IL100485A - Compositions for degrading hydrocarbons and bacterial enzymes therefor - Google Patents

Description

6.038 100 , 48513 NON POLLUTING COMPOSITIONS TO DEGRADE HYDROCARBONS AND BACTERIA FOR USE THEREOF 100 , 485 / 2 A B S R A C T The present invention relates to compositions containing bacteria capable of degrading hydrocarbons, such as petroleum or petroleum products, and utilizing complex insoluble organic nitrogen sources wherein the organic nitrogen molecules of the complex nitrogen-containing material is not utilizable by most soil and water microorganisms, a method of enhancing the b i odegr ada t i on of petroleum and biologically pure cultures of bacteria. 100,485/2 BACKGROUND OF THE INVENTION The present invention relates to compositions containing bacteria capable of degrading hydrocarbons, such as petroleum or petroleum products, and complex nitrogen-containing materials such as ur e a- f orma 1 dehyde resins and other compatible polymers as the source of nitrogen, a method enhancing the bi odegr ada t i on of hydrocarbons and biologically pure cultures of bacteria.

Bacterial degradation of petroleum hydrocarbons has been known and recognized for decades. The subject has been reviewed comprehensively in the literature, examples being CRC Critical Reviews in Biotechnology, Volume 3, Issue 3, "Microbial Sur f ac t an t s " by E. Rosenberg and "Report on the 1991 Oil Spill Conference", San Diego, California, 4-7 March 1991 and references cited therein, whose contents are incorporated by their mention.

Reports have been published that show the b i or emed i a t i on of hydrocarbons in closed vessels (as described in US 3,941,692) is effective, using almost any source of water-soluble inorganic nitrogen and phosphorous. However, studies have shown that bio- remediation of oil on the open seas or on oil-polluted beaches was still a major problem. Initial growth is stopped or slowed down by the natural tendency of most nutrients and fertilizers to diffuse into the water or the adjacent ground and are, thus, under-used by the microorganisms. Said water-soluble nutrient and fertilizers also suffer from the fact that they enable the uncontrolled growth of numerous naturally-occurring microor anisms in the soil and water which then, themselves, become a serious cause of water pollution. -2 - 100, 48513 Various methods have been employed up to the present as described in US 4,401,762, US 4,460,692 and the references cited in the above described report from 1991. While some successes were obtained in the prior reports, the processes of bi odegr ada t i on occurred very slowly over a period of months .

SUMMARY OF THE INVENTION In accordance with the present invention, there are provided novel bacteria which are effective as bi or emed i a t i on agents for petroleum pollution. There is also provided a composition of at least one microorganism capable of degrading hydrocarbons and utilizing complex insoluble organic nitrogen sources, as a source of nitrogen and optionally phosphorous wherein the organic nitrogen molecules of the complex nitrogen - containing material is not utilizable by most soil and water microor anisms in a mixture with a suitable carrier. There is further provided a method of enhancing the biodegradat ion and/or bio- emu 1 s i f i ca t i on of petroleum, which comprises contacting the petroleum with said composition so that the microor anism utilizes the petroleum as a source of carbon, thereby degrading the petroleum, without any substantial growth of the indigeneous competing microorgani sms .

In a specific embodiment of the present invention the said composition contains at least one of the novel bacteria effective as a bi oremed i a t i on agent for petroleum; and uses a ur ea- f orma 1 dehyde resin or other compatible polymers as a source of nitrogen. - 3 - 100, 485 /3 DETAILED DESCRIPTION OF THE INVENTION The novel bacteria were obtained by an enrichment culture procedure using crude oil as the carbon and energy source and a ur ea- f orma 1 dehyde resin, such as UF-1, as the source of nitrogen and phosphorous and sea water or tap water as the source of other minerals. In the case of sea water this afforded a mixed culture. Three different colony types were isolated by restreaking. These strains are referred to as ER-RL3, tentatively identified as Pseudomonas alicaligenes or Al i ca 1 i gene s , ER-RL4, tentatively identified as Pseudo-monadaceas genus Pseudomonas and ER-RT, tentatively identified as Pseudomonadaceae genus Gluconobac t er . 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 on 16 December 1991, under Accession Numbers NCIMB-40464, NCIMB-40465 and NCIMB-40466 for ER-RL3 , ER-RL4 and ER-RT, re spec t i ve 1 y .

In the case of using tap water a mixed culture was also obtained. Two different colony types were isolated by restreaking. These strains are referred to as ER-RLD and ER-RLX, both tentatively identified as strains of Ac i ne t obac t er calcoacet icus . 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 on 8 June 1992, under Accession Numbers NCIMB-40506 and NCIMB-40507 for ER-RLD, ER-RLX respectively. 4 100,485/4 The urea- f orma 1 dehyde (UF) resins of the novel compositions of the present invention can be almost any type of UF f er t i 1 i zer . Ex ampl e s are described in Controlled Release Fertilizers by Sarah P. Lande 1 s . A . Leder and N.Takei, Chemical Economic Handbook, SRI International, 1990, whose contents are incorporated by mention, and especially pages 535.800W - 535.8001F. The UF resins preferably contain 10%-40% nitrogen, 0%-34% phosphorous and 0%-12% potassium, where any UF fertilizer which contains insoluble nitrogen in the range of 5% -40% is most preferred. Examples of preferred fertilizers are UF-1 (12-12-12), Triazin (18-3-4), Fluf (16-2-4), Fluf (10-0-10) plus 10 mM K2HP04 and Haifatert (29-0-0) plus Haifatest (0-34-9).

It has also been discovered that certain cultures produced enzymes - which rapidly converts insoluble high molecular weight UF resins into low molecular weight utilizable nitrogen and phosphorous compounds. These factors can act in the absence of the microbial cells after the cells are disrupted. It is, therefore, a further modification of the invention, that the cell-free supernatant, which is obtained after the cells are harvested from the fermented mixture and are then disrupted, and which contain the UF degrading factor, can be used to provide utilizable nitrogen and phosphorous compounds. It is also part of the present invention, that the cell-free supernatant containing the UF degrading factors in crude or purified form, can be produced in suitable fermentation equipment .

