RU2482179C1 - Bacillus atropheus BACTERIA STRAIN - OIL AND OIL PRODUCT DECOMPOSER - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The Bacillus atropheus IPNG - ELA-2 bacteria strain, having recycling capacity with respect to oil and oil products, is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under registration number VKPM V-10592 and can be used to clean water and soil from oil.

EFFECT: invention enables to cut the duration of recycling oil and oil products.

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Description

The invention relates to the field of biotechnology and relates to the production of a new strain of bacteria that can be used to purify water and soil from oil.

Known strains of Rhodococcus erythropolis, Rhodococcus luteus, Micrococcus flavus (SU 1493666), Flavobacterium tirrennicum (SU 1409594), which carry out the destruction of oil products in water, but are not able to clean the soil from oil.

Acinetronacter sp. HB-1 (RU 2077579); Acinetronacter sp. JN-2 (RU 2064017), Pseudomonas alcaligenes E7 (RU 2134723), Pseudomonas putida 36 (SU 1076446), suitable for cleaning the soil from oil, but the process of disposal slows down sharply when the temperature drops below 20 ° C, which makes them difficult to use in conditions North.

The strain Rhodococcus erythroplis E-15 (RU 2019527) showed great efficiency at low positive temperatures, but the process of oil biodegradation proceeds slowly and only with the addition of nitroammophos or nitroammophos and sodium nitrate.

The objective of the invention is the expansion of the nomenclature of strains having a utilizing ability in relation to oil and oil products.

The problem is solved by the fact that the obtained strain of Bacillus atropheus IPNG-ELA-2, with a utilizing ability in relation to oil and oil products, which can be used to clean soils and water bodies contaminated with oil and oil products in a wide temperature range (from +8 to + 30 ° C).

The bacterial strain Bacillus atropheus IPNG-ELA-2 was isolated from oil-contaminated soil in the territory of the Amga Oil Depot Branch (Amga village, Amga district, Republic of Sakha Yakutia) from a depth of 10 cm.

The strain is deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika (VKPM) (Moscow, 1st Dorozhny pr., D.1) under registration number VKPM V-10592.

The resulting strain is characterized by the following features.

Morphological and cultural characteristics.

Gram-positive spore bacilli, 0.8-1.5 microns in size, forming centrally located endospores, are located singly or in short chains.

On meat and peptone agar (wt.%: Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2) with the addition of 1% glucose grows in the form large, flat, matte beige color of the colonies, whose diameter is 0.3-6.0 cm. The edge of the colonies is slightly wavy, with small developed folds in the central upper part. After a few days (3-4), the colonies release brown pigment into the nutrient substrate. In older cultures, colonies are also stained dark (brown).

On Saburo medium (wt.%: Fish meal hydrolyzate - 1.0; pancreatic casein 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 large rounded wrinkled colonies of cream or off-white color, the maximum diameter of which is 1.5 cm.

The strain grows on the mineral environment of Muntz with oil and oil products (diesel fuel) of the following composition: (wt.%) KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; KH ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; agar - 2.0; oil or diesel fuel - 1.0; distilled water - the rest, pH 7.2, in the form of dry, opaque opaque colonies, 1-3 mm in diameter, cream or off-white in color.

In meat and peptone broth (wt.%: Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2) grows in the form of a wrinkled film on top of the broth.

Physiological and biochemical characteristics

The strain grows at temperatures from +8 to + 37 ° C, under aerobic conditions. Under anaerobic conditions, the strain does not die immediately, but develops more slowly. Optimum growth + 30 ° C. Grows at pH 6.0-8.0. It grows in salt broth with the addition of 0.1-2.0% NaCl. It has catalase activity. Hydrolyzes gelatin and casein. Lecithinase does not form. Does not hydrolyze starch. Oxidizes glycerol, glycogen, salicin. Does not ferment D-arabinose, inulin, mannitol.

The strain is not virulent, not toxic, not toxigenic (tested on white mice).

The strain can be maintained by regular reseeding (once every 10-14 days) on mowed meat and peptone agar or in a mineral medium with oil and oil products or stored in a lyophilized state in ampoules at 4 ° C.

To store the strain, you can also use the method of immobilization of a suspension of microorganisms on the surface of zeolite chips with a fraction of 1.0-3.0 mm Immobilized microorganisms are stored at + 4 ° C in the refrigerator for 24 months. Control passages are carried out 4 times a year (every 3 months), pre-emulsifying fractions of zeolite with immobilized bacteria in sterile distilled water (1 g of zeolite crumbs with immobilized microorganisms in 9 ml of sterile water). Sowing is carried out in Petri dishes on the surface of 3% MPA.

