RU2703142C1 - Pseudoalteromonas arctica strain for decomposition of oil and oil products - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Disclosed is Pseudoalteromonas arctica strain capable of decomposition of oil and oil products. Strain is deposited under registration number VKPM V-13085.

EFFECT: invention provides for purification of water areas of water bodies, shoreline from oil and oil products at temperature from +20 to -2½ °C and salinity 30±10 g/l in nutrient poor conditions.

1 cl, 3 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of biotechnology, microbiology, ecology, environmental protection and is a new bacterial strain of Pseudoalteromonas arctica ARC 39 VKPM B-13085, with the ability to degrade oil and oil products. The strain can be used to clean the waters of water bodies, coastlines contaminated with oil and oil products at temperatures from +20 to -2.5 ° C and salinity of 30 ± 10 g / l.

State of the art

A known strain of Rhodococcus sp. - a destructor of petroleum hydrocarbons (RF patent No. 2518349, priority 04/11/2013, publ. 06/10/2014.), which utilizes oil, fuel oil, kerosene, naphthalene, pentadecane, tetradecane, hexadecene, phenyldecane, prystan for 5 days at 20-30 ° C. This strain showed a high degree of destruction of hydrocarbons at a temperature of + 30 ° C. However, this strain is not capable of producing hydrocarbon utilization at low temperatures.

Known strain of Bacillus subtilis "Kolyma-7 / 2k" (RF patent 2446900, priority 13.07.2010, publ. 10.04.2012), which can be used in the Far North. The use of such a microbial preparation allows reducing the oil concentration to 0.48% over 3 months of the summer period. The disadvantage of this strain is that it utilizes hydrocarbons in soil conditions.

The known strain of Exiguobacterium mexicanum VKPM B-11011 (RF patent No. 2523584, priority 03/27/2013, publ. 20.07.2014). The strain can be used to clean soils and water bodies contaminated with oil and oil products in a wide temperature range from +4 to + 37 ° С. Also known is a strain of Serratia plymuthica VKM B-2819D (RF patent No. 2560272, priority 09/22/2014, publ. 08/20/2015), which has the ability to quickly dispose of oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), can be used to clean soils and water bodies contaminated with oil and oil products in a wide temperature range from +4 to + 30 ° С. The disadvantage of the strains is that they are not able to utilize oil pollution at high salinity of water up to 30 g / l.

The strain Rhodococcus erythropolis is deposited under registration number VKM AC-2613D (RF patent 2553336 priority 08/20/2013 opul. 06/10/2015). The strain is used to clean freshwater, brackish and marine objects and bottom sediments. The microorganism ensures the destruction of oil in water bodies at temperatures from +2 to + 25 ° C and salinity up to 2.7%. The disadvantage of the invention is that the strain utilizes hydrocarbons in conditions rich in nutrients (the composition of the medium includes peptone 5 g / l and sucrose 15 g / l). Under real conditions of hydrocarbon pollution utilization, the amount of nutrients is significantly reduced.

The closest analogue is the strain Nocardia coeliaca ARC 12 VKPM Ac-1990 (RF patent 0002624067, priority 06/30/2017, publ. 06/30/2017), which is capable of degradation of oil and oil products. The strain can be used to clean the waters of water bodies, coastlines contaminated with oil and oil products, at temperatures from +20 to + 4 ° C and salinity of 30 g / l, in conditions of nutrient-poor conditions. The disadvantages of the drug include the fact that the claimed temperature range of strain activity is in the region of positive temperatures. Also, the limits of salinity in which it remains active are not indicated for the preparation.

Disclosure of invention

The objective of the present invention is to obtain a bacterial strain capable of degrading oil and oil products at temperatures from +20 to -2.5 ° C and salinity of 30 ± 10 g / l under conditions poor in nutrients.

The problem is solved in that a strain of Pseudoalteromonas arctica ARC 39 VKPM B-13085 is obtained, capable of degrading oil and oil products, which can be used to clean water bodies of water bodies, coastlines contaminated with oil and oil products at temperatures from +20 to -2, 5 ° С and salinity of 30 ± 10 g / l in conditions poor in nutrients.

The strain was isolated from the Kara Gates Strait, near Morozov Island, in August 2015. The strain was deposited at the All-Russian Collection of Industrial Microorganisms of the Federal State Unitary Enterprise GosNIIgenetika (VKPM, Moscow, 1st Dorozhny pr., D.1) under registration number VKPM B-13085.

The strain was identified based on a study of its cultural-morphological and physiological-biochemical characteristics, as well as on the basis of a study of the 16S rRNA gene sequence.

The resulting strain is characterized by the following features.

Cultural and morphological characters.

The colonies are yellowish, shiny, smooth, round, convex, with a smooth edge. Cells are small rods, 2-4 microns in size, motile. Gram stain is negative.

Physiological and biochemical characteristics

The strain grows at a temperature of +20 to -2.5 ° C, under aerobic conditions. Grows at pH 4.0-8.0. It grows on solid and liquid nutrient media with a NaCl content of 30 ± 10 g / l. Able to grow in a liquid medium with hydrocarbons as the sole source of carbon and energy.

