RU2270063C2 - Bacterium kurthia zopfii strain capable to recover oil, petroleum products and aromatic polycyclic hydrocarbons - Google Patents

Abstract

FIELD: microbiological industry, in particular purification of soil, water and surfaces.

SUBSTANCE: new Kurthia zopfii 7-204 strain isolated from soil and having resistance in continuous process of cleaning from petroleum may be cultivated in wide pH range: from 5.0 to 10. Kurthia zopfii 7-204 strain is capable to oxidize high percent of hard-oxidized aromatic hydrocarbons, containing in wide paraffin cut and hard oils as well as chemically pure polycyclic compounds at 20-30°C.

EFFECT: strain for continuous and discontinuous effective purification.

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Description

The invention relates to the microbiological industry, in particular to a strain capable of oxidizing oil, oil products and aromatic polycyclic hydrocarbons in a wide pH range of 5.0-10.0 units for use in cleaning soil, water and surfaces.

Known aerobic oil-oxidizing bacteria Micobacterium flavescens VKPM-B-6000, Micobacterium sp. IL-4, Rhodococcus sp. 56e, Acinetobacter sp. HB-1 (BI No. 30, 2002, RF patents No. 2191752 and No. 2191753, class 7 C 02 F 3/34, C 09 C 1/10, C 12 N 1/26). The disadvantage of these cultures is a narrow range of pH (6.8-8.0), inability to oxidize aromatic polycyclic hydrocarbons.

Known bacterial strains of Pseudomonas alcaligents B-1, Pseudomonas putida and Pseudomonas alcaligents E-7, used to clean water and soil from oil and oil products (BI No. 21, 23 - 1999, patents RU No. 2133770, class 6 C 12 N 1 / 20, С 02 F 3/34, В 09 С 1/10, BI No. 23, 1999, patents RU No. 2134722 and No. 2134723, class 6 С 12 N 1/20, C 02 F 3/34, В 09 C 1/10).

These bacterial strains are also incapable of oxidizing polycyclic aromatic hydrocarbons and have a narrow pH spectrum of the fermentation medium of 6.8-7.2 to 7.5-8.0 units.

The closest in technical solution to the present invention is a consortium of bacterial strains Acinetobacter calcoaceticus VKPM-B-7179, Pseudomonas fluorescens VKPM-B-7152, Alcaligenes faccalis VKPM-B-7180 (BI No. 9, 1998, RF patent No. 2107722, class RF 6 С 12 N 1/20, С 02 F 3/34, С 12 R 1/27, 1/01), designed for cleaning soil, water, tanks and wastewater of industrial enterprises from oil pollution. A disadvantage of the known consortium of bacteria is the low level (5-8%) of paraffin utilization with a high content of aromatic hydrocarbons with a narrow pH of 6.8-7.0 units, low temperature of 6-15 ° C fermentation, and the inability to oxidize polycyclic aromatic hydrocarbons.

The technical result of the invention is a wider pH range of the fermentation medium of 5.0-10.0 units, the ability to oxidize aromatic polycyclic hydrocarbons, a higher oxidation rate of hydrocarbon contaminants. To do this, it is proposed to use the new active natural bacterial strain Kurthia zopfii 7-204, grown in a mineral medium of the following composition (in g / l): (NH ₄ ) ₂ SO ₄ - 3.0; KH ₂ PO ₄ - 6.5; MgSO ₄ - 0.7 and trace elements (12.5 mg / L each): FeSO ₄ × 7H ₂ O; MnSO ₄ × 5H ₂ O; ZnSO ₄ × 7H ₂ O and 3 mg / L CuSO ₄ × 5H ₂ O.

The origin of the strain

The proposed strain of Kurthia zopfii 7-204 isolated from soil samples of the Nizhnevartovsk field in 1999, is not toxic, not pathogenic. The strain was identified by Bergey's “Guide to Bacteria” (translation edited by Academician G.A. Zavarzin, 1997).

Generic and species name of the strain

The isolated strain belongs to the genus Kurthia and to the species Zopfii.

Cultural and morphological features of the strain

On meat-peptone agar (MPA) it forms round, shiny, creamy white colonies, the edge is smooth, the structure is homogeneous, and the consistency is pasty. The diameter of the young colony is 2-2.5 mm, the old - 4-5 mm. The bacteria are gram-positive, non-acid-resistant, non-spore forming short thick rods 0.8-1.2 × 2.0-3.0 μm in size, single, sometimes in V-shaped pairs, cocci 0.7 × 0.8-1 in size are found , 4 × 1.6 μm. Old cultures usually consist of coccoid cells, they do not form mycelium. Cells are motionless or weakly mobile.

Physiological, biochemical and biotechnological characteristics

As the sole carbon source, n-paraffins obtained by urea dewaxing with a high content of aromatic hydrocarbons are used; oil from different regions of the country; polycyclic aromatic hydrocarbons; naphthalene, phenanthrene. Aerobes. Metabolism of the respiratory type with the formation of a small amount of acid with growth on glucose, xylose, glycerin, mannose. Gas does not form. The bacteria are catalase-positive, they do not dilute gelatin, they do not clot milk, its peptonization (weak) is observed on day 12. Nitrates do not recover. Nitrate nitrogen is not consumed. On a slice of potato - abundant growth of culture (beige color). With growth in BCH - moderate turbidity, loose sediment. Bacteria grow well in a wide range of pH 5.0–10.0 units; at pH 5.0–6.0, the oxidation of n-paraffins obtained by urea dewaxing is at the level of 30–33%; at pH 7.0 -8.0 units - 88.4-95.0%, with a pH of 8.0-10.0 units, biodegradation is 95.0-85.5%. Thus, the proposed strain relates to alkalophilic cultures.

