RU2157839C1 - Bacterial strain serratia odorifera a 3b for oxidation of petroleum hydrocarbons and derivatives - Google Patents

Abstract

FIELD: biotechnological methods for oil pollution removal. SUBSTANCE: bacterial strain isolated from soil polluted by petroleum derivatives is cultured for 72 h in liquid nutrient medium containing mineral salts, soya been flour, and molasses at pH 6.8-7.2 and 28 C under aerobic conditions. Destructive activity of strain ranges between 90.3 and 94.5 for paraffin fractions of mazut and between 88.7 and 93.0% for olefin fractions, efficiency of strain being high at temperatures between 15 and 25 C within a wide pH range: from 5.0 to 8.0. EFFECT: enhanced oil destructive activity. 2 tbl, 2 ex

Description

 The invention relates to biotechnology, in particular to the cleaning of environmental objects from pollution by oil and oil products using microorganisms, and can be used to obtain biological products for cleaning soils and surface water from oil pollution, as well as to eliminate accidental oil spills.

 Known oil-oxidizing strains of bacteria Acenitobacter (see RF patent N 2053204 in class C 02 F 3/34 from 04/12/94,) and Mycobacterium (see RF patent N 2053206 in class C 02 F 3/34 of 29.09. 94 g.), Used for biodegradation of hydrocarbons of petroleum products in the environment. However, these strains are not effective enough.

The closest technical solution (prototype) are strains of Pseudomonas putida PI K _o -1, Pseudomonas fluorescens P-896 and Micrococcus PI K _y -1, which are used as aerobic oil-oxidizing bacteria to create a biological product for cleaning from oil and oil products. The strains have sufficient activity at a pH of from 4.5 to 8.5 and a temperature of from 10 to 40 ^o C (see RF patent N 2122980 according to class C 02 F 3/34 from 04/12/94).

 The disadvantage of these strains is the relatively low oil-oxidizing activity against heavy macromolecular compounds that make up hydrocarbons of petroleum products, in particular paraffins and olefins that make up fuel oil.

 The objective of the present invention is to obtain a strain of bacteria with high oil-oxidizing activity against heavy macromolecular compounds that are part of hydrocarbons of petroleum products, in particular paraffins and olefins.

 To solve this problem, we propose a bacterial strain Serratia odorifera A 3b GKM VIZR N 100, which has high destructive activity against heavy macromolecular compounds, in particular, paraffins and olefins that are part of fuel oil.

 The bacterial strain Serratia odorifera A 3b was deposited in the State Collection of microorganisms of the All-Russian Scientific Research Institute for Plant Protection (GKM VIZR) under number 100 dated 03/12/99. The strain was identified by the Identifier of Microorganisms by Bergi (1980).

 The bacterial strain Serratia odorifera A 3b isolated from soil contaminated with fuel oil, in the village. Kishkenay (Republic of Lithuania) on the territory of the Klaipeda branch of TOG JSC. The strain is isolated on a selective nutrient medium containing hydrocarbons of petroleum products as the sole source of nutrition.

 The strain is selected and stabilized on the basis of hydrocarbon-oxidizing activity in relation to oil and oil products.

Cultural and morphological features
On MPA and SPA culture forms opaque white or light cream colonies with a smooth edge. Microscopy reveals rods that are motile with peritrichous flagging and do not form spores. Cell sizes (0.6 - 0.8) • (1.5 - 3.0) microns. The strain grows at a temperature of from 5 to 40 ^o C, the optimum growth temperature is 25 - 32 ^o C. The strain is stored on jambs of MPA or SPA at a temperature of 2 - 5 ^o C or in lyophilized form in ampoules.

Physiological and biochemical characteristics
Gram-negative (for alkali). Optional anaerobic with respiratory and enzymatic type of metabolism. Does not need growth factors. The oxidase test is negative, the catalase test is positive. Does not produce fluorescent pigment. The Voges-Proskauer reaction is positive. It has nitrate-reducing ability. Hydrogen sulfide does not form, indole does not form, does not destroy urease. Arginine dihydrolase is absent, tryptofandeaminase is absent. Lysine decarboxylase and ornithine decarboxylase are present. Utilizes citrate, gelatin hydrolyzes. Forms acid from cellobiose. Ferments sugars: glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiosis, amygdalin, arabinose. It utilizes nitrates, ammonium salts, amino acids, peptides, and yeast hydrolyzate as a source of nitrogen.

To grow the strain and obtain a biological product, a seed and fermentation medium of the following composition was used:
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.

 The grown culture was used in experiments to evaluate the destructive activity of the bacterial strain Serratia odorifera A 3b in relation to paraffin and olefin fractions of fuel oil, which are typical representatives of heavy macromolecular compounds that make up hydrocarbons in petroleum products.

 The oil-oxidizing activity of the strain was evaluated by IR spectrometry using the AN-2 instrument to reduce the concentration of hydrocarbons in petroleum products in samples containing paraffin and olefin fractions of fuel oil.

 Example 1

 Isolation and fractionation of heavy fractions of petroleum products from oil-contaminated soil to obtain environmental samples was carried out as follows. Oil products were extracted from the soil with carbon tetrachloride, after which the solvent was distilled off. The obtained oil products were dissolved in petroleum ether, and after a day, asphaltenes precipitated in a dark precipitate were filtered from the solution. Monoaromatic hydrocarbons were sorbed from the filtered solution onto alumina used as a sorbent. The sum of the paraffin and olefin fractions thus obtained was separated on columns with alumina impregnated with silver nitrate. For this, the paraffin fraction was eluted with hexane, the olefin fraction was displaced from the column with carbon tetrachloride; then the hexane solution of the paraffin fraction was concentrated and re-applied to the column with a small amount of alumina, and the paraffin fraction remaining on the column was displaced with hexane.

The isolated paraffin fraction of fuel oil was added to the liquid 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 - 3 g / l
Water - Up to 1 L
The bacterial strain Serratia odorifera A 3b was seeded in flasks containing 50 ml of medium, the initial concentration of paraffin fractions of fuel oil was 32-35 mg / l, the pH of the medium ranged from 5.0 to 8.0. The strain was cultivated on a rocking chair in the temperature range from 15 to 25 ^o C for 30 days. The paraffin content in the medium was monitored weekly. The results are presented in Table 1.

 Example 2

Isolated, as described in example 1, the olefin fraction of fuel oil was added to the liquid nutrient medium as described in example 1 composition. The bacterial strain Serratia odorifera A 3b was seeded in flasks containing 50 ml of medium, the initial concentration in the medium of olefin fractions of fuel oil was 23 - 25 mg / l, the pH of the medium ranged from 5.0 to 8.0. The strain was cultivated on a rocking chair in the temperature range from 15 to 25 ^o C for 30 days. The olefin content in the medium was monitored weekly. The results are presented in Table 2.

The experiments confirmed a very high destructive activity of the claimed strain in relation to high molecular weight paraffin (up to 94.5%) and olefin (up to 93.0%) fractions of fuel oil. Moreover, the strain is effective at temperatures (T = 15 - 25 ^o C) in a wide pH range (pH 5.0 - 8.0).

 A biological product based on the bacterial strain Serratia odorifera A 3b can be obtained industrially using standard biotechnological equipment using available raw materials.

Claims (1)

 The bacterial strain Serratia odorifera A 3b GKM VIZR 100 for the oxidation of hydrocarbons and petroleum products.

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