AU667695B2 - Non polluting compositions to degrade hydrocarbons and microorganisms for use thereof - Google Patents

Description

E an~irY~a~ -lqXI?7~- Our Ref: 453531 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT

B

o Applicant(s): a

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Ramot University Authority for Applied Research and Industrial Development 32 University Street Ramat Aviv TEL AVIV

ISRAEL

DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Non polluting compositions to degrade hydrocarbons and microorganisms for use thereof Address for Service: Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 r r 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 urea-formaldehyde resins and other compatible polymers as the source of nitrogen, a method enhancing the biodegradation 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 Surfactants" 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 bioremediation 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 microorganisms in the soil and water which then, themselves, become a serious cause of water pollution.

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2 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 biodegradation occurred very slowly over a period of months.

SUMMARY OF THE INVENTION In accordance with present invention, there are provided novel bacteria which are effective as bioremediation 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 microorganisms in a mixture with a suitable carrier. There is further provided a method of enhancing the biodegradation and/or bio-emulsification of petroleum, which comprises contacting the petroleum with said composition so that the microorganism utilizes the petroleum as a source of carbon, thereby degrading the petroleum, without any substantial growth of the indigenous competing microorganisms.

S'o According to one embodiment of the present invention there is provided a composition for degrading hydrocarbons which comprises at least one microorganism characterized by a UFUi°o" 20 activity greater than 200 units per 10 ml and capable of degrading hydrocarbons by utilizing a complex nitrogen-containing polymer material which contains organic nitrogen in a state v. h is not utilizable by most soil and water microorganisms wherein said polymer has a Si,.jlecular weight of at least 12,000.

In a specific embodiment of the present invention the said composition contains at least one of the novel bacteria effective as a bioremediation agent for petroleum; and uses a ureaformaldehyde resin or other compatible polymers as a source of nitrogen.

i D:llRAMOT:3034592,PAT;NI:6 February 1996 The novel bacteria were obtained by an enrichment culture procedure using crude oil as the carbon and energy source and a urea-formaldehyde resin, such as UF-I, 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 Alicaligenes, EIR-RL4 ten atively identified as Pseudomonadaceas genus Pseudomonas and EH-RT, tentatively identified as Pseudomonadaceae genus Gluconobacter. 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, respectively.

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 Acinetobacter ca coacet i cus. These strains were deposited with the National Collections of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen, Scotland, pursuant to the provisions of the B:.dapest 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.

i; 4 The urea-formaldehyde (UF) resins of the novel compositions of the present invention can be almost any type of UF fertilizer. Examples are described in Controlled Release Fertilizers by Sarah P. Landels, 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 Fluf Fluf (10-0-10) plus 10 mM K.HPO 4 and Haifatert (29-0-0) plus Haifatest (0-34-9).

It has also been discovered that certain cultures produced factor(s) probably an enzyme or 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 ureaformaldehyde utilization activity (UFU) of 200 units, most preferably at least about 400 units, where the UFU activity 5 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 1 0 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 invention.

6 EXAMPLE 1 Isolation of petroleum-utilizing bacteria from sea water using UL-1 as the source of nil rogen 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 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 tile mixture at 250C 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 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-LIF medium.

Three pure cultures, able to grow on CO-UFJ 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).

l-~liitUiilyll-- 7 After incubation for 3 days at 250C, 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 m lium.

EXAMPLE 2 Isolation of petroleum-utilizing bacteria from tap water using UF-1 as the source of ni rogen and phosphorous A mixed bacterial culture was oblained 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 cuntain- ing 0.5% crude oil 0.1% UF-1 and 0.04% MgSO4 (CO-UF1-2 medium). The source of bacteria was oil-contaminated soil. The soil used was obtained from d fferent locations in the U.S.A. and in Israel. The enrichment was carried out by inoculating 10 ml of the CO-F1-2 medium in a 125 ml flask and incubating the mixture at 250C 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.

C 8 Several pure cultures able to grow on CO-UF1-2 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 restreaking on agar. These strains are referred 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-UF1-2 medium.

