RU2307869C2 - Method of biological restoration of oil-polluted land - Google Patents

Abstract

FIELD: oil pollution elimination.

SUBSTANCE: invention relates to biotechnology, soil science, and environmental protection and provides a method for biological restoration of oil-polluted land under extreme north conditions. Method consists in preliminary liming, introducing mineral fertilizers, and treating polluted areas with oil-destroying biological preparation containing (i) microbial fertilizer-substrate in the form of fermented agricultural production wastes and (ii) bacterial culture immobilized on the above substrate, after which sowing and harrowing are performed. Treatment is executed with dry preparation once in the season during warm period in dose 100 g/m² using as bacterial culture strain Bacillus cereus 3 K with titer 10¹² cells/g substrate. Biopreparation is spread during or before sowing followed by harrowing to a depth of 5-10 cm.

EFFECT: simplified restoration procedure, improved soil restoration processes, and shortened restoration time.

3 tbl, 3 ex

Description

The invention relates to soil science, land use and ecology, and in particular to a method for carrying out biological reclamation of oil-contaminated soil using biological preparations-oil destructors based on microbial fertilizers (fermented agricultural waste) and based on microorganisms-destructors (bacteria of the genus Bacillus), and is used to clean contaminated oil and oil products of the soil while restoring its physico-chemical properties and natural biocenosis.

Bacterial cultures of p. Bacillus are known, which are characterized by the ability to produce antibiotic substances and inhibit the growth of phytopathogenic microorganisms, see Junge H., Krebs S., Frier K. // Mitt.Biol. Land - und Frostwirt. Berlin-Dahlem. - 1990. - No. 226.

Bacterial fertilizer is known, including a humic-containing base and a bacterial supplement from the group of bacteria: Bacillus subtilis, Bacillus cereus, Bacillus polymyxa, RU No. 2186049 C2, C05F 11/08, C12N 1/20, C12R 1/07, 2002.07.27. In the bacterial additive used cultures of bacteria of the strain Bacillus cereus, strain 138D in an amount of 5.0 · 10 ⁶ cells / g basis.

Known bacterial strains used to clean the soil and water from oil pollution, in particular Bacillus sp., RU No. 2083667 C1, C12N 1/20, B09C 1/10, C02F 3/34, 1997.07.10; RU No. 21115629 C1, C02F 3/34, B09C 1/10, 1998.07.20; RU No. 2182529 C1, B09C 1/10, C02F 3/34, C12N 1/20, C12N 1/26, 2002.05.20; RU No. 2204597 C2, C12N 1/20, C02F 3/34, B09C 1/10, C12N 1/20, 2003.05.20.

A known method of cleaning the soil from contamination by oil and oil products, including the introduction into peat culture of microorganisms and mineral additives containing nitrogen and phosphorus, RU No. 2137559 C1, B09C 1/10, B09C 1/08, 1999.09.20.

A known method of cleaning the soil from oil and oil products, including the introduction of a bacterial culture in a mixture with fertilizer, RU No. 2041172 C1, C02F 3/34, EW 15/04, C12N 1/20, 1995.08.09.

A known method of purifying water and soil from oil pollution, including the introduction of biomass of oil-oxidizing bacteria and fertilizers, RU No. 2039714 C2, C02F 3/34, C12N 1/20, 1995.07.20.

There is a method of reclamation of soil contaminated with petroleum products by adding additives in the form of activated sludge to biological treatment plants, RU No. 2183142 C2, B09C 1/10, 2002.06.10.

A known method of reclamation of soils contaminated with oil and oil products by treating the soil with a bacterial preparation based on a natural strain of bacteria of the genus Bacillus, RU No. 2077397 C1, B09C 1/10, C09K 3/32, C12N 1/20, 1997.04.20.

All known methods require long periods of reclamation, and the biological products used have a low destructive ability.

A known method of cleaning oil-contaminated soils, including liming, fertilizing, treatment of contaminated areas with a biological product-oil destructor containing microbial fertilizer-carrier in the form of fermented agricultural waste and bacteria culture, immobilized on a carrier, seed application and harrowing, Arkhipchenko I.A., Zagvozdkin V.K. Purification of oil-contaminated soils using biological products based on microbial fertilizers // Ecology and Industry of Russia. Special issue. 2004. S. 16-18.

This technical solution is made as the closest analogue of the present invention.

However, it has a disadvantage.

The oil-destroying activity of the applied microbial fertilizers is insufficient to conduct biological reclamation in areas with a short thermal period.

The present invention is based on the solution of a technical problem, which makes it possible to increase the adaptability of biological reclamation and to reduce the time of reclamation of oil-contaminated soils in the Far North, through the use of a biological product-oil destructor of complex action:

- with high oil destructive activity,

- with the positive properties of mineral and organic fertilizers.

According to the invention, this problem is solved due to the fact that the method of biological reclamation of oil-contaminated soil in the natural conditions of the Far North includes liming, applying mineral fertilizers, treating contaminated areas with a biological product-oil destructor containing microbial fertilizer-carrier in the form of fermented agricultural waste and a bacterial culture immobilized on carrier, seed application and harrowing.

The treatment is carried out with a dry biodegradation oil product once a season during a warm period at a dose of 100 g / m ² , and a Bacillius cereus 3 K. strain with a titer of 10 ¹² cells / g of carrier is used as a bacterial culture, while the biodegradation product is scattered with seed placement or in front of him, followed by harrowing to a depth of 5-10 cm.

The applicant has not identified sources containing information about technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "novelty."