The invention is illustrated by the following examples.

Example 1. Isolation of a strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592.

The strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592 was isolated from oil-contaminated permafrost soil in the territory of the Branch of the Amginsky oil depot of OJSC Sakhaneftegazsbyt (Amga settlement, Amginsky district, Central Yakutia) from a depth of 10 cm, as follows:

1.0 g of soil was introduced into 250 ml of the Muntz mineral medium of the following composition (wt.%): KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0 08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; agar - 2.0; distilled water - the rest; the pH of the medium is 7.2. Oil was used as the sole source of carbon and energy, which was introduced into the medium in an amount of 0.16 wt.%. The incubation was carried out for 14 days on a thermostatic rocker UVMT-12-250 at 200 rpm and 29 ± 1 ° C. Bacterial growth was observed after 7-10 days of incubation for the formation of a whitish emulsion and the disappearance of oily stains of oil.

A pure bacterial culture was obtained by culturing (under the above conditions) and repeated reseeding (more than 10) of the accumulative culture on Petri dishes with agar from a fish meal hydrolyzate (GRM agar) with the addition of 1% glucose. Crops were incubated under stationary conditions in a thermostat at a temperature of + 30 ° C. After 48 hours, the appearance of a dry cream-colored colony was observed on the surface of the timing agar, which were identified as the Bacillus atropheus IPNG-ELA-2 strain by cultural-morphological and biochemical characteristics, as well as by the analysis of the sequences of the variable regions of 16S rDNA.

Example 2. Preparation of a bacterial isolate and a preparation based on cells of the strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592

The isolated colonies obtained in example 1 are subcultured on beveled meat and peptone agar and incubated for 48 hours at a temperature of + 30 ° C. The cells are washed off with a 0.9% NaCl solution and the initial microbial suspension of the bacterial isolate is prepared, 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. AM Tarasevich.

To obtain the preparation, they prepare Stepin's liquid nutrient medium (RU 2053293) of the following composition, wt.%: Hydrochloric acid hydrolyzate of casein - 1.4; fodder yeast extract - 0.3; MgSO ₄ · 7H ₂ O - 0.020; NaCl - 0.4; (NH ₄ ) ₆ · Mo ₇ O ₂₄ · 4H ₂ O - 0.05; distilled water - the rest, pH 7.3-7.4. The medium is sterilized at 0.5 atm for 20 minutes. Under sterile conditions, 2.0 cm ³ emulsions of the Perftoran preparation (OJSC NPF Perftoran, Russia) and 10.0 cm ³ suspensions of the bacterial isolate are added to 100.0 cm ^{3 of the} prepared medium and cultured in flasks on a rocking chair for 48 hours , at a temperature of 29 ± 1 ° C. A liquid turbid cell culture of the Bacillus atropheus IPNG-ELA-2 VKPM B-10592 strain is obtained, with a titer of at least 1 × 10 ⁹ cells / cm ³ , which can be used as a preparation for treating soil contaminated with oil and oil products.

Example 3. The study of the oil-oxidizing activity of a strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592 in relation to crude crude oil.

In a cone with a volume of 200.0 cm ³ , each containing 100.0 cm ³ of the Münz mineral medium, a suspension of the bacterial isolate obtained in Example 2 is added, the concentration is adjusted to 1 × 10 ⁹ microbial cells per 1 cm ³ according to the optical standard GISK named after AM Tarasevich and add 1% oil.

As a control, use the same flask with medium, oil, but without bacteria (to determine the total (natural) losses). The experiment is carried out in triplicate. The flasks are cultivated on a shaker at 200 rpm, at temperatures + 8 ° C; + 20 ° C and + 30 ° C for 7 days.

The oil content in the medium is determined using a concentrator "IKN-025" (FR.1.31.2007.03234). The degree of degradation of petroleum products is calculated as the ratio of the difference between the initial and residual content of petroleum products in water to their initial content, expressed as a percentage.

The results shown in table 1 show that in the mineral environment the proposed strain already on the fifth day at a temperature of + 8 ° C utilizes 56.4% of the oil; at + 20 ° C - 96.5%; at + 30 ° C - 97.13%.

