Preparation for cleaning of soil from oil and oil products

FIELD: biotechnologies.

SUBSTANCE: preparation containing biodestructor of oil contamination represents centrate of cultural liquid of microbial mass of consortium of oil-oxidising microorganisms immobilised on peat carrier. Consortium composition includes the following: Rhodococcus eqvi SRI MCC B-1115 bacteria strain, Rhodotorula glutinis SRI MCC Y-1113 yeast and Rhodotorula glutinis ARSRI MCC Y-1114 yeast strain in the ratio of 1:1:1 respectively, which have been grown jointly.

EFFECT: invention allows improving quality of soil cleaning from oil and oil products under conditions with reduced temperatures.

2 tbl

 

The invention relates to the oil industry and ecology and can be used for cleaning oil-contaminated soil.

One of the most dangerous pollutants are oil and oil products, annual penetration of which into the environment is estimated at hundreds of millions of tons. In practice reclamation works when cleaning oil-contaminated soil using biological oxidizing action is not the last place.

Known drug-biodestruction oil pollution microbial-enzymatic Microsim(TT) "PETRO THREAT" (http://www.microzym.ru/oilspills.htm), producer LLC RSA trading, sanitary-epidemiological conclusion No. D from 11.03.2005. The drug represents a completely natural biological destructor petroleum hydrocarbons intended for environmentally safe cleaning soil and water bodies from oil pollution. As the active elements of the drug contains the consortium of (12) strains of living of hydrocarbon-oxidizing microorganisms in the form of concentrate dry spores concentration equal to 4×1012CFU/g, the set of natural microbial enzymes, mineral salts of nitrogen, potassium, phosphorus, organic feeding media from food corn flour, organic fertilizer.

The drug is intended for the biological is tion clean soil covers, sand, sludge, wastewater, water bodies and other non-aggressive environment from pollution by oil and oil products: gasoline, diesel fuel, fuel oil, motor oils, crude oil.

Disadvantages PETRO TRITA are:

- increasing production costs, as in the preparation contains more strains of microorganisms;

carrier PERTO TRITA food is corn flour - less accessible in remote Northern regions and therefore more expensive to import;

- the drug is not able to carry out the cleanup of oil and oil products in environmentally adverse conditions.

The basis for the production of biological products are strains of crops oil-oxidizing microorganisms. In most cases, the target product of the process of cultivation in the production technology of the bacterial preparation is microbial mass, and the remainder after division, spent culture liquid (centrate)is a waste of production. After centrifugation, the centrate is collected in special containers where subjected to heat treatment, after which it merges. Draining of supernatant entails environmental pollution and costs associated with environmental events. As centrate, enriched with biologically active substances, can be used for bi the remediation of contaminated oil and petroleum products land.

The task of the invention to provide a new drug capable of disposal of oil and oil products from the soil, when environmental conditions are unfavorable to the activity of microorganisms. Under no conditions implied low temperature, high humidity, Cromartie soil etc.

The technical result is to improve the quality of cleaning soils from oil and oil products in Arctic conditions, the possibilities for use in regions with low temperature environment, in contrast to microbial agents.

The technical result is achieved by the fact that the drug for cleaning soil from oil and oil products containing biodestruction oil pollution, according to the invention represents centrate cultural liquid microbial mass consortium of oil-oxidizing microorganisms immobilized on thoroseal, and the consortium is composed of: a bacterial strain Rhodococcus eqvi B-l 115, a strain of the yeast Rhodotorula glutinis Sri ECR Y-1114, a strain of yeast research Institute ECR Rhodotorula glutinis Y-1113, taken in the ratio 1:1:1, grown together.

Centrate, which is in the industrial production of microbial agents is a waste, is a culture fluid containing metabolic products of bacteria, including extracellular enzymes, vydeleny the environment in the process of growing microbial mass, which are resistant to adverse conditions compared to biomass without centrate. Immobilization centrate on thoroseal (adsorption mixing and drying of the supernatant with peat) creates additional resistance to inhibitors and is a condition for storage and transport of extracellular enzyme complex.

Due to resistance to adverse environmental factors (under unfavorable environmental factors include low temperature, high humidity, Cromartie soil etc.) extracellular enzymatic activity centrate allows the degradation of petroleum hydrocarbons in the soil, while for microbial mass, which is the basis of biological data on environmental factors is negative.

