RU2489485C1 - STRAIN Rhodococcus Erythropolis, USED FOR DECOMPOSITION OF OIL - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus erythropolis 1-KP, extracted from podzol soil contaminated with oil from the territory of the Kolsky peninsula, having high speed of oil utilisation, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01142 and may be used for treatment of contaminated soils from oil.

EFFECT: improved quality of soil cleaning from oil.

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Description

The invention relates to the microbiological industry and relates to a new culture of microorganisms that destroy oil, and which can be used to clean contaminated soils.

A known strain of Rhodococcus erythropolis for the decomposition of oil and oil products (RF Patent 2257409, MKI C12N 1/20, B09C 1/10, publ. 2004). The strain is deposited as Rhodococcus erythropolis VKPM AC-1668. The strain is isolated from a sample of oil-contaminated soil of the Republic of Bashkortostan, grows on many natural and synthetic media and has a relatively high rate of utilization of oil and oil products.

It should be noted that a significant drawback of this strain is that this microorganism does not have the ability to biological oxidation of “heavy” and aromatic hydrocarbons and other types of harmful chemical compounds, such as alcohols, aldehydes, industrial oils, fats, etc.

A known consortium of strains of hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis IT BKM Ac-1769 and Pseudomonas fluorescens BKM B-6847 (RF Patent 2428471, C12N 1/26, C02F 3/34, B09C 1/10, publ. 2010), which are part of the ecobiopreparation -MMS ”, designed to clean hydrocarbon pollutants, primarily oil and fuels and lubricants, and to improve the efficiency of cleaning environmental objects from oil and oil products.

However, the disadvantage of this invention is that the cultivation of microorganisms of different taxonomic and species affiliation, as well as differing in growth rate, substrate specificity, temperature optimum growth leads to their competition and reduces their survival, and as a result complicates and makes the growing process more material and energy consuming.

The objective of the invention is to expand the arsenal of funds and obtain a new strain, characterized by a high rate of oil utilization.

To achieve this goal, the proposed strain Rhodococcus erythropolis 1-KP. The strain is deposited in the departmental collection of beneficial microorganisms for agricultural purposes of the Russian Academy of Agricultural Sciences (RCAM) under the number RCAM01142.

The strain Rhodococcus erythropolis 1-KP isolated from oil-contaminated podzolic soil of the Kola Peninsula.

The strain was isolated by culturing the initial sample with oil-contaminated soil in a liquid mineral medium with the addition of hydrocarbons, followed by sieving on meat peptone agar (MPA) to obtain isolated colonies.

The strain was identified based on a study of its cultural-morphological and physiological-biochemical characteristics in accordance with the description given in the Bergey bacterial identifier (Bergey's. Manual of Systematic Bacteriology. 1986. 8 ^th ed. V.2. Baltimore-Londone, William & Wilkins ), as well as based on the study of the 16S rRNA gene sequence.

Cultivation in the laboratory was carried out on nutrient media of the following composition:

1) MPA medium (sucrose - 5.0 g / l; glucose - 5.0 g / l; MPA - 50 g / l; agar-agar - 8.0 g / l), pH of the medium 7.3-7, 5 before sterilization.

2) Mineral environment with C ₁₄ -C ₁₇ to preserve the practical value of the culture:

(K ₂ HPO ₄ - 0.5 g / l; KH ₂ PO ₄ - 0.5 g / l; MgSO ₄ - 0.3 g / l; NH ₄ H ₂ PO ₄ - 1.0 g / l; peptone - 2.0 g / l; agar-agar - 15.0; after the medium is added to the tubes before sterilization, layered ~ 0.8 ml of the fraction of liquid hydrocarbons C ₁₄ -C ₁₇ ), the pH of the medium is 7.1-7.2 before sterilization .

Growing conditions: on MPA for 96 hours at 28 ° C, on a mineral medium with hydrocarbons 96-120 hours at 28 ° C.

