CA1285510C - Bacterial composition for the recovery of oil-polluted water and soil and a method for the recovery of water and soil - Google Patents

Abstract

ABSTRACT

A bacterial composition contains as an active biocomponent the strain Pseudomonas putida-36 deposited under No. B-2443, and a mineral component which contains the following mixture of salts, in percent by weight:
34.26 - 37.12 of KNO3, 28.66 - 31.28 of NH4Cl, 25.42 -28.71 of NH4H2PO4, and 2.89 - 11.66 of NH4NO3. The weight ratio of the biocomponent to the mineral com-ponent is from 1 to 26-32. The content of living cells in the biocomponent is from 1.8 x 1010 to 3.6 x 1012 per 1 g of dry biomass. The number of cells of the biocomponent is from 104 to 106 per 1 ml.
The concentration of the mineral component is from 0.07 to 0.08% by weight. The composition may be used either with or without a carrier which may include a solid inert material or water. A method for the recovery of water or soil by treating them with the above bacterial composition in an amount of 350 to 800 mg per 1 m2 of the surface to be treated.

Description

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BACTERIAL COMYOSITION FOR RECOVERY OF OIL-PO~IUTED WATER A~D SOIL

The pre~ent invention relate~ to environmental protec-tion, refers to biological recovery of oil-polluted water and soil and, more particularly, to a bacterial compo~i~ion intended for this specific purpose.
The wide-spread pollution of water and ~oil with oil component~ observed at present i8 attributed to the ever-growing volumes of output9 transportation, refining and uti-lization o~ oil and oil products. Such traditional methods of recovery aR mechanical, chemical and phyaical fail to provide a sufficient degree o~ recovery of oil-polluted water and 80ilo ~herefore, it has been suggested to achie~e this aim by resorting to the resources of Mature itself9 i.e. the effect o~ microorganisms capable of assimilating the hydrocarbons of oil. It ha~ been e~tablished by research thst in the oil spilled over the sea surface in the form of a film 0.1 - 0.4~um thick the nonvolatalized components of oil are attacked by various microbes which degrade a con-siderable proportion of oil within 2 - 3 months. Besides, it i8 emphasi~ed by ~cientific re~earch that the amount of oil decomposed by other orgsnisms or by natural o~ida-tion amounts to one tenth the amount degraded by microbes.
~ owever, in case o~ heavy pollutions, the process of natural 0elf-recovery of water basins may stretch over a long time, reaching several scores of ye~rs. It has bee~ cal-culated that the rate of biodegradation i~ 0.5 kg~ha/day so that degradation of 64 00 t of oil spilled after a tanker wreck may take as long as 20 years approxi-mately. (Microbial hydrocarbon degradation within intertidal zones impacted by the Amoco Cadiz oil spillage. Atlas R.M., Bronner A. "Amoco Cadiz.
Consequences pollut. accident. hydrocarbons. Actes Collog. Int., Brest, 19.22 Nov. 1977 Paris, 1981, pp.
251-256).
Known in the prior art are methods for removal of oil from the water surface by the use of microorganisms and stimulating their activity by introducing the sources of nitrogen and phosphorus into the polluted medium (US Patent No. 4042495, US
Patent No. 4087356). However, the population density of microorganisms of natural biocenoses is so low that even intensive mineral feeding failed to bring about the satisfactory degree and time of oil degradation on water.
A more promising proposition in this field was constituted by application to the polluted area of bacterial cells of -the hydrocarbon-oxidizing microorganisms either in the form of pure isolated cultures or a combination of several genera and species of said microorganisms (Microbiological J.
47, No. 2, publ. 1985, E.I. Kvasnikov, T.M.
Klushnikova, S.L. Kuberskaya, V.S. Zalevsky, G.F.
Smirnova, T.P. Kasatkina, V.I. Svarnik, A.A. Koval "The use of bacteria associations for recovery of oil-polluted bilge water", pp. 12-14).
The difficulties of controlling the quantitative and qualitative composi-tion of bacterial associations denied the possibility of launching the industrial employment of this method.
The wide~t recognition wa~ given to the use o~ micro-organi~m~ mutants produced by the gene engineering methoda.
