosphorus are added to the water, preferably in the form selected from KH₂PO₄, K₂HPO₄, NH₄Cl, (NHL_t)₂SO₄, NH₃, NaNO₃, urea, and the like. The required concentration of the nitrogen and phosphorous depends on the approximate concentration of hydrocarbon in the water or the amount of hydrocarbon on a surface: i.e. a range of about 1 gram to about 25 gram of a phosphorous source and about 4 gram to about 100 gram nitrogen source per 100 gram hydrocarbons to be degraded. In some embodiments the range is about 2.5 gram to about 10 gram phosphorous and about 10 gram to about 40 gram nitrogen per 100 gram hydrocarbon. Preferably, in order to degrade 100 gram hydrocarbons, about 5 gram phosphorous and about 20 gram nitrogen are added to the sea water. Depending on the culture(s) and process, it may also be desirable to add small amounts of carbohydrate sources such as glucose, starch and the like, and small amounts of other nitrogen sources such as corn steep liquor, peptone, meat extract, yeast extract, fish solubles and the like. The mixture of hydrocarbons, nutrients and water is then used without sterilization. A preferred suitable bacterial inoculum is then added and the fermentation carried out. In some embodiments aeration is provided. It is important that a suitable bacterial inoculum is available so that the fermentation results in a relatively rapid growth of the bacteria concentration and consequently a very rapid degradation of the hydrocarbons.

The fermentation is carried out for a period of approximately 4 hours to about 6 days, during which time the microbial organisms multiply and the hydrocarbons present in the mixture degraded.

Aeration of the water can be carried out directly in the compartment or by a circulating system connected to the compartment. After a suitable period of microbial growth, the hydrocarbons in the mixture are converted, all or in part, to non-oily products. The fermented mixture is then discharged into the sea, compliant with international treaty at ppms below the permitted discharge levels, and thus without evidence of a typical oil slick in the wake of the vessel. Land based use of the process would discharge either into coastal sea waters, at or near the salt/fresh water interface in rivers, or into coastal settling or seepage ponds with the consequence that the water reaches the aquifers.

The portions of the compartment wall in contact with the fermenting mixture are relatively clean, without any typical sludge, and with only a small amount of hydrocarbons, which is easily washed off with a low pressure water hose.

Alternatively, the fermented mixture in the compartment is not immediately emptied, and serves as ballast during the return voyage. The fermented mixture-ballast is discharged prior to the end of the voyage, and the compartments given a low pressure water rinse to prepare them for the next load of petroleum or oil, or in preparation for "gas free' repairs at dry-dock. Combining the tank cleaning and the ballast step into one economical and safe operation is a very desirable feature of the invention.

In one embodiment the oil from the engine room serves as a substrate for the bacterial strains of the present invention. Oil from the engine room can be washed into the bilge tank, slop tank or another compartment and treated with one or more of the isolated bacterial strains having 16S rRNA selected from SEQ ID NO: 1-4.

In another modification of the invention, the bacteria present in the fermented mixture are harvested by any of the procedures known to the art, as for example, centrifugation, and the clarified supernatant discharged. Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES Example 1 : Novel Bacteria for the Treatment of Oil Polluted Seawater

Four novel bacterial strains were isolated and shown to be effective in the degradation of hydrocarbons.

Isolation of Bacteria

Fresh Mediterranean seawater off the coast of Tel Aviv, Israel served as the source of bacteria. 100 ml the fresh seawater was mixed with 100 ml drainage water containing 180 ppm TPH (total petroleum hydrocarbon). The method used to determine TPH throughout this research was the standard method (EPA 418.1). To the 200 ml mixture in a 1 -liter flask 10 mg urea and 5 mg K₂HPO₄ were added. The enrichment medium flask was aerated by shaking at 150 rpm at 25⁰C for 4 days. The liquid that initially appeared brown turned colorless after 3 days. Microscopic observation indicated that the 4 day culture contained ca. 3 x 10⁷ bacteria per ml. The culture was diluted l:10⁵ in sterile seawater and 0.1 ml spread on seawater agar (about 88% seawater, 10% distilled water, 2% agar, 0.1% urea and 0.02% K₂HPO₄). The Petri dish was inverted and 0.1 ml crude oil placed on the lid. After incubation for 5 days at 25⁰C, colonies were picked, re-streaked on seawater agar and tested for their ability to grow on the crude oil/seawater enrichment medium (described above). The four most abundant strains that grew well on the enrichment as pure cultures are referred to herein ASHl (SEQ ID NO:1), ASH2 (SEQ ID NO:2), OKI (SEQ ID NO:3) and ASH4 (SEQ IDNO:4). Bacterial Classification and Deposit

The genomic DNA from 2 ml of an overnight culture of each of the strains ASHl₅ ASH2, OKI and ASH4 was obtained using the Wizard DNA genomic purification kit (Promega Corp., Madison, WI). The extracted DNA was purified by the drop dialysis procedure and was used as the template in a PCR reaction to amplify the 16S rRNA DNA. The 50 μl reaction consisted of 5 μl 10 x buffer, 1 μl 2.5 mM total dNTP mixture, 5 μM of each primer, 10 ng (nanogram) template DNA and 2.5 units Ex Taq DNA polymerase (Takara Shuzo). The primers used were 5'-AGAGTTTGATCMTGGCTCAG-S' and 5'- TACGGYTACCTTGTTACGACTT-3'. Amplification conditions for the PCR included an initial denaturation step at 95°C for 3 min followed by 30 cycles at 94°C for 1 min, 55°C for 1 min and 72⁰C for 1 min and a final extension step at 72°C for 10 min. Reaction products were checked for size and purity on 1% agarose gels and the DNA fragments were then recovered with a QIAquick PCR purification kit (Qiagen). DNA sequencing was performed using the chain-termination method in an ABI Prism (model 377, version 2.1.1) automatic sequencer. Primers used for the sequencing reaction were complementary to the conserved regions of the 16S rRNA DNA. Sequences were subjected to the BLAST basic local alignment search tool in the GenBank database (web site: www.ncbi.nlm.nih.gov/blast^') to determine the most likely identity of the strain.

Based on their 16S rRNA sequences, strains ASHl, ASH2, OKI and ASH4 are novel species (SEQ ID NOS: 1-4), belonging to the Idiomarina, Rhodobacteriacea, Alteromonas and Alcanivorax genera, respectively. The bacterial strains have been deposited with the NCIMB Depository and have been assigned deposit reference numbers NCIMB 41371 (ASHl, SEQ ID NO: 1); NCIMB 41372 (ASH2, SEQ ID NO:2); NCIMB 41370 (OKI, SEQ ID NO:3); and NCIMB 41373 (ASH4, SEQ ID NO:4). Example 2: Treatment of Drainage Water with the Mixture of ASHl. ASH2. ASH4 and OKI

Drainage water was removed from petroleum storage tanks by the Eilat-Ashkelon Pipeline Company. One cubic meter of drainage water in a plastic tank was inoculated with a mixture of ASH 1-4 so that the initial concentration of each strain was present at 10⁴ per ml. Urea (50 g) and K₂HPO₄ (20 g) were added and the tank aerated at the rate of 10 1/min. The temperature in the tank varied from about 24°C to about 26⁰C. The experiment was carried out in duplicate with two different batches of drainage water, one that contained initially 180-ppm soluble and insoluble hydrocarbons and the other 10-ppm. A control in which no bacteria were added was also performed. The Results are provided in Table 1.

Table 1. Microbiological treatment of drainage water

In addition to the efficient degradation of TPH (total petroleum hydrocarbons), the water, which was initially brown, became colorless. TPH includes both the soluble and insoluble fractions of hydrocarbons.

Example 3: Cleansing of hydrocarbon-contaminated surfaces

Hydrocarbon contaminated surfaces can be cleaned by contacting the surface with a bacterial inoculum and salt water. The bacterial inoculum is preferably diluted into a volume of saltwater in a hydrocarbon-contaminated tank. In a non-limiting example, a 20 meter deep tank is filled with salt water to about 50 cm deep. Bacterial inoculum is added to the saltwater and circulated through the tank by a water pump that carries the water to a sprayer, which rotates and sprays the mixture on the ceiling and sides of the tank. As the hydrocarbons are exposed to the bacteria-salt water mixture they are degraded into non-toxic material.

Example 4: Cleaning of a cargo tank The starboard slop tank (Tank A) and the port slop tank (Tank B) of a 120,000 ton oil carrier are carefully cleaned prior to taking on a cargo of crude oil. These slop tanks measure about 12 meters x 5 meters x 25 meters (depth). After cleaning, and prior to taking on the oil cargo, Tank A is fitted with a rotating spray system. Tank B is fitted with an aeration system. The tanker discharges its cargo of crude oil at Eilat in the normal manner, and Tanks

A and B are not cleaned. Seawater was added to both tanks. The total volume of liquid in Tank A was 10 cubic meters and that in Tank B 121 cubic meters. A thick layer of oil could be seen floating on the surface of the solutions both tanks.

Urea (20 kilograms) and K₂HPO₄ (1 kilogram) are dissolved in seawater and added to Tank A, and similar amounts to Tank B. Tank A and Tank B are inoculated with a suspension of bacteria containing a total of 10¹² bacterial cells. Air is introduced into Tank B at the rate of 1-3 cubic meters per minute. The spray system sprays the seawater throughout tank A and recycles the water. As the water flows down the side of the tank the water is siphoned into the sprayer again. The tanks are sampled at the start of the test, and every day thereafter.

While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein.

Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

Claims

1. An isolated bacterial strain having a 16S rRNA polynucleotide sequence set forth in SEQ ID NO:1; the bacterial strain having accession number NCIMB 41371.

2. An isolated bacterial strain having a 16S rRNA polynucleotide sequence set forth in SEQ ID NO:2, the bacterial strain having accession number NCIMB 41372.

3. An isolated bacterial strain having a 16S rRNA polynucleotide sequence set forth in SEQ ID NO:3, the bacterial strain having accession number NCIMB 41370.

4. An isolated bacterial strain having a 16S rRNA polynucleotide sequence set forth in SEQ ID NO:4, the bacterial strain having accession number NCIMB 41373.

5. A isolated variant bacterial strain having a 16S rRNA polynucleotide sequence at least 99.5% homologous with the sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

6. A bacterial inoculum comprising at least one bacterial strain according to any of claims 1-5.

7. The bacterial inoculum according to claim 6 comprising a plurality of bacterial strains according to any one of claims 1-5.

8. The bacterial inoculum according to claim 6 comprising the bacterial strains having 16S rRNA sequences according to SEQ ID 1-4.

9. The bacterial inoculum according to claim 6 wherein the inoculum is provided in a form selected from liquid or powder.

10. A method for degrading hydrocarbons in salt water, said method comprising the steps of

a) providing a bacterial inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4, a variant thereof having at least 99.5% sequence identity and mixtures thereof; and

b) contacting said hydrocarbons with said bacterial inoculum under conditions suitable to disperse said hydrocarbon.

11. The method according to claim 10 further comprising the step of adding a source of nitrogen and a source of phosphorous.

12. The method according to claim 10 further comprising the step of adding an oxygen source.

13. The method according to claim 10 wherein the bacterial inoculum comprises four bacterial isolates having 16S rRNA sequences set forth in

SEQ ID NO-.l ; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4 and variants thereof having at least 99.5% sequence identity.

14. The method according to claim 10 wherein hydrocarbons are selected from soluble and insoluble hydrocarbons.

15. The method according to claim 14 wherein the insoluble hydrocarbons are selected from crude petroleum and refined petroleum.

16. The method according to claim 10 wherein the hydrocarbon contaminated seawater is selected from the group consisting of bilge water, ballast water and drainage water.

17. The method according to claim 16 wherein the hydrocarbons are degraded to a final hydrocarbon concentration of less than about 50 ppm.

18. The method according to claim 16 wherein the final hydrocarbon concentration is less than about 20 ppm.

19. The method according to claim 16 wherein the final hydrocarbon concentration is equal to or less than 10 ppm.

20. The method according to claim 10 wherein the method is carried out at a temperature in the range of about 10°C to about 35⁰C.

21. A method for the degradation of hydrocarbons on hydrocarbon contaminated surfaces comprising the steps of

a) exposing the hydrocarbon contaminated surface to salt water; and b) contacting said hydrocarbon-surface with a bacterial inoculum, the bacterial inoculum comprising at least one bacterial isolate having a 16S rRNA sequence selected from the group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4 under conditions suitable to degrade said hydrocarbons.

22. The method according to claim 21 wherein the surface is selected from the group consisting of a cargo tank, a slop tank and a coastal tank.

23. The method according to claim 21 wherein the tank is filled to about 50% volume capacity.

24. The method according to claim 21 wherein the tank is filled to about 20% volume capacity

25. The method according to claim 21 wherein the tank is filled to about 5% volume capacity.

26. The method according to claim 21 further comprising the step of circulating the salt water in the tank.

27. The method according to claim 26 wherein circulating the salt water containing the bacteria comprises circulation by means of a spraying mechanism

28. The method of claim 27 wherein the spraying mechanism comprises a rotary sprayer.

29. The method according to claim 219 wherein the method is carried out at a temperature in the range of about 10°C to about 35°C.

30. The method according to claim 21 wherein the method is carried out at a temperature in the range of about 15°C to about 30°C.

31. The method according to claim 21 wherein the method is carried out at a temperature in the range of about 20°C to about 28⁰C.