Strain of bacteria bacillus vallismortis - destructor of oil and oil products

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus vallismortis VKPM V-11017 is proposed - destructor of oil and oil products. Strain may within short period of time in the wide range of temperatures from +8 to +37°C degrade oil by 78.3%.

EFFECT: strain may be used to clean soil and water contaminated by oil and oil products, such as diesel fuel, motor oil, gas condensate.

3 tbl, 5 ex

 

The invention relates to the field of Microbiology and represents a new bacterial strain of the bacteria Bacillus vallismortis VKPM B-11017, with the ability to quickly dispose of oil and oil products (diesel fuel, motor oil, gas condensate). The strain can be used for cleanup of soils and water contaminated with oil and oil products, in a wide temperature range from +8 to +37°C.

The level of technology

Known strains: Rhodococcus erythropolis, Rhodococcus luteus, Micrococcus flavus (1. Klyuchnikova M., Smirnova GF, Kubersky S.L., Slabospitsky A.T., Zalewski B.C., Kasatkina, ETC., Eliseeva G.S., A.S. SU 1493666-A1. Cleaning method podslantsevyh wastewater from petroleum products. Application 4280617, 09.07.1987. Publ. 15.07.1989); Flavobacterium tirrennicum (2. Novozhilov M.I. Popova LE, Nedovodiyev ETC., A. Kalamkarov LI As. SU 1409594. The strain of bacteria Flavobacterium tirrennicum used to clean podslanevye and industrial wastewater from oil and oil products. Application 4066152, 24.03.1986. Publ. 15.07.1988), which can decompose the hydrocarbon oil.

The disadvantage of the above strains is that they carry out the degradation of petroleum products only in water and not able to clean the soil from oil.

Known strains: Pseudomonas putida 36 (3. Dyadechko V.N., Tolstokorova LE, Morozova so-CALLED. Strain @ @ 36 used for purification of water and soil from oil and oil products. A.S. USSR 1076446. For the WHC 3474745, 22.07.1982. Publ. 28.02.1984); Acinetronacter sp.-1 (4. Zhirkov N.A., cholodenko's VP Strain of bacteria ACINETOBACTER SPECIES used for purification of water and soil from oil and oil products. RF patent 2077579. Application 9404331, 06.12.1994. Publ. 20.04.1997); Acinetronacter sp.JN-2 (5. Ushkov S. B., Elias, NV Strain ACINETOBACTER SPECIES used for purification of water and soil from oil and oil products. RF patent 2064017. Application 93009595, 23.02.1993. Publ. 20.07.1996); Pseudomonas alcaligenes E7 (6. Ermolenko SM, cholodenko VP, Chugunov V. bacterial Strain PSEUDOMONAS ALCALIGENES E7 used for purification of water and soil from oil and oil products. RF patent 2134723. Application 98105707, 25.03.1998. Publ. 20.08.1999) and Pseudomonas alcaligenes B-1 (7. Ermolenko SM, cholodenko VP, cast Irons, VA, Kobelev B.C., Akimova N.A. bacterial Strain PSEUDOMONAS ALCALIGENES IN-1, used for purification of water and soil from oil and oil products. RF patent 2133770. Application 98105709, 25.03.1998. Publ. 27.07.1999).

The disadvantage of strains is that they disposed of the oil at relatively high temperatures.

Greater efficiency at low positive temperatures showed the strain Rhodococcus erythroplis E-15 (8. There are regional laws T.V., Arakelyan AI, Komarova T.N., Ilyinsky V.V. Method of cleaning soil from oil pollution. RF patent 2019527. Application 93017464, 3.04.1993. Publ. 15.09.1994), but the process of biodegradation of oil flows slowly and only adding ammonium nitrate or ammonium nitrate and sodium nitrate.

As about the tipa is the bacterial strain Bacillus subtilis Kolyma 7/2" (9. Neustroev BTW, Tarabukina, NP, Neustroev, M., Sazonov, N., Greenhouse, S., Stepanova A.M. purification Method permafrost soils from oil spore-forming bacterium BACILLUS SUBTILIS. RF patent 2446900. Application 2010129158, 13.07.1010. Publ. 13.07.2010), with the ability to reduce the concentration of oil to 0.48%, i.e. the level of oil from 135143 mg/kg reduced to 645 mg/kg within 3 months of the summer period. However, the strain carries out the degradation of oil and only in the soil conditions of the model experiment and not capable of cleaning water from oil and dispose of petroleum products (diesel fuel, motor oil, gas condensate), including in conditions of wide temperature range from +4 to +37°C.

