RU2617949C1 - Method of soil and water protection from oil - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of purifying soil and water pollution by oil and petroleum products comprises applying the bacterial preparation consisting of a cell strain Stenotrophomonas maltophilia SNBS-3, deposited in the Russian National Collection of Industrial Microorganisms under accession number VKPM B-12247 at a concentration of 1⋅10⁹ cells/cm³ suspension in contaminated soil or an aqueous environment.

EFFECT: invention improves the effectiveness of cleaning soil and water from oil and petroleum products in a relatively short time and a wide temperature range.

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Description

The method of purification of soil and water from oil and oil products

The invention relates to the field of ecology and environmental protection, namely, methods for cleaning the soil and the aquatic environment from oil and oil products.

Known strains of microorganisms used to purify water and soil from oil and oil products: Pseudomonas putida 36 (see SU No. 1076446, IPC C02F3 / 34, B09C1 / 10, C12N1 / 20 C02F3 / 34, C02F101: 32, C12N1 / 20, C12R1 : 40, publ. 02.28.1984), Acinetronacter sp. HB-1 (see RU No. 2077579, IPC C12N1 / 20, C02F3 / 34, B09C1 / 10, C12R1: 01, C12N1 / 20, B09C101: 00, published on 04/20/1997), Pseudomonas alcaligenes E7 (see RU No. 2134723, IPC C12N1 / 20, C02F3 / 34, B09C1 / 10, C12N1 / 20, C12R1: 38, publ. 08/20/1999).

A disadvantage of the known strains is that they utilize oil at relatively high temperatures.

The strain Rhodococcus erythroplis E-15 showed great efficiency at low positive temperatures (see RU No. 2019527, IPC C02F3 / 34, E02B15 / 04, C09K17 / 00, published September 15, 1994), but the process of oil biodegradation proceeds slowly and only at the addition of nitroammophos or nitroammophos and sodium nitrate.

A known consortium of oil-oxidizing microorganisms in the composition of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ky-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 at a weight ratio of 3-12 wt.% Each microorganism, while risotorfin is introduced into the contaminated medium in an amount of 30-120 g / m ² (cm . RU No. 2191643, IPC B09C1 / 10, C12N1 / 20, C12N1 / 20, C12R1: 01, published on October 27, 2002).

A disadvantage of the known solution is that when preparing a working suspension of microorganisms, the temperature is maintained at + 18 ... + 22ºС, i.e. microorganisms that are part of the consortium are not capable of degradation of oil products at low positive temperatures (+ 5ºС).

A bacterial strain “Bacillus subtilis Kolyma 7/2” (see RU No. 2446900, IPC B09C1 / 10, publ. 10.04.2012), which has the ability to reduce the oil concentration to 0.48%, that is, the oil level from 135143, is used as a prototype mg / kg is reduced to 645 mg / kg during the 3 months of the summer period. However, the strain degrades only oil and only in soil and is not able to utilize oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), including in an aqueous medium under a wide temperature range from +5 to + 37 ° С.

The problem to which the claimed invention is directed is expressed in expanding the range of effective methods for cleaning environmental objects (in particular, soil and water) from oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate) in a relatively short time, in a wide temperature range from +5 to + 37 ° С.

To solve this problem, the method of purification of soil and water from oil and oil products is characterized in that a bacterial preparation consisting of cells of the Stenotrophomonas maltophilia strain SNBS-3 in a concentration of 1 · 10 ⁹ cells / cm ^{3 of} suspension is used to clean the water from oil and oil products .

A comparative analysis of the features of the claimed solution with the signs of the prototype and analogues indicates the conformity of the claimed solution to the criterion of "novelty."

The features of the characterizing part of the claims provide a solution to the assigned functional tasks.

The proposed bacterial strain Stenotrophomonas maltophilia SNBS-3 was isolated on February 16, 2015 from frozen soil samples contaminated with diesel fuel, taken near the containers of the Momsky Distribution Zone of Sakhaenergo OJSC, Northeast Yakutia, Momsky District, Honuu Village.

