Strain pseudomonas aeruginosa rcam01139 for decomposition of oil and diesel fuel

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.

3 tbl

 

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

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, VC 1/10, publ. 2007), which was isolated from samples of soil and groundwater karst cavities, selected on the territory of Perm region and used for the destruction of oil and petroleum products.

It should be noted that growing a mixture of crops, in this case a consortium of two strains, possibly with high material and energy consumption, in comparison with increasing biomass, single cell culture of microorganisms.

Known bacterial strain Pseudomonas aeruginosa CHR-25 (RF Patent No. 2270806, C12N 1/20, C02F 3/34, C12R 1/385, publ. 2006), which was isolated from penasaran soil Sterlitamak petrochemical plant by cultivation of the soil sample in a saline environment with subsequent selection of the most active forms, and used for wastewater treatment chemical companies from aromatic compounds.

Presented in the patent data, it follows that the spectrum of the strain Pseudomonas aeruginosa XP-25 is directed to the utilization of aromatic compounds, in particular phenol is, and does not apply to other heavy hydrocarbon fractions and toxicants, such as alcohols, aldehydes, technical oils, fats and other

The objective of the invention is the expansion of the means and obtaining a new strain, characterized by a high rate of utilization of oil and diesel fuel, which are the sole sources of carbon and energy.

To achieve the objectives proposed by the strain Pseudomonas aeruginosa 12-R, selected from a sample contaminated soil tank farm "Streams", St. Petersburg. The strain is deposited in the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM) number RCAM01139.

The selection of the strain was carried out by culturing the original sample is contaminated soil in liquid mineral medium with the addition of hydrocarbons with subsequent sieving on mastopathy agar (MPA) to obtain isolated colonies.

Identification of the strain was carried out on the basis of a study of its cultural-morphological and physiological-biochemical characteristics in accordance with the description given by the determinant of bacteria Bergey (Bergey's. Manual of Systematic Bacteriology. 1986. 8thed V.2. Baltimore-Londone, William & Wilkins), as well as on the basis of a study of the sequence of 16S rRNA genes.

Cultivation in the laboratory is Torno conditions was carried out on nutrient medium of the following composition:

1) Environment king B (glycerol - 10.0 g/l; peptone - 20,0 g/l; K2HPO41.5 g/l; MgSO41.5 g/l; agar-agar - 15.0 g/l), pH ~7.6 to sterilization.

2) Mineral medium with C14-C17for practical conservation of cultural values:

(K2HPO4- 0.5 g/l; KH2PO4- 0.5 g/l; MgSO40.3 g/l; NH4H2PO4- 1.0 g/l; peptone - 2.0 g/l; agar-agar - 15,0; after editing environment in a test tube prior to sterilization naslovom ~0.8 ml fractions of liquid hydrocarbons From14-C17), pH of 7.1 to 7.2 before sterilization.

Growing conditions: medium king B - 72 hours at 28°C in mineral medium with hydrocarbon - 96-120 hours at 28°C.

Storage of strain produced by periodic subculture to fresh medium 1 time per 3 months.

The morphological characteristics of the strain is characterized as gram-negative straight rod with rounded ends, the size of 0.5-0.7)×(1-3 microns), it is well painted all aniline dyes. In smears is single, in pairs or short chains. Mobile has a single flagellum. The dispute does not form, capsules no, but produces mucus, which is a thin layer surrounds the microbial cell.

This strain is characterized by the following cultural-morphological features. Colony milky white, round, convex, with smooth edges, on the environment, king B (peptone 20 g/l, Ksub> 2HPO41.5 g/l, MgSO4×7H2O - 1.5 g/l, glycerol 10 g/l) gives a yellow-green pigmentation. Pseudomonas aeruginosa 12-R is an aerobe. Chemoorganotrophs, grows in a wide temperature range from 6 to 45°C, a pH range from 4.5 to 9.0. Optimum temperature for growth is 37°C, pH of 7.2-7.5, but also grows well at a temperature of 42°C. To nutrient environments undemanding, grows well in MPA and mesopatamia broth (hereinafter - BCH). On the MPA through the day formed a fairly large (3-5 mm) translucent colonies milky color with a pearl shade. The center of the colony darker than the periphery, edges smooth, clear. On the sloped agar culture of Pseudomonas aeruginosa 12-R gives a subtle shiny plaque. A characteristic property of an appearance by the end of the first day of blue-green staining of the cultures with subsequent penetration of the pigment (pyocyanin) in a nutrient medium.

This strain is characterized by the following biochemical characteristics. Pseudomonas aeruginosa 12-R has weak sacharolytica activity: splits only the glucose with the formation of acid without gas. More pronounced proteolytic activity: liquefies gelatin and a collapsed blood serum, hydrolyzes casein. Roll litmus milk and then breaks down the clot. Restores the nitrate to nitrite and then to nitrogen. Does not form indole and H2S, gives neg the optimum reaction of Voges-Proskauer. Test for oxidase positive. Utilizes 2-ketogluconic.