The objective of the invention is to obtain a bacterial strain with high scavenging ability towards both oil and oil products in a relatively short time, in a wide temperature range from +8 to +37°C, which can be used for cleanup of soils and water contaminated with oil and oil products (diesel fuel, motor oil, gas condensate).

The implementation of the invention

The proposed bacterial strain Bacillus vallismortis VKPM B-11017 isolated from oil-contaminated soil route of oil pipeline "Eastern Siberia - Pacific Ocean", 1338,5 km, Lensky district, Republic of Sakha (Yakutia), may 19, 2010. Stamp fee is deposited by the all-Russian collection of industrial microorganisms in FSUE gosniigenetika - PMBC, 117545 Moscow, 1st Road travel, 1. Collection number VKPM B-11017.

The strain Bacillus vallismortis VKPM B-11017 recycles oil and oil products (diesel fuel, motor oil, gas condensate) as the sole carbon source. The process goes in a wide range of temperatures (from +8 to +37°C).

The use of the strain Bacillus vallismortis VKPM B-11017 in vivo for treatment of frozen soil from oil, at an average soil temperature from +8 to +14°C, allows to reach 78.3% of degradation of oil for 60 days.

Destruction of oil and oil products in water at a water temperature of +20°C is 50.8 per cent, for 2 months.

Destruction of oil and oil products in the aquatic environment with the addition of mineral components after 7 days at a temperature of +8°C 18.5 - 56.4 per cent, at a temperature of +20°C to 31.7 and 67.8%at a temperature of +30°C - 44,7 - 74,0%, at a temperature of +37°C - 50,0 is 84.7%, depending on the contaminant type.

The resulting strain is characterized by the following features.

Morphologically signs.

Gram-positive spore-forming bacilli in smears are single and short chains.

On mesopatamia agar (wt.%: enzymatic peptone - 1.0; sodium chloride and 0.5; agar - 1,0; water meat rest, pH 7.0 to 7.2) supplemented with 1% glucose grows in the form of a smooth dry beige colonies, after 1-3 days the colonies are stained in brown C is no, the substrate is not pigments.

On the surface beveled mycopathologia agar without the addition of 1% glucose grows in the form of a dry wrinkled films beige colour.

On the environment Saburo (wt.%: hydrolyzed fish meal - 1,0; pancreatic hydrolysate of casein to 1.0; yeast extract and 0.2; sodium phosphate one-deputizing - 0,2; D-glucose - 4,0; agar - 1,0; distilled water - the rest; pH of 6.0±3) forms large colonies from dirty brown to marsh-brown shade with a diameter of 1 cm or more.

In mesopatamia broth (wt.%: enzymatic peptone - 1.0, sodium chloride and 0.5, water meat - rest; pH 7.0 to 7.2) grows in the form of wrinkled films beige colour on top of the broth.

Mineral environment of Muntz (10. Kersten D.K. Morphological and cultural properties of indicator microorganisms oil and gas shooting - Microbiology, 1963, No. 5 - S-1030) with oil and oil products (diesel fuel, motor oil, gas condensate) of the following composition, wt.%: KNO3- 0,4; MgSO4·7H2O - 0,08; NaCl - 0,1; K2HPO4to 0.14; KH2PO4- 0,06; agar - 2,0; oil or petroleum products - 1,0; distilled water - the rest, pH 7,2, in the form of opaque matte beige-grey colonies with a diameter of 0.1-0.2 see

Physiological and biochemical characteristics.

The strain grows well at temperatures from +8 to +37°C in aerobic conditions. At temperatures below +8°C in anaerobic conditions, the strain did not immediately die, but progresses more slowly. The optimum growth +30°C. Grows at a pH of 6.0 - 8.0. Grows in salt broth with the addition of 0.1-2.0% NaCl.

Strain catalanopolonesa, oxidization. The oxidative metabolism type. Biochemical activity expressed weakly. Not absorbed lysine and ornithine. Does not consume citrate and malonate sodium. Gelatin is not hydrolyses. Starch is not decompose. Indoleacetate. Does not form hydrogen sulfide. Reaction Voges-Proskauer negative. Phenylalaninamide negative. Ferments beta-galactosidase.

