RU2489482C1 - STRAIN Pseudomonas aeruginosa RCAM01139 FOR DECOMPOSITION OF OIL AND DIESEL FUEL - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Pseudomonas aeruginosa RCAM01139 is proposed for decomposition of oil and diesel fuel.

EFFECT: strain is characterised by high efficiency in processes of oil and diesel fuel utilisation, which are the only sources of carbon and energy.

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Description

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

A known consortium of strains of hydrocarbon-oxidizing bacteria Pseudomonas aeruginosa ND K3-1 and Pseudomonas fluorescens ND K3-2 (RF Patent No. 2312719, C12N 1/26, B09C 1/10, publ. 2007), which is isolated from samples of soil and groundwater of karst cavities, selected in the Perm region. and used for the destruction of oil and oil products.

It should be noted that the cultivation of a mixture of cultures, in this case a consortium of two strains, is possible at high material and energy costs, in comparison with the growth of biomass, consisting of one culture of microorganism cells.

A known bacterial strain Pseudomonas aeruginosa XP-25 (RF Patent No. 2270806, C12N 1/20, C02F 3/34, C12R 1/385, publ. 2006), which was isolated from phenol-contaminated soils of the Sterlitamak petrochemical plant by culturing a soil sample in a mineralized medium followed by the selection of the most active forms, and used for wastewater treatment of chemical plants from aromatic compounds.

From the data presented in the patent, it follows that the spectrum of action of the strain Pseudomonas aeruginosa XP-25 is aimed at the disposal of aromatic compounds, in particular phenol, and does not apply to other "heavy" fractions of hydrocarbons, as well as ecotoxicants, such as alcohols, aldehydes, industrial oils, fats, etc.

The objective of the invention is to expand the arsenal of funds and obtain a new strain, characterized by a high rate of utilization of oil and diesel fuel, which are the only sources of carbon and energy.

To achieve this objective, the proposed strain Pseudomonas aeruginosa 12-P, isolated from a sample of oil-contaminated soil at the Ruchi oil depot, St. Petersburg. 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 RCAM01139.

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) King B medium (glycerin - 10.0 g / l; peptone - 20.0 g / l; K ₂ HPO ₄ - 1.5 g / l; MgSO ₄ - 1.5 g / l; agar-agar - 15.0 g / l), pH ~ 7.6 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 King B - 72 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-negative straight stick with rounded ends, size (0.5-0.7) × (1-3) microns, is well stained with all aniline dyes. In smears is located singly, in pairs or short chains. It is mobile, has one flagellum. It does not form a dispute, it does not have a capsule, but it produces mucus, which surrounds a microbial cell with a thin layer.

This strain is characterized by the following cultural and morphological features. Milk-colored colonies, round, convex with even edges, on King B medium (peptone - 20 g / l, K ₂ HPO ₄ - 1.5 g / l, MgSO ₄ × 7H ₂ O - 1.5 g / l, glycerin - 10 g / l) gives a yellow-green pigmentation. Pseudomonas aeruginosa 12-P is an aerobic. Hemoorganotroph grows in wide temperature ranges from 6 to 45 ° C, pH range from 4.5 to 9.0. The optimum growth temperature is 37 ° C, pH 7.2-7.5, but also grows well at a temperature of 42 ° C. It is undemanding to nutrient media, grows well on MPA and meat and peptone broth (hereinafter - MPB). On the MPA, after a day, fairly large (3-5 mm) translucent milky-colored colonies with a pearlescent shade are formed. The center of the colony is darker than the periphery, the edges are even, clear. On mowed agar, a Pseudomonas aeruginosa 12-P culture produces a thin, shiny coating. Its characteristic property is the appearance by the end of the first day of blue-green staining of the culture with the subsequent penetration of the pigment (pyocyanin) into the nutrient medium.

This strain is characterized by the following biochemical characteristics. Pseudomonas aeruginosa 12-P has weak saccharolytic activity: it breaks down only glucose to form acid without gas. Proteolytic activity is more pronounced: it dilutes gelatin and clotted blood serum, hydrolyzes casein. Coagulates litmus milk and then cleaves a clot. Restores nitrates to nitrites and further to nitrogen. It does not form indole and H ₂ S, it gives a negative Voges-Proskauer reaction. The test for oxidase is positive. Disposes of 2-ketogluconate.

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

The following are examples of the biological oxidation of oil and diesel fuel from contaminated soil with the Pseudomonas aeruginosa 12-P strain.

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. Soil pollution with oil and oil products was carried out directly in glasses. Diesel fuel and two oil samples, the qualitative composition of which are given in Table 1, were chosen as the oil pollutant. 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 and diesel fuel, 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 and diesel fuel was carried out in laboratory conditions. Microorganisms with a titer of 105-108 cells / ml were introduced into the 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 mother culture of bacteria Pseudomonas aeruginosa 12-P 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 NRA 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 (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 dilutions 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 Pseudomonas aeruginosa 12-P strain of contaminants, 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 Pseudomonas aeruginosa 12-P 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 Diesel fuel 0,0 +2.1 +2.4 -5.4 -41.5 -67.8 Oil 1 0,0 -22.4 -36.6 -47.0 -57.4 -62.1 Oil 2 0,0 -15.7 -26.6 -42.8 -59.0 -63.4

According to the data in Table 2. for 13 weeks of the duration of the experiments, the content of petroleum products in oil-contaminated soil samples decreases by 62.1-67.8% of their initial content. A decrease in the content of petroleum products in samples contaminated with diesel fuel is observed from 6 weeks, and in samples contaminated with oil, from 2 weeks from the start of the experiments. It is noteworthy that the oxidation of petroleum products in diesel fuel was observed only after 6 weeks of continuing experiments. The explanation for this can be the active oxidation of diesel fuel by hydrocarbon-oxidizing microorganisms with the formation of intermediate products. This is supported by a high titer of hydrocarbon-oxidizing microorganisms in soil contaminated with diesel fuel.

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 Pseudomonas aeruginosa 12-P.

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 Pseudomonas aeruginosa 12-P.

According to the results of the analysis, the CBA content in the experimental samples of oil-contaminated soil ranges from 1.38-1.84% with a test duration of 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 40.43% rel. As a part of this pollution, the content of asphaltenes, methane-naphthenic, mono- and biaromatic hydrocarbons decreased.

Based on the above data, it follows that the strain Pseudomonas aeruginosa 12-P RCAM01139 is highly effective in the processes of oil and diesel fuel utilization, and, as a result, can be used for biological treatment of soils from these pollutants.

Claims (1)

Strain Pseudomonas aeruginosa RCAM01139 for the decomposition of oil and diesel fuel.