Such "~upermicrobe~" exhibiting a hlgh oil-oxidizing activity are capable o~ degrading amall amount~ of spilled oil in a ~hort time but fail in cases o~ massive pollutions which call for longer period~ o~-oil degradation. Thua9 it i8 known, that the life apan of the mutant, genus Pseudomonas, i8 but 7.5 days from the ~tart of multiplication of desth, which limits its employment only to a cxude oil content in water not exceeding 50~ mg~1 (European Patent No. 000774223 IPC Cl2 ~ 1/209 Sept. 26, 1984). In addition9 it has been ~ound that the mutants are un~-tsble and apt to lo~e quickl~
the properties inoculated to them in natural conditions.
The pure cultures i~olated from the natural envlronment prove to have more stable propertie~. ~or example, there iB
a known ~train Pseudomonas putida-36,~eparated from an oil-polluted ares of 80il that has remained ~n~ecovered for a long time; said strain i~ depo~ited under No~ B-2443 in the Central Museum of Industrial Microorganisms of the Re-search Institute "VNIIGENETIKA". Thie ~train i~ utilized for the recovery o~ oil-polluted water and soil (USSR InYentor'~
Certificate ~o. 1076446, IPC C12 N 15/00, February 28~ 84), The prolo~ged e~istence of the strRin under extreme con-dition~ ha~ adapted it perfectly not only to B high degree of oil pollution but alao to the preaence of highly-to~ic chemic~ls in the habitat ~nd to ~harp variation~ of tempera-ture. ~he high resi~tance of the atrain and the ctability of its oxidizing properties made it useful in recovery of natural fresh and sea-water basins, industrial sewages of oil refineries, oil storage and transport tanks and oil-polluted soils.
Ilowever, the degree of water and soil recovery from various kinds of oil remained insufficient, being 68.4% for aromatic and 74.5% for paraffin oils.
*hus, the problem of removal of oil and oil products from the surface of water and soil remains pressingly urgent and calls for elaboration of new, more efficient methods and means.
An object of -the present invention is to provide a bacterial composition for the recovery of oil-polluted water and soil which would provide for the highest degree of recovery while being cheap, readily available and simple in preparation and industrial utilization.
The above object has been achieved by developing a bacterial composition for the recovery of oil-polluted water and soil, the active biological componen-t of said composition being the strain Pseudomonas putida-36 deposited under No. B-2443 which, according to the invention, is characterized in that its mineral component is a mixture of mineral salts in the following proportions, mass %:
KN03 34.26 - 37.12 NH4C1 28.66 - 31.28 NH4H2P04 25.42 - 28.71 NH4N03 2.89 - 11.66 The claimed composition makes for a maximum elimination of polluting oil from water and ~oilO
A good practice i~ to u~e the ~train Pseudomona~ pu-tida-36 containing from 1.8 . 101 to 3.6 . 1012 living cells per g of dry biomass.
The composition containin~ the above strain with 1.8 101 to 3.6 . 1012 living cells per g of dry bio-mass proves to be most effective.
It i8 practicable that the maos rBtiO in ~aid composi-tion between the biocomponent and mineral component~ i.e~
the mixture of said ~alts VJOUld be 1:26 - 32.
The above ratio i~ ~uf~icient for maintaining the ac-tive vitalit~ of said strai~.
The claimed composition may be used with or without a carrier, solid or liquid one. Water may be used as a li-quid carrier while talc, diatomite, kaoline, paraffin, etc.
as a ~olid one, ~ he content of said strain in the compo~ition with li-quid (water) carrier i~ 104 to 106 cells/ml and that o~
~he mi~ture of ~aid salts, 0.07 - 0~08 mas~ %O ~hi~ amount of the biocomponent is mo~t rational because the content of biocomponent cells below 104 will prove ineufficient for normal growth and development of cells while the content of the mixture o~ said salt~ below .07 mas~ ~0 at the gi~en content of biocomponent cells will be insuf~icient for the normal growth of the biocomponent; at the same time, the content of biocomponent cells exceeding 106 i~ inexpedient ~ince in presence of hydrocarbon~ in oil pollution~ their content increa~es due to natural multiplic~tion.