EXAMPLE 3 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 mixea culture had a doubling time of approximately 2 hours, reaching stationary phase after hours at 2x10 0 cells/ml. Growth preceded emulsification by approximately 5 hours. The p1H 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 mostly because of emulsification of the oil.

EXAMPLE 4 Dependence of growth of mixed culture from sea water on UlP-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 9 with each of the three pure cultures: growth and emulsification 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 UF-1 as the nitrogen source. Similar results were obtained with ER-RL-X: Growth and emulsification depended upon the presence of both crude oil and UF-1.

Table 1.

Utilization of crude oil and UF-l *by mixed culturea from sea water is UF- I 0.1 0. 1 0. 1 0.1 0. 1 0. 1 0 0.05 0.2 0. 5 1.6 Crude oil

M~

0 0.05 0.02 0. 05 0.1 0. 5 1.0 0. 5 0.5 0. 5 0.5 0. 5 0. 5 Turbidi ty 24 75 97 256 435 1320 1400 22 800 1200 1250 2000 2650 Cell density (CFU/mI) 1 0 X10 6 1. 3 x 10 7 5. 5 X10 7 6. 0X 10 7 7. 8 X 10 7 1. 6 x18 6 2. O x 108 3. O X 10 6 1. 4 x10 8 1. 5 x10 8 1. 5 x 108 4. O x 108 4. 0 x10 8 a =The experiment was carried out as described in Figure 1, except that the concentrations of UF-i and crude oil were varl'ed as indicated and the sample time was 48 hours.

11 TABLE 2 1Jt1lzation of crude oilI and UF- I byv ER-RL-D.

UF- I

M%

Crude Oil Turbid! ty k.u.) Cell density (CFU/mlI) 0.1 0 24 1 .0 X 106 0o). 22 3.0 x 106 0.1 0.5 1300 2.0 x 188 _I 1_1 I_! 12 EXAMPLE 6 Characterization of marine strains with UFU activity From the mixed culture in sea water, three pure strains that could grow on crude oil and UF-1 (see Figs. 2, 3 and 4) were isolated and characterized. 'the properties of these strains are described in Table 3. Figs. 2, 3 and 4 show the kinetics of growth and emulsification of the three strains ER-RL3, ER-RL4 and ER-RT, respectively. All the strains have the ability to grow and emulsify oil, using UF-1 resin as a nitrogen aiid phosphorus source. The strains showed similar doubling times of approximately 3 hr. Strain ER-RT reached the highest growth density (2 x 10 u ml), although its emulsification (1000 ability was almost the same as the two other strains. The pH in the growth media in all the strains decreased from an initial value of 7.8 to about 7 at the end of the growth experiment after 72 hours. Here, as in the case of the mixed culture, the turbidity of the cultures came mostly from the emulsified oil.

EXAMPLE 7 Characterization of fresh water strains with UFU activity From the mixd 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. 13oth strains have 'he ability to grow and emulsify oil, using UF-1 resin as a nitrogen and phosphorus source.

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e 13 Table 3 Characterization of strains from CO-UF-I medium S t r a i n Property S t r a i n ER-RL3 Colony of marine agar diameter 2 mm color white Bacterial shape rod Dimensions of cells (pm) from marine agar (16h) 0.3/1.5 from broth on oil (72h) 0.5/1.6 Motility Flagellar arrangement from marine agar (16h) peritrichal from broth on oil (72h) none Growth temperature, OC (100h) 200C-250C (24h) 370C (24h) 410C (100h) NaCI tolerance NaCI requirements for growth Lipase Oxidase Catalase Starch hydrolysis Urease Plasmids ER-RL4 <1 mm white rod 0.6/1.2 0.5/2.2 polar polar

ER-RT

1-2 mm yellowish rod 0.6/1.0 0.44/1.1 none none 2>60 kb 1 3 kb 2>60 kb Antibiotic sensitivity ampicillin tetracycline penicillin G erythromycin nalidixic acid Utilization of carbon source decane n-hexane (vapour) toluene 4 xylene naphthalene hexadecane tetradecane glucose acetate lactate succinate citrate ethanol maltose lactose starch crude oil solar iso-octane L_ 14 Table 3 (cont.) Ten tativye c las s ifica tion: ER-RL4 Pseudomonadaceae genus Pseudomonas.