The strain was identified and deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), Federal State Unitary Enterprise GosNIIgenetika, 117545, Moscow, 1st Dorozhny pr-d, 1, registration number VKPM V-12247.

Initial screening using a database of GenBank and RDP-II showed that the studied strain belongs to the following systematic groups of Bacteria; Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Stenotrophomonas.

When sequencing the variable regions of 16S rDNA, the following assembled nucleotide sequence was obtained for the studied strain:

Sequence processing results are presented in graphical form.

According to the analysis of RDP II 16S rRNA, a phylogenetic tree was constructed with homologous strains of Stenotrophomonas maltophilia SNBS-3 (see drawing).

The criterion for classifying a microorganism to a particular species is considered to be at least 97% homology.

According to the results of the analysis of sequencing of variable regions of genes encoding 16S rRNA, the test strain is assigned to the species Stenotrophomonas maltophilia (100%).

The resulting strain is characterized by the following features.

Morphological features

Gram-negative rods, in smears are located singly or in random clusters. Spore and capsules do not form.

Cultural signs

On meat peptone agar (MPA) (wt.%): Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2, forms translucent yellowish colonies with a smooth edge with a diameter of 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared. Older crops may form a brownish or grayish pigment.

On nutrient agar based on a fish meal hydrolyzate (wt.%): A fish meal hydrolyzate - 1.2; enzymatic peptone - 1.2; sodium chloride - 0.6; agar - 1.0; distilled water the rest; pH 7.1 - 7.5, forms translucent yellowish colonies with a smooth edge with a diameter of 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared. Older crops may form a brownish or grayish pigment.

On the environment of Saburo (wt.%): Hydrolyzed fish meal - 1.0; casein pancreatic hydrolyzate - 1.0; yeast extract - 0.2; monosubstituted sodium phosphate - 0.2; D-glucose 4.0; agar - 1.0; distilled water - the rest; pH 6.0 ± 3, forms yellowish colonies, in old cultures it can form a brownish or gray pigment, the diameter of the colonies is 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared.

In meat and peptone broth (wt.%): Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2, forms turbidity.

On the mineral environment of Münz (see Kersten DK Morphological and cultural properties of indicator microorganisms of oil and gas imaging. - Microbiology, 1963, No. 5. P.1024-1030) with oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate, gasoline) of the following composition (wt.%): KNO₃- 0.4; MgSO_four7H₂O - 0.08; NaCl - 0.1; K₂HPO_four–0.14; Kh₂PO_four–0.06; agar-2.0; oil or petroleum products - 1.0; distilled water - the rest, pH 7.2, grows in the form of translucent grayish-yellow small colonies with a diameter of 1 mm.

Physiological and biochemical characteristics

The aerobic strain grows at a temperature of +5 to + 37 ° C, grows more rapidly + 10 + 30 ° C. It grows at pH 5.0 - 8.5. Optimum pH 6.5 - 8.0. It grows in salt broth with the addition of 0.1 - 2.0% NaCl.

Used carbon sources: sorbitol, mannitol.

Proven unused carbon sources: lactose, glucose, sodium citrate, sodium malonate. The strain is indole negative. The Voges - Proskauer reaction is negative. Phenylalanine deoxaminase is negative. Does not ferment beta galactosidase. Does not recycle inositol. Product synthesized by strain: surfactants capable of degrading oil. Uses petroleum hydrocarbons and oil products as an energy source.

The strain is not toxic to higher plants (biotesting was carried out on the seeds of oats ordinary variety "Skakun").

The strain can be maintained by regular reseeding (once every 10-14 days) on beveled meat and peptone agar or stored in a lyophilized state in ampoules at a temperature of + 4 ° С.

The invention is illustrated by the following examples.