The cell-free supernatant from the bacteria have a urea-formaldehyde utilization activity (UFU) of 200 units, most preferably at least about 400 units, where the UFU activity 5 100 ,485/2 is defined as the catalytic breakdown of high molecular weight insoluble UF resins into low molecular weight units which are water soluble and dialyzable.

While the invention will now be described in connection with certain preferred embodiments in the following examples, it will be understood that it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives modifications and equivalents, as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of 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 procedures as well as of the principles and conceptual aspects of the i nven t i on . 6 100 , 485 /2 EXAMPLE 1 Isolation of petroleum-utilizing bacteria from sea water using UF-1 as the source of nitrogen and phosphorous A mixed bacterial culture was obtained by enrichment culture procedure using crude oil as the carbon and energy source, UF-1 resin as the nitrogen and phosphorous source and sea water as the source of other minerals. The enrichments were carried out in CO-UF1 medium which consists of 0.5% crude oil (v/v), 0.1% UF-1 and sterile filtered sea water. The enrichment was carried out by inoculating 10 ml of the CO-UF1 medium in a 125 ml flask with tar (collected from Tel Baruch Beach, Tel Aviv) and incubating the mixture at 25°C with reciprocal shaking (100 strokes per minute). After 3 days, 1 ml of the culture was transferred to 19 ml of sterile CO-UF1 medium and incubation was carried out as above. The procedure was repeated two additional times, after the third transfer a turbid culture was obtained after inoculation and overnight growth. This culture is referred to as "the mixed culture". Microscopic examination indicated that the mixed culture consisted of several different types of motile and non-motile bacteria. The mixed culture was maintained by weekly transfers to fresh sterile CO-UF1 medium.

Three pure cultures, able to grow on CO-UF1 medium, were obtained from the mixed culture by spreading a dilution of the mixed culture onto marine agar (18.7 g marine broth MA.2216, 1.6 g agar, both from Difco Laboratories, Detroit, Mich., and 10 g NaCl per liter deionized water). 100,485/3 After incubation for 3 days at 25°C, three different colony types were isolated by restreaking on marine agar. These strains are referred to as ER-RL3 , ER-RL4 and ER-RT. Each of the pure strains was able to grow on and emulsify crude oil in CO-UF1 medium.

EXAMPLE 2 Isolation of petroleum-utilizing bacteria from tap water using UF-1 as the source of nitrogen and phosphorous A mixed bacterial culture was obtained by enrichment culture procedure using crude oil as the carbon and energy source, and UF-1 as the nitrogen and phosphorous source. The enrichment was performed in a medium which consisted of tap water containing 0.5% crude oil (v/v), 0.1% UF-1 and 0.04% MgS04 (CO-UFl-2 medium). The source of bacteria was oi 1 -con t amina t ed soil. The soil used was obtained from different locations in the U.S.A. and in Israel. The -6-1995 enrichment was carried out by inoculating 10 ml of the CO-Fl-2 medium in a 125 ml flask and incubating the mixture at 25°C with reciprocal shaking (100 strokes per min). After 3 days, 1 ml of the culture was transferred to 19 ml of sterile medium and incubation was carried out as above. The procedure was repeated two additional times, after the third transfer, turbid cultures were obtained after inoculation and overnight growth. These cultures were referred to as "the mixed cultures". Microscopic examination indicated that each mixed culture consisted of several different types of motile and non-motile bacteria. The mixed cultures were maintained by weekly transfers to fresh sterile medium. 100, 485 /3 Several pure cultures able to grow on CO-UFl-2 medium were obtained from the mixed cultures by spreading a dilution of the mixed culture onto nutrient agar (from Difco Laboratories, Detroit, Mich.). Two different colony types were isolated by res treaking on agar. These strains are referred -6-1995 to as ER-RL-D and ER-RL-X, the latter isolated from US soil. Each of the pure strains was able to grow on and emulsify crude oil in CO-UFl-2 medium.

EXAMPLE Growth of the mixed culture from sea water The kinetics of growth of the mixed culture on crude oil is shown in Fig. 1. The mixed culture had a doubling time of appro imately 2 hours, reaching stationary phase after 40 hours at 2xl08 cells/ml. Growth preceded emul s i f i c a t i on by appro imately 5 hours. The pH of the growth medium decreased from an initial value of 7.8 to a minimum of 6.8 at 24 hours, and then increased to a final value of 7-7.2. The turbidity of the culture rose sharply up to 3200 k.u., mostly because of emul s i f i ca t i on of the oil.

EXAMPLE Dependence of growth of mixed culture from sea water on UF-1 and crude oil The results summarized in Table 1 indicate that growth of the mixed culture is dependent on crude oil as carbon source and on UF-1 as nitrogen source. Similar results were obtained 100 , 485 / 3 with each of the three pure cultures: growth and emul s i f icat ion depended upon the presence of both crude oil and UF-1.

EXAMPLE Dependence of growth of mixed cultures of UF-1 and crude oil.

The results summarized in Table 2 indicate that growth of ER-RL-D is dependent on crude oil as the carbon source and on -6-1995 UF-1 as the nitrogen source. Similar results were obtained with ER-RL-X: Growth and emul s i f icat ion depended upon the presence of both crude oil and UF-1. 10 100,485 / 2 ble 1. ilizatlon of crude oil and UF-1 mixed culture9 from sea water UF-1 Crude oil Turbidity Cell density (%) (%) Ck.u. ) (CFU/ml ) 0, 0 24 OxlO6 0 0.05 75 3x107 0 0.02 97 5 x 10 0 0.05 256 O lO? 0 0.1 435 8xl07 0 0.5 1320 6xl88 0 1.0 1400 0x10" 0 0 , 22 OxlO6 0.05 0 , 800 4xl08 0.2 0 1200 5x108 0.5 0 1250 5 108 1.0 0 2000 OxlO8 1.6 0 2650 OxlO0 a = The experiment was carried out as described in Figure 1, except that the concentrations of UF-1 and crude oil were varied as indicated and the sample time was 48 hours. 11 100, 485/3 TABLE 2 Utilization of crude oil and UF-1 by ER-RL-D.