ER-RL3 Pseucdomornas alcaligeties or Alcaligenes (has several degenerate peritrichous flagella) ER-RT Pseudomonadaceae genius (iluconobacter

TABLE

Characterization of strains from CO-UF--l-2 mediumt S Ir a i n Proper ty ER-RI.-D ER-RL-X Colony on salt-ethanol agar plates di aieter 2 mm 2 mm colour White white Bacterial shape short rod short rod Mo ti l t y Growth temperature, OC (l100h) 20-250 (24h) 370 (24h) 4i10 (100h) oxidase Urea se +1 Reduction of nitrates Reduction of nitrates Indole production Glucose acidification Arginine dehydrogenase Gelat ine hydrolysis+ Assimilation of C-sources glucose Rsarabinose mannose manni tol N-ace ty 1- gluc os a mne k maltose gluconate c apr ate adipate ma late c itr a te Antibiotic sensitivity penicillin G ampicillin tetracycline S S palidixic acid S S chloramphenicol S S kanamycin S S bk EM 16 Table 4 (cant.

Tentative classi fication: Acinetobacter calcoaceticus The DNA of both strains was able to transform competent auxotrophic cells of Acinetobacter calcoaceticus BD413 to prototrophy (Juni and Hanick, Journal of Bacteriology 98:281-288, 1969).

17/ Table Growth Of Pure Cultures from Sea Water On Commercial LiP Fertilizersa Fertilizer (N-P-K) S t r a i n s ER- RL.3 CPU /m I E R- R L 4 Eb CP U /M I H R- RI Eb CUM I 1. UP-i (12-12-12) 4x107 2. Triazen (18-3-4) 2X.10 8 3 x10 8 3 x10 8 4 x10a <1 05 3. Fluf (16-2-4) 4. Fluf (10-0-10) mM K2H 7 P04 4 x 10 7 4 x 10 7 7 x10 7 4 x10 6 2 x10 4 x10 6 5 X10 7 1-afatest (29-0-0) plus lHaifatest (0-34-9) 2x10 8 5x10 8 a -Inocula were grown on the same media, but With different fertilizers. The fertilizer was used at a concentration of 0.1 mg/mI nitrogen and a minimuz of 0.01 mg/mi of phosphorus. Colony forming units per ml (CPU/mi) were determined every 24 hours.

b E Emulsification of oil.

nb -i-3i- CI-~"N'II*CrYY~; I 1 EXAMPLE 8 Range of polymers utilized by strai: ER-RL3, ER-RL and ER-R'I 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 Strain ER-RL3 grew on all the UF- fertilizers tested. The best growth was obtained on Triazone and a mixture of high nitrogen UF fertilizer (20-0-0) with a high lu phosphorous slow release fertilizer Emulsification 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 1 5 supplemented with inorganic phosphate. Strain ER-RT showed a preference for UF-I.

EXAMPLE 9 Range of Polymers Utilized by the Microorganisms as Nitrogen and Phosphorous Sourcesi Different commercial urea-formaldehyde 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 nh- n 19 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 smaler than 12,000 molecular weight. In addition, both ER-RL-D and ER-RL-X used triazone as a source of nitrogen and phosphorous.