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An objeot of the invention al~o lie~ in providing a method of recovery of oil-polluted water and ~oil compri~-ing the use of said compo~ition without a carrier, applied at the rate of 350 - 800 mg/m2 of the polluted area~
~ hlB amount of the COmposition is enough for an all-round recovery of water polluted by up to 25 kg/m3 of oil and of ~oil containing up to 10 kg/m2 of oil. -~ he polluting agents in said methods may be crude oiland its products such aa mazouts, lubricants, oils, fuels.
The claimed invention improves the recovery of oil-polluted water and soil by 22 - 32 % a~ compared with the use of the strain covered by the USSR I~ventor's Certificate No. 1076446 and permit~ con~erting the hydrocarbons to the eeologically neutral compound~ thus attaining a high en-vironmental effect.
The claimed composition iB innocuous to man and e~vir-onment being active both in fresh and sea water and on land.
~ he composition features a high resi~tance to chemic-al pollution of water and soil with toxic elements of dril-ling muds and other chemical reagent~.
The~e and other advantage~ of the invention will be-come apparent from the detailed de~cription that follows.
The strain Pseudomonas putida-36 used in the composi-tion iB isolated from the wa~te drilling mud on organic ba~-iR (oil) in the Tyumen oil fields, USSR, and deposited und-er ~o. I~M n M B-2443 in the Central ~useum of Industrial microorgani~ms of the Research Institute "VNIIGE~ETIKA".
~ he Pseudomonas putida-~6 strain has the following characteriatic~
Morpholo~ical and culture_ propertie~
Size of one-day culture ~ 1.7 - 2.4 ~ O.3 - O.5Jum.The prevailing ahape of cells iG ~traight, rodlike. Gram-nega-tive. The colonies growing on pl~in agar are of the convex, round colourless nsture7 3 - 7 mm in ~ize. On beef-e~tract broth it forms cloudy gas-evolving sediment. Cultivation on potato agar re~ult~ in intensive growth with prominent continuous streak0 and formation of mucu~. Doee not liquefy gelatin. Curdle~ and aci~i~ies milk.

Aerobe, growa &t 28 - 42C, ceasee to grow at tempera-tures below 4C.
Relation to car~ohydrate~: as~imilate~ lactose, gluco~e and maltose with evolution o~ ga~. Doe~ not a~eimilate ars-binoseO
Relation to hydrocarbon~: a~similates naphthalene, camphor,octane, hexane, benzene, toluene, xylene~ para~in, asphaltenee. Para~in oils aa~imilated better than aromatic type 8 .
Relation to nutrient sources: aaaimilates nitric ni-trogen.
The strain requires no special nutrients, being capable of growing on hydrocarbon-depleted media, e.g. stratal wat-er mineralized to not over 10 g/l, is viable at an ~mbient temperature from 70C down to minu 50C and its activity is not aYfected by varyin~ ~eather condition~.
On being dried, the cella retain their viability and hydrocarbon-oxidizing ability which is superior to that of the source strain. Observation of -the titer of a lyophili~ed culture over a 24-month storage period has conformed its stability.
The claimed strain relates to nonpa-tho-genic microorganisms. Peroral, intranasal, intraabdominal and intravenous administration to white mice, keratoconjunctival and epicutaneous application to rabbits have revealed its practical harmlessness and nonpathogenicity. The clinical observation of the medical staff handling said strain revealed no allergies nor bacterioses.
According to the invention the disclosed strain is used in combination with a mixture of mineral salts. Inasmuch as the strain Pseudomonas putida-36 is specifically noted for assimilation of nitrates, nitrate compounds have been given a special preference in selecting the mineral salts.
It has been found, that the optimum relation of the main sources of mineral feeds such as N, P205, K20 is 1:1. Such a relationship can be obtained with the following composition of mineral salts, mass %:
KNO334.26 - 37.12 NH4C:L28.66 - 31.28 NH4~2PO425.42 - 28.71 4 32.89 - 11.66 Thus, the mineral component of the claimed composition delivers 17% N into the habitat of bacteria (10.2% in the form of NH~Cl, NH4NO3, NH4H2PO4 and 6.8% in the form , :: ' - ..