UF-1 Crude Oil Turbidity Cel 1 dens i ty -6-1995 ( ) (%) EXAMPLE 7 Characterization of fresh water strains with UFU activity From the mixed culture obtained on CO-UF-1-2 medium, two pure strains that could grow on crude oil and UF-1 were isolated and characterized. The properties of these gram-negative strains are described in Table 4. Both strains have the ability to grow and emulsify oil, using UF-1 resin as a nitrogen and phosphorus source. 13 100 , 485 / 2 /fable 3 Characterization of strains from CQ-UP-1 medium Prooertv S t r a i n ER-KL3 ER-RL4 ER-RT Colony of marine agar d i ame t er 2 mm < 1 mm 1-2 mm color whi t e wh i t e ye 11 owi s I Bacterial shape rod rod rod Dimensions of cells (μτα) from marine agar (16h) 0.3/1 0.6/1 0.6/1.0 from broth on oil (72'h) 0.5/1 0.5/2 0.44/1.1 Mo t i 1 i t y + + + Flagellar arrangement from marine agar (16h) peri trichal po 1 ar none from broth on oil (7 h) none po 1 ar none Growth temperature, °C 4°C (lOOh) 20°C-25°C (24h) + 37°C (24h) + 41°C (100h) NaCl (6%) tolerance + + NaCl requirements for growth + + Lipase + + Ox i da se + + Ca t a 1 a s e + + Starch hydrolysis + + Urease Plasmids 2>60 kb 2>60 kb 1 3 kb Antibiotic sensitivity ampi c i 11 i n S S tetracycline R R peni c i 11 in G S R er y t hr omyc i n S S nalidixic acid R R Utilization of carbon source dec ane + n-hexane (vapour) + t o 1 uene xylene naphthalene hex adecane + t e t r adecane ++ lucose + acetate + + lactate + + succinate + + citrate + + ethanol + + ma 1 t o s e + + lactose starch + crude oil + + solar + + i so-oc t ane + 14 100,485 / 2 Table 3 ( con t . ) Tentative classification: HR-RL4 - Pseudomonadaceae genus P s eudomona s .

ER-RL3 - Pseudomonas alcaligenes or Al c a 1 i g ene s (has several degenerate peri trichous fiagella) ER-RT - P s eudomonadaceae genus G 1 ucono bac t er 15 100, 485 /3 TABLE Characterization of strains from CO-UF-1-2 medium Strain Proper t y ER-RL-D ER-RL-X Colony on salt-ethanol agar plates di ameter 2 mm 2 mm co 1 our whi t e whi t e Bacterial shape short rod short r Mo t i 1 i t y Growth temperature, °C 4° (lOOh) 20-25° (24h) + + 37° (24h) + + 41° (lOOh) Cytochrome oxidase Urease + / Reduction of nitrates Reduction of nitrates -6-1995 Indole production Glucose acidification Arginine dehydrogenase Gelatine hydrolysis Assimilation of C-sources g lucose ar abinose mannose manni t o 1 N-acetyl-glucosamine mal tose g lucona t e capr a t e ad i pa t e ma 1 a t e + citrate + Antibiotic sensitivity penici 11 in G + / + /- ampi c i 11 i n + / + /- te t racyc 1 ine S s palidixic acid s chlorampheni col s kanamyc i n s 16 100, 485 /3 Table 4 (cont . ) Tentative classification: Ac ine t obac t er calcoaceticus The DNA of both strains was able to transform competent auxotrophic cells of Ac i ne t obac t er calcoaceticus BD413 to prototrophy (Juni and Hanick, Journal of Bacteriology 98:281-288, 1969). 17 100, 48513 Table 5 Growth Of Pure Cultures from Sea Water On Commercial UF Fertilizers8 Fertilizer (N-P-K) S t r a i n s ER-RL3 ER-RL4 ER-RT CFU/ml Eb CFU/ml E*> CFU/ml E*> 1. UF-1 (12-12-12) 4xl07 ++ 3xl08 ++ 7xl07 ++ 2. Trisert (13-3-4) 2x108 + 3xlOB + 4x10* 3. Fluf (16-2-4) 4xl07 ++ 4x108 2x105 4. Fluf (10-0-10) + 10 mM K2HP04 4xl07 <105 4x106 5. Haifatest (29-0-0) plus Haifatest (0-34-9) 2x108 + 5x108 5xl07 Inocula were grown on the same media, but with different fertilizers. The fertilizer was used at a concentration of 0.1 mg/ml nitrogen and a minimum of 0.01 mg/ml of phosphorus. Colony forming units per ml (CFU/ml) were determined every 24 hour s .

E = Emu 1 s i f i ca t i on of oil. 18 100, 4851 3 EXAMPLE 8 Range of polymers utilized by strains ER-RL3. ER-RL4 and ER-RT as N and P sources Different commercial urea formaldehyde slow release fertilizers could support the growth and crude oil utilization ability of the strains ER-RL3, ER-RL4 and ER-RT (Table 5). Strain ER-RL3 grew on all the UF- fertilizers tested. The best growth was obtained on Trisert and a mixture of high nitrogen UF fertilizer (20-0-0) with a high phosphorous slow release fertilizer (0-34-0). Emu 1 s i f i ca t i on of the crude oil was obtained in each case, the strongest obtained with UF-1 resin and Fluf (16-2-4) resin. Strain ER-RL4 grew well on crude oil on all UF fertilizers except the one containing no phosphorous (10-0-10), which was supplemented with inorganic phosphate. Strain ER-RT showed a preference for UF-1.

EXAMPLE 9 Range of Polymers Utilized by the Microorganisms as Nitrogen and Phosphorous Sources; Different commercial urea- forma 1 dehyde and triazone slow release fertilizers could support the growth and crude-oil utilization ability of strains ER-RL3, ER-RL-4, ER-RT, ER-RL-X and ER-RL-D.

