Bacillus atropheus bacteria strain - oil and oil product decomposer

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.

4 tbl, 6 ex

 

The invention relates to the field of biotechnology and the receipt of a new strain of bacteria, which can be used for purification of water and soil from oil.

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

Known strains Acinetronacter sp. HB-1 (EN 2077579); Acinetronacter sp. JN-2 (EN 2064017), Pseudomonas alcaligenes E7 (EN 2134723), Pseudomonas putida 36 (SU 1076446), suitable for cleaning soil from oil, but the recycling process drastically slows down when the temperature drops below 20°C, which hampers their use in conditions of the North.

Greater efficiency at low positive temperatures showed the strain Rhodococcus erythroplis E-15 (EN 2019527), but the process of biodegradation of oil flows slowly and only adding ammonium nitrate or ammonium nitrate and sodium nitrate.

Object of the invention is to widen the range of strains utilizing ability in relation to petroleum and petroleum products.

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

The bacterial strain Bacillus atropheus IPNG-ELA-2 isolated from oil-contaminated soil site Branch amginskii tank farm (Pampa, amginskii district, Republic of Sakha (Yakutia) from a depth of 10 cm

The strain is deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika (VKPM) (Russia, Moscow, 1st Road Ave, 1) under the registration number VKPM B-10592.

The resulting strain is characterized by the following features.

Morphological and cultural characteristics.

Gram-positive spore-forming bacilli, size of 0.8-1.5 μm, forming a centrally-located endospores, are arranged singly or in 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 large, flat, matte beige colonies, whose diameter is 0.3 to 6.0 see the edge of the colonies undulating, with small developed folds in the Central upper part. After a few days (3-4) colonies isolated in the nutrient substrate brown pigment. In old cultures the colony also painted in dark (brown) color.

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 a large rounded wrinkled colonies cream or off-white color, the maximum diameter of which is 1.5 cm

The strain grows on mineral environment of Muntz with oil and oil products (diesel fuel) of the following composition: (wt.%) KNO3- 0,4; MgSO4·7H2O - 0,08; NaCl - 0,1; KH2HPO4to 0.14; KH2PO4- 0,06; agar - 2,0; oil or diesel fuel - 1,0; distilled water - the rest, pH 7,2, in the form of a dry opaque opaque colonies with a diameter of 1-3 mm, cream or off-white color.

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 a wrinkled film on top of the broth.

Physiological and biochemical characteristics

The strain grows at temperatures from +8 to +37°C in aerobic conditions. In anaerobic conditions, the strain does not die immediately, but has been slower. 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. Has a catalase activity. Hydrolyzes gelatin and casein. Lecithinase does not form. Not hydrolyzes starch. Oxidizes glycerol, glycogen, salicin. Not ferments D-arabinose, inulin, mannitol.

The strain is not virulent, non-toxic, non toxigenic (test conducted on white mice).

The strain can be maintained by regular transfers (1 in 10 to 14 days) on the beveled mesopatamia agar or on mineral medium with oil and oil sludge is stored in a dried state in vials at 4°C.

To store strain can also use the method of immobilization of the suspension of microorganisms on the surface of zeolite crumb fraction 1,0-3,0 mm Immobilized microorganisms stored at a temperature of +4°C in the refrigerator for 24 months. Control transfers are conducted 4 times a year (every 3 months), pre-amblyraja fraction of zeolite with immobilized bacteria in sterile distilled water (1 g of zeolite chips with immobilized microorganisms in 9 ml of sterile water). Sowing is carried out in Petri dishes on surface 3% MPA.

The invention is illustrated by the following examples.

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

Strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592 isolated from oil-contaminated soil soil site Branch amginskii tank farm JSC "sahaneftegazsbyt" (Pampa, amginskii district, Central Yakutia) from a depth of 10 cm, as follows:

1.0 g of soil were made in 250 ml of mineral medium of Muntz of the following composition (wt.%): KNO3- 0,4; MgSO4·7H2O - 0 08; NaCl - 0,1; K2HPO4to 0.14; KH2PO4- 0,06; agar - 2,0; distilled water - the rest; the pH of the medium was 7.2. As the sole source of carbon and energy used oil, which was made on Wednesday in the amount of 0.16 wt.%. Incubation was performed 14 days on a thermostatted shaker WVMT-12-250 at 200 Rev/mi and 29±1°C. Bacterial growth was observed after 7-10 days of incubation on education whitish emulsion and the disappearance of oily stains of oil.

Pure culture of bacteria was obtained by cultivation (when 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. Crops were incubated under stationary conditions in a thermostat at a temperature of +30°C. After 48 hours on the surface RM-agar observed the occurrence of dry cream shade colonies that cultural-morphological and biochemical characteristics, as well as the results of the analysis of sequences of variable regions of 16S rDNA identified as a strain of Bacillus atropheus IPNG-ELA-2.

Example 2. Preparation of bacterial isolates and drug-based cells of the strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592

Isolated colonies obtained in example 1, subcultured on beveled mastopathy agar and incubated for 48 hours at a temperature of +30°C. Cells are washed with 0.9% NaCl solution and prepare the initial microbial suspension of bacterial isolate, volume 25 cm3with a concentration of 1×109microbial cells/cm3optical standard gisk named after. Amerasia.