L 1 1 1 20 TablIe 6 Growth of Individual Strains on UF-I of Low and High Molecular Weighta S t r a i n ER- RL3 ER- RL 4

ER-RT

ER1 RL- D ER- RL-X CFU/mi after 412 ioirn nf cyrow'th ilgh M. W. res In 1. 5 x10 9 6 0 (x 10 9 8i. 2 x10 9 1. 0 x10 0 5. 0x 1080 Low M.W. resin Tfri a zone 2. OX10 9 2. 5x 10 9 2. Ox 'L09 2. 5 x10 B 2. 5x10 8 I x 107 6 x 1U 7 8 Cells were grown in the standard medium previously described (CO-UFI for the sea water medium and CO-UF1-2 for the tap water medium), containing 0.1% crude oil and 0.1%Z fertilizer. The initial cell concentration was 1 X 106. To obtain high and [ow molecular weight UF-1, the UP-I was suspended in waler* (sea water for L-R-RL-3, ER-RL-4, and ERH, RE and tap water for EIR-RL-D and ER-RL-X) and dialyzed against the water for 4 days. The UP-i remaining in the dialysis bag is the high molecular weigh, fraction and the material pass ing the membrane is the low molecular weight fraction.

21 EXAMPLE A simulated open system experiment demonstrating adherence of UF-1 to the oil: Using microorganisms grown in CO-Up-1 medium One of the major problems with supplementing a contaminated oil spill in the sea with water-soluble 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 I he fert lizer UF-1. The procedure that was used compared growth and emulsification (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 S 2C 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 Sstrains 13l-RL3, ER-RL4 and 1HR-RT and the mixed culture grew after the dilution procedure, reaching more than 107 cell/ml and high values of turbidity. The mixed culture showed the highest ability to grow, reaching I x 100 cells/ml and 900 k.u. Strain ER-RL3 was almost as effective as the mixed culture in growth yield and emulsification of the crude oil.

C----YIX1~*IEII~WPi~i 22 Table 7 Growth of ER-RL4, ER-RL3 and ER-RT on crude oil and UF-1 Following Removal of Waler Soluble Nutrientsa Strain Cell Density Turbidity time 120 h (CFU/ml) time 0 t i me 1.20 h UF- 1 AS UF-1 AS UF-1 AS ER-RL4 ER-RL3

ER-RT

Mixed culture 5.0x10 5 6.0x10 5 9.0x10 7 4.0x10 5 8.0x10 5 4.0105 9.0x10 7 2.0x10 6 3.0x10 5 4.0x10 5 6.OxlO' 2.0x10 6 3.0x10 5 5.0x10 5 1.Ox108 1.0x10 6 550 22 860 490 900 23 a UF-1 medium contained 0.2% fertilizer (containing 0.2% nitrogen.

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 pi of a pure or a mixed culture and incubated for 120 hours at 250C with shaking.

Viable counts were determined after inoculation (time 0) and at 120 hours. Turbidity was measured at 120 hours.

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23 EXAMPLE 11 Secretion of extracellular emulsifier Cells of ER-RL-X were grown in medium containing ammonium sulfate as a nitrogen source and ethanol as a carbon source. When the culture reached the stationary phase of growth (about 109 cells per ml) the cells were removed by centrifugation. The supernatant (7.5 ml) was incubated with crude oil (100 p1) with shaking. The oil was emulsified, indicating the presence of an extracellular emulsifier.

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24 EXPERIMENT 12 UFU (Urea-formaldehyde utilization) activity: Part al Characterizat ion and Puri f icat ion from ER-RL3 Determination of UFU act ivi ty: principle UFU activity is defined as the catalytic breakdown of high molecular weight UP (not soluble) into low molecular weight units, which are water soluble and dialyzable.

Bioassay for UFU activity Princ pie: The bioassay is based on determining the breakdown of Ui-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 qu ntitated by growth of HR-RL3 bacteria utilizing it as a sole nitrogen source.

Procedure: Step A Dialysis (molecular weight exclusion 12,000) of sample to be tested (bacteria, extract etc.) for 24 hours at 40C to remove low molecular weight nitrogen and phosphorus. The high molecular weight fraction will be called "Retentive A".

I- Step B Step C Dialysis of UF-I, same as in step The high molecular weight insoluble fraction will be called "Retentive B".

Retentive A is incubated together with Rententive B for 24 hours at 300C in a dialysis bag (as in Step 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".

Step D- Dialyzate C is now used as N and P source for an indicator culture. This is performed by adding to this sea water 0.5% sodium acetate as carbon source aiid 105 bacteria of strain ER-RL3.