Table 1 The degree of oil utilization after 7 days of cultivation of the strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592 t ° C, + Before the experiment, mg After 7 days, mg Destruction,% thirty 1000,0 28.64 97.13 twenty 1000,0 34.32 96.56 8 1000,0 405.78 56.42 The control 1000,0 1000,0 0,0

Example 4. Purification of ice water from crude oil using cells of a strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592

3.0 dm ^{3 of} ice water, 30 g of crude crude oil and 30.0 cm ^{3 of the} bacterial isolate of the Bacillus atropheus IPNG-ELA-2 strain (according to Example 1) containing 1.0 · 10 are added to a 5.0 dm ³ desiccator. ⁹ cells / cm ³ suspension. As a control, ice water is used in an amount of 3.0 dm ³ with the addition of 30 g of oil and without introducing a microbial suspension of the bacterial isolate.

table 2 The dynamics of the destruction of oil in water after making the drug based on the strain of Bacillus atropheus IPNG-ELA-2 B-10592 Experience option The amount of oil before introducing the cells of the strain, g The amount of oil 2 months after the introduction of the cells of the strain, g The degree of utilization of oil,% Water + oil + cells of the Bacillus atropheus IPNG-ELA-2 strain thirty 0.4718 98.42 Water + oil thirty thirty 0,0

The experiment is carried out for 2 months, maintaining the water temperature in the range + 15-20 ° C.

The results obtained (table 2) showed that in ice water the destruction of oil under the influence of the Bacillus atropheus IPNG-ELA-2 strain for 2 months was 98.42%. There was no destruction in the control tank (table 2).

Example 5. Purification of soil from oil using cells of the bacterial strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592

The experiment on the cleaning of permafrost soil contaminated with oil was carried out in vivo on the oil-contaminated territory of the branch of the Amga oil depot of OJSC Sakhaneftegazsbyt (Amga, Central Yakutia). The temperature of the soil, during the entire period of the experiment, at a depth of 20 cm, averaged + 8 ° C; at a depth of 10 cm - + 14 ° C.

The liquid preparation obtained in example 2, containing a cell culture of a strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592, with a titer of at least 1 × 10 ⁹ cells / cm ³ contribute to the soil contaminated with oil at the rate of 1 liter of the drug per 1 m ² oil-contaminated soil with an oil penetration depth of up to 30 cm. The soil is thoroughly mixed with a shovel and exposed under natural conditions for 2 months.

The content of petroleum products in the soil is determined in accordance with (RD 52.18.647-2003. Guidelines for determining the mass fraction of petroleum products in soils. Measurement method by gravimetric method).

Table 3 The dynamics of oil degradation in soil treated with a preparation based on the Bacillus atropheus IPNG-ELA-2 B-10592 strain Experience option The oil content before the introduction of the strain, mg / kg The oil content, 2 months after the introduction of the strain, mg / kg Oil destruction in 2 months,% Soil + oil + cells of the strain of Bacillus atropheus
IPNG-ELA-2 186079 80343 56.82 Soil + oil 5960 5561 6.69

The research results (table 3) show that the proposed strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592 for 2 months, at an average soil temperature of + 8-14 ° C, degrades crude oil by 56.82% at that the time as the natural loss of oil in permafrost soil not treated with the preparation was 6.69% (table 3).

Example 6. Cleaning the soil from diesel fuel using cells of the bacterial strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592

The experiment on the cleaning of permafrost soil contaminated with diesel fuel was carried out in vivo in the emergency area of the Momsky branch of the State Unitary Enterprise Housing and Public Utilities in the PC (Yakutia) (Honuu village, North-East Yakutia). The temperature of the soil, during the entire period of the experiment, at a depth of 20 cm, averaged + 8 ° C; at a depth of 10 cm - + 14 ° C. Oil penetration depth up to 15 cm.

The preparation according to example 2 is applied to the soil contaminated with oil, at the rate of 1 liter of the preparation per 1 m ^{2 of} oil-contaminated soil. The soil is thoroughly mixed with a shovel and exposed under natural conditions for 45 days.

The research results shown in table 4 show that after 45 days the destruction of oil products amounted to 89.53%.

Table 4 Destruction of diesel fuel after cleaning the soil Experience option pH Humidity% The content of oil products, mg / kg Destruction after 45 days,% before cleaning after cleaning Soil + diesel fuel + Bacillus atropheus IPNG-ELA-2 B-10592 bacterial strain 7.01 46.8 23420 2450 89.53 Soil + Diesel 6.99 49.5 25823 21247 17.7

Thus, the claimed strain quickly utilizes oil and oil products, in particular diesel fuel, in water and soil in a wide temperature range (from +8 to + 30 ° C).

Claims (1)

The bacterial strain Bacillus atropheus VKPM B-10592 is a destructor of oil and oil products.