Strain pseudomonas citronellolis, used for decomposition of oil and diesel fuel

FIELD: biotechnologies.

SUBSTANCE: strain pseudomonas citronellolis 48-U, having high speed of oil and diesel fuel recycling, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VIISHM) under the registration number RCAM 01441 and may be used for treatment of contaminated soils from oil and diesel fuel.

EFFECT: improved quality of soil cleaning from oil and diesel fuel.

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 the 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 XP-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 the dryer is La, 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 citronellolis 48, isolated from sediment of a water reservoir located in the ravine Bear under the warehouse lubricants. The Ulyanovsk region. The strain is deposited in the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM) number RCAM01141.

The selection of the strain was carried out by culturing the original sample contaminated oil sludge reservoir 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 placentas the activity of genes 16S rRNA.

Cultivation in laboratory 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 with14-C17to preserve the practical value of culture: (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 hydrocarbons 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 a gram-negative Bacillus with rounded ends measuring 0.5×(1-1,5) microns, movable with one polar flagellum, does not form spores, capsules has not.

The strain Pseudomonas citronellolis 48-culture is characterized by the following morphological features. Colony bright, milky white, transparent, round, convex, with smooth edges, on the environment, king B (peptone 20 g/l, K2HPO41.5 g/l, MgSO4×7H2O - 1.5 g/l, Glazer the n - 10 g/l) gives a yellow-green pigmentation, grows at pH 5-6, grows in propstei 1-4% NaCl, 1% sodium lactate, guanidine HCl, in the presence of K2TeO3, sodium butyrate. Obligate aerobe (anaerobic growth in the presence of nitrate), optimum temperature for growth is 30°C. Can grow at 41°C.

This strain is characterized by the following biochemical characteristics. Gives a positive reaction with tetradecyl sodium sulfate and tetrazolium purple, disposes of α-D-glucose, D-fucose, guideway acid, glycerin, glycyl-L-Proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, L-pyroglutamic acid, D-gluconic acid, amide glucuronic acid chinayou acid, β-hydroxyphenylarsonic acid, L-lactic acid, citric acid, L-malic acid, D-malic acid, α-keto-glutaric acid, β-hydroxy-D,L-butyric acid, γ-aminobutyric acid, propionic acid, acetic acid, is sensitive to nalidixic acid, aztreonam, troleandomycin, rapamycin SV, lincomycin, vancomycin and minocycline.

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 whom whom oxidation of oil and diesel fuel contaminated soil strain of Pseudomonas citronellolis 48-U.

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 - naphthenic-aromatic heavy oil field "Russian".

Table 1
The qualitative composition of the original oil pollutants and chloroform extracts (HBA) soil samples prepared for the experiments to study the effectiveness of the treatment
№ p/pClicks the set of technical documents Group composition HBA, wt.%Hydrocarbon composition of oils, weight%
oilbenzene resinspiropentane
nye resin
asphaltenesmethane-naphthenic fractionmonoaromatic hydrocarbonsMicromatics
such hydrocarbons
birth
Polyaromatic
ical hydrocarbons
1The original diesel fuel89,023,105,732,1581,8710,51a 7.62-
2The chloroform bitumoids α, soil contaminated with diesel fuel88,571,506,933,0073,4419,493,004,07
3Source what I oil 71,9517,898,941,2264,5218,3814,84of 2.26
4The chloroform bitumoids α, soil contaminated with oil73,3111,5511,953,1956,3517,9625,69-

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 titer of 105-108cells/ml Uterine culture bacteria Pseudomonas citronellolis 48-Have been grown in 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%

K2HPO4- 0,15%

MgSO4- 0,15%

Glycerol - 1%

Tap water - the rest is up to 100%

pH before sterilization 7,6

2. Masop penny broth with glucose (0.5%) and sucrose (0.5 percent).

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, P, 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 was conducted in the conventional method of limiting dilution 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 strain of Pseudomonas citronellolis 48-contamination, the composition of which is summarized in table 1. Given in wt.% relative to their original content in oil-contaminated soil.

Oil content (NP) in contaminated soil in the cleaning process strain of Pseudomonas citronellolis 48-
Table 2
PollutantThe content of the NP, wt.% from the initial content in the soil
The duration of treatment, weeks
0246813
Oil
Diesel fuel0,0+7,7+5,7-15,6-46,3-72,2
Oil0,0-19,1-36,3-48,1-59,9-60,3

According to Table 2, for 13 weeks duration experiments of oil content in oil-contaminated soil samples decreased 60.3-72,2% of the original content. The decrease in the content of oil products in samples contaminated with diesel fuel the m 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 hydrocarbon oxidizing bacteria 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 citronellolis 48-U.

Data quality HBA - (of chloroform bitumen (a) samples of contaminated soil prior to cleaning, are 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 and in the process of biological oxidation strain of Pseudomonas citronellolis 48-U.

According to the results of the analysis, the content of HBA in experimental models of oil-contaminated soil 2,69% when the duration of the experiments 91 days. Has changed dramatically and qualitative composition of HBA samples of soil, compared with doopity composition X is A. 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 42,71-70,12% Rel.

Based on the data presented above it follows that the strain Pseudomonas citronellolis 48-RCAM 01141 characterized by high efficiency in the processes of decomposition of oil and diesel fuel and, consequently, can be used for biological treatment of soils from these pollutants.

The strain Pseudomonas citronellolis, deposited in the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM) (GNUS ARRIAM) under registration number RCAM 01441 and used for the decomposition of oil and diesel fuel.



 

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