RU2192462C1 - Strain of bacterium burkholderia caryophylli jap-3 for oxidation of polycondensed aromatic hydrocarbons - Google Patents

Abstract

FIELD: biotechnology, microbiology, ecology. SUBSTANCE: invention relates to treatment of objects of environment from oil and petroleum products pollution using microorganisms. Invention proposes the strain of bacterium Burkholderia caryophylli Jap-3 showing high oil-oxidizing activity with respect to polycondensed aromatic hydrocarbons. Strain is deposited in collection VIZR at = 151. Strain of bacterium Burkholderia caryophylli Jap-3 can be grown by industrial method using standard equipment and available raw. Strain can be used as the base for preparing biopreparations for treatment of soils and surface waters from petroleum pollutions containing high concentrations of polycondensed aromatic hydrocarbons. EFFECT: high activity of bacterial strain. 2 tbl

Description

 The invention relates to biotechnology, in particular to the cleaning of environmental objects from oil and oil products pollution using microorganisms and can be used to obtain biological products for cleaning soils and surface waters from oil pollution containing high concentrations of polycondensed aromatic hydrocarbons.

 Known oil-oxidizing strains of bacteria Acenitobacter, see RF patent 2053204 for class. With 02 F 3/34 from 04/12/94, and Mycobacterium, see RF patent 2053206 for CL. C 02 F 3/34 dated 09/29/94, used for biodegradation of hydrocarbons of petroleum products in the environment. However, these strains are not effective enough.

Also known strains of Pseudomonas putida PI K ₀ -1, Pseudomonas fluorescens and P-896 _have Micrococcus PI K -1, which is used as an aerobic oxidizing bacteria to create a biological product for the purification of oil and petroleum products. The strains have sufficient activity at a pH of from 4.5 to 8.5, see RF patent 2122980 for class. C 02 F 3/34 dated 04/12/94

 A known bacterial strain Bacillus cereus B 36b, with high destructive activity against hydrocarbons of oil and oil products at pH from 3.0 to 8.5, see RU 2157843 from 05.31.1999.

 This technical solution is made as a prototype of the present invention.

 The disadvantage of the prototype is the relatively low oil oxidizing activity at high concentrations of polycondensed aromatic hydrocarbons.

 The objective of the present invention is to obtain a strain of bacteria with high oil-oxidizing activity under environmental pollution by polycondensed aromatic hydrocarbons.

 To solve this problem, a bacterial strain Burkholderia caryophylli Jap-3 is proposed, which has high oil-oxidizing activity against polycondensed aromatic hydrocarbons.

 The bacterial strain Burkholderia caryophylli Jap-3 was deposited in the State collection of microorganisms of the All-Russian Scientific Research Institute of Plant Protection (GKM VIZR) under the number 151 from 06/18/2001. The identification of the strain was carried out according to the "Identifier of microorganisms" Bergi (1997).

 The bacterial strain Burkholderia caryophylli Jap-3 was isolated from soil contaminated with polycondensed aromatic hydrocarbons in the prefecture of Tokyo, Japan.

 The strain was isolated on a selective nutrient medium containing polycondensed aromatic hydrocarbons as the sole source of nutrition.

 The strain is selected and stabilized on the basis of high hydrocarbon-oxidizing activity against polycondensed aromatic hydrocarbons.

 Cultural and morphological characteristics of the strain: movable straight sticks of 0.8-0.9x0.9-2; 1.5x1.5 microns, located singly and in clusters. On standard nutrient media (MPA, BBL, Becton Dickinson) forms round colonies 2 mm in diameter, convex, smooth, shiny, mucous, the edges are even, white, opaque. Gram stain and Gram reaction by the Gregensen method (1978) are gram-negative.

Biochemical signs of the strain. Aerobe. Hemoorganotroph. Does not need growth factors. The oxidase test is positive. Catalase test is positive. Does not form a fluorescent pigment. It does not grow at +4 ^o C. It grows at +41 ^o C. It has the ability to denitrify with the formation of N ₂ . Gelatin does not hydrolyze. Starch does not hydrolyze. Uses glucose, glutamate, ethanol, succinate, xylose, mannitol as the sole carbon source. Uses hydrocarbons as a growth source.