Due to the implementation of the distinguishing features of the invention / in combination with the features indicated in the restrictive part of the formula /, important new properties of the object are achieved.

In the proposed technical solution:

- the use of a biological product-oil destructor increases the adaptability of biological reclamation;

- the presence of a biological product-oil destructor high oil destruction activity contributes to the improvement of soil restoration processes and reduce the timing of reclamation;

- immobilization of a bacterial culture of a strain of Bacillus cereus 3 K. on a microbial fertilizer carrier increases the oil destructive activity and provides a promising technology for the creation of dry bulk biological products-oil destructors highly effective and easy to use;

- the spreading of a biological product-oil destructor once when placing seeds or in front of him simplifies the technology of biological reclamation;

- liming the soil and the application of mineral fertilizers improves the process of biological reclamation;

- harrowing after introducing a biological product-oil degrader to a depth of 5-10 cm helps to increase destruction and create favorable conditions for the work of oil-oxidizing bacteria;

- the used dose of 100 g / m ² biological product-oil destructor is optimal.

The applicant is not aware of any publications that would contain information on the influence of the distinguishing features of the invention on the achieved technical result, in this regard, according to the applicant, it can be concluded that the claimed technical solution meets the criterion of "inventive step".

The restoration of oil-contaminated soils in the Far North is a complex and lengthy process. This is due to the climatic conditions of the region: a short growing season with low average daily temperatures.

Oil pollution of soils is accompanied by changes in their chemical and agrochemical properties.

The study of remediation technologies was carried out at oil spill sites in the Far North and at emergency diesel fuel spills in the Leningrad Region.

The invention consists in the following.

Liming and mineral fertilizing are carried out, after which biological reclamation is carried out.

The treatment of contaminated sites is carried out with a dry biological product-oil destructor.

The biological product-oil destructor contains a microbial fertilizer carrier and a culture of bacteria.

A carrier microbial fertilizer is used in the form of fermented agricultural waste.

As a culture of bacteria using a strain of Bacillius cereus 3 K. with a titer of 10 ¹² cells / g of carrier.

Processing is carried out once a season during the warm period.

The introduction of a biological product-oil destructor is carried out in a dose of 100 g / m ² .

The biological product-oil destructor is scattered when applying grass seeds or in front of it, followed by harrowing to a depth of 5-10 cm.

As a microbial fertilizer carrier, the Bamil microbial fertilizer or the Omug microbial fertilizer are used.

Bamil microbial fertilizer in the form of dried biomass of microorganisms of activated sludge was formed during the processing of pig-breeding complexes and contains native microorganisms: Bacillus pumilus, Bacillus sphaericus, Micrococcus hylae, Arthrobacter viscosus, Bacillus licheniformis.

Immobilization of the bacterial culture of the strain of Bacillus cereus 3 K. on the microbial fertilizer Bamil is as follows:

- mix manure-containing wastewater with recirculated activated sludge;

- carry out aerobic processing at a load of sludge 70-225 mg CPG / g h and a growth rate of sludge of 0.01-0.02 h ^-1 until the wastewater pollution by COD is 40-70%;

- carry out the separation of excess activated sludge, its compaction and drying at a temperature of 170-190 ° C;

- bacteria of the strain Bacillus cereus 3 K. are growing on solid dry nutrient agar for 3 days;

- create an aqueous suspension of bacteria of a strain of Bacillus cereus pcs. 3 K. with a titer of 10 ¹³ cells / g and distributed on the surface of 1 m ² 1 kg of dry microbial fertilizer Bamil;

- spray 100 ml of a suspension of bacteria of a strain of Bacillus cereus pcs. 3 K. with a titer of 10 ¹³ cells / g.

The obtained biological product-oil destructor contains a bacterial culture of a strain of Bacillus cereus 3 K. with a titer of 10 ¹² cells / g of Bamil microbial fertilizer.

The immobilization of the bacterial culture of the strain of Bacillus cereus 3 K. on the microbial fertilizer Bamil contributes to the creation of a dry bulk biological product-oil destructor.

The microbial fertilizer Omug in the form of a fermented litter of bird droppings contains native microorganisms: Clavibacter michiganese, Bacillus amyloliquefaciens, Micrococcus varians.

Immobilization of the bacterial culture of the strain of Bacillus cereus 3 K. on the microbial fertilizer Omug is as follows:

- carry out aerobic fermentation of thermophilic bacteria waste poultry farms (litter of bird droppings), which activates the growth of aerobic microflora;

- add mineral fertilizers (NPK) weighing about 2% by weight of the processed material;

- provide activation of vital activity by optimizing the moisture content of the initial mixture (40-60%), maintaining high porosity (20-30%);

- maintain the processing temperature of the mixture due to the heat released during the oxidation of the organic matter of the processed material;

- bacteria of the strain Bacillus cereus 3 K. are growing on solid dry nutrient agar for 3 days;

- create an aqueous suspension of bacteria of a strain of Bacillus cereus 3 K. with a titer of 10 ¹³ cells / g and distribute on the surface of 1 m ² 1 kg of dry microbial fertilizer Omug;

- 100 ml of a suspension of bacteria of a strain of Bacillus cereus 3 K. are sprayed with a titer of 10 ¹³ cells / g.

The obtained biological product-oil destructor contains a bacterial culture of a strain of Bacillus cereus 3 K. with a titer of 10 ¹² cells / g of Omug microbial fertilizer.

The immobilization of the bacterial culture of the strain Bacillus cereus 3 K. on the microbial fertilizer Omug contributes to the creation of a dry bulk biological product-oil destructor.

Bacterial culture of a strain of Bacillus cereus pