RU2489483C1 - STRAIN Rhodococcus fascians, USED FOR OIL DECOMPOSITION - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus fascians 4-G, extracted from soil contaminated with black oil and sampled from the territory of a boiler plant located in the settlement Gorelovo, Leningrad region, 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 RCAM01140 and may be used for treatment of contaminated soils from oil.

EFFECT: invention makes it possible to increase quality of soil treatment 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, a strain of Rhodococcus fascians (4-G), isolated from fuel oil-contaminated soil taken from the boiler house located in the village of Gorelovo, Leningrad Region, was proposed. 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 RCAM01140.

The strain was isolated by culturing the initial sample of soil contaminated with fuel oil 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 medium with C ₁₄ -C ₁₇ to maintain 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, layering ~ 0.8 ml of the C ₁₄ liquid hydrocarbon fraction -C ₁₇ ), pH 7.1-7.2 before sterilization.

Growing conditions: on the MPA medium 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 rod with rounded ends with a size of (0.6-0.7) × (1-1.5) microns, does not form a spore, immobile.

The strain Rhodococcus fascians 4-G is characterized by the following cultural and morphological features. The colonies are bright orange, round, opaque, convex, small with smooth edges, grows at 37 ° C, grows at pH 5-6, grows in the presence of 1-4% NaCl, 1% sodium lactate, in the presence of LiCl, NaBr, K ₂ TeO ₃ , has urease. Strict aerob. The Voges-Proskauer reaction is negative.

This strain is characterized by the following biochemical characteristics. It utilizes stachyose, D-raffinose, α-D-lactose, D-melibiosis, D-fructose, D-galactose, D-serine, D-sorbitol, D-mannitol, D-arabitol, glycerin, pectin, D-gluconic acid, quinic acid, D-sugar acid, L-lactic acid, citric acid, L-malic acid, bromosuccinic acid, β-hydroxy-D, L-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, acetic acid, tween-40, is sensitive to aztreonam.

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 a strain of Rhodococcus fascians 4-G.

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. As a pollutant, naphthenic-aromatic oil was selected, 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.

Table 1 presents the results of chemical and bituminological analysis of the average sample of soils contaminated with oil, wt.%. Oil is the naphthenic-aromatic heavy oil of the Russkoye field.

An experimental assessment of the ability of a strain of microorganisms to biodegrade oil and diesel fuel 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 fascians 4-G 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 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 (an average of 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 with a 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 fascians 4-G 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 0,0 -27.3 -40.0 -48.3 -57.7 -60.5

According to the data in Table 2, over 13 weeks of the duration of the experiment, the content of oil products in contaminated soil samples is reduced by 60.5% 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 fascians 4-G.

Data on the qualitative composition of CBA - (chloroform bitumen α) samples of contaminated soil before treatment, are presented in Table 1, in comparison with the data on the experimental samples presented in Table. 3, allow us to analyze the nature of changes in the group and hydrocarbon composition of the chloroform bitumen α in the course of biological oxidation by the strain Rhodococcus fascians 4-G.

According to the results of the analysis, the CBA content in the experimental samples of oil-contaminated soil is 1.92% with a test duration of 91 days. The qualitative composition of the CBA of the experimental soil sample has sharply changed, compared with the experienced composition of the 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 42.50% rel.

Based on the above data, it follows that the strain Rhodococcus fascians 4-G RCAM01140 is characterized by high efficiency in the processes of oil utilization, and as a result can be used for biological treatment of soils from this pollutant.

It should also be noted that the considered strain of Rhodococcus fascians 4-G RCAM01140 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 fascians, deposited in the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (RCAM) (GNU VNIISCHM) under registration number RCAM 01140 and used for the decomposition of oil.