RU2128221C1 - Strain arthrobacter species for petroleum and petroleum products decomposition - Google Patents

Abstract

FIELD: technical microbiology, ecology. SUBSTANCE: strain of bacterium Arthrobacter sp. OS-1 is isolated from water polluted with petroleum and deposited in VKPM of "VNII-genetics" Institute at number Ac-1556. Biomass yield on industrial medium is (2-3) x 10¹⁰ cells/ml, its activity on Raimond's petroleum-containing medium is evident at dilution degree 10¹-10¹⁰ on 5-th day of petroleum biodegradation. Biodegradation degree by crude petroleum in the presence of the strain indicated above for 30 days is 98-99%. Strain can be used for removal of petroleum pollution from the soil and reservoir surfaces. The proposed strain that decomposes petroleum and petroleum products can be used for treatment of polluted surfaces. EFFECT: enhanced effectiveness of the strain proposed. 1 tbl, 2 ex

Description

 The invention relates to the world biological industry and relates to a new culture of microorganisms that destroy oil and oil products, which can, for example, be used to clean 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) / ав. St. 1493666, C 02 F 3/34, 1989 /.

 The use of these cultures of microorganisms is possible only in aeration tanks. Cultivation of a mixture of cultures is possible at high material and energy costs.

 The closest analogue is the bacterial strain Rhodococcus erythropolis BKM Ac-1339D, used to clean water and soil from oil and oil products / av. St. 1805097, C 02 F 3/34, 1993 /. The disadvantages of the known strain are 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 aim of the 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, OC-1, isolated from a sample of oil-contaminated water obtained from the Van Egan field, was proposed. The strain was deposited in VKPM Institute "VNIIgenetiki" under the number Ac - 1556.

 The strain was isolated by plating the initial water samples on meat-peptone agar with 0.5% oil with preliminary passivation of the initial samples through Raymond's liquid medium with oil.

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.

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

 The culture is mobile, gram-stable short rod-shaped small cells and round cells, located mainly in pairs, as well as individually, less often connected in chains of 3 or more cells. Culture is polymorphic. There are branching chains and rods located at an angle to each other, as well as individual smaller cocciform cells and cysts. The cells are motile, form a capsule, do not form spores. Cell sizes vary between (0.7-0.8) - (0.8-2.5) microns.

The strain grows on many natural and synthetic environments. On the MPA, after 2 days of growth at 30 ^° C, it forms flat, round colonies with a dull slightly raised center and a translucent, even opalescent edge in the light. On MPA, containing instead of glucose 0.5% oil, it forms strongly convex, round, shiny, whitish-cream-colored colonies. As a rule, when growing on solid media for 2-3 days, a continuous growth is found along the line in the form of convex mucous formations of a whitish-cream color, with an oily sheen.

 The culture is not lysogenic; after storage on solid media for 2-3 weeks, negative spots were not detected.

On a meat-peptone broth at 30 ^o C after 3 days, a turbidity of the medium, a whitish precipitate, the formation of a thin wall ring, as well as a small amount of agglomerates of cells in the thickness of the medium in the form of flakes, a film on the surface of the medium are noticeable. The optimum growth at 28-30 ^o C, pH 6.8-7.2.

 On the Raymond mineral medium with glucose forms a whitish thin 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, a slight opalescent haze is observed in the thickness of the medium.

 The strain activity was determined by Raymond’s environment with oil visually according to the degree of oil degradation.

The composition of the medium for the propagation of the strain is as follows; g / l:
Meat-peptone hangar:
Meat Water - 100
Agar - 20
NaCl - 5
Peptone - 10
Glucose - 7
Tap water - The rest is up to 1 liter
Raymond agar medium with oil:
Na ₂ CO ₃ - 0.1
CaCl - 0.01
MnSO ₄ - 0.02
FeSO ₄ - 0.01
MgSO ₄ - 0.2
Na ₃ Cl - 2.0
NaCl - 5.0
Na ₂ HPO ₄ - 1.5
KH ₂ PO ₄ - 1,0
Agar - 20.0
Meat-peptone broth - 500.0
Distilled water - The rest is up to 1 liter
The cultivation conditions on the fermentation medium: temperature 28-30 ^o C, pH 6.8-7.2, the duration of cultivation 22 hours

The composition of the fermentation medium,%:
Fodder Yeast (BVK) - 2
Corn Extract - 1
Culture Arthrobacter sp VKPM Ac - 1556 is not pathogenic for warm-blooded animals.