Strains of microorganisms obtained by extraction from oil-contaminated soil in a nutrient medium of čapek, of the following composition: sucrose 30 g, agar 20 g, NaNO3- 5,0 g, MgSO4- 0.5 g KCl, 0.5 g, KH2PO4to 3.0,

Strains of the yeast Rhodotorula glutinis 55-1-P, Rhodotorula glutinis u-CB # 26 and bacterial strain Rhodococcus eqvi 34-1(28-99/2) deposited in the research culture Collection of microorganisms" fsri SRC VB "Vector". Registration number: Y-1113, Y-1114, fill 15, respectively.

Source selection strain of the yeast Rhodotorula glutinis 55-1-P: oil-contaminated soil Usinsk district republics of the Komi vozeiskoe oil fields, 1999. Colony bright pink, convex, smooth, shiny slimy, smooth edge. Cells oval, 3-7 X4 - 15 μm, polar budding.

Source selection strain Rhodotorula glutinis u-CB # 26: oil-contaminated soil Usinsk district of the Komi Republic SLE. 203 Verkhnevizeiskogo oil field, 1995. Colony bright pink, convex, smooth, shiny slimy, smooth edge. Cells awalnya, 3-7 X 4-15 μm, polar budding.

The source selection of the bacterial strain Rhodococcus eqvi 34-1(28-99/2): oil-contaminated soil Usinsk and vozeiskoe deposits of the Republic of Komi, soil, fresh and old oil spill. Colonies on nutrient agar round, convex, up to 5 mm in diameter, smooth, shiny, cream-colored, with age - pale pink; cell life cycle - sticks-cocci.

The method of obtaining the supernatant includes:

- joint to improve the main cultures of microorganisms: Rhodotorula glutinis 55-1-P, Rhodotorula glutinis u-CB # 26, Rhodococcus eqvi 34-1(28-99/2), isolated from oil-contaminated soil in the flasks 100 ml Chapek's medium with addition of oil, within 3 days, in the ratio of 1:1:1;

- obtaining the supernatant by dividing jointly developed cultures of microorganisms: Rhodotorula glutinis 55-1-P, Rhodotorula glutinis u-CB # 26, Rhodococcus eqvi 34-1(28-99/2), by centrifuging for microbial mass and the supernatant 2,7 thousand revolutions per minute for 8-10 minutes (narab is determined jointly microorganism cultures spread across the tubes and separated in a centrifuge to sediment-microbial mass and the culture fluid is centrate);

- immobilization centrate on thoroseal (adsorption mixing of effluents and peat at a rate of 1 cm2× 100 cm2(15 g per 1 kg) with subsequent drying).

In the laboratory of the Institute was tested method of manufacturing the supernatant, comprising the following steps:

Step 1. Joint to improve the main cultures of microorganisms: Rhodotorula glutinis 65-1-P, Rhodotorula glutinis u-CB # 26, Rhodococcus eqvi 34-1(28-99/2), isolated from oil-contaminated soil in the flasks 100 ml Chapek's medium with addition of oil for 3 days:

culture of microorganisms have been made in liquid medium of čapek bacteriological loop in the ratio of 1:1:1 to 10 μl at room temperature under sterile conditions;

oil added to the flask as an additional power source in the amount of 0.5 ml;

- zarabatyvanie biomass of microorganism cultures were performed in temperature-controlled setting the microorganism growing WVMT-12-250 at a temperature of 30°C and 180 rpm for 3 days.

Step 2. Obtaining the supernatant by centrifugation of the culture fluid with microbial mass:

- jointly developed within 3 days of the microbial biomass was poured into centrifuge tubes were placed in a centrifuge (the COUNTRY CLMN-P10-02 and perform the separation of the biomass at 2.7 thousand rpm for 8-10 minutes on centrate and microbial mass;

the separation of supernatant (supernatant in the centrifuge tube) from microbial biomass (residue in the centrifuge tube) was performed in a sterile box by draining the supernatant into a sterile tube. At the bottom of the centrifuge tube remained settled cells microbial mass.

Step 3. Immobilization of supernatant adsorption binding thoroseal:

- Centrate was infused into peat, with constant stirring at a rate of 1 cm2× 100 cm2(15 g of supernatant for 1 kg of peat). Characteristics of peat are presented in table 1:

Table 1
Agrochemical characteristics of peat
pHP2About5(mg/100 gK2O, mg/100 gCA, mg/mEq 100 gMg, mg/mEq 100 gN%S with%
peat2.822.4±4.529.5±4.451.3±3.86.61±0.50.9±0.1415.8±1.6

- Drying of peat took place at room temperature T=21-23°C to air-dry state.