The strain is stored at periodic reseeding on freshly prepared media once every 3 months.

According to morphological characteristics, the strain is characterized as a gram-positive bacillus with a size of (0.6-0.8) × (2.3-2.5) microns, does not form a spore, immobile.

This strain is characterized by the following cultural and morphological features. Colonies on meat-peptone agar when cultivated at T = 28 ° C are shiny, convex, smooth, beige-colored mucous membranes with a smooth edge. The size of the colonies does not exceed 5.0 mm three days after seeding. It is an aerobic. It grows in the temperature range from 4 to 35 ° C. The optimum temperature is 28 ° C. It grows at pH from 3.5 to 9.0, the optimal pH = 6.9.

The strain Rhodococcus erythropolis 1-KP is characterized by the following biochemical characteristics. Assimilates maltose, mannose, fructose, sucrose, glucose, hydrocarbons, ethyl alcohol, glycerin. Consumes acetic, propionic, butyric, citric, salicylic acid. Assimilates ammonia nitrogen, nitrate nitrogen. It does not require the addition of growth factors and vitamins to the growth medium. It does not dilute gelatin, starch does not hydrolyze. Acid formation with growth on sugars and hydrocarbons is weak. Indole and hydrogen sulfide does not form. Catalase positive. Resistant to cefazidine, polymyxin, cephalexin.

The practical application of this strain is determined by its physiological and biochemical characteristics. The strain is designed to eliminate the effects of oil pollution of the soil.

The following are examples of the biological oxidation of oil by the strain Rhodococcus erythropolis 1-KP.

The garden soil prepared for the experiments was packaged in 1.5 kg each in laboratory battery glasses with a capacity of 5 liters each. Oil pollution of the soil was carried out directly in glasses. Two oil samples were selected as the oil pollutant, the qualitative composition of which is given in Table 1. A portion of the oil pollutant was introduced into the soil by the gravimetric method and amounted to ~ 30 g, or ~ 2.0 wt.% To the soil. Glasses with oil-contaminated soil were kept in the laboratory with periodic loosening. This contributed to a more uniform distribution of the oil pollutant in the soil and, if possible, the evaporation of its light fractions.

In Tab. 1 presents the results of chemical and bituminological analysis of an average sample of soils contaminated with oil samples, wt.%. Oil 1 - naphthenic-aromatic heavy oil of the Russkoye field. Oil 2 - methane-naphthenic light oil of the West Tebuk field.

An experimental assessment of the ability of a strain of microorganisms to biodegrade oil was carried out in laboratory conditions. In prepared soil samples, pre-packaged 1.5 kg in laboratory glass beakers with a capacity of 5 liters each, and contaminated with oil and diesel fuel, a microorganism culture with a titer of 10 ⁵ -10 ⁸ cells / ml was introduced. The mother culture of bacteria Rhodococcus erythropolis 1-KP was grown on a shaker (200 rpm) at 28 ° C for 48-72 hours in flasks with 100 ml of one of the media of the following composition, wt.%:

1. Peptone - 1%

K ₂ HPO ₄ - 0.15%

MgSO ₄ - 0.15%

Glycerin - 1%

Tap water - the rest is up to 100%,

pH before sterilization of 7.6;

2. Meat-peptone broth with glucose (0.5 wt.%) And sucrose (0.5 wt.%).

pH before sterilization of 7.6.

An experimental assessment of the ability of a strain of microorganisms to biodegrade oil was carried out in laboratory conditions. Prepared soil samples, pre-packaged 1.5 kg in laboratory glass beakers with a capacity of 5 liters each, and contaminated with oil, introduced a culture of microorganisms with a titer of 10 ⁵ -10 ⁸ cells / ml. The mother culture of bacteria Rhodococcus erythropolis 1-KP was grown on a shaker (200 rpm) at 28 ° C for 48-72 hours in flasks with 100 ml of one of the media of the following composition, wt.%:

1. Peptone - 1%

K ₂ HPO ₄ - 0.15%

MgSO ₄ - 0.15%

Glycerin - 1%

Tap water - the rest is up to 100%,

pH before sterilization of 7.6;

2. Meat-peptone broth with glucose (0.5 wt.%) And sucrose (0.5 wt.%).

pH before sterilization of 7.6.