The strains utilize formaldehyde-urea polymers of both low molecular weight (soluble in water) and high molecular weight (insoluble in water), as demonstrated in Table 6. The 19 100 , 485 / 2 separation between low molecular weight (soluble) polymers and high molecular weight (insoluble) polymers was performed by dialysis, the pore size allowing the passage of molecules smaller than 12,000 molecular weight. In addition, both ER-RL-D and ER-RL-X used triazone as a source of nitrogen and phosphorous . 20 100,485/3 Table 6 Growth of Individual Strains on UF-1 of Low and High Molecular Weight8 Strain CFU/ml after 42 hours of growth Hi gh M. W. res in Low M.W. resin Tr i s er t ER-RL3 1.5x10» 2.0x109 ER-RL4 6.0x10» 2.5x109 ER-RL 1.0x108 2.5x108 1 107 1 107 -6-1995 ER-RL 5.0x108 2.5x10» 6x107 Cells were grown in the standard medium previously described (CO-UF1 for the sea water medium and CO-UFl-2 for the tap water medium), containing 0.1% crude oil and 0.1% fertilizer. The initial cell concentration was 1 x 106. To obtain high and low molecular weight UF-1, the UF-1 was suspended in water (sea water for ER-RL-3, -6-1995 ER-RL- 4 , and ER, RE and tap water for ER-RL-D and ER-RL-X) and dialyzed against the water for 4 days. The UF-1 remaining in the dialysis bag is the high molecular weight fraction and the material passing the membrane is the low molecular weight fraction. 21 100 , 485 / 2 EXAMPLE 10 A simulated open system experiment demonstrating adherence of UF-1 to the oil: Using microorganisms grown in CQ-UF'-l medium One of the major problems with supplementing a contaminated oil spill in the sea with water-solu le nitrogen and phosphorous compounds is, that they will not be concentrated near the oil, where the growth must occur. Rather the compounds will spread and dilute in the open system. It was therefore interesting to test whether or not this problem could be overcome by use of the fertilizer UF-1. The procedure that was used compared growth and emu 1 s i f i c a t i on (turbidity) after the fertilizer or soluble nutrients and oil were mixed and the aqueous phase was removed and replaced with fresh sea water. The data (Table 7) clearly show, that UF-1 resin can support growth on crude oil even after the aqueous phase had been removed and replaced twice with fresh sea water containing no additions. None of the cultures that received ammonium sulfate and phosphate salts in place of UF-1 resin and treated in a similar manner grew significantly. This demonstrates that the soluble nutrients were efficiently removed by the wash out process. The pure strains ER-RL3, ER-RL4 and ER-RT and the mixed culture grew after the dilution procedure, reaching more than 1U7 cell/ml and high values of turbidity. The mixed culture showed the highest ability to grow, reaching 1 x 10u cells/ml and 900 k.u. Strain ER-RL3 was almost as effective as the mixed culture in growth yield and emulsif ication of the crude oil. 22 100 , 485 / 2 Table 7 Growth of ER-RL4, ER- RL3 and ER-RT on crude oil and UF-l Following Removal of Water Soluble Nutrients3 Strain Ce 11 Dens i t y Tur b i d i t y ( k. u. ) (CPU /ml ) t ime 0 time 120 h time 120 h UF-l AS UF-l AS UF-l AS ER-RL4 5.0x105 ό.ΟχΙΟ5 9.0xl0/ 4.0x105 550 22 ER-RL3 8.0x105 4.0x10s 9.0x10"' 2.0x10* 860 25 ER-RT 3.0x105 4.0x105 6.0xl07 2.0xl06 490 20 Mixed 3.0x10s 5.0x105 1.0x10» l.OxlO* 900 23 cul tur e a = UF-l medium contained 0.2% fertilizer (containing 0.2% ni t rogen .

AS medium contained 0.1% ammonium sulfate (equivalent to 0.02% nitrogen) and 10 mM phosphate buffer.

Both media contained 0.5% crude oil as a carbon source . b = Each 20 ml medium was mixed well and then allowed to stand for 5 minutes, allowing the crude oil and the aqueous phase to separate. The aqueous phase was then removed as completely as possible and replaced with 20 ml fresh sea water. The procedure of removal of the remaining water soluble nutrients was repeated once more. Each flask was then inoculated with 40 μΐ of a pure or a mixed culture and incubated for 120 hours at 25°C with shaking.

Viable counts were determined after inoculation (time 0) and at 120 hours. Turbidity was measured at 120 hour s . 23 100.4851 3 EXAMPLE 11 Secret ion of e tracellular emulsifier Cells of ER-RL-X were grown in medium containing ammonium sulfate (0.1%) as a nitrogen source and ethanol (0.5%) as a carbon source. When the culture reached the stationary phase of growth (about 109 cells per ml) the cells were removed by cent r i fuga t i on . The supernatant (7.5 ml) was incubated with crude oil (100 μΐ) with shaking. The oil was emulsified, indicating the presence of an extracellular emulsifier. 24 100, 48512 EXPERIMENT 12 UFU ( Urea- forma 1 dehyde utilization) activity: Partial Characterization and Purification from ER-RL3 Determination of UFU activity: principle UFU activity is defined as the catalytic breakdown of high molecular wei ht UF (not soluble) into low molecular weight units, which are water soluble and dialyzable.

Bioassay for UFU activity Principle: The bioassay is based on determining the breakdown of UF-1 to low molecular weight molecules. The low molecular weight (soluble) fraction is separated by dialysis, as it passes the dialysis bag and is found in the dialyzate. It is then quantitated by growth of ER-RL3 bacteria utilizing it as a sole nitrogen source.

Procedure : Dialysis (molecular wei ht exclusion = 12,000) of sample to be tested (bacteria, extract etc. for 24 hours at 4°C to remove low molecular weight nitrogen and phosphorus. The high molecular weight fraction will be called "Retentive A". 25 100 , 485 / 2 Step B - Dialysis of UF-1, same as in step A. The high molecular weight insoluble fraction will be called "Retentive B" .

Retentive A is incubated together with Rententive B for 24 hours at 3U°C in a dialysis bag (as in Step A), placed in x200 its volume of sea water.

The sea water, which contain - at the end of the incubation - the soluble fraction derived from Retentive B by the cellular fraction of Retentive A, is called "Dialyzate C" .

Dialyzate C is now used as and P source for an indicator culture. This is performed by adding to this sea water 0.5% sodium acetate as carbon source and 10 bacteria of strain ER-RL3.

Growth was followed for 16-24 hours at 30°C.

Definition: One unit of UFU (urea formaldehyde utilization) activity supports a xlO multiplication of 10s cells of ER-RL3 under the conditions described a bove .