Example 1. Isolation and cultivation of a strain of Stenotrophomonas maltophilia SNBS-3

The bacterial strain Stenotrophomonas maltophilia SNBS-3 was isolated from frozen soil samples contaminated with diesel fuel by the accumulative culture method in Munz with oil, followed by reseeding on MPA.

For this, 1.0 g of frozen soil from the above object was added to 250 ml of the Muntz mineral medium of the following composition: (wt.%) KNO₃- 0.4; MgSO_four7H₂O - 0.08; NaCl - 0.1; K₂HPO_four- 0.14; Kh₂PO_four- 0.06; distilled water - the rest, pH - 7.2, Oil was used as the sole source of carbon and energy, which was introduced into the Münz environment in the amount of 1000 mg per 1.0 dm³Wednesday. At the first stage, the incubation was carried out under rocking conditions at 200 rpm and a temperature of + 20 ± 1 ° С. Bacterial growth was observed after 3 days of incubation by the formation of a turbid emulsion and the disintegration of the oil layer.

A pure bacterial culture of Stenotrophomonas maltophilia SNBS-3 was obtained by culturing under the conditions listed above and repeated reseeding (more than 10) of the accumulation culture in Petri dishes with MPA.

Further, crops were incubated under stationary conditions at various temperatures from +5 to + 37 ° C. After 72 hours, the appearance of translucent yellowish-gray colonies with a diameter of up to 5 mm was observed on the MPA surface, which, according to the cultural-morphological and biochemical characters, as well as the results of the analysis of the nucleotide sequences of the 16S rRNA gene and key phenotypic characters according to the taxonomic descriptions given in Bergi's determinant (see Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact .; July 1985, p. 408) has been identified as a Stenotrophomonas maltophilia strain of SNBS-3.

Example 2. Preparation of a bacterial preparation based on cells of the Stenotrophomonas maltophilia SNBS-3 strain.

The isolated colonies obtained in example 1 were subcultured on a beveled MPA and incubated for 48 hours at a temperature of + 20 ± 1 ° C. MPA-grown cultures of Stenotrophomonas maltophilia SNBS-3 were washed off with 0.9% NaCl; prepared the initial microbial suspension of the bacterial isolate with a titer of 1 · 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. A.M. Tarasevich.

To obtain the preparation, a liquid mineral medium was prepared according to Example 1 and cultured in flasks under rocking conditions for 48 hours, at a temperature of 20 ± 1 ° C, at 200 rpm. The resulting liquid mixture of cells of the Stenotrophomonas maltophilia SNBS-3 strain with a titer of 1 · 10 ⁹ cells / cm ^{3 was} used as a preparation for treating soil and water contaminated with oil and oil products.

Example 3. The study of the oil-oxidizing activity of a strain of Stenotrophomonas maltophilia SNBS-3

The cells of the Stenotrophomonas maltophilia SNBS-3 strain are washed with 0.9% NaCl from beveled meat-peptone agar. Prepare the initial microbial suspension of the bacterial isolate, with a volume of 25 cm ³ , with a concentration of 1 · 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. A.M. Tarasevich.

The obtained bacterial isolate of the strain Stenotrophomonas maltophilia SNBS-3 was cultured in an aqueous medium with a mineral composition according to example 1.

100.0 cm ^{3 of the} finished mineral medium and 1000 mg of oil are introduced into cones with a volume of 250.0 cm ³ . Flasks are seeded with cells of the strain to a concentration of 1 · 10 ⁹ cells / cm ³ . As a control, put the same flask with medium, oil and without bacteria to determine the total (natural) losses. The experiment is carried out in three repetitions. Flasks are cultured at different temperatures (from + 5 ° C to + 37 ° C) for 3-5 days.

Biodegradation of oil and oil products is determined by the spectrometric method using an IKN-025 concentrator (see FR.1.31.2007.03234 MVI 02/01/117 Method for measuring the mass concentration of oil products in drinking, natural and waste waters by the IR spectrometric method using an IKN concentrator -025 ").

