RU2257411C1 - Strain pseudomonas graminis for decomposition of petroleum and petroleum products - Google Patents

Abstract

FIELD: biotechnology, industrial microbiology.

SUBSTANCE: invention relates to treatment of environment objects from pollutions with petroleum and petroleum products using microorganisms. The strain is deposited as Pseudomonas graminis VKPM B-8615. The strain is isolated from petroleum-polluted water sample obtained from the West Siberian deposit and grows in many natural and synthetic media. The strain Pseudomonas graminis shows the high utilization rate of petroleum and petroleum products.

EFFECT: valuable properties of strain.

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Description

The invention relates to the microbiological industry and relates to a new culture of microorganisms that destroy oil and oil products, and which can be used, for example, for cleaning contaminated surfaces.

A known mixture of specially selected strains of bacteria Rhodococcus erythropolis VKPM B-3078 (36a-1), Rhodococcus luteus VKPM B-3077 (25) and Micrococcus flavus VKPM B-3079 (53) (St. USSR No. 1493666, C 02 F 3 / 34, 1989), used for the destruction of oil and oil products.

The use of these cultures of microorganisms is possible only in aeration tanks. The cultivation of a mixture of cultures is possible with large material and energy costs.

Known bacterial strain Rhodococcus erythropolis BKM Ac-1339D, used for the decomposition of oil and oil products, which is used to purify water and soil from these products. A disadvantage of the known strain is the insufficient rate of oil utilization and the inability to dispose of oil and petroleum products at a high content to the volume of the contaminated object.

The closest analogue is the consortium of microorganisms Devoroil, which actively oxidizes oil products both in the contact zone with water and directly in the oil film. The consortium includes bacterial and yeast cells. The composition is isolated from produced water of an oil field (US Pat. RF 2023686).

The aim of this invention is to obtain a new strain, characterized by a high rate of utilization of oil and oil products, which are the only sources of carbon and energy.

To achieve this goal, the proposed strain Pseudomonas graminis T-7 VKPM B-8615, isolated from a sample of oil-contaminated water obtained from the West Siberian field (certificate of deposit attached).

The strain was isolated by sieving the initial sample with oil-contaminated water on meat and peptone agar with 0.5% oil and preliminary passage of the initial sample through Raymond’s liquid medium with 0.5% oil.

The strain was identified based on a study of its cultural-morphological and physiological-biochemical characteristics in accordance with the description given in the definition of bacteria Bergey / Bergey Smanual of Systematic Bacteriology vol. 1. 1984 p. /, And also based on a study of the 16S rRNA gene sequence according to Edwards U. Et all.

The strain is stored on agarized nutrient media at a temperature of 6-8 ° C for a month. Longer storage of the strain is carried out in hermetically sealed ampoules in a freeze-dried state.

The composition of the medium for storage of the strain:

Meat peptone agar (g / l):

Meat water - 100.0

Agar - 20.0

NaCl - 5.0

Peptone - 10.0

Glucose - 7.0

Tap water - up to 1 liter

Raymond agar medium with oil (g / l):

Na ₂ CO ₃ - 0.1

CaCl ₂ - 0.01

MnSO ₄ - 0.02

FeSO ₄ - 0.01

MgSO ₄ - 0.2

NH ₄ Cl - 0.2

NaCI - 3.0

Na ₂ HPO ₄ - 1.5

KN ₂ PO ₄ - 1,0

Agar - 20.0

Meat peptone broth - 500

Oil - 0.5% vol.

Distilled water - up to 1 liter

This strain is characterized by the following morphological and cultural characteristics.

The culture is a mobile, single, rod-shaped straight or slightly curved cells with a size of (0.5-1.0) × (1.5-4.0) microns. Gram-negative. Form a capsule. Aerobe.

The strain grows on many natural and synthetic environments. On MPA after 18-20 hours of growth at 30 ° C, the culture forms colonies, rounded, flat, with uneven edges, of dense consistency with a rough surface. R-forms.

As a rule, when growing on solid media for 2–3 days, a continuous growth along the line is detected.

The culture is not lysogenic; after storage on solid nutrient media for 2-3 weeks, no negative spots were detected (at T = 30-35 ° C).

On meat and peptone broth at 30 ° C under stationary conditions, after 18-20 hours, growth in the form of a film on the surface of the medium is observed. The optimum growth is 29-31 ° C, pH 6.8-7.2.

On the Raymond mineral medium with glucose forms a whitish precipitate, slightly opalescent turbidity in the thickness of the medium and a thin wall ring.

On Raymond's mineral medium with oil as the only carbon source, slightly opalescent turbidity is observed in the thickness of the medium. Visually, the oil significantly degraded - with shaking, a suspension forms in the form of a finely dispersed phase of brown color, which is completely not typical for native oil.

The propagation of the culture was carried out by reseeding on meat peptone agar or agar medium of Raymond, the composition of which is presented above.

The cultivation conditions on the fermentation medium: temperature 29-31 degrees, pH 6.8-7.2, the duration of cultivation 22-24 hours.

The composition of the fermentation medium (% wt.):

BVK feed yeast - 2

Corn Extract - 1

Tap water - up to 100

The strain revealed a high level of sequence similarity of 16S rRNA (99.3-99.6) with the species Pseudomonas graminis.

The activity (productivity) of the strain, as well as other production indicators: on a production nutrient medium, the biomass culture productivity is (2.5 ± 0.5) × 10 ⁹ cells / ml, activity on Raymond medium - on the 5th day of biodegradation of oil (when containing oil 10%) is observed in a dilution of 10 ^-1 -10 ^-8 , the degree of oil consumption is 80%. The degree of oil consumption with a soil content of 10% oil on day 15 is 65%.

The activity was determined on a Raymond medium with oil visually according to the degree of oil degradation, as well as by the gravimetric method and gas-liquid chromatography.

The results of studies of the pathogenicity of the claimed strain show that the average virulent dose of the strain with a single intraperitoneal administration to white mice was more than 10 ⁹ cells per animal.

The strain does not have the ability to translocation and dissemination into the internal organs and blood of animals.

The strain does not have toxic and toxigenic properties.

Observation of the animals for 30 days did not reveal any clinical signs of the disease, and a study of the internal organs did not reveal pathological changes recorded morphologically.

The studied microorganisms do not have an irritating effect on the mucous membrane of the eye, do not penetrate the natural barriers of the upper respiratory tract, do not disseminate into the internal organs upon intranasal entry into the body.

Case Studies

The practical application of this strain is determined by its physiological and biochemical characteristics. The inventive strain is intended to eliminate the effects of water and soil pollution with oil and oil products.

Example 1

The culture was grown in 250 ml flasks with a working volume of filling 30 ml of nutrient medium on a shaker at 350 rpm, which provides an oxygen mass transfer coefficient of K _1a = 90 h ^-1 . The duration of cultivation is 22-24 hours, the final concentration of cells in the culture fluid is an average of 3 × 10 ⁹ cells / ml.

Example 2

The experimental assessment of the ability of a strain of microorganisms to biodegrade oil was carried out in laboratory conditions, in pilot and field experiments comprehensively by the following indicators: a decrease in the amount of hydrocarbons, a change in the fractional composition of oil during biodegradation, a change in the area of residual oil relative to the total area of the contaminated surface.

The culture fluid after growing the strain of bacteria-oil destructors was concentrated by separation to a cell concentration of 1 × 10 ⁹ -10 ¹⁰ cells / ml. For bioprocessing of water surfaces, the culture fluid of the claimed strain was diluted to a final cell concentration of 1x10 ⁶ cells / ml in fresh water, 0.2% nitroammophoski was added. The prepared solution was applied at the rate of 10 l / m ^{2 of} the soil surface. After 10-15 days, repeated bioprocessing of the contaminated surface was carried out. If necessary, in the case of a high level of contamination, a third bioprocessing was carried out 10-15 days after the second. On the soils, when the treated surface dries, watering and loosening were carried out, if possible. The soil and water in the pilot tests were carried out from garden watering cans, in the field - using an irrigation or fire truck with a capacity of 5-10 m ³ .

In laboratory conditions, the treatment of soil artificially contaminated with oil was carried out in laboratory glassware with a volume of 0.25-0.50 liters. Laboratory experiments were performed at room temperature.

Pilot tests were carried out on the surface of water bodies with an area of 1-2 m ² or more or on accidentally contaminated soils, where the pollution level was previously determined.

Field tests were carried out at natural sites after emergency oil spills on them. Both pilot and field tests were carried out in the summer-autumn period.

Samples for analysis were initially and dynamically taken according to a five-point scheme using a standard sampler.

Mass analysis of the initial level of pollution and further monitoring of the process of cleaning soil objects and water from oil pollution was carried out by the gravimetric method. The qualitative composition of the contaminant was carried out by gas-liquid chromatography. The quantitative composition of the oil fractions was determined using the peak area, and the qualitative composition was determined by the retention time. As a control, native oil that was not subjected to biodegradation was used.

The effectiveness of biodegradation of pollution was estimated by the loss of oil hydrocarbons in the process of bioprocessing, taking as 100% the initial concentration of pollution.

The number of living cells of microorganisms in all experiments was taken into account by the generally accepted method of limiting dilutions on elective media.

Tables 1-3 show the results of testing the biodegradable ability of the strain in laboratory conditions, field and pilot experiments.

Table 1 shows the results of comparative testing of the degrading ability of the strain Pseudomonas graminis T-7 and the preparation Devoroil on a soil model in laboratory conditions. As a model, a sample of contaminated soil from the Paveletsky enterprise providing petroleum products was used. The control was a soil sample contaminated with hydrocarbons from the same site without introducing any additional microorganisms.

Table 2 presents the results of field tests of the biodegradable ability of the Pseudomonas graminis T-7 strain at a natural site (Chernaya River) contaminated with hydrocarbons (HC) as a result of accidental oil spills.

Table 3 presents the results of testing the strain Pseudomonas graminis T-7 in comparison with the domestic drug in pilot experiments, which were put on chronic oil pollution.

Table 3 No. day Oil content Degree of purification g / kg % % 1 0 178.2 100 0 Devoroil fifteen 131.2 74 26 thirty 88.3 47 53 2 0 178.2 100 0 Pseudomonas fifteen 55.9 30.5 73.5 graminis T-7 thirty 42,2 19.3 80.7

Thus, the results obtained indicate that with the help of the Pseudomonas graminis T-7 strain intense degradation can be achieved with a high content of it in the object of contaminated substrate and it can be used to remove oil pollution from the surface of the soil and water bodies.

Claims (1)

The strain Pseudomonas graminis VKPM B-8615 for the decomposition of oil and oil products.