Growth was followed for 16-24 hours at 300C.

Definition: One unit of UFU (urea formaldehyde utilization) activity supports a x10 multiplication of 5 cells of ER-RL3 under the conditions described above.

The results for ER-RL3 appear in Table 8.

Ci_ 26 Table 8 UFU Activity Of Cells Fraction used for bioassay (Sr (added to dialysis bag in step C) i. Control no cell added II. Washed whole cells grown of UF-i III. Extracellular fluid of II concentrated x5 by (NH 4 2 S0 4 .0 precipitation (70% saturation) UFLJ Activity owth of ER-RL3 Units 1 9X 10 7 1. 6X109 1. OX 1013 190 16,000 1,000 i 27 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 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.

L 28 TABLE 9 UFU activity of cells UFU activity Fraction used for bioassay (added to dialysis bag in s tep C) I. Control no cells added II. Washed cells of ER-RL-D grown on UF-1 111 Washed cells of ER-RL-X grown on UF-1 Growth of RU3 8. 5 x 105 6 x 106 2 .3 x 109 Unit s 8. 6,000 23,000

IL.

S 29 EXAMPLE 14 Partial purification of UFU activity of Sea Water Microorganisms Procedure Growth of cells: A preculture was prepared by inoculating one loopful of cells from a single colony on plate into a 100 ml flask containing 20 ml of SWA medium (sea water containing sodium acetate and 0.1% UF-1) and incubated at 300C for 16-24 hours on a reciprocal shaker (100 strokes per minute).

20 ml of this preculture were inoculated into a 2 liter flask and incubated at 300C for 3 days on a reciprocal shaker.

Extraction of protein: Cells were collected by centrifugation for 10 minutes at 10,000 x g at 40C 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 Ammonium sulfate precipitation: Ammonium sulfate was added to the supernatant with stirring to a final concentration of of saturation. The solution was kept overnight at The precipitate was collected by centrifugatlon 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 40 C.

30 Ultra-filtration: Protein fractions were ultra-filtered 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 fihe separation of amimonium sulfate precipi t ates by molecular weight appear in Table 11.

31 fa blIe IleSUIt S of Pur if ic at ion Cellular Fraction ER- RL3 Cell extract Supernatant activi ty after (NH 4 ),.S0 4 .0 precipitation ER-RL4 Cell extract Supernatant activi ty after (NH 4 2 S0 4 precipitation

ER-RT

Cell extract Supernatant activity after (NH 4 2 S0 4 0 precipitation No protein added UFU Activity Growth of ER-RL13 Protein ___mg/mI .2 x10 8 13 3 x10 6 0 .125 8X 107 0. 65 1.5X 10 6 0. 06 3. 0 x10 0 0. 9 2.5XI0 6 0.065 2 X10 6 0 Uni t s 4 ,200 870 3,000 t 32 Table 11 Separation of Ammnonium Sulfate Precipitates By Molecular Weight Molecular Weiglit ER-RL3 total protein >300, 000 30 0 ,00 0 100,000> <300,000 50,000 .0 50,000 Con trol Units of Activity 1,296 34 900 799 41 28 C S 33 EXAMPLE Enhance Biodegradation of Hydrocaruon Contaminated Beach Sand Using Microorganisms isolated from Sea Water Experimental Procedures A. Site selection and characteristics Two plots of 50 m 2 were selected, one for the experiment and the second for the control. They were located approximately m from the water line, about 3 km north of Zvulun Beach (Kiryat Yam). The plots were chosen because they were representative of the oil polluted sands. The oil polluted about 5 cm in depth. The initial concentration of oil (pentane-extractable) 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 The unpacked density of the sand was 1.27 g/cm 3 The maximum temperature of the sand was 360C at noon.