The results for ER-RL3 appear in Table 8. 26 100,485 / 2 Table 8 UFU Ac t i vi t v Of Cel 1 s UFU Ac t i vi t v Fraction used for bioassay Growth of ER-RL3 Units (added to dialysis bag in step C) I. Control - no cell added 1.9x10"' 190 II. Washed whole cells grown of UF-1 1.6Χ109 · 16,000 II . Extracellular fluid of 11 1.0xl0u 1,000 concentrated x5 by ( H >2S04 precipitation (70% saturation) 27 100,485/3 EXAMPLE 13 UFU (Urea-formaldehyde utilization activity) Partial Characterization and Purification from ER-RL-D and ER-RL-X.

Following the method described in Example 12 except 1993 substituting fresh water in place of sea water, the UFU activity of the strains ER-RL-D and ER-RL-X was determined, The results appear in Table 9. 28 100,485/3 / TABLE 9 UFU activity of cells UFU activi ty Fraction used for bioassay Growth of RL3 Units (added to dialysis bag in s tep C) I. Control - no cells added 8.5 x 105 8.5 -6-1995 II. Washed cells of ER-RL-D grown on UF-1 6 x 108 6,000 III. Washed cells of ER-RL-X grown on UF-1 2.3 x 109 23,000 29 100 , 485 I 2 EXAMPLE 14 Partial purification of UFU activity of Sea Water Microorganisms Procedure Growth of cells; A precuiture was prepared by inoculating one ioopful of cells from a single colony on plate into a 100 ml flask containing 20 ml of SWA medium (sea water containing 0.5% sodium acetate and 0.1% UF-1) and incubated at 30°C for 16-24 hours on a reciprocal shaker (100 strokes per minute). 20 ml of this precuiture were inoculated into a 2 liter flask and incubated at 30°C for 3 days on a reciprocal shaker.

Extraction of protein; Ceils were collected by centrifu-gation lor 10 minutes at 10,000 x g at 4°C and the pellet was re-suspended in 3 ml or 0.05M sodium phosphate buffer, pH=7.6. The cell suspension was sonicated for 1 minute in an ice bath, 5 times with 1 minute intervals. The sonicated cell suspension was then centrifuged for 60 minutes at 30,000 x g at 4°C.

Ammonium sulfate precipitation; Ammonium sulfate was added to the supernatant with stirring to a final concentration of 70% of saturation. The solution was kept overnight at 4°C. The precipitate was collected by centrifugation and then dissolved in 3 ml of 0.05 M sodium phosphate buffer, pH=7.6. The solution was dialyzed twice against 5 liters of water at 4°C. 10U , 485 / 2 Ultra- filtrati on; Protein fractions were u 1 t r a- f i 1 t er ed by use of the apparatus and appropriate molecular weight filters of Amicon.

The results of the purification of each strain appear in Table 10. The results of the separation of ammonium sulfate precipitates by molecular weight appear in Table 11. 31 100 , 485 / 2 Table 10 Results of Purification Cellular Fraction UFU Activity Growth of ER-KL3 Protein m g / m 1 ER-RL3 Cell extract 4.2x10» 1 4,200 Supernatant activity after (NH-iJzSC precipitation 3xl06 0.125 30 ER-RL4 Cell extract 8x10 0.65 870 Supernatant activity after ( H4)2S04 precipitation 1.5x106 0.06 15 ER-RT Cell extract 3.0xl0« 0.9 3,000 Supernatant activity after (Nt ^SC precipitation 2.5xl06 0.065 25 No protein added 2x10* 0 20 100,485 / 2 Ta le 11 Separation of Ammonium Sulfate Precipitates By Molecular Weight Molecular Weight Units of Activity RL3 total protein >300 , 000 <300 , 000 ioo,ooo> <30o,ooo 20 > 50,000 799 < 50,000 41 Con t r o 1 28 33 100, 48512 EXAMPLE 15 Enhance Bi odegr ada t i on of Hydrocarbon Contaminated Beach Sand Using Microorganisms isolated from Sea Water Experimental Procedures A . Site selection and characteristics Two plots of 50 m2 were selected, one for the experiment and the second for the control . They were located appro imately 30 m from the water line, about 3 km north of Zvulun Beach (Kiryat Yam) . The plots were chosen because they were representative of the oi l polluted sands. The oi l polluted about 5 cm in depth. The initial concentration of oil ( pen t ane-ex t r ac t a b 1 e ) in the upper 10 cm was 0..23% and 0.38% in the control and in the experimental plots, respectively. The moisture content of the sand was 4%-5%. The unpacked density of the sand was 1.27 g/cm3. The maximum temperature of the sand was 36°C at noon.

B. initial Trea tment The experimental plot was inoculated with 20 1 of a mixed bacterial culture containing strains ER-RL3 , ER-RL4 and ER-RT. UF-1 (38 kg in the form of a fine powder) was added as the source of ni trogen and phosphorous. The plot was then watered with sea water from the adjacent sea (water temperature was 2/°C). The plot was then 34 100, 485 / 2 tilled by hand with the help of a simple rake to the depth of about 5 cm.

The control plot was left undisturbed.

C . Da i 1 v t rea tmen t After the first day, the experimental plot was watered daily between 15:00-16:00 with appro imately 1.5 m sea water with the help of a SUB 3000 GR submergible pump attached to a hose. After watering, the plot was raked to a depth of about 5 cm .

D . Sampling procedure Ten random core samples ( 5 cm diameter x 10 cm depth) were taken prior to the daily watering from both the experimental and control plots., The samples were mixed thoroughly in the field, placed in a plastic bag and brought to the laboratory. The sand was either extracted the same day or stored overnight at 4°C.

E . Determination of residual petroleum in the sand For each time point, triplicate 50 g samples were extracted and the results presented are the average of the three values. Each 50 g sample was placed in a 500 ml bottle that contained 50 ml n-pentane. After shaking vigorously for 5 minutes, 25 ml of the pentane extract were transferred to a 100 ml flask containing anhydrous CaCl2- After standing for 35 100 , 485 / 3 5 minutes with occasional stirring, the dried pentane extract was filtered through Whatmann 2V paper and then evaporated in vacuo (water aspirator) at 30°C. The residual hydrocarbon was dissolved in 3 ml n-pentane and placed in small glass vials in a chemical hood. The weight of the pen t ane-ex t r ac t -able hydrocarbon was determined by weighing to constant weight at room temperature on an analytical balance. A control of n-pentane taken through the same procedure contained 0.54 mg residues per 100 ml pentane. Thus, the contribution of the non- vo 1 a t i 1 e s in the pentane to the values reported was 0.006 mg/g sand, which is negligible. The values reported were, however, corrected for the moisture content of the specific sand sample.