The experimental data show that in an aqueous medium with mineral components, under conditions of constant aeration, the proposed strain already utilizes 4.4 - 5.5% of oil and oil products after 3 days at a temperature of + 5 ° C; at a temperature of + 20 ° C - 56.4 - 59.8%; at a temperature of + 30 ° C - 62.0 - 63.4%; at a temperature of + 37 ° C - 67.0 - 69.6%; depending on the type of xenobiotic (see table 1).

Example 4. Purification of tap water from oil using cells of the strain Stenotrophomonas maltophilia SNBS-3

3.0 dm ^{3 of} tap water was added to a desiccator with a volume of 5.0 dm ³ , which had settled for one day at room temperature. 30000 mg of crude crude oil and 30.0 cm ^{3 of the} bacterial isolate of the Stenotrophomonas maltophilia strain SNBS-3 (according to Example 2) containing 1.0 · 10 ⁹ cells / cm ³ suspension are added to water. As a control, water is used in an amount of 3.0 dm ³ with the addition of 30,000 mg of oil and without introducing a microbial suspension of the bacterial isolate.

The experiment is carried out for 45 days at a temperature of + 20 ± 1 ° C.

The results obtained (see table 2) showed that in water the destruction of oil under the influence of the Stenotrophomonas maltophilia strain SNBS-3 for 45 days was 53.0%. The destruction in the control tank has not changed significantly.

Example 5. Cleaning the soil from oil pollution using cells of the strain Stenotrophomonas maltophilia SNBS-3

The experiments using cells of the Stenotrophomonas maltophilia SNBS-3 strain for cleaning the soil from oil and oil products were carried out on sandy soil under the conditions of a model experiment artificially contaminated with oil and diesel fuel. The initial oil content (NP) in the soil before soil treatment with cells of the Stenotrophomonas maltophilia strain SNBS-3 was 3000 mg / kg, which corresponded to the average level of contamination in accordance with regulatory documents (see Letter No. 04-25 of the Ministry of Natural Resources of Russia dated December 27, 1993). / 61-5678 "On the procedure for determining the amount of damage from land pollution by chemical substances; Methodological recommendations for identifying degraded and polluted lands. Committee of the Russian Federation for Land Resources dated March 27, 1995 No. 3-15 / 582, 29 pp.).

The effectiveness of the use of the Stenotrophomonas maltophilia SNBS-3 strain was evaluated by the degree of oil pollution degradation in soil samples taken for analysis before and after the application of the microbial suspension based on the cells of the Stenotrophomonas maltophilia SNBS-3 strain.

Assessment of the residual oil content in the soil before and after the experiment was carried out by the gravimetric method according to the instructions, generally accepted documents (see RD 39-0177098-015-90 “Instructions for monitoring the state of soils at the facilities of enterprises of the Ministry of Oil and Gas Industry”).

For this, soil samples before and after treatment with a biological product were subjected to extraction with carbon tetrachloride in a Soxhlet apparatus, after which the extractant was removed by distillation with a reflux condenser, bringing the residue to a constant weight. The moisture content of the samples was determined by the generally accepted method (see GOST 28268-89 “Soils. Methods for determining moisture, maximum hygroscopic humidity”), after which the content of oil products was recalculated on dry soil.

The experiments were carried out in a refrigerator at a temperature of + 5 ± 1ºС; in room conditions at a temperature of + 20 ± 1 ° C and in thermostatically controlled conditions at a temperature of + 30 ± 1 ° C and a temperature of + 37 ± 1 ° C.