In the laboratory trials were conducted made of supernatant, immobilized on thoroseal for the optimization process, the money is Adachi oil contaminated soil substrates. Immobilized on peat centrate was made in the soil substrates, we determined the degree of degradation of petroleum hydrocarbons after certain intervals of time. Used centrate composition of microorganisms Rhodotorula glutinis, Rhodococcus erythropolis, Rhodococcus eqvi, taken in the ratio 1:1:1, jointly developed. For comparison felt appropriate microbial mass. As objects of study used sand, clay and peat substrates, the oil in concentrations of 50 and 150 mg/year Experienced substrates were contaminated with oil.

On contaminated substrates (table 2) were made centrate immobilized on the peat at a rate of 1 cm × 100 cm2(15 g/kg), and (for comparison) microbial mass with a titer of cells in the working solution of 1.2·109(the solution was brought to 5 ml/kg of air-dry soil). Additionally made of mineral fertilizer - ASFC (N16P16K16) (350 kg/ha (35 g/1000 cm2in aqueous solution, NMat the rate of 100 kg/ha (10 g/1000 cm2). Once samples were taken for tests: enzyme activity (catalase, dehydrogenase, lipase), the intensity of CO2residual oil content in the soil. Further analysis samples were taken 3 days after the start of the experiment, 15 days, 30 days and 6 months. The research results presented in table 1, taken from the source and destination parameters.

The action of lipases aimed at splitting of ester bonds between glycerol and high molecular weight fatty acids with formation of free fatty acids, di - and monoglycerides and glycerol. Change lipase activity is associated with the accumulation of low-and non-degradable biological substances formed during biodegradation of crude oil, but also from the fact that in the degradation of lipids participate enzyme system, very similar to the systems of oil biodegradation.

In self-cleaning oil-contaminated substrates leads to a minor change in the enzymatic activity by the end of the experiment. Basically there is a decrease in catalase, dehydrogenase activity and lipase activity. The introduction of oil-contaminated substrates as centrate with mineral fertilizers and microbial mass increases biochemical processes. In clayey and sandy substrates increased respiratory activity in the variants with the introduction of supernatant.

Table 1 shows the experimental results and comparative analysis. Adopted the following conventions - Control - soil, uncontaminated oil and neopentanoate and microbial mass, 1 - oily soil without treatment of the centrate and microbial mass, 2 - contaminated soil + centrate on thoroseal and mineral fertilizer, 3 - contaminated soil + microbial mass and mineral fertilizer, I - raw data (enzymatic activity, the intensity of selection WITH2in the beginning of the experiment), II - final data (enzymatic activity, the intensity of selection WITH2after 6 months of production experience).

As can be seen from table 1, by the end of the experiment the substrates is a noticeable reduction in the level of oil pollution (10-40%) as with the introduction of supernatant, and with the introduction of microbial mass.

Sandy substrate unlike clay and peat is characterized by low biological activity, low content of biogenic elements, i.e. the most environmentally unfavorable for purification from oil products. The introduction of oil increases lipolytic processes and dehydrogenation processes, but the degree of purification in the polluted substrate 6 months low. Introduction microbial mass or centrate improves biochemical activity. When making centrate amplifies the intensity of the allocation of CO2. The greatest degree of cleaning in sandy substrate in the variant with the introduction of supernatant.

Thus, due to resistance to adverse environmental the practical factors of biodestruction oil pollution can enhance the degradation of oil in soil at high oil load and at low ambient temperature, even in situations where the activity of microbial destruction of oil is minimized by these factors. The drug allows you to change the composition and structure of oil pollution and make it more accessible to microbial attack. Competitive advantages of the product is the availability and cheapness in manufacture, based on the use of waste products and is available for Northern conditions thoroseal.

Preparation for cleaning soil from oil and oil products containing biodestruction oil pollution, representing centrate cultural liquid microbial mass consortium of oil-oxidizing microorganisms immobilized on thoroseal, and the consortium is composed of: a bacterial strain Rhodococcus eqvi Institute of PFC In-1115, the strain of yeast Rhodotorula glutinis Sri ECR Y-1114 and the strain of yeast Rhodotorula glutinis Sri ECR Y-1113, taken in the ratio 1:1:1, respectively, grown together.



 

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