The strain is not virulent, non-toxic, non toxigenic, not phytotoxic (test conducted on white mice and seeds of higher plants (oat variety "Roller" and the wheat cultivar "Prilenskoe-19").

Storage conditions:

1. In dried form in vials at +4°C.

2. The strain can be maintained by regular re-seeding (1 every 2 weeks) on the beveled MPA.

The invention is illustrated by the following examples.

Example 1. Cultivation of a strain of Bacillus vallismortis VKPM B-11017

The proposed strain of Bacillus vallismortis VKPM B-11017 was isolated from oil-contaminated soil route of oil pipeline "Eastern Siberia - Pacific Ocean", 1338,5 km, Lensky district, Republic of Sakha (Yakutia), may 19, 2010.

Cultivation of the strain was performed in a mineral environment of Muntz of the following composition (g/DM3): KNO3- 4,0; MgSO4The 7H 2O - 0,8; NaCl to 1.0; K2HPO4- 1,4; KH2PO4- 0,6; agar - 20,0; distilled water 1,0 DM3; pH of the medium was 7.2. To which 1.0 g of contaminated soil with the above object was made in 250 ml of mineral medium of Muntz. As the sole source of carbon and energy used oil, which was made on Wednesday of Muntz in the amount of 1000 mg 1.0 DM3environment. Incubation was carried out in a thermostatted katalozhnyh conditions at the "WVMT-12-250" at 200 rpm and a temperature of 29±1°C. bacterial Growth was observed after 7-10 days of incubation on education turbid whitish emulsion and the disappearance of oily stains of oil. A pure culture of bacteria was obtained by cultivation under the above conditions and multiple transfers (more than 10) cumulative culture on Petri dishes with agar from the hydrolyzate, fish meal (RM-agar) supplemented with 1% glucose. Further crops were incubated under stationary conditions in a thermostat at 30°C. After 48 hours on the surface RM-agar appeared dry, beige colonies that cultural-morphological and biochemical characteristics, as well as the results of the analysis of nucleotide sequences of 16S rRNA gene and the key phenotypic characteristics according to taxonomic descriptions given in the Determinant of Berga (11. Bergey''s Manual of Systematic Bacterioloy Book Review Int. J. of Syst. Bact.; July 1985, p.408), identified as a strain of Bacillus vallismortis.

Isolated colonies were perseval on beveled mastopathy agar. After 48 hours incubation at a temperature of +30°C cells of the strain Bacillus vallismortis VKPM B-11017 with beveled agar used to determine activity in the process of destruction of oil and petroleum products.

Example 2. The study Neftekhimiya activity of the bacterial strain Bacillus vallismortis VKPM B-11017

Cells of strain Bacillus vallismortis VKPM B-11017 with beveled mycopathologia agar washed with 0.9% NaCl solution, prepared the initial microbial suspension of bacterial isolate, volume 25 cm3with a concentration of 1×109 microbial cells/cm3optical standard gisk named after. A.M. Tarasevich.

The obtained bacterial isolate of the strain Bacillus vallismortis VKPM B-11017 cultivated in an aqueous medium with mineral composition, similar in composition to the environment of Muntz, the composition of which is described in example 1.

In flasks with a volume of 200.0 cm3contribute 100,0 cm3ready mineral environment and 1000 mg of oil. The flask is inoculated with cells of strain up to a concentration of 1·109cells/ cm3. As a control, put the same bulb with the environment, oil and without bacteria to determine the total (natural) losses. The experience carried out in three replications. Bulb cultivated in katalozhnyh conditions at the "WVMT-12-250" at 200 rpm, when the temperature is +8°C; +20°C +30°C and +37°C for 7 days.

The biodegradation of crude oil and petroleum products are determined by a spectrometric method using concentrator "TSC-025" (12. FR. 1.31.2007.03234 MIM 01.02.117 the Method of measurement of mass concentration of petroleum products in drinking water and wastewater IR-spectrometric method using concentrator "TSC-025").

The experimental data shows that in the aquatic environment with mineral components of the proposed strain by 7 days at a temperature of +8+37°C utilizes 18,59-52,82% of oil; 25,46-82,26% diesel fuel; 28,19-50,04% - motor oil; 56,40-84,73% of gas condensate (table 1).