It was established that the degree of biodegradation of oil and oil products in experimental samples 45 days after soil treatment with a bacterial suspension based on cells of the Stenotrophomonas maltophilia strain SNBS-3 was 6.6-7.3% at a temperature of + 5 ± 1 ° C; at a temperature of + 20 ± 1 ° C - 54.8-58.0%; at a temperature of + 30 ± 1 ° C - 60.0-66.6%; at a temperature of + 37 ± 1 ° C - 60.6-63.3%, depending on the type of xenobiotic (see table 3).

Thus, the effectiveness of the method for cleaning soil and water from oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), a bacterial preparation consisting of cells of the Stenotrophomonas maltophilia strain SNBS-3 at a concentration of 1 · 10 ⁹ cells / cm ³ suspensions, in a relatively short time (from 3 to 45 days), in a wide temperature range from +5 to + 37 ° C.

Table 1

The degree of utilization of oil products by the strain Stenotrophomonas maltophilia SNBS-3 in an aqueous medium with mineral components

Experience option Control sampling The oil content, mg / DM ³ t ° C +5 +20 +30 +37 Water + mineral components + strain +
oil to experience 1000,0 1000,0 1000,0 1000,0 after experience 956.0 404.0 380.0 304.0 % destruction 4.4 59.6 62.0 69.6 Water + mineral components + strain +
diesel fuel to experience 1000,0 1000,0 1000,0 1000,0 after experience 950.0 436.0 366.0 330.0 % destruction 5,0 56.4 63,4 67.0 Water + mineral components + strain +
motor oil to experience 1000,0 1000,0 1000,0 1000,0 after experience 948.0 402.0 380.0 322.0 % destruction 5.2 59.8 62.0 67.8 Water + mineral components + strain +
hydraulic oil to experience 1000,0 1000,0 1000,0 1000,0 after experience 954.0 402.0 380.0 312.0 % destruction 4.6 59.8 62.0 68.8 Water + mineral components + strain +
gas condensate to experience 1000,0 1000,0 1000,0 1000,0 after experience 945.0 404.0 376.0 320,0 % destruction 5.5 59.6 62,4 68.0

table 2

The destruction of oil in water by a strain of Stenotrophomonas maltophilia SNBS-3

The oil content, mg / DM ³ The degree of utilization of oil,% Experience option to experience after experience Water + oil + strain 30,000.0 15 900,0 53.0 Control: Water + Oil 30,000.0 29,300.0 2,3

Table 3

The destruction of oil and oil products in the soil under the influence of cells of the strain Stenotrophomonas maltophilia SNBS-3

Experience outline The oil content, mg / kg Oil degradation,
% before cleaning after cleaning Experience at a temperature of + 5 ± 1ºС Control (soil + oil) 3000,0 2940.0 2.0 Soil + oil + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 2800.0 6.6 Control (soil + diesel fuel) 3000,0 2900.0 3.3 Soil + diesel fuel + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 2780.0 7.3 Experience at a temperature of + 20 ± 1ºС Control (soil + oil) 3000,0 2820.0 6.0 Soil + oil + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1356.0 54.8 Control (soil + diesel fuel) 3000,0 2800.0 6.6 Soil + diesel fuel + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1260.0 58.0 Experience at a temperature of + 30 ± 1ºС Control (soil + oil) 3000,0 2794.0 6.8 Soil + oil + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1,200.0 60.0 Control (soil + diesel fuel) 3000,0 2750.0 8.3 Soil + diesel fuel + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1000 66.6 Experience at a temperature of + 37 ± 1ºС Control (soil + oil) 3000,0 2760.0 8.0 Soil + oil + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1,180.0 60.6 Control (soil + diesel fuel) 3000,0 2700.0 10.0 Soil + diesel fuel + cell suspension of the strain Stenotrophomonas maltophilia SNBS-3 3000,0 1,100.00 63.3

Claims (1)

The method of purification of soil and water from oil and oil products, characterized in that a bacterial preparation is used, consisting of cells of the strain Stenotrophomonas maltophilia VKPM B-12247 at a concentration of 1 × ^{10 9} cells / cm ³ suspension.

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