The strain is maintained by reseeding 1 time in 14 days on a solid nutrient medium modPCA of the following composition (g / l): K ₂ HPO ₄ - 1.5; KH ₂ PO ₄ 0.75; MgSO ₄ - 1.0; (NH ₄ ) ₂ SO ₄ - 4.0; NaCl - 30; casein hydrolyzate - 5.0; yeast extract - 2.5; D-glucose - 1.0; agar - 14; pH 7.0 or liquid nutrient medium No. 1 of the following composition: K ₂ HPO ₄ - 1.5; KH ₂ PO ₄ 0.75; MgSO ₄ - 1.0; (NH ₄ ) ₂ SO ₄ - 4.0; NaCl - 30; casein hydrolyzate - 0.5; yeast extract - 0.1; pH 7.0. In a liquid nutrient medium, a mixture of marketable oil of the Vozeyskoye field (density 37.9 ° API, sulfur content 0.66%, characterized as heavy, viscous, with a high content of resins and asphaltenes) and diesel fuel in a 1: 1 ratio was used as a hydrocarbon substrate volume.

Also, the strain can be stored at + 6 ± 2 ° C in a lyophilized state using skimmed milk powder as a protective medium.

For the preparation of the drug, a nutrient premix based on various organic components in various proportions (such as skimmed milk powder, flour of various plant cultures, milk whey, molasses, etc.), inorganic substances stimulating microbial growth can be added to the microorganism culture in the first place. a turn containing such macrocells as nitrogen and phosphorus, iron, etc. (various complex fertilizers). In addition, in dry form, the preparation contains protective media used to increase the survival of microorganisms during drying, for example, sugar solutions, complex media, such as a solution of skimmed milk powder, dried proteins, and others.

The implementation of the invention

The present invention is illustrated by specific examples of execution, which are not the only possible, but clearly demonstrates the possibility of achieving the desired technical result.

Example 1. Isolation and cultivation of the strain Pseudoalteromonas arctica ARC 39 VKPM B-13085.

The strain was isolated from seawater samples taken in the water area of the Kara Gate, near Morozov Island, in August 2015. A sample weighing 5 ± 2 g was added to 100 ml of nutrient medium No. 1. Cultivation was carried out at a temperature of + 4 ° C, on an orbital shaker (130 rpm). The cultivation time is 7 days. The resulting accumulative culture was made in the amount of 10 ml in 100 ml of nutrient medium No. 1. At least 30 passages of the accumulative culture were carried out. The strain was isolated from the accumulative culture on modPCA medium; the resulting pure culture was further cultivated on medium No. 1.

Example 2. The study of the oil-oxidizing activity of the strain Pseudoalteromonas arctica ARC 39 VKPM B-13085.

The strain grown in liquid nutrient medium No. 1, was introduced as inoculum in 100 ml of nutrient medium No. 1. As a control, a culture medium was used without adding culture. After cultivation at a temperature of -2.5 ° C, 130 rpm for 30 days, hydrocarbons were extracted with hexane according to GOST 31953-2012, a chromatographic assessment of hydrocarbon losses was carried out on a Crystal 2000 m chromatograph (Chromatek) with a DB- capillary column 1 (Agilent) [length - 30 m, diameter - 0.25 mm, layer thickness - 0.25 μm] with a flame ionization detector (PID). The temperature of the detector is 350 ° C; evaporator temperature - 300 ° С; carrier gas is helium; gas flow - 1.0 ml / min; gas flow rate - 20.0 ml / min; hydrogen consumption - 20 ml / min; air consumption - 200 ml / min. Measurements are carried out under conditions of a temperature gradient from 100 to 300 ° C with a gradient speed of 15.0 ° C / min.

The total decrease for the medium-boiling and high-boiling fractions was not less than 30% for 30 days of cultivation at -2.5 ° C and salinity of the medium 30 g / L.

The total decrease for the medium-boiling and high-boiling fractions was not less than 30% for 30 days of cultivation at -2.5 ° C and a salinity of 40 g / L.

Example 3. A method of manufacturing a preparation based on a strain of Pseudoalteromonas arctica ARC 39 VKPM B-13085.

The strain Pseudoalteromonas arctica ARC 39 VKPM B-13085, grown on modPCA at a temperature of + 4 ° C for 7 days, contribute to the liquid nutrient medium of the following composition (g / l): K ₂ HPO ₄ - 1.5; KH ₂ PO ₄ 0.75; MgSO ₄ - 1.0; (NH ₄ ) ₂ SO ₄ - 4.0; NaCl - 30; casein hydrolyzate - 5.0; yeast extract - 2.5; D-glucose - 1.0; pH 7.0. Then cultivated at a temperature of + 10 ° C on an orbital shaker for 3 days to a final titer of 10 ⁹ CFU / ml. A protective medium (10% skimmed milk powder) is added to the resulting cell suspension in a nutrient medium. Next, the resulting suspension is frozen and dehydrated by lyophilization. The resulting dry biomass of the strain Pseudoalteromonas arctica ARC 39 VKPM B-13085 is mixed with nutritious premix (skimmed milk powder) in a ratio of 1:10.

Thus, a strain is obtained that is capable of degrading oil and oil products at a temperature of -2.5 ° C and salinity of 30 ± 10 g / l under conditions poor in nutrients.

Claims (1)

The strain Pseudoalteromonas arctica VKPM B-13085 for the destruction of oil and oil products in an aqueous medium at a temperature of +20 to -2.5 ° C and salinity of 30 ± 10 g / l in conditions poor in nutrients.