To obtain drug producing liquid nutrient medium Stepin (EN 2053293) of the following composition, wt.%: salancik the th casein hydrolysate - 1,4; fodder yeast extract and 0.3, MgSO4·7H2O - 0,020; NaCl to 0.4; (NH4)6·Mo7O24·4H2O - 0,05; distilled water - the rest, 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 cm3the emulsion of the drug "Perftoran" (JSC SPF "Perftoran", Russia) and 10.0 cm3suspensions of bacterial isolates and cultured in flasks on a shaker for 48 hours at a temperature of 29±1°C. Receive liquid unclear cell culture of the strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592, with a titer of at least 1×109cells/cm3that can be used as a drug for treatment of soil contaminated by oil and oil products.

Example 3. Research-oxidizing activity of the strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592 in respect of raw crude oil.

In flasks with a volume of 200.0 cm3containing 100,0 cm3mineral environment Muntz, make a suspension of bacterial isolates, obtained according to example 2, bring the concentration up to 1×109microbial cells on 1 cm3optical standard gisk named after. Amerasia and add 1% of oil.

As a control use the same flask with the environment, petroleum products, but without bacteria (to determine the total (natural) losses). The experience carried out in three replications. Flasks were cultured on SC is alke at 200 rpm, at temperatures of +8°C; +20°C and +30°C for 7 days.

The oil content in the environment is determined by concentrator "TSC-025" (FR). The degree of degradation of oil is calculated as the ratio of the difference between initial and residual oil in the water to their original content, expressed as a percentage.

The results in table 1 show that in the mineral environment of the proposed strain already on the fifth day when the temperature is +8°C disposes of 56.4% of oil; at +20°C to 96.5%; at +30°C - 97,13%.

Table 1
The degree of utilization of oil after 7 days of cultivation of the strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592
t°C +To experience mgAfter 7 days mgDestruction, %
301000,028,6497,13
201000,034,3296,56
81000,0405,7856,42
Control the 1000,01000,00,0

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

In the desiccator volume of 5.0 DM3make 3,0 DM3ice water, 30 g of raw crude oil and 30.0 cm3bacterial isolate of a strain of Bacillus atropheus IPNG-ELA-2 (example 1)containing 1,0·109cells/cm3suspension. As a control, use ice water in an amount of 3.0 DM3with the addition of 30 g of oil and without making microbial suspension of the bacterial isolate.

Table 2
The dynamics of degradation of oil in water after application of the drug on the basis of the strain Bacillus atropheus IPNG-ELA-2-10592
Version of experienceThe amount of oil to make cells of strain, gThe amount of oil in 2 months after making the cells of the strain, gThe degree of utilization of oil, %
Water + oil + cells of strain Bacillus atropheus IPNG-ELA-2300,471898,42
Water + oil 30300,0

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

The results obtained (table 2) showed that in ice water, the destruction of oil under the influence of the strain Bacillus atropheus IPNG-ELA-2 for 2 months amounted 98,42%. Destruction in the control tanks were absent (table 2).

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

Experiment on cleaning permafrost soils contaminated with oil, conducted under natural conditions in contaminated territories branch amginskii tank farm JSC "sahaneftegazsbyt" (Pampa, Central Yakutia). The soil temperature during the entire period of the setting of the experiment, at a depth of 20 cm, averaged +8°C; at a depth of 10 cm to +14°C.

Liquid preparation obtained according to example 2, containing the cell culture of the strain Bacillus atropheus IPNG-ELA-2 VKPM B-10592, with a titer of at least 1×109cells/cm3bring in soil contaminated with oil at the rate of 1 liter of the drug per 1 m2oil-contaminated soil with oil penetration depth up to 30 cm Soil thoroughly mixed with a shovel and exhibit in vivo within 2 months.

The oil content in the soil is determined in accordance with (RD 52.18.647-2003. Methodological decree the Oia determine the mass fraction of oil in the soil. Measurement technique gravimetric method).

Table 3
The dynamics of degradation of oil in soil treated with Metarhizium strain of Bacillus atropheus IPNG-ELA-2-10592
Version of experienceThe oil content to make strain, mg/kgThe amount of oil that is 2 months after the Deposit of the strain, mg/kgDestruction of oil for 2 months, %
Soil + oil + cells of strain Bacillus atropheus
IPNG-ELA-2
1860798034356,82
Soil + oil596055616,69

The results of the studies (table 3) show that the proposed strain of Bacillus atropheus IPNG-ELA-2 VKPM B-10592 for 2 months when average soil temperature +8-14°C, carries out the degradation of crude oil on 56,82% in that time, as a natural loss of oil in permafrost soil not treated with the drug, was 6.69 per cent (table 3).

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

Experiment to clean the ke permafrost soil, contaminated diesel fuel, carried out in natural conditions at the emergency site Momsky branch of the state unitary enterprise "housing and communal services in the PC (Yakutia)" (Schonau, North-Eastern Yakutia). The soil temperature during the entire period of the setting of the experiment, at a depth of 20 cm, averaged +8°C; at a depth of 10 cm to +14°C. the Depth of oil penetration up to 15 cm

The preparation according to example 2 applied to the soil contaminated with petroleum products, based on 1 liter of the drug per 1 m2oil-contaminated soil. The soil is thoroughly mixed with a shovel and exhibit in vivo within 45 days.

The results of the studies summarized in table 4, show that after 45 days, the degradation of petroleum products amounted to 89,53%.

Table 4
Destruction of diesel fuel after cleaning soil
Version of experiencepHHumidity, %The content of oil products, mg/kgDestruction within 45 days %
before cleaningafter cleaning
Soil + diesel + strain bacter the th Bacillus atropheus IPNG-ELA-2-10592 7,0146,823420245089,53
Soil + diesel6,9949,5258232124717,7

Thus, the claimed strain quickly disposes of crude oil and petroleum products, particularly diesel fuel, water and soil in a wide range of temperatures (from +8 to +30°C).

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



 

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