RESULTS The experimental data are presented in Tables 12 and 13. On day zero (September 1, 1991), the core samples were taken prior to any treatment. At the beginning of the experiment, the experimental plot contained significantly more hydrocarbon (3.80 mg/g sand) than the control plot (2.3 mg/g sand). There was only a small decrease in the first day. However, by the fourth day 30% of the hydrocarbon had been degraded. The bio-degradation continued, reaching 50% on day 9 and 84.5% on day 25, when the experiment was concluded.

The control plot showed a 18% degradation by day to day were not due to the pentane extraction procedure, because the average standard deviations were very low (0.03 mg/g sand, corresponding to less than ±2%). Therefore we assume that the core sampling was probably the source of the day to day variation. 36 100 , 485/2 S U M M A R Y Enhanced b i ode r ada t i on (bacteria + nutrient UF-1) of the contaminated sands was successful, reaching 84.5% degradation after 25 days, compared to an untreated control, which leveled off at less than 20%.

Table 12 Enhanced b i ode r ad a t i on of hydrocarbon- contaminated beach sand.

Day Pen t ane-ex t r ac t ab 1 es a % degradation mg / gram sand 0 3.80 0 1 3.70 2.6 4 2.76 30 9 1.89 50 14 0.88 77 21 1.40 63 25 0.59 84.5 a = Each value is the average of three determinations.

The average standard deviation was 0.1 mg/gram sand for the experimental value and 0.15 mg/gram for the control value. 37 100 , 485 / 2 Table 13 Natural bi odegr ada t i on of hydrocarbon-contaminated beach sand Day Pen t ane-e t r ac t abl e s a % degradation mg / gram sand 0 2.30 0 4 2.53 0 9 1.88 18 14 1.70 26 21 1.94 15. 6 25 1.95 15. 6 a - Each value is the average of three determinati on s The average standard deviation was 0.1 mg / gr am sand for the experimental value and 0.15 m / r am for the control value.

Claims (12)

1. 38 100,485/3 1. A composition for degrading hydrocarbons which comprises one or more bacteria characterized by a UFU activity greater than 200 units per 10ml capable of degrading hydrocarbons by utilizing polymeric ni troeen containing material which contains organic nitrogen in a state which is not utilizable by most soil and water micro - organisms wherein said polymer has a molecular weight of at least 12,000.

2. A composition in accordance with Claim 1 also containing phosphate either in a free or bound for m .

3. A composition in accordance with Claim^ 1 or Claim 2 wherein the polymeric nitrogen-containing material is urea- forma 1 dahyde resin.

4. A composition in accordance with Claim 3 wherein the urea - formaldehyde polymer comprises a range of 5% to 40% nitrogen 0% to 34% phosphorous, and 0% to 12% potassium.

5. A composition in accordance with any of Claims 1 to 4 wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml.

6. A composition in accordance with Claim 4 wherein the urea - formaldehyde resin contains 10% to 35% insoluble nitrogen.

7. A composition in accordance with any of Claims 1 to wherein the bacteria is designated ER-RL3 (NCIMB Accession No.40464).

8. A composition in accordance with any of Claims 1 to 6, wherein the bacteria is designated ER-RL4 (NCIMB Accession No. 40465).

9. A composition in accordance with any of Claims 1 to 6, wherein the bacteria is designated ER-RT (NCIMB Accession No.40466). 100,485/3 - 39 CLAIMS CONTINUED

10. A composition in accordance with any of Claims 1 to 6 wherein the bacteria is a mixture of two bacterias chosen from the group consisting of ER-RL3 , ER-RL4 and ER-RT.

11. A composition in accordance with any of Claims 1 to 6, comprising in combination the bacteria ER-RL3 , ER-RL4 and ER-RT.