B. Initial Treatment The experimental plot was inoculated with 20 1 of a mixed bacterial culture containing strains BR-RL3, ER-RL4 and ER-RT. UF-1 (38 kg in the form of a fine powder) was added as the source of nitrogen and phosphorous. The plot was then watered with sea water from the adjacent sea (water temperature was 270C). The plot was then 34 tilled by hand with the help of a simple rake to the depth of about 5 cm.

The control plot was left undisturbed.

C. Daily treatment After the first day, the experimental plot was watered daily between 15:00-16:00 with approximately 1.5 m 2 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 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 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 minutes, 25 ml of the pentane extract were transferred to a 100 mi flask containing anhydrous CaCtI.. After standing for

_C

minutes with occasional stirring, the dried pentane extract was filtered through Whatmann 2V paper and then evaporated in vacuo (water aspirator) at 300C. The residual hydrocarbon was dissolved in 3 ml n-pentane and placed in small glass vials in a chemical hood. The weight of the pentane-extractable 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-volatiles in the pentane to the values reported was 0.006 mg/g sand, which Is negligible. T!ie 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 coiita!ned significant- ly more hydrocarbon (3.80 mg/g sand) than the control plot (2.3 mg/g S 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 Therefore we assume that the core sampling was probably the source of the day to day variation, r -i I 36

SUMMARY

Enhanced biodegradation (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 Table 12 Enhanced biodegradation of hydrocarboncontaminated beach sand.

Day 0 1 Pentane-extractablesa mp/eram sand degradation 3. 3.70 2. 76 1.89 0.88 1.40 0.59 2.6 14 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.

Table 13 Natural biodegradation of hydrocarbon-contaminated beach sand Pentan-extractables a mg/gram sand Day degradation 2.30 2.53 1.88 1,70 1.94 1.95 0 0 18 26 15,6 15,6 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, o 0 r o 0i o i 0 00 00 0 0:it 000 0 20 EXAMPLE 16 Two bacterial strains designated RM1 and PU1 (belonging respectively to the genus Agrobacter and Xanthomonas) were isolated following the procedures of the aforementioned examples, particularly Examples 1, 3 and 4.

Results of analysis of these strains are presented in Tables 14, 15 and 16, A third strain was isolated (designated RM2), This strain has not yet been used in studies on the breakdown of petroleum in pure cultures, Table 14 PU1 RM1 Xantlhomonas strain Agrohacter strain QMIO:QhAOT: 343092VIAT.NII:6 Vebruary 96 38 Both strains were identified by Api 20 ne test produced by Bio Merieux.

RM1 PUl

NO

3 TRP GLV ADH URE ESC GEL PNG GLV ARA MNE MAN NAG MAL GNT CAP ADI MLT CIT PAC Ox 4, C 4 4 900 0 0004 4400 00 00 0 4 0 4 4 4944 4 4 4 9 0490 40 20 9' I I 4 £9 C 44*4 4090 4 *4 44 B9 0 4 4s Table Ability to use (gro on) different hydrocargon I-Lydrcarbon RM1 PUl Usage Crude Parafin oil Solar (Gas- oil) D10:lRAhOT:3034S92,I'AT:NII:6 lVebtuaty 1996 39 Hydrcarbon.

He.,,adecdine Cyclohexane Naphthalene Anchracene Toluene RM1 PUl Table 16 Kinetics of growth and oil degradation by strains RM1 and PUi in the presence of 0.5% crude oil and 0.1% F-i fertilizer in sea water at Days 0 2 7

CFUI/ML

0.8 x 106 2.0 x t07 4.6 x 101 5.1 x 108 Strain RM1 Oil Degradation 0 45

CFU/ML

2,3 x 106 6.06 x 101 1.0 xr i0, 9.0 x 108 Strain PUl Oil Degradlatation 0 43 0 0~' 00 0 00 QO 00 0 0 0 0 0 0 0 00 00 00 0 0 0 00 00 0 00 0 0000 C000 0 00 0 00 00 00 0 0 00 0 00 0 I

IC

00 I 000 I)1O;IRAMOT:3O345q2,PA-r:NI h6 Poebriary 1996

Claims (43)

1. A composition for degrading hydrocarbons which comprises at least one microorganism characterized by a UFU-activity greater than 200 units per 10 ml and capable of degrading hydrocarbons by utilizing a complex nitrogen-containing polymer material which contains organic nitrogen in a state which is not utilizable by most soil and water microorganisms 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 form.