Table 1
The degree of utilization of oil and petroleum products by a strain of Bacillus vallismortis VKPM B-11017 after 7 days of cultivation
Version of experiencet°C
Xenobiotic, mg/DM3Period+8+20+30+37
Oilto experience1000,0 1000,01000,01000,0
after the experience814,1425,0393,0and 471.8
% degradation18,59of 57.560,752,82
Diesel fuelto experience1000,01000,01000,01000,0
after the experience745,4393,7171,0177,4
% degradation25,4660,6382,982,26
Motor oilto experience1000,01000,01000,01000,0
after the experience718,1682,8553,0499,6
% 28,1931,7244,750,04
destruction
Gas condensateto experience1000,01000,01000,01000,0
after the experiencekm 436.0321,2259,4152,7
% degradationof 56.467,8874,0684,73

Example 3. Preparation cell-based strain Bacillus vallismortis VKPM B-11017

Bacterial isolate obtained by the method described in example 2, is transferred into a flask with liquid sterile environment, similar to the composition of the environment Stepin and other (13. Stepin A.A., Samygin V.M., kukhtin VP, Vladeltsev IV, Aga L.K., Caplaw VI Method of producing biomass bacilli. RF patent 2053293. Application 5036470, 09.01.1992. publ. 27.01.1996), g/DM3: hydrochloric acid hydrolysate of casein - 14,0; fodder yeast extract 3,0; MgSO4·7H2O - 02; NaCl - 4,0; (NH4)6Mo7O24·4H2O - 0,5; distilled water to 1.0 DM3; pH 7.3 to 7.4. The medium is sterilized at 0.5 psi for 20 minutes

Under sterile conditions, 100,0 cm3ready environment contribute 2.0 cm3emulsion "Perftoran" (JSC SPF "Perftoran", Russia) and 10.0 cm3suspensions of bacterial isolate. Cultivated in flasks on a shaker for 48 hours at a temperature of 29±1°C. Receive liquid turbid culture, with a titer of at least 1 billion cells/cm3used as a drug for the treatment of soil or water from oil pollution.

Example 4. Water purification from oil using drug-based cells of the strain Bacillus vallismortis VKPM B-11017

In the desiccator volume of 5.0 DM3made 3,0 DM3water and 30.0 cm3drug-based bacterial strain Bacillus vallismortis VKPM B-11017, obtained as described above (example 3). To create an oil film capacity was added 30000 mg of raw crude oil. As control was used the water in an amount of 3.0 DM3with the addition of 30 000 mg of oil and without making microorganisms.

The experiments were carried out within 2 months when the water temperature +20±1°C.

The biodegradation of oil determine spectrometric method using concentrator "TSC-025" (12. FR. 1.31.2007.03234 MIM 01.02.117. The method of measurement of mass concentration is AI petroleum products in drinking, natural and waste waters IR-spectrometric method using concentrator "TSC-025").

It is established that the destruction of oil in water for 2 months amounted to 50.8 per cent. Destruction in the control tank for 2 months did not change significantly (table 2).

Table 2
The dynamics of degradation of oil in water after application of the drug on the basis of the strain Bacillus vallismortis VKPM B-11017
Version of experienceThe amount of oil to make cells of strain mgThe amount of oil in 2 months after making the cells of strain mgThe degree of utilization of oil, %
Water+oil+cells of strain Bacillus vallismortis VKPM B-1101730 00014 736,250,80
Control30 00029 334,6of 2.21

Example 5. Clean-up of soil from oil using cells of the bacterial strain Bacillus vallismortis VKPM B-11017

Experiments for the purification of permafrost soils contaminated with oil, using cells of the strain Bacillus vallismortis VKPM B-11017. The experience made in the natural the conditions. The average soil temperature during the entire period of the setting of the experiment, on the horizon of 20 cm was +8°C; on the horizon 10 cm to +14°C.

For the experiment the cells of the strain Bacillus vallismortis VKPM B-11017 with beveled mycopathologia agar obtained by the method described in example 1, washed with 0.9% NaCl solution, preparing a microbial suspension of bacterial isolates with a concentration of 1×109microbial cells/cm3optical standard gisk named after. A.M. Tarasevich.