12. A composition in accordance with any of Claims 1 to 7, 10 or 11 comprising a cell -free extract which is a product of ER-RL3. A composition in accordance wi any of Claims 1 to 6,8,10 or 11 , compr i s i ng a cell free extract, which is a product of ER-RL4. A composition in accordance with any of Claims 6, or 9 to 11, comprising a cell - free extract which is a product of ER-RT. 15. A composition in accordance with any of Claims 1 to 6, wherein the bacteria is designated ER-RLD ( NCI MB Accession No.40506). -1995 16. A composition in accordance with any of Claims 1 to 6, wherein the bacteria is designated ER-RLX (NCI MB Accession No.40507). 17. A composition in accordance with any of Claims 1 to 6, wherein the bacteria is a mixture of ER-RLD and ER-RLX. 18. A composition in accordance with any of Claims 1 to 6, 15, or 17, comprising a cell-free extract, which is a product of ER-RLD . 19. A composition in accordance with any of Claims 1 to 6, 16 or 17, comprising a cell - free extract which is a product of ER-RLX. - 40 - 100,485/4 CLAIMS CONTINUED A composition for degrading hydrocarbons which comprises one or more bacteria, selected from the group consisting of pseudamonas, ac ine t obac t er and aerobacter characterized by a UFU activity greater than 200 units per 10ml capable of degrading hydrocarbons by utilizing polymeric nitrogen containing material which contains organic nitrogen in a state which is not utilizable by most soil and water micro-or anisms wherein said polymer has a molecular weight of at least 12,000. A composition in accordance with Claim 20 also containing phosphate either in a free or bound form A composition in accordance with of Claims 20 or 21 wherein the polymeric nitrogen-containing material is a urea formaldahyde resin. A composition in accordance with of Claims 22 wherein the ur ea- f orma 1 dehyde polymer comprises a range of 5% to 40% nitrogen, 0% to 34% phosphorous, and 0% to 12% potassium. A composition in accordance with any of Claims 20-23, wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml. A composition in accordance with Claim 23 wherein the ur ea - for ma 1 dehyde resin contains 10% to 35% insoluble nitrogen. A composition accordance with any of Claims 20 to 25 wherein the bacteria designated ER-RL3 (NCIMB Accession No.40464). A composition in accordance with any of Claims 20 to 25 wherein the bacteria is designated ER-RL4 (NCIMB Accession No.40465). A composition in accordance with any of Claims 20 to 25, wherein the bacteria is designated ER-RT (NCIMB Accession No.40466). 100,485/3 - 41 CLAIMS CONTINUED A composition in accordance with any of Claims 20 to 25, wherein the bacteria is a mixture of two bacterias chosen from the group consisting of ER-RL3, ER-RL4 and ER-RT. A composition in accordance with any of Claims 20 to 25, comprising in combination the bacteria ER-RL3. ER-RL4 and ER-RT. A composition in accordance with any of Claims 20 to 26,29, or 30 comprising a cell -free extract which is a product of ER-RL3. A composition in accordance with any of Claims 20 to 25,27,29, or 30, comprising a cell - free extract, which is a product of ER-RL4. A composition in accordance with any of Claims 20 to 25, or 28, to 30, comrpising a cell - free extract, which is a product of ER-RT. ith any of Claims 20 s designated ER-RLD ith any of Claims 20 s designated ER-RLX ith any of Claims 20 s a mixture of ER-RLD ith any of Claims 20 cell-free extract, ith any of Claims 20 cell - free extract - 42 - 100,485/3 CLAIMS CONTINUED 39. A composition for degrading hydrocarbons which comprises one or more bacteria selected from the group consisting of ER-RL3 (NCIMB Accession No.40464). ER-RL4 (NCIMB Accession No.40465), ER-RT (NCIMB Accession No.40466), ER-RLD (NCIMB Accession No.40506) . and ER-RLX (NCIMB Accession No.40507). characterized by a UFU activity greater than 200 units per 10ml capable of degrading hydrocarbons by utilizing complex nitrogen - containing material which contains organic nitrogen in a state which is -6-1995 not utilizable by most soil and water micro-organisms wherein said polymer has a molecular molecular weight of at least 12,000. 40. A composition in accordance with Claim 39 also containing phosphate either in a free or bound form. 41. A composition in accordance with Claims 39 or 40, wherein the polymeric nitrogen- containing material is a urea - formaldehyde resin. 42. A composition in accordance with Claim 41 wherein the ur ea- f orma 1 dehyde polymer comprises a range of 5% to 40% nitrogen, 0% to 34% phosphorous, and 0% to 12% potass ium . 43. A composition in accordance with any of Claims 39-42 wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml . 44. A composition in accordance with Claim 24 wherein the urea - formaldehyde resin contains 10% to 35% insoluble nitrogen. 100,485/3 - 43 - CLAIMS CONTINUED A method of enhancing the b i odegr ada t i on and/or bioemulsification of petroleum or petroleum fractions, which comprises contacting the petroleum with a composition containing at least one bacteria characterized by a UFU- activity greater than 200 units per 10ml and capable of degrading petroleum or petroleum fractions by utilizing polymeric nitrogen-containing material which contains or anic-nitrogen in a state which is not utilizable by most oil and water micro-or anisms wherein said poylmer has a molecular weight of at least 12,000 46. A method in accordance with Claim 45 wherein the composition also contains phosphate either in a free or bound form. 47. A method in accordance with either Claims 45 or 46, wherein the polymeric nitrogen - containing material is a urea formaldehyde polymer. 48. A method in accordance with any of Claims 45 to 47, wherein the petroleum comprises an oil slick on the sur face of water. 49. A method in accordance with any of Claims 45 to 47, wherein the petroleum contaminates sand, soils, rocks and shel 1 s . 50. A method in accordance with any of Claims 45 to 47, for treatment of petroleum contaminated objects chosen from the group consisting of cargo holds, ballast tanks, pipelines and oil storage tanks. o 51. A method in accrodance with any of Claims 45 to 50, wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml. 52. A method in accordance with any of Claims 45 to 51, wherein the urea - formaldehyde resin comprises a range of 5% to 40% nitrogen, 0% to 34% phosphorous and 0% to 12% potassium. 44 - 100.485/4 CLAIMS CONTINUED 53. A method in accordance with any of Claims 45 to 52, wherein the ur ea- f or ma 1 dehyde resin contains 10% to 35% insoluble nitrogen. 54. A method in accordance with any of Claims 45 to 53, wherein the bacteria is designated ER-RL3 (NCIMB Accession No.40464). 55. A method in accordance with any of Claims 45 to 53, wherein the bacteria is designated ER-RL4 (NCIMB Accession No.40465). 56. A method in accordance with any of Claims 45 to 53, wherein the bacteria is designated ER-RT (NCIMB Accession No.40466). 57. A method in accordance with any of Claims 45 to 53, wherein the bacteria is a mixture of two bacterias, bacteria chosen from the group consisting of ER-RL3, ER-RL4 and ER-RT. 58. A method in accordance with any of Claims 45 to 53, comprising in combination with bacteria ER-RL3, ER-RL4 and ER-RT. 59. A method in accordance with any of Claims 45 to 50, 54,57 and 58 comprising a cell-free extract, which is a product of ER-RL3. 