3. A composition in accordance with claims 1 or claim 2 wherein the complex nitrogen- containing polymer is a urea-formaldehyde 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. o ~e o o so LIR O Q j i) O O D 0 (1 Il O Uu ULI OO 0~ d O D r L 20 5. A composition in accordance with any of claims 1 to 4, wherein the microorganism is characterized by an UFU activity of greater than about 400 units per 10 ml. £r o 88 4 8 88

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 6, wherein the microorganism is designated ER-RL3 (NCIMB Accession No 40464).

8. A composition in accordance with any of claims 1 to 6, wherein the microorganism is designated ER-RL4 (NCIMB Accession No 40465). "A V DIO:RAKIOTt3O34592A'ATftNII:6 Vbruaty 1996 A aawaaaa~-- -sl~i~ 13111~--- I 41

9. A composition in accordance with any of claims 1 to 6, wherein the microorganism is designated ER-RT (NCIMB Accession No 40466). A con tposition in accordance with any of claims 1 to 6, wherein the microorganism is a mixture of two microorganisms 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 microorganisms 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.

13. A composition in accordance with any of claims 1 to 6, 8, 10 or 11, comprising a 15 cell-free extract, which is a product of ER-RL4.

14. A composition in accordance with any of claims 1 to 6, or 9 to 11, comprising a cell- 0 0 0S free extract, which is a product of ER-RT. 0 0 0 20 15. A composition in accordance with any of claims 1 to 6, wherein the microorganism is designated F. RLD (NCIMB Accession No 40506). o o u e

16. A composition ,n accordance with any of claims 1 to 6, wherein the microorganism is designated ER-RLX (NCIMB Accession No 40507).

17. A composition in accordance with any of claims 1 to 6, wherein the microorganism 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. j DIO:RAMOT:3034592.'AT:NII;6 February 1996 1 r -I 42

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. A method of enhancing the biodegradation and/or bioemulsification of petroleum or petroleum fractions, which comprises contacting the petroleum with a composition containing at least one microorganism characterized by a UFU-activity greater than 200 units per 10 ml and capable of degrading petroleum or petroleum fractions by i utilizing a complex nitrogen-containing polymer material which contains nitrogen molecules which are insoluble in water as its source of nitrogen and where said complex nitrogen containilig polymer has a molecular weight of at least 12,000.

21. A method in accordance with claim 20 wherein the composition also contains a phosphate either in a free or bound form. o o

22. A method in accordance with either claim 20 or claim 21 wherein the complex nitrogen-containing polymer is a urea-formaldehyde polymer. i Xo° 23. A method in accordance with any of claims 20 to 22, wherein the petroleum 20 o comprises an oil slick on the surface of water.

24. A method in accordance with any of claims 20 to 22, wherein the petroleum contaminates sand, soils, rocks and shells. A method in accordance with any of claims 20 to 22, for treatment of petroleum contaminated objects chosen from the group consisting of cargo holds, ballast tanks, pipelines and oil storage tanks.

26. A method in accordance with any of claims 20 to 25, wherein the microorganism is characterized by an UFU activity of greater than about 400 units per 10 ml. DIO;RAMor305S92,PAT;Nl!:S reruaty 1996 43

27. A method in accordance with any of claims 20 to 26, wherein the urea-formaldehyde resin comprises a range of 5% to 40% nitrogen, 0% to 34% phosphorous and 0% to 12% potassium.

28. A method in accordance with any of claims 20 to 27, wherein the urea-formaldehyde resin contains 10% to 35% insoluble nitrogen.

29. A method in accordance with any of claims 20 to 28, wherein the microorganism is designated ER-RL3 (NCIMB Accession No 40464). o oo o o 0o o o o o o o o t, o o So o 9 0 0 0 a 0 0 oa oa a G 00o a a 00 o a a aI 000 A method in accordance with any of claims 20 to 28, wherein the microorganism is designated ER-RL4 (NCIMB Accession No 40465).