of KN03 and NH4~03); 17% P205 (includin~ 15% of water-~o-luble P205) in the form of ~H ~2P04 and 17% o~ ~ 0 in the ~orm of KN03.
Said balance of ~, P205 a~d K20 ensures the most effective utilization of the strain Pseudomona putida-36 for the recovery of water and 80il polluted by oil and oil products .
The culture of microorgani~ms ma~ be used both in the native and dry form.
To produce the biomas3 of the cells, the strain Pse~do-monas putida-36 is grown in liquid or solid nutrient media containing the source~ of nitroge~, pho~phorus and potassium in presence of hydrocarbons. Cultivation i~ carried out in aerobic condition~ at 30. In these condition~ the cell~ of microorganism~ gro~ and develop vigorously. If need be, the obtainRd biomass of the cells ca~ be dried by lyophilizi~g or dry-air spr~ying.
The obtained biomas~ should contain from 1.8 . 101 to 3.6 1012 living cells per 1 g o~ dry mas~.
The composition can be prepared either dry or as an aqueous aolution.
Preparation of the dry composition, whether with or without the carrier consists in ~imple mechanical miging o~ the biocomponent, a mixture of mineral salt~ and, if ne-ces~ary, a carrier.
~ he cl~imed compo~ition containing a biolo~ical and a mineral components is a light free-~lowing yellowish powd-er easily soluble in water9 oil-polluted too.

Preparation of the composition in the form of an aqueou~
solution i~ confined to mixing the biocomponent and ~aid com-bination of mineral ~alt~ in water.
The dry comp3sition a3 a whole csn be mixed with water;
alternatively, each component, i.eO biocomponent and mineral component csn be mixed with water sepsrately.
The obtained aqueous compo~ition i6 traneparen-t and slightly opaque due to the presence of the biological com-ponent.
The recommended ma~ ratio of the biological component and mineral salt mixture i~ 1:26 - 32. In other word6,there are 26 - 32 ma~s part~ of the mineral component (mixture of salt~) to one part of the biological component. This re-lationship is goYerned by the fact that the amount of miner-al component below 26 ma3s parts will be in~ufficient ~or e~fective activity of bac~eria while its amount exceeding 32 mass parts ~ails to provide additio~al ~timulus to the vital activity of cells and brings about an unwarranted and undesirable expenditure of the mineral component.
The term "oil pollution" 3hould be understood as the presence of crude oil or oil products in a water-dissolYed, emul~ified and film state in the natural and artificial water basins, in 6ea water and on area6 of land.
The dry composition ~hould be given preference ~or the recovery o~ local pollutions of va3t areas or small ~tret-che~ of damp 30il.
The dry composition i6 readily dis~olved in water 80 that the bacterial cells are quickly and uniformly di~tri-"~ ~

buted in the water-oil contact zone and are given acce~
to the nutrient sub~trate (hydrocarbons) thus eneuring rapid growth of the cells and all-round elimination o~ oil pollu-tions as proved by vi~ual observation~ and control analy~e~.
It is expedient that the wide area~ of both water and land ~h~ll be recovered ~xo~ oil pollution~ by mean~ of a composition with a carrier, either liquid, e.g. watar~ or solid, e.g. talc, diatomite~ paraffin, kaoline and other inert, light, solid materials. The employment of fil~ers makes for a more uniform di~tribution of a relat~vely small amount of the compo~ition over large polluted areas from airplanes and helicoptersO If a solid carrier is used9 it~
ratio in the claimed composition is 30 - 50 to 1.
The preferable liquid cPrrier i8 a~y kind of water ~ince it i8 generally available, neutral and innoc~ou~ to-ward~ the object o~ pollution, and becau~e bacteria tend to grow more intensivel~ in ~ater which proves to b~ moot esQential when dealing with oil pollutions on dry lands, sandy and stony areast ~uch ae beaches And ~ea-front~.
Concentrati~n of the biocomponent in an aqueous 80-lution ranges from 104 to 106 cells/ml. If thi~ concentra-tion is under 104, it will prove insu~icient ~or the norm-al growth and development of cells while co~centration above 106 ie unrea~onable since the amount of biocomponent cell~
increa~ee due to their natural multiplication in the pre-eence o~ hydrocar~ons in oil pollutione.
The above concentration of cells in the aqueous eolu-tion ie obtained by dissolving 2.5 - 25 g of the biological ~s~