The practical application of this strain is due to its physiological and biochemical characteristics. The strain is designed to eliminate the effects of soil contamination with oil and diesel fuel.

Below are examples of biological oxidation of oil and diesel fuel contaminated soil strain of Pseudomonas aeruginosa 12-R.

Prepared for the experiments of garden soil was put up to 1.5 kg in laboratory battery glasses with a capacity of 5 litres each. Soil pollution by oil and oil products was performed directly in the glasses. As oil pollutants were selected diesel fuel and two samples of oil, the qualitative composition of which is given in table 1. Linkage of oil pollutants in the ground made by the gravimetric method and was ~30 g, or ~2.0 wt% on the soil. Glasses with oil-contaminated soil was maintained in laboratory conditions with periodic loosening. This contributed to a more uniform distribution of oil pollutants in the soil and, if possible, the evaporation of its light fractions.

In table 1 presents the results of chemical bitumen analysis of an average sample of soil contaminated by oil and diesel fuel, wt.%. Oil 1 - naphthenic-aromatic heavy oil field "Russian". Oil 2 methane-naphthenic light oil field "Western Tabuk".

Experimental evaluation of the ability of microorganisms to biodegradeable oil and diesel fuel were carried out in laboratory conditions. In the prepared soil samples, pre-packaged in 1.5 kg in laboratory battery glasses with a capacity of 5 liters each, and contaminated oil and diesel fuel have made the culture of microorganisms with a title 105-108 cells/ml Uterine culture bacteria Pseudomonas aeruginosa 12-R were 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%

To2NRA4- 0,15%

MgSO4- 0,15%

Glycerol - 1%

Tap water - the rest is up to 100%

pH before sterilization 7,6

2. Mastopathy broth with glucose (0.5%wt.) and sucrose (0.5 weight %). pH before sterilization 7,6.

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

Accounting for the number of living cells of microorganisms in the experiment were conducted common method of limiting the spy who s on elective environments.

Monitoring of ongoing processes of purification of 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 sampling soil on these kinds of analyses was typically 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 of the content of petroleum products (NP), wt.% from the initial content in the soil when cleaning the strain Pseudomonas aeruginosa 12-P impurities, the composition of which is summarized in table 1. Given in wt.% relative to their original content in oil-contaminated soil.

Table 2
Oil content (NP) in contaminated soil in the cleaning process strain of Pseudomonas aeruginosa 12-P
PollutantThe content of the NP, wt.% from the initial content in the soil
The duration of treatment, weeks
0246813
Oil
Diesel fuel0,0+2,1+2,4-5,4-41,5-67,8
Oil 10,0-22,4-36,6-47,0-57,4-62,1
Oil 20,0-15,7-26,6-42,8-59,0-63,4

According to Table 2. for 13 weeks duration experiments of oil content in oil-contaminated soil samples is reduced by 62,1 and 67.8% of the original content. The decrease in the content of oil products in samples contaminated with diesel fuel, observed from 6 weeks, and in samples contaminated with oil, with 2 weeks from the beginning of the experiments. Noteworthy that the oxidation of the oil in diesel fuel was observed only after 6 weeks of continuing the experiments. The explanation for this can be the active oxidation of diesel fuel hydrocarbons is kislowski microorganisms with the formation of intermediate products. This is also evidenced by the high titer of hydrocarbon-oxidizing microorganisms in soil contaminated with diesel fuel.

In Table 3 shows the content and qualitative composition of HBA oil-contaminated soil samples Table 1 through 91 days from the beginning of the experiments cleanup strain of Pseudomonas aeruginosa 12-P.

Data quality HBA - (chloroform of bitumen-α) samples of contaminated soil prior to cleaning, is presented in table 1, in comparison with experimental samples, are presented in Table. 3, allow the analysis of changes in group and hydrocarbon composition of chloroform bitumen-α in the process of biological oxidation strain of Pseudomonas aeruginosa 12-R.

According to the results of the analysis of the content of HBA in experimental models of oil-contaminated soil varies in the range of 1.38-1.84 per cent during the duration of the experiments 91 days. Has changed dramatically and qualitative composition of HBA samples of soil, compared with doopity composition of HBA. After 91 days after the start of the experiments on the utilization of oil-contaminated soils has decreased the most accessible for microorganisms its fractions. The most available for the oxidation process in the composition of oil pollution is the fraction oils. The oil content decreased to 40,43% Rel. Part of this pollution has decreased content of the asphalt is new, methane-naphthenic mono - and barometrically hydrocarbons.

Based on the data presented above, it follows that the strain Pseudomonas aeruginosa 12-R RCAM01139 characterized by high efficiency in the recycling processes of oil and diesel fuel, and, consequently, can be used for biological treatment of soils from these pollutants.

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



 

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1 tbl, 2 ex

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3 tbl, 4 ex

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4 tbl, 3 ex

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3 tbl

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