RU2142996C1 - Strain arthrobacter sp for degradation of crude oil and petroleum products - Google Patents

Abstract

FIELD: microbiological industry and spilt oil removal. SUBSTANCE: when consuming only carbon of crude oil, strain is not pathogenic and can be used to clean water, soil and other oil-polluted surfaces. EFFECT: accelerated oil consumption. 3 tbl, 2 ex

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) / a.c. USSR N 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 VKPM As-1339D, used for the decomposition of oil and oil products, which is used to clean 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 Arthrobacter sp., OC-1 strain, isolated from a sample of oil-contaminated waters obtained from the Van Egan field. The strain was deposited in VKPM Institute "VNIIGenetiki" under the number Ac-1556. / pat. RF 2128221 /.

 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, a strain of Arthrobacter sp. MIA-74, isolated by exposure to cosmic radiation on a strain of Arthrobacter sp., OS-1, VKPM As-1575 (a certificate of deposit is attached).

 Isolation of the strain was carried out by sieving samples of strain OS-1 after exposure in space flight.

 Identification of the strain was carried out on the basis of a study of its cultural-morphological and physiological-biochemical characteristics in relation to the description given in the definition of bacteria Bergey / Bergey Smanual of Systematic Bacteriology vol. 1.184 p. / ..

The strain is stored on agarized nutrient media at a temperature of 6-8 ^o 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
NaCl - 3.0
Na ₂ HPO ₄ - 1.5
KH 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 consists of mobile, gram-short short rod-shaped small cells and round cells arranged in pairs or several in a row, as well as individually, located at an angle to each other, as well as individual cocciform cells. The cells are motile, they do not form spores. The cell size is in the range of 0.7 - 0.8 - 2.5 microns.

The strain grows on many natural and synthetic environments. On MPA after 2 days of growth at 30 ^o C, the culture forms round colonies, not very large, slightly convex, with a matte surface and smooth edges, slightly leathery consistency, with pink pigment. As a rule, when grown on solid media for 2–3 days, a continuous growth along the line in the form of a pink color is found.

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

On a meat and peptone broth at 30 ^o C after 3 days, a turbidity of the medium, a whitish precipitate, the formation of a thin wall ring, a small amount of agglomerates of cells in the thickness of the medium in the form of flakes, a weak film on the surface of the medium are noticeable. The optimum growth is 28 - 30 ^o 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 is 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.

Cultivation conditions on a fermentation medium: temperature 28 - 30 ^o C, pH 6.8 - 7.2, the duration of cultivation 22 hours.

 The composition of the fermentation medium.

 BVK fodder yeast - 2%.

 Corn extract - 1%.

 Physiological and biochemical characteristics.

Optional anaerobic grows in the temperature range from 18 to 30 ^o C.

 Starch weakly hydrolyzes, milk does not peptidize, gelatin does not thin, casein does not hydrolyze, non-acid-resistant, catalase-positive. It grows well on BCH diluted with Raymond medium in a ratio of 1: 1, with NaCl at a concentration of 0.5 and 1.0%. When grown on media with 5% NaCl, a very slight clouding of the medium and the formation of a wall ring are observed. At a concentration of NaCl of 10, 15, 20%, there is no growth.

 Relation to carbon sources.

 The strain assimilates carbohydrates with the formation of acids: from hexoses - glucose, from polysaccharides - slightly starch and from alcohols - mannitol. In this case, acidification of the medium from pH 7.0 and 5.5 is observed. Poorly assimilates sucrose, maltose, rhamnose. On media with arabinose and lactose, growth was not observed.

This strain shows good growth ability on hydrocarbon substrates: paraffin (C ₁₀ - C ₁₃ ), hexadecane and crude oil, and growth in this case is mainly observed at the phase boundary. In a liquid environment, Raymond is able to absorb oil and paraffins in a concentration of up to 20%.

 Attitude to nitrogen sources.

The strain grows well on environments with ammonium and nitrate nitrogen (NaNO ₃ , NH ₄ NO ₃ , NH ₄ Cl _[CA1] , when oil acts as the sole source of carbon.

 Culture Arthrobacter sp. MIA-74 is non-pathogenic for warm-blooded.

In a production nutrient medium, biomass culture productivity is 2–3 • 10 ⁹ cells / ml, activity in sand with oil - on the 7th day of biodegradation of oil, a dilution of 10 ¹ - 10 ⁸ is noted. Activity was determined in sand with oil by spectrophotometric and chromatographic analysis by the amount of residual oil.

The results of studies of the inventive 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 translocate and disseminate into the internal organs and blood of animals after 30 days of observation.

 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 intake. With multiple intranasal administration, it was found that microorganisms have a weak sensitizing and moderately pronounced dysbiotic effect.

As a result of the work, it was found that the culture of Arthobacter sp. MIA-74 most actively consumes heavy oil fractions (C ₂₄ - C ₃₃ ). During the life of microorganisms, secondary metabolites are formed in the form of fatty acids with a chain length of C ₁₄ - C ₂₀ , they can contribute to the quantitative assessment of the fraction.

 Examples of specific use.

 The practical application of this strain is determined by its physiological and biochemical characteristics, namely, the ability to actively grow on media with hydrocarbons (oil, paraffin, hexadecane).

 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 under 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 biological treatment of water surfaces, the culture fluid of the claimed strain was diluted to a final cell concentration of 1 • 10 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 was 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 processing 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 level of contamination 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 efficiency of pollution biodegradation was estimated by the loss of oil hydrocarbons in the process of bioprocessing, taking the initial concentration of pollution as 100%.

 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.

 In the table. 1, 2, 3 shows the results of testing the biodegradable ability of the strain in laboratory conditions, field and pilot experiments.

 In the table. 1 shows the results of comparative testing of the degrading ability of the strain Arthrobacter sp MIA-74 and the preparation Devoroil on a soil model in laboratory conditions. As a model, a sample of contaminated soil from the Paveletsky oil products supply company was used. The control was a soil sample contaminated with hydrocarbons from the same site without introducing any additional microorganisms.

In the table. 2 presents the results of field tests of the biodegradable ability of strain MIA-74 at a natural site (Chernaya river) contaminated with hydrocarbons (HC) as a result of accidental oil spills
In the table. 3 presents the results of testing the strain MIA-74 in comparison with the domestic drug in pilot experiments, which were put on stale oil pollution.

 Thus, the results obtained indicate that using the strain Arthobacter sp. MIA-74 can be achieved intense degradation with its high content 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)

 Strain Arthrobacter sp. MIA-74 VKPM As-1575 for the decomposition of oil and oil products.

Images