The obtained cell suspension of strain Bacillus vallismortis VKPM B-11017 applied to the soil, the contaminated oil in the amount of 1 liter of suspension for 1 cubic meters of contaminated soil. The soil is thoroughly mixed and exhibit within 60 days.

For the analysis of soil samples taken before the start of the experiment and after 60 days after its termination. The efficiency of biodegradation of oil offered by the strain estimate gravimetric method (14. RD 39-0147098-90. Instructions to monitor the condition of soils at the sites of enterprises Minneftegazstroy. - 1990). The research results show that the proposed strain within 60 days, at an average soil temperature +8+14°C, carries out the degradation of oil by 78.3%, while natural loss of oil under the same environmental conditions in the soil not treated with the drug, was to 7.32% (table 3).

td align="right" namest="c0" nameend="c3"> Table 3
The dynamics of degradation of oil in permafrost soils treated with suspension cell-based strain Bacillus vallismortis VKPM B-11017
Version of experienceThe oil content to make strain, mg/kgThe oil content after 60 days after application of strain, mg/kgDestruction of oil for 60 days %
Soil+oil+drug on the basis of the strain Bacillus vallismortis VKPM B-110173886843to 78.3
Control1377112762to 7.32

Technical and economic efficiency

Thus, the advantage of the proposed bacterial strain BACILLUS VALLISMORTIS VKPM B-11017 is that it has a high scavenging ability towards both crude oil and petroleum products and in a relatively short time (from 7 to 60 days)in a wide temperature range from +8 to +37°C carries out the degradation of oil by 78.3%, while natural loss of oil under the same environmental conditions in the soil not treated with the drug, are to 7.32%. The strain can be used for cleanup of soils and on isdi water, contaminated by oil and oil products (diesel fuel, motor oil, gas condensate).

The strain of bacteriaBacillus vallismortisVKPM B-11017 - destructor of oil and oil products.



 

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3 ex

FIELD: process engineering.

SUBSTANCE: set of invention relates to water treatment. Proposed method comprises immobilisation of biomass bearing oil-oxidisers taken in effective amount into organic hydrophobic peat-based sorbent and drying it. Note here that said organic hydrophobic sorbent is prepared by low-temperature pyrolysis in vacuum of peat minced to 0.2-0.5 mm size at 210-250°C for 60-90 minutes. Said immobilisation is performed by adding sorbent into biomass suspension during its fermentation at retardation of oil-oxidising microorganisms growth. Note here that mass of added sorbent is 6-10 times larger than that of microorganisms contained in biomass suspension at the moment of sorbent application. Said oil-oxidising microorganisms represent yeast culture Candida maltosa All-Russian collection of industrial microorganisms Y-3446 or culture of bacteria Dietzia maris All-Russian collection of industrial microorganisms Ac-1824. Besides, this invention covers biosorbant for water treatment thus produced.

EFFECT: faster preparation, higher absorption capacity and efficiency.

2 cl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention may be used to produce a new biopreparation for cleaning of water, soil, industrial drains from pesticides resistant to decomposition and selected from chlorophenoxyacetic acids, such as 2,4-dichlorophenoxyacetic acid (2,4-D), trichlorophenoxyacetic acid (2,4,5-T), chlorophenoxyacetic acid (CPAA), phenoxyacetic acid (PAA), - 2,4-dichlorophenoxy-α-propionic acid, 2-methyl-4-chlorophenoxy-α-propionic acid, 2,4,5-trichlorophenoxy-α-propionic acid, 2,4-dichlorophenoxy-α-butyric acid, methyl-[1-(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate, 2,4-dichlorophenol, imidoclapride, hexachlorohexane, and also phenol. The biopreparation is an association of strains of bacteria Pseudomonas putida VKPM V-10997, Bacillus cubtilis VKPM V-10999 and Rhodococcus erythropolis VKPM - As-1882 at the weight ratio of (1-2):(1-2):1. At the same the biopreparation, as a rule, contains additionally a sorbent, organic, mineral and stimulating additives and has activity that stimulates growth of plants and fungicide properties. The produced biopreparation is introduced as an aqueous solution by means of sprinkling into polluted soil or industrial drain in efficient amount.

EFFECT: invention makes it possible to improve cleaning of water, soil, industrial drains from pesticides resistant to decomposition.

21 tbl, 29 ex

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.

EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.

5 tbl, 6 ex

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