Strain of bacteria pseudomonas panipatensis vkpm v-10593 - destructor of oil and oil products

FIELD: biotechnologies.

SUBSTANCE: strain of bacterial Pseudomonas panipatensis VKPM V-10593 is proposed, which is capable of quick recycling of oil and oil products, in particular, diesel fuel. Strain may be used to clean soil and water reservoirs contaminated with oil and oil products, in a wide range of temperatures from +8 to +30°C.

EFFECT: improved properties of a strain.

2 tbl, 5 ex

 

The invention relates to the field of Microbiology, and can be used in the cleanup of soil and groundwater when released into the environment of oil and oil products (diesel fuel).

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 Pseudomonas panipatensis S 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 is the region of temperatures (from +8 to +30°C).

The strain Pseudomonas panipatensis C71 isolated from water open water (lake of Hali-Balls, village Capellan, amginskii district, Republic of Sakha (Yakutia)), contaminated Arctic diesel fuel, with samples taken from a depth of 10 cm, in the destruction of 1 m from the shore.

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-10593.

The resulting strain is characterized by the following features.

Cultural and morphological characteristics.

This microscopic characteristics (appearance under a microscope). Gram-negative motile Bacillus size of 2.0 to 3.0×0.6 to 0.8 μm. Spores and capsules do not form. Smears are placed randomly, single or in short chains (2-3).

On nutrient agar (wt.%: hydrolyzed fish meal - 1,2; peptone is an enzymatic - 1,2; sodium chloride - 0,6; agar - 1,0; distilled water - the rest; pH 7,1-7,5) and mesopatamia agar (wt.%: enzymatic peptone - 1.0; sodium chloride and 0.5; agar - 1,0; water meat rest, pH 7.0-7.2, for 24 hours, the strain forms a disc-shaped, moist, pasty consistency S - colony grayish or dirty bluish color with a diameter of 1.5-5 mm, an easily removable from the agar. At temperatures above +20°C, the strain forms a diffusible pigment green color, which is when t is mperature +4°C can be lost and recovered at a temperature of from +20°C.

On the environment Endo (wt.%: pancreatic hydrolysate fish meal - 1,2; Baker's yeast extract - 0.1; sodium chloride - 0,34; sodium sulfite to 0.08; sodium phosphate two substituted - 0.05; basic fuchsin - 0,02; lactose - 1.0; agar - 1,0; distilled water - else) at pH 7.4±2 forms a round with blue tint colony of 0.5-3.0 mm in diameter, convex, smooth, with a dense center and a flat edge, shiny, translucent, eventually acquiring a matte surface.

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 6.0±3) forms a round with a grayish tint of the colony of 1.0-5.0 mm in diameter, convex, smooth, shiny, opaque, through 24-72 hours acquiring matte surface and a green pigment.

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; K2HPO4to 0.14; KH2PO4- 0,06; agar - 2,0; oil or diesel fuel - 1,0; distilled water - the rest, pH 7.2, wet opaque bluish colonies with a diameter of 1-2 mm

In mesopatamia broth (wt.%: enzymatic peptone - 1.0, sodium chloride and 0.5, water Masna is - else; pH 7.0 to 7.2) grows in the form of diffuse opacities.

Physiological and biochemical characteristics

Strain is an aerobe. Quickly (within 24-72 hours) growing on media with addition of hydrolyzed fish meal, at a temperature from 8°C to +41°C. the Optimum growth +30°C. Grows at pH 6.0 to 8.0. Grows in saline medium with addition of 0.1-2.0% NaCl. Has oxidase and catalase activity. As a carbon source consumes acetate and citrate. Restores nitrates. Denitrification activity is absent. Not hydrolyzes gelatin. Has urease activity. Not hydrolyzes starch. Lecithinase does not form. Indoleacetate. Oxidises maltose and D-glucose. Not ferments glycerol, Inositol, mannitol, rhamnose, sorbitol, raffinose, arabinose. Does not oxidize sucrose, mannitol, xylose, arabinose, fructose, galactose. Does not require additional growth factors.

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 be stored in a lyophilized state in vials at 4°C.

The invention is illustrated by the following examples.

Example 1. The selection of a strain of Pseudomonas panipatensis C71 VKPM B-10593

The strain Pseudomonas panipatensis C71 VKPM B-10593 isolated from the lake water challah-Balls (scapular, amginskii district. the Central Yakutia), as follows:

1.0 cm3the investigated water rubbing with a spatula on the surface apparitional nutrient medium of Muntz of the following composition (wt.%): KNO3- 0,4; MgSO4·7H2O - 008; NaCl - 0,1; K2HPO4to 0.14; KH2PO4- 0,06; agar - 2,0; distilled water - the rest; pH to 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 in a thermostat at a temperature of +8, +20 and +30°C for 1-10 days. Education bluish moist colonies of the strain Pseudomonas panipatensis C71 on the environment Muntz oil occurs after 1-3 days at a temperature of +30°C, after 4-6 days at a temperature of +20°C, 7-10 days at a temperature of +8°C.