On a production nutrient medium, biomass culture productivity is (2-3) • 10 ¹⁰ cells / ml, activity on Raymond medium with oil - on the 5th day, biodegradation of oil is noted with a dilution of 10 ¹ -10 ¹⁰ . Raymond's activity with oil was determined visually by the degree of oil degradation.

 Physiological and biochemical characteristics.

Optional anaerobic grows in the range of 18-30 ^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; of polysaccharides, slightly starch; and of alcohols, mannitol. In this case, acidification of the medium from pH 7.0 to 5.5 is observed. Poorly assimilates sucrose, maltose, rhamnose. On media with arabinose and lactose, growth was not observed.

This strain exhibits good growth ability on hydrocarbon substrates: paraffin (C ₁₀ -C ₁₃ ), hexadecane and crude oil; moreover, growth in this case is observed mainly 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), when oil acts as the sole source of carbon.

 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 ability of the Arthrobacter sp VKPM Ac-1556 strain to grow and form metabolites (fatty acids) when it was grown on a mixture of liquid paraffins (C ₉ -C ₂₂ ) and hexadecane was analyzed. Hydrocarbons were sterilized separately at 1 at 30 minutes and were added sterilely at a concentration of 5% to Raymond agar medium indicated above.

The medium was poured into rocking flasks with a capacity of 300 ml of 100 ml of medium. The flasks were inoculated on a shaker with 350 rpm suspension of the Arthrobacter sp strain, obtained by flushing from mowed agar and containing 2.5 × 10 ¹⁰ cells / ml.

The cultivation was carried out in a shaker at a temperature of 30 ^o C for 14 days. It was found that the inventive strain actively forms fatty acids when grown on hydrocarbons in the following amounts:
in culture fluid, mcg / l
S _{16: 0} - 99.8
S _{16: 1} - 2.20
S _{18: 0} - 111.70
C _{18: 1} - 8.0
in hydrocarbons, mcg / l
S _{16: 0} - 382
S _{16: 1} - 115
S _{18: 0} - 763
S _{18: 1} - 167
It should be noted that most of the C ₁₆ -C ₁₈ fatty acids are dissolved in hydrocarbons.

 The pH value at the end of cultivation decreased from the initial 7.1 to 5.0, which additionally indicates the active vital activity of the strain with growth on hydrocarbon substrates, which confirms the presence of active growth of microorganisms in an oil-containing medium (see table).

 Example 2

Experiments were carried out to determine the degree of biodegradation of crude oil under the influence of the strain Arthrobacter sp. A culture containing 9.5 × 10 ⁹ cells / ml was seeded with Raymond medium (100 ml of Raymond medium in 300 ml of conical flasks) with 5% oil from the Sosnovsky field and incubated for 30 days on a rocking chair.

 After 30 days, the oil in the flasks changed its former consistency, although the culture fluid was clear.

 The pH of the culture fluid decreased from 7.1 to 5.5, which confirms the viability of microorganism cells actively developing in oil.

 The destruction results were taken into account by the disappearance of the oil film.

 After 30 days, a 98-99% disappearance of the oil film was observed. The results of the destruction are shown in the table. During the experiment, the content of oil and hydrocarbon changed.

 Thus, the results obtained indicate that, using the Arthrobacter sp VKPM Ac-1556 strain, intense oil degradation can be achieved at a high oil content (up to 15%) to the volume of contaminated substrate and it can be used to remove oil contaminants from the soil surface and water bodies.

Claims (1)

 Bacterial strain Arthrobacter sp. VKPM As-1556, used for the decomposition of oil and oil products.

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