The experiments were carried out at room temperature (on average 18-20 ° C) for 91 days with periodic mixing and moistening as the soil dries. Humidification was carried out with sterile water; approximately every 3 weeks, moistening was carried out with a mineral medium containing K, P, N and having a pH of 7.1–7.2 before sterilization. The soil in the control experiments was moistened only with sterile water.

The number of living cells of microorganisms in the experiment was taken into account by the generally accepted method of limiting dilution on elective media.

Monitoring of the ongoing processes of cleaning oil-contaminated soil was carried out according to the following parameters:

- oil content, mg / kg.

- the content and qualitative composition of the chloroform extract.

The frequency of soil sampling for these types of analyzes was, as a rule, 2 weeks; the analysis was carried out according to GOST 17.1.4.01-80 and RD 52.18.575-96.

Table 2 shows the results of the monitoring - the content of petroleum products (NP), wt.% Of the initial content in the soil when cleaning the strain of pollution, the composition of which is given in Table. 1. Given in wt.% Relative to their initial content in oil-contaminated soil.

table 2 The content of oil products (NP) in contaminated soil during the cleaning process with strain Rhodococcus erythropolis 1-KP Pollutant The content of NP, wt.% Of the initial content in the soil Duration of cleaning, weeks 0 2 four 6 8 13 Oil products Oil 1 0,0 -13.7 -32.1 -47.2 -62.4 -62.5 Oil 2 0,0 -16.9 -20.1 -42.5 -65.0 -71.0

According to the table, for 13 weeks of the duration of the experiment, the content of oil products in contaminated soil samples decreases by 62.5-71.0% of their initial content. A decrease in the content of petroleum products in samples contaminated with oil is observed from 2 weeks from the start of the experiments.

Table 3 shows the content and qualitative composition of the CBA of oil-contaminated soil in the samples of Table 1 after 91 days from the beginning of the cleaning experiments with the strain Rhodococcus erythropolis 1-KP.

Data on the qualitative composition of CBA - (chloroform bitumen α) samples of contaminated soil before treatment, presented in Table 1, in comparison with data on experimental samples presented in Table 3, allow us to analyze the nature of changes in the group and hydrocarbon composition of chloroform bitumen α in the process biological oxidation by a strain of Rhodococcus erythropolis 1-KP.

According to the results of the analysis, the CBA content in the experimental samples of oil-contaminated soil ranges from 1.33-2.43% with the duration of the experiments 91 days. The qualitative composition of CBA in experimental soil samples has sharply changed, compared with the experienced composition of CBA. 91 days after the start of experiments on the disposal of oil pollution of the soil, a decrease in its most accessible fractions for microorganisms was noted. The most accessible for biooxidation in the composition of oil pollution is the fraction of oils. The oil content decreased to 38.55-42.25% rel.

Based on the data presented above, it follows that the strain Rhodococcus erythropolis 1-KP RCAM01142 is characterized by high efficiency in oil recovery processes, and as a result can be used for biological treatment of soils from these pollutants.

It should also be noted that the considered strain of Rhodococcus erythropolis 1-KP hydrocarbon-oxidizing microorganism can be used in the biological oxidation of petroleum products, such as diesel fuel. The basis for this conclusion is that the composition of diesel fuel and other petroleum products, as well as the composition of oils, includes various classes of hydrocarbons that undergo biological degradation by microorganisms.

Claims (1)

The strain Rhodococcus erythropolis, deposited in the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (RCAM) (GNU VNIISCHM) under registration number RCAM 01142 and used for the decomposition of oil.