The optimum cultivation temperature is 20-30 ° C, when the temperature drops below 15 ° C and increases above 35 ° C, growth slows down, and at a temperature below 5 ° C and above 40 ° C, growth stops completely. Antibiotic sensitivity was studied using paper disks containing standard concentrations (see Table 1).

Table 1
The resistance of the strain to antibiotics Antibiotic The antibiotic content in the disk, mcg The diameter of the zone of inhibition of growth, mm Interpretation of Results Ampicillin 10 32 Steadily sensitive Carbenicillin 25 16 Moderately sensitive Oxacillin 10 17 Moderately sensitive Streptomycin thirty eighteen Sensitive Kanamycin thirty twenty Sensitive Chloramphenicol thirty 16 Moderately sensitive Rifampicin 5 16 Sensitive Tetracycline thirty thirty Steadily sensitive Erythromycin fifteen thirty Steadily sensitive

The invention is illustrated by the following examples.

Example 1. Culture Kurthia zopfii 7-204 is grown periodically in a rocking flask with a volume of 750 ml at a temperature of 20-25 ° C, pH 7.5-8.0 on mineral medium No. 10 of the following composition (g / l): (NH ₄ ) ₂ SO ₄ - 3.0; KH ₂ PO ₄ - 6.5; MgSO ₄ - 0.7 and trace elements (12.5 mg / L each): FeSO ₄ × 7H ₂ O; MnSO ₄ × 5H ₂ O; ZnSO ₄ × 7H ₂ O and 3 mg / L CuSO ₄ × 5H ₂ O. Oil is used as the sole carbon source with a total hydrocarbon content of 59.2%, including aromatic hydrocarbons - 31.3%, volatile hydrocarbons - 18, 6%, resins - 1.3%, asphaltenes - 0.6%. The concentration of crude oil in the nutrient medium is 1% (by weight). Sowing is carried out by optical density, which is 0.2 at a wavelength of 540 nm. After 10 days of cultivation, oil recovery is 40%.

Example 2. Culture Kurthia zopfii 7-204 is grown batchwise for 72 hours in rocking flasks with a volume of 750 ml in the mineral medium No. 10 indicated in Example 1, inoculating by optical density = 0.1 unit at a wavelength of 540 nm and pH of medium 5 , 0-6.0 and an optimal temperature of 25-30 ° C. A broad fraction paraffin in a concentration of 1% weight is used as a carbon source. the following composition: C ₁₀ -C ₁₂ = 12%, C ₁₄ -C ₁₈ = 50.4%, C ₁₉ -C ₂₈ = 31.4%, aromatic hydrocarbons 6.2%. After 72 hours, the utilization of urea paraffin was 30-33%.

Example 3. A culture of Kurthia zopfii 7-204 is grown in a batch manner under the same conditions as in Example 2 — utilization at a pH of 7.5 units was 91.8%.

Example 4. The strain Kurthia zopfii 7-204 is grown under similar conditions as in example 2 at a pH of 8.0-9.0 units. Disposal after 72 hours of fermentation was 91.8-95.0%.

Example 5. The strain Kurthia zopfii 7-204 is grown under similar conditions of example 2 at pH 9.0-10.0 units, after 72 hours of fermentation, utilization was 91.1-85.5%.

Example 6. The strain Kurthia zopfii 7-204 was grown batchwise in rocking flasks with a volume of 750 ml in mineral medium No. 10 (see example No. 1). Phenanthrene at a concentration of 0.1% was used as a carbon source. Cultivation was carried out for 15 days at a temperature of 25-30 ° C, the pH of the medium was 7.5 units, inoculation of the culture by optical density was 0.1 units at a wavelength of 540 nm. The utilization of chemically pure polycyclic aromatic hydrocarbon - phenanthrene - amounted to 39.0%.

Thus, the proposed alkalophilic culture of Kurthia zopfii 7-204 grows in a wide range of pH: from 5.0 to 10.0 units, an optimum of 7.5-10.0 units and is capable of oxidizing difficultly oxidizable aromatic hydrocarbons contained in broad fraction paraffins , heavy oils and chemically pure compounds of a polycyclic nature at an optimal cultivation temperature of 20-30 ° C. The strain is stable and steady in a batch and continuous process.

Deposition and storage of the strain. The strain is deposited in the culture collection of LLC PKF "BIGOR" under the number 7-204. The culture is maintained on meat-peptone agar (MPA), replanted once every 2 months. In addition, the culture is stored in a lyophilized state on Faibich medium at a temperature of + 4 ° C.

Claims (1)

The bacterial strain Kurthia zopfii 7-204, which has the ability to utilize oil, petroleum products and aromatic polycyclic hydrocarbons.