The strain is stored on the jambs of the MPA at (2-5) ^o C or in lyophilized form in ampoules.

To grow the strain and obtain a biological product used inoculum and fermentation nutrient medium of the following composition:
KH ₂ PO ₄ - 2 g / l
Na ₂ HPO ₄ - 3 g / l
MgSO ₄ • 7H ₂ O - 0.5 g / l
FeCl ₃ • 6H ₂ O - 0.05 g / l
CaCl ₂ • 2H ₂ O - 0.01 g / l
(NH ₄ ) ₂ SO ₄ - 2 g / l
Soya flour - - 5 g / l
Molasses - 20 g / l
Water - Up to 1 L
The seed was grown in flasks, and the cultivation process was carried out in fermenters V = 100 l at T = 28 ^o C, pH = 6.8-7.2 for 72 hours under aerobic conditions. 0.01% crude oil was used as an inductor. Then the deep culture of the microorganism (10%) was introduced into sterile peat simultaneously with mineral additives and was grown at T = 3 ^o C for 120 hours.

As mineral additives used:
Urea - 0.6 wt.%;
Double superphosphate - 0.3 wt.%;
Potassium sulfate - 1.5 wt.%
The titer of the obtained biological product is 1 • 10 ¹² CFU / g.

 The oil-oxidizing activity of the strain was evaluated by IR spectrometry using the AN-2 instrument to reduce the concentration of hydrocarbons in oil products in soil samples containing high concentrations of mobile forms of salts of heavy metals. The change in the concentration of polycondensed aromatic hydrocarbons was studied on a Hewlett-Packard chromatography-mass spectrometer, which includes an HP-5890 gas chromatograph, an HP-5972 mass-selective detector and a Vectra 5/100 workstation with data processing software and databases of mass spectral information WILEY138. L and NBS75K.L. To detect individual compounds in the samples under study, the obtained chromatograms for the total ion current were reconstructed from several mass numbers of ions that are most characteristic for these compounds. The data on the chemical composition of the compounds given in the examples were obtained both directly and by searching for specific compounds. The calculations were performed by the method of internal normalization for one of the most characteristic mass numbers of the analyzed component, taking into account the conversion factor of the intensity of the selected line to the total intensity of the full mass spectrum.

Example 1
A biological product based on the bacterial strain Burkholderia caryophylli Jap-3 was introduced into ceramic vessels filled with soil with an oil content of 30.8 to 39.0 g / kg. The total amount of aromatic compounds in the soil is 15.9 wt. %, the content of benzopyrene derivatives and isomers - 0.58 wt. % The amount of biological product introduced is 1 g per 1 kg of soil. The vessels were kept in thermostats in the temperature range from 3 to 45 ^o C for 90 days. The hydrocarbon content in the soil was monitored once every 30 days. The experiment was carried out in triplicate. The results are presented in table 1.

Example 2
In the second example, a biological product based on the bacterial strain Burkholderia caryophylli Jap-3 was introduced into glass vessels filled with soil with an oil content of 40.8-59.5 g / kg. The total amount of aromatic compounds in the soil is 18.9 wt.%, The content of anthracene and phenanthrene derivatives is 1.98 wt. % The experiment was carried out in triplicate. The results are presented in table 2.

As a result of the experiments, it is possible to state a very high destructive activity of the drug based on the claimed strain (up to 90%), including with a high content of polycondensed aromatic carbohydrates. Moreover, the strain is effective in a wide range of temperatures (T = 3-45 ^o C).

 The bacterial strain Burkholderia caryophylli Jap-3 can be grown industrially using standard biotechnological equipment using available raw materials.

Claims (1)

 The bacterial strain Burkholderia caryophylli Jap-3 (deposited in the State collection of microorganisms VIZR under 151) for the oxidation of polycondensed aromatic hydrocarbons of petroleum products.

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