31. A method in accordance with any of claims 20 to 28, wherein the microorganism is 15 designated ER-RT (NCIMB Accession No 40466).

32. A method in accordance with any of claims 20 to 28, wherein the mnicroorganism is a mixture of two microorganisms chosen from the group consisting of ER-RL3, ER- RL4 and ER-RT.

33. A method in accordance with any of claims 20 to 28, comprising in combination the microorganisms ER-RL3, ER-RL4 and ER-RT.

34. A method in accordance with any of claims 20 to 25, 29, 32 and 33, comprising a cell-free extract which is a product of ER-RL3. A method in accordance with any of claims 20 to 25, 30, 32 and 33, comprising a cell-free extract, which is a product of ER-RL4.

36. A method in accordance with any of claims 20 to 25 and 31 to 33, comprising a cell- free extract, which is a product of ER-RT. DIO:1RAMOT:303492.PAT;NII6 rbruary 1996 A 44

37. A method in accordance with any of claims 20 to 28, wherein the microorganism is designated ER-RLD (NCIMB Accession No 40506).

38. A method in accordance with any of claims 20 to 28, wherein the microorganism is designated ER-RLX (NCIMB Accession No 40507).

39. A method in accordance with any of claims 20 to 28, wherein the microorganism is a mixture of ER-RLD and ER-RLX.

40. A method in accordance with any of claims 20 to 25, 37 or 39, comprising a cell-free extract which is a product of ER-RLD.

41. A method in accordance with any of claims 20 to 25, 38 or 39, comprising a cell-free 0, o extract, which is a product of ER-RLX.

42. A biologically pure culture of a microorganism designated ER-RL3 (NCIMB Accession No 40464). ,o 2o 43. A biologically pure culture of a microorganism designated ER-RL4 (NCIMB 20 Accession No 40465).

44. A biologically pure culture of a microorganism designated ER-RT (NCIMB Accession No 40466).

45. A mixed culture comprising two microorganisms chosen from the group consisting of ER-RL3, ER-RL4 and ER-RT.

46. A mixed culture which comprises in combination microorganisms ER-RL3, ER-RL4 and ER-RT.

47. A biologically pure culture of a microorganism designated ER-RLD (NCIMB Accession No 40506). 1) D10:flAMO'r:3034592.I 'rA:NII;:6 I:tWlary 1996

48. A biologically pure culture of a microorganism uesignated ER-RLX (NCIMB Accession No 40507).

49. A mixed culture comprising the microorganisms ER-RLD and ER-RIX. A composition according to claim 1 which additionally comprises at least one cold- water insoluble nitrogen-containing polymer.

51. A composition according to claim 50 wherein said polymer is a urea-formaldehyde resin.

52. A method according to claim 20 which comprises contacting the petroleum with at t 0 Sleast one cold-water insoluble nitrogen-containing polymer. o 0 X 15 53. A method according to claim 52 whetein said polymer is a urea-formaldehyde resin. o 0

54. A composition which comprises at least one microorganism characterized by a UFU- activity greater than about 200 units per 10 ml capable of degrading hydrocarbons, 0:"0 methods of using such compositions to enhance the biodegradation and/or 0 0 20 bioemulsification of petroleum or petroleum fractions, and biologically pure cultures of the microorganisms which can be used in these compositions substantially as 0 hereinbefore described with reference to the examples. DATED this 6th day of February 1996. RAMOT UNIVERSITY AUTHORITY FOR APPLIED RESEARCH AND INDUSTRIAL DEVELOPMENT By their Patent Attorneys DAVIES COLLISON CAVE D10:RAMOT:3034592.l'AT: 1:16 Pebruary 1996 r ~multCLLPluli~---;--- 'C A B S T' R A C T The present invention relates to composi- tions 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 biodegradation of petroleum and biologically pure cultures of bacteria. r