Example 2. Preparation of bacterial isolates and drug-based cells of the strain Pseudomonas panipatensis C71 VKPM B-10593

Isolated colonies of the strain Pseudomonas panipatensis C71 obtained in example 1, subcultured on beveled mastopathy agar and incubated for 24 hours at a temperature of +30°C. the 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 risk imagemedia.

15.0 cm3the suspension obtained bacterial isolates contribute 150 cm3medium composition (wt.%): K2HPO42PO4to 0.14; NaCl to 0.2; glycerol - 2,0; peptone - 0,4; MnSO4·7H2O - 0,002 and cultured in flasks on a shaker for 48 hours at a temperature of 29±1°C (pH 7,2).

Get liquid culture greenish tint with a titer of at least 1 billion cells/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 Pseudomonas panipatensis C71 VKPM B-10593 in laboratory conditions

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 risk imagemedia, and add 1% oil or diesel fuel.

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 in shaker at 200 rpm and at temperatures of +8°C, +20°C and +30°C for 5 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 is aniu, expressed in percent

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 79.6% oil and 40.0% of diesel fuel; at +20°C - 94.5% of oil and 48.5% of the diesel fuel; at +30°C - 96.4% of oil and 67.7% of diesel fuel.

Table 1
The degree of utilization of oil after 5 days of cultivation of the strain Pseudomonas panipatensis C71-10593
t°C (+)OilDiesel fuel
To experience mgAfter 5 days mgDestruction, %To experience mgAfter 5 days mgDestruction, %
301000,035,6296,431000,0322,9667,70
201000,0EUR 54.1594,591000,0 514,1848,58
81000,0203,9679,601000,0599,5440,05
Control1000,01000,00,01000,01000,00,0

Example 4. Cleaning the ice water from the crude oil using cells of the bacterial strain Pseudomonas panipatensis C71 VKPM B-10593

In the desiccator volume of 5.0 DM3make 3,0 DM3ice water and 30.0 cm3microbial suspensions of the bacterial isolate Pseudomonas panipatensis C71-10593 (example 1)containing 1,0·109cells/cm3suspension. To create the oil film in the vessel, add 30 g of raw crude oil. As a control, use ice water in an amount of 3.0 DM3with the addition of 30 g of oil and without making microorganisms.

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

Table 2
The dynamics of degradation of oil in the water after making the drug-based strain Pseudomonas panipatensis C71-10593
Version of experienceThe amount of oil to make cells of strain, gThe amount of oil within 60 days after making the cells of the strain, gThe degree of utilization of oil, %
Water + oil + cells of strain Pseudomonas panipatensis C71-10593300,499698,33
Water + oil30300,0

The results obtained (table 2) showed that the destruction of oil strain of Pseudomonas panipatensis C71-10593 2 months amounted 98,33%. Destruction in the control tank is missing.

Example 5. Cleaning the soil from petroleum products using cells of the bacterial strain Pseudomonas panipatensis C71 VKPM B-10593

Experiment on cleaning permafrost soils contaminated Arctic diesel fuel, carried out in natural conditions on the territory of the Momsky branch of the state unitary enterprise "housing and communal services in PC (I)" (Phono, North-Eastern Yakutia). The soil temperature during the whole period of the setting of the experiment was on average at a depth of 20 cm +8°C; at a depth of 10 cm +13°C. the Depth of penetration of oil products averaged 20 see

Drug-based cells of the strain Pseudomonas panipatensis C71 VKPM B-10593 on what to example 2 applied to the soil, contaminated by petroleum products, based on 1 liter of the drug per 1 m2contaminated soil. The soil is thoroughly mixed, moistened to 60% of the total capacity and exhibit in vivo within 2 months. The content of nefteproduktov in the soil is determined in accordance with RD 52.18.647-2003. HOWTO determine the mass fraction of oil in the soil. Measurement technique gravimetric method.

The research results shown in table 3, show that when the soil temperature in the range from +8 to +13°C declared strain for 2 months made degradation of Arctic diesel fuel 91,71%

Table 3
The dynamics of degradation of oil in soil contaminated Arctic diesel fuel
Version of experienceThe oil content to make strain, mg/kgThe content of oil products, 2 months after the Deposit of the strain, mg/kgThe destruction of the spilled diesel fuel for 2 months, %
Soil + diesel fuel + cells of strain Pseudomonas panipatensis C71-10593183511521 91,71
Soil+diesel3260190758,50

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

The bacterial strain Pseudomonas panipatensis VKPM B-10593 - destructor of oil and oil products.



 

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