60. A method in accordance with any of Claims 45 to 50, 55, 57 and 58, comprising a cell-free extract, which is a product of ER-RL4. A method in accordance with any of Claims 45 to 50, and 47 to 56, comprising a cell-free extract, which is a product of ER-RT. -6-1995 A method in accordance with any of Claims 45 to 53, wherein the bacteria is designated ER=RLD (NCIMB Accession No. 40506). A method in accordance with any of Claims 45 to 53, wherein the bacteria is designated ER-RLX (NCIMB Accession No. 40507). CLAIMS CONTINUED 64. A method in accordance with any of claims 45 to 53, wherein the bacteria is a mixture of ER-RLD and ER-RLX. 65. A method in accordance with any of Claims 45 to 53, 43 or 45 comprising a cell-free extract which is a product of ER-RLD. 66. A method in accordance with any of Claims 45 to 53, 44,45, comprising a cell-free extract, which is a product of ER-RLX. 67. A method of enhancing the bi odeg r ada t i on and/or bioemulsification of petroleum or petroleum fractions, which comprises contacting the petroleum with a composition containing at least one bacteria chosen from the group consisting of pseudamonas, acintobacter and aerobacter characterized by a UFU-activity greater than 200 units per 10ml and capable of degrading petroleum or petroleum fractions by utilizing polymeric nitrogen - containing material which contains organic nitrogen in a state which is not utilizable by most oil and water micro-organisms wherein said polymer has a molecular weight of at least 12,000. 68. A method in accordance with Claim 67 wherein the composition also contains phosphate either in a free or bound form. 69. A method in accordance with either Claims 67 or 68, wherein the polymeric nitrogen-containing material is a urea- forma 1 dehyde polymer. 70. A method in accordance with any of Claims 67 to 69, wherein the petroleum comprises an oil slick on the surf ace of water. 71. A method in accordance with any of Claims 67 to 69, wherein the petroleum contaminates sand, soils, rocks and she 11 s . - 46, - 100,485/3 CLAIMS CONTINUED A method in accordance with any of Claims 67 to 69, for treatment of petroleum contaminated objects chosen from the group consisting of cargo holds, ballast tanks, pipelines and oil storage tanks. A method in accordance with any of Claims 67 to 72, wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml. A method in accordance with any of Claims 67 to 73, wherein the urea-formaldehyde resin comprises a range of 5% to 40% nitrogen, 0% to 34% phosphorous and 0% TO 12% potassium. A method in accordance with any Claims 67 to 74, wherein the urea - formaldehyde resin contains 10% to 35% insoluble nitrogen. A method in accordance with any of Claims 67 to 75, wherein the bacteria is designated ER-RL3 . A method in accordance with any of Claims 67 to 75 wherein the bacteria is designated ER-RL4 (NCIMB Accession No.40465). 78. A method in accordance with any of Claims 67 to wherein the bacteria is designated ER-RT (NCIMB Accession No. 40466). A method in accordance with any of Claims 67 to 75, wherein the bacteria is a mixture of two bacterias ER-RL3, ER-RL4 and ER-RT. 80. A method in accordance with any of Claims 67 to 75 comprising in combination the bacteria ER-RL3 , ER-RL4 and ER-RT. 81. A method in accordance with any of Claims 67 to 72,74,78, and 79 comprising a cell - free extract which is a product of ER-RL3. 100,485/3 - 47 - CLAIMS CONTINUED 82. A method in accordance with any of Claims 67 to 72,76,78 and 79, comprising a cell- ■ free e tract which is a product of ER-RL4. 83 A method in accordance with any of Claims 67 to 72, and 69 to 78, comprising a cell-free extract, which is a product of ER-RT. 84 A method in accordance with any of Claims 67 to 75, wherein the bacteria is designated ER-RLD (NCIMB Accession No.40506). 85 A method in accordance with any of Claims 67 to 75 wherein the bacteria is designated ER-RLX (NCIMB Accession No.40507). 86 A method in accordance with any of Claims 67 to 75, wherein the bacteria is a mixture of ER-RLD and -6-1995 ER-RLX. 87 A method in accordance with any of Claims 67 to 75, 84 or 86 comprising a cell-free extract which is a product of ER-RLD. 88, A method in accordance with any of Claims 67 to 75, comprising a cell-free extract, which is a product of ER-RLX. 89. A method of enhancing the bioremedi at ion and/or bi oemu 1 s i f i ca t i on of petroleum or petroleum fractions, which comprises contacting the petroleum with a composition containing one or more bacterias selected from the group consisting of ER-RL 3 (NCIMB Accession No.40464), ER-RL4 (NCIMB Accession No.40465), ER-RT (NCIMB Accession No.40466), ER-RLD -6-1995 (NCIMB Accession No.40506). and ER-RLX (NCIMB Accession No.40507) characterized by a UFU activity greater than 200 units per 10ml and capable of degrading petroleum or petroleum fractions by utilizing polymeric nitrogen-containing material which contains organic nitrogen in a state which is not utilizable by most soil and water micro-organisms wherein said polymer has a molecular weight of at least 12,000 100,485/3 ■48- CLA I MS CONT I NUED A method in accordance with Claim 89 wherein the composition also contains phosphate either in a free or bound form. A method in accordance with either Claim 89 or Claim 90, wherein the polymeric nitrogen-containing material is a ur ea- f orma 1 dehyde polymer. A method in accordance with any of Claims 89 to 91, wherein the petroleum comprises an oil slick on the surface of water. A method in accordance with any of Claims 89 to 91, wherein in the petroleum contaminates sand, soils, rocks and she lis. A method in accordance with any of Claims 89 to 91 for treatment of petroleum contaminated objects chosen from the group consisting of cargo holds, ballast tanks, pipelines and oil storage tanks. A method in accordance with any of Claims 87 to 94 wherein the bacteria is characterized by an UFU activity of greater than about 400 units per 10ml. An enzyme derived from the biologically pure cul ture of a micro-organism desginated ER-RL3 (NCIMB Accession No.40464), that is greater than about 200 units per 10ml. An enzyme derived from the biologically pure culture of a micro-organism designated ER-RL4 (NCIMB Accession No.40465), that is greater than about 200 units per 10ml. An enzyme derived from the biologically pure culture of a micro-organism designated ER-RT (NCIMB Accession No.40466), that is greater than about 200 uni t s per 10ml . An enzyme derived from the biologically pure culture of a micro-organism designated ER-RLD (NCIMB Accession No.40506). that is greater than about 200 units per 10ml. - 49 - 100,485/3 CONTI UED 100. An enzyme derived from the biologically pure culture of a micro-organism designated ER-RLX (NCIMB Accession No.40507). that is greater than -6-1995 about 200 units per 10ml. 101 The enzyme of any one of Claims 96 to 100 that is greater than about 400 units per 10ml. 102. A composition in accordance with Claims 1 to 44 which comprises at least one bacteria characterized by a UFU - activity greater than about 200 units per 10ml capable of degrading hydrocarbons, methods of using such compositions to enhance the biodegradat ion and/or bioemulsiiication of petroleum or petroleum fractions, and biologically pure cultures of the micro-organisms which can be used in these compositions substantially as herein before described with reference to the examples.