component containing 1~8 . 101 to 3.6 ~ 1012 living cellsin one litre water.
Another object of the invention iB a method of recovery of oil-polluted water a~d ~oil comprising the application of said composition to thepolluted areas. The composition in any form (whether dry or a water ~olution) i~ Qpplied mostly once to the polluted area where the above-cited pa~
rameters, i.e~ content of biocomponent cells and the amount of the mineral ~alt mixture make for an active growth of biocomponent cells and ensure their active vitality. Depend-ing on the intensity of oil pollution, the amount of appli-ed compo~ition varies from 350 to 800 mg per ~q.m of the treated surface. Thi~ amount is sufficient for eliminating oil pollution of up to 25 kg/m3 of water and up to 10 kg~m -of soil.
~ he di~clo~ed i~ventio~ is by far more profitable commercially than the m~thod~ known in the priOr art.
~ he invention proYides a radical sQlution of the environ-ment protection problem~ particularly the recovery of oil-polluted water and soil and, what for its technical and eco-nomical lndices, it outclasses the known methods pur~uing similar purposes.
An emphatic advantage of the disclosed compo~ition and the method of its application i~ the increase in the degree of water and soil recovery to an sb~olute reli~bility aft-er they have been massiYely polluted with oil and oil pro-ducts.
The disclosed composition ~implifieL the prQcess of ~2 ~5~

recovery due to its ready-to-u~e form whereas the use of a pure culture in~olve~ additionsl complications caused bg the necessity for growiDg the ba~teria on liquid nutrient media, carrying them to the point of application, additional control of the concentration of living bacterial cellsl etc.
The disclo~ed composition and the method of application thereof can be readily realized under industrial conditions since they do not require any special equipment and special-ly-trained personnel. ~he method i~ ~ucce~s~ully applicable in the field.
The disclo~ed composition doe~ not require any parti-cular storage conditions and it~ application is not a~fected by weather and climate. The compositlon is noted for stabi-lity of its properties.
The disclosed composition i3 harmles~ for man and en-vironment) i-t i8 explosion- and fire-proof.
The discloeed composition can be succes~fully used in a num~er o~ media, such as aea and ~resh water, induRtrial sewages, areas of 50il polluted with crude and bunker oil and the products of itB refining, e.g. die~el fuel, ma~out,lu-bricants, etc.
The best compo~ition ie the one containing the strain Yseudomonas putide-36 deposited under No. B-2443 and a mix-ture of mineral salts, mass %: KN03 - 36.53; ~H4Cl - 30.83;
NH4H2P04 - 27.53 and NX4N03 - 5.11. The content of livi~g cells in the biological component is about 3.6 ~ 1012 per gram of dry biomass. ~he ratio of biologicsl to mineral com-ponents i~ 1:28. The composition is prepared by mi~ing it~

~ 8 ~

components in the ma~s ratios ~pecified above. The compo~i-tion i~ a li~ht finely-disper~ed powder, It is practlcable that said composition be u~ed as an aqueous solution..
This composition iB mo~t effective in comparison with other formulas since it requires a lower expenditure o~ the biocomponent and, consequently, permits using the Pame amo-unt for treating a rnuch larger area of pollution. Thus, in case of a composition who~e biocomponent contains 1.8 . 101 living cells per g of dry biomass, its consumption rate i8 25 mg per m2 while the biocomponent having 3.6 1012 living cells per g of dry biomass ca~ be used at the rate of a mere 205 g per m2 which reduces the consumption of the biocompo-nent 10 times or, in other words, increases the treated area 10 times.
This ver~ion curtail~ considerably the expenditures ~or tran~portation of the compo~ition.
The present invention will become more apparent from the appended examples of which e~amples 1 through 3 illustrate the preparation of biocomponent, examples 4 through 6 des-cribe the preparation of the dry composition without a car-rier, examples 7 through 9 describe its preparation in ~n aqueous solution, example~ 10 to 12 egplain the p~eparation of the dry compo~ition with a carrier, examples 13 through illustrate the u~e of the claimed composition for the reco-very of oil-polluted water a~d soil, and example 18 ~how~
the u~e of a pure strain, mixture of mineral ~alts and of the di~closed composition :E~camDle 1 _. .
~his e~ample illustrate~ the preparation of biomass s~

from the cells of the active component of the compo~itiQn.
The source ~train P~eudomonas putida 36 is grown on a liq~id nutrient medium in the pre~ence of hydrocarbons, e.g. crude oil by the deep culti~ation method in a fermenter on the nutrient medium of the following formula, g:
~P4 10.0 K~2P4 1.0 4N3 2.0 H20 1000.0 Cultivation is carried out under aerobic condition3 at 30C.
The prepared biomass is concentrated and dried by the method of dry-air spraying at +60C~ The number of living cell~
found by the titration method has been 108 10 per g of dry matter~
xample 2.
The biomas~ i8 prepared by the technique~ laid down in Example 1. The obtained bioma~ dried by the dry-air ~praying method at 40C. ~he produced bioproduct contains 2.4 ~ 1011 cell~ per g of dry matter.
Exampl~
The biomass prepared as described in Example 1 i9 dried by the method of lyophilization. ~he obtained product con-taine 3.6 1012 living celle per g of dry matter.
ExamPle 4.
1 g of the dry biomass prepared 88 de~cribed in Example 1 is mixed with 25 g mineral salt mixture of the following composition, maQs ~ 03 - 34.26, ~H4Cl - 28-66, NH4H2P04 -- 25.42, The prepared composition has the form of a finely ~ - 16 -~8g~
-dispersed yellowish powder.
Example 5 1 g of the dry biomass prepared as in Example 2 is mixed with 28 g of a mineral salt mixture of the following composition, mass%: KN03 -36.53, NH4Cl - 30.83, NH4H2P04 - 27.53, NH4N03 -5.11. The produced composition has the form of a finely-dispersed yellowish powder.
Example 6 To increase the concentration of the composition, 1 g of the dry composition prepared as in Example 3, containing 3.6 10 living cells per g of dry matter is mixed with 32 g of a mineral salt mixture comprising 37.12% KN03, 31.28% NH4Cl, 28.71%
NH4H2R04 and 2.89% NH4N03. The produced composition has the form of a finely-dispersed yellowish powder.
Example 7 To make a water solution, 362.5 mg of a dry composition prepared according to Example 4 should be mixed with 0.5 1 water. The produced liquid composition contains 10 living cells per ml and 0.07% of a mineral salt mixture. The prepared composition is transparent and slightly opaque due to the presence of the biological component.
Example 8 To prepare an aqueous solution, 540 mg of the dry composition produced according to Example 5 should be dissolved in 0.75 1 water. The obtained liquid composition contains 10 cells per ml and 0.07% of the mineral salt mixture. The composition is transparent and slightly opaque due to the presence of the biocomponent.

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Example 9 To prepare an aqueous solution, 825 mg of the dry composition produced according to Example 6 should be dissolved in 1 1 water. The obtained aqueous composition contains 10 living bacterial cells per 1 ml and 0.08% of the mineral component.
Example 10 ~ 0 prepQre a granular composition, 26 g of the dry com-position containing 1 g of biocomponent and 25 g of a miner-al ~alt mixture should be mixed with 780 g of paraffin car-rier. ~he ma~s ratio of the dry compo~ition to the carrier iB 1: 30. The carrier ~hould first be ground to gran~les 0~05 - 0.1 mm in ~ize and placed into a plate gr~nulator together with the dry composition. ~he obtai~ed granular composition i8 recommended for the recovery of oil-polluted water ~urfaces.
Example 11 To prepare a dry composition, 29 g of the dry compo-sition produced according to Example 5 and containing 1 g of biocomponent and 28 g of a mineral ~alt ~ture should be mixed with 1160 g of kaoline carrier at a ratio o~ 40 parts of the filler to I masa part of the dry compo~ition. The mlxture then should be vigorously mixed by mechanical agit-ators. The obtained compo~ition i8 a light, free-flowing white powder.
Example 12 To prepare a dry compo~ition, 33 g of the dry composi-tion produced according to Example 6 and containing 1 g of biocomponent and 32 g of a mineral ealt mi~ture ~hould be mixed with 1690~of diat ~ te carrier. The ratio o* dry composition and carrier is 1:50 mas~ parts. A thoroughly mixed compo-~ition with the carrier is a light-grey free-flowing light powder. l'he obtained compo~ition is recommended for the re-covery of large oil-polluted areas of bogged and water-logg-ed ~oil~.
E~amPle 13 An area of soil 50 OOO m2 ha~ been polluted by a doae of 10 ~g of crude oil per m2. In other words, the total amount of oil in the area wa~ 500 ton~. To eliminate the oil pollution, 50 m3 o~' the aqueou~ ~olution according to Example 9 wa~ u~ed. Each litre of the compo~ition contains 825 g of a mixture of biocomponent with mineral ~alt~. The composition i8 spread uniformly at the rate of 1 l/m by spraying from ~elf-propelled ~prayers equipped with pre~sure pumps capable of delivering the pulverized jet o* liquld to a di~tance of 35 - 45 m. On expiration o~ 2 months after a ~ingle treatment, the amount of destroyed oil was 460 t.
The experiment was conducted in sunny weather alte~nating with rain ~ the temperature varyi~g from ~26 to 0~C. Ob~er-vation~ revealed active plant vegetation in the polluted area.
Example 14 A 9ection of a natural fre~h-water bàsin within the territory of an oil field has been polluted with crude oil over an area of 25 OOO m2 at the rate of 23.3 l per m3 which mean~ that the total amount of crude oil in the section was 581.5 t~ To eliminate oil pollution a water composition pro-duced according to Example 8 was u~ed. Said composition contained 543.75 mg of dry composition in which 18.75 mg of biocomponent and 525~g of a mineral salt mixture were pre-~ent at a ratio of 1:28 and the water solution contained s~

105 li~ing bacterial cell per ml a~d 0.07% of the mineral salt mixture- The CompOBitiOn Was prepared in the amount of 18.75 m3 at the rate of 0.75 1 per 1 m of the polluted BUr-face. The water composition was applied once by uniform aeri-al spraying. On expiration of 2 months after treatment the basin wae completely recovered from oil.
Example 15 An object of treatment is open ~edimentation basin~
for industrial s0wa~e of ~n oil refinery. The total area of the basin is 800 000 m2, the volume of water, 1.5 million m3. The content o~ oil products (v~ater-~oluble, emulsified and in a ~ilm state) amount~ to 75 mg/l.
~ he oil pollution i~ eliminated b~ the use of a compo-~ition proùuce~ accordi~g to Example 7 and containing 2.5 kg of biocomponent and 70 kg of a mineral salt mixture dis~olv-ed in 400 m3 o~ water; this composition cont~ins 104 living bacterial cells a~d 0007% of the mineral ~alt mixture per ml water.
The composition i8 di~tributed uniformly over the en-tire aree of the water basin at the rate of O.5 1/m2 from a fire truck equipped with a tank and a carriage-mounted fire hose.
14 day~ after a single treatment the content of oil producta in water has dropped to 0.5 mg/l, remaining stable at this level within the following 14 day~ in ~pite of a con~tant inflow of the indu~trial sewage laden with 75 mg/l of oil product~.

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Example 16 A glas~ reservoir (1.25 m long, 0.8 m wide, 0.15 m high) is filled with 100 l of watex ~rom the Kara Sea and 1720 g of crude oil.The oil film i8 removed by u~ing 22.5 g of the composition prepared in accorda~ce with E~amplè 10.
This composition contain~ 25 mg of biological component, 700 mg of mineral component and 21.75 g o~ paraffin carrier.
At a room temperature of 18C the sea water wa~ completely puri~ied of oil within 14 days after a single treatment.
Example 17 A 200 m boggy area of ~oil polluted with 60 kg o~ oil (0~3 kg/m ) was treated ~ith 7 500 g of the dry compo ition prepared according to Example 12. The dry composition wa~
uniformly sp ayed oYer the polluted area at the rate o~
37.5 g per m . This dose contained 25 mg of the biocompo~
nent, 750 mg of a mineral salt mixture and 36,75 g of the inert diatomite carrier. 22 day~ after a single treatme~t the area wa~ fully cleaned from oil.
E~ample 18 ~ he given example illustrates the employment of a pure strain Pseudomona~ putida 36, or of a mineral salt mix-ture or of their combination9 i.e. the claimed composition for elimlnation of oil pollutiona. The results gained are ~um-marized in the Table below.
It can be seen from the Table that the pure strain P~eudomonas putida-36 decomposes 68-76% of the initial amo-unt of oil; a mixture of mineral salt~ (w/o strain), 6.4 - 8.13~; and the claimed composition, 98.9 - 99.9~.

_ 21 -Table Re~ults of rTe Bt8 of Strain Pseudomonas Putida-36, I~ture of Mineral Salts and Claimed Compo~ition for RecoYery o~ Oil-Polluted Water ~nd ~oil Condition~ Oil de~radation ef~i-of Treating material Sample _ cienoy, %
experiment ~0 aromatic paraflin Oil-pollut- Strain P~eudomo- 1 69~1 72~8 et water~ nas putida-36 2 87 ~ 5 76 ~ 7 l~o YOl. 3 6~3o72 7309 Duration of 68. 44 74 ~ 46 experiment ~ineral ~alt mix~ 1 6D7 ' 7~1 96 - 120 h. ture, %:
~KNO3 36053 2 6.9 5.8 ~I4Cl 30.83 3 5.6 6.7 N~4E2P04 27 ~ 53 6.4* 6.53 4 3 5~ 1 Claimed compo~ition containing strain 1 98.6 99.8 Pseudomona~ putida- 2 98.9 100.0 36 and mixture o~ 3 99.7 * 10090 *
aaid mineral ~alta 99.06 99.93 Oil-polluted Strain Paeudomonaa 1 73 . 9 75 ~oil,10% vol putida-36 . 2 68.2 74 9 3 72.7 79.2 Duration of 71 ~ 6 76.4 experiment 20 - 30 daya Mineral ~alt mix- 1 7.7 7.9 ture, % 2 7.9 8.0 3 6.9 8.5 7.5 8.13 KN03 36.53 NH4Cl 30~83 H2P04 27~53 4 3 5.11 Claimed composition 1 98.4 99.8 containing atrain 2 98.7 10000 Pssudomonas putida- 3 99.6 99.9 36 and mixture of ~ *
~aid mi~eral ~alts 98-9 99-9 , ..... . , . . , . ,~
* mean value The u~e of the mineral salt mixture in combin~tion with the pure ~train P~eudomonas putida-36 raises the efficiency of sQid ~train 1~31 - 1.45 time~ on the average and i~ cap-able of ensuring practically 100% recovery of the media pol-luted with oil and oil product~.

Claims (13)

1. A bacterial composition for cleaning water and soil from oil pollution comprising the bacteria Pseudomonas putida-36 as an active biocomponent, deposited under No. ?M?M B-2443, and a mineral component comprising a mixture of mineral salts, having a percent by weight of the mixture of from 34.26 - 37.12% KNO3; 28.66 - 31.28% NH4Cl; 26.42 -28.71 NH4H2PO4 and 2.89 - 11.66% NH4NO3, wherein the ratio of the biocomponent to the mineral component being from 1:26 to 1:32.

2. A composition of claim 1, wherein the content of living cells in said biocomponent is from 1.8 x 1010 to 3.6 x 1012 per gram of dry solid.

3. A composition of claim 1 containing an inert filler.

4. A composition of claim 3, wherein the inert filler is a solid composition selected from the group consisting of talc, paraffin, kaolin and diatomaceous earth, and is present in a ratio of filler to mixture of biocomponent and mineral com-ponent is from 30:1 to 50:1.

5. A composition of claim 3, wherein the inert filler is water.

6. A composition of claim 5 comprising from 0.07% to 0.08% by weight of the mineral salts and 104 to 106 living cells of the biocomponent per milli-liter.

7. A method of cleaning water and soil from oil pollution, which comprises contacting the water or soil with a composition comprising a bacteria Pseudomonas putida-36 deposited under No. ?M?M
B-2443 as an active biocomponent and a mineral component comprising a mixture of salts in a percent by weight of 34.26 - 37.12% KNO3; 28.66 - 31.28%
NH4Cl; 26.42 - 28.71 NH4H2PO4 and 2.89 - 11.66%
NH4NO3, the weight ratio of the biocomponent to the mineral component being 1:26 to 1:32.

8. A method of claim 7, wherein the number of living cells in the biocomponent is from 1.8 x 1010 to 3.6 x 1012 per gram of a dry biomass.

9. A method of claim 7, wherein the composi-tion is in admixture with an inert filler.

10. A method of claim 9, wherein the inert filler is a solid selected from the group consisting of talc, paraffin, diatomaceous earth and kaolin in a weight ratio of the inert filler to the composition of from 30:1 to 50:1.

11. A method of claim 8, wherein from 350 to 800 milligrams of the composition is applied to a square meter of surface to be treated.

12. A method of claim 9, wherein the composi-tion is in admixture with water to provide a mixture containing from 0.07 to 0.08% by weight mineral component and from 104 to 106 living cells per milliliter of admixture.

13. A method of claim 12, wherein the admix-ture is applied in an amount of 0.5 to 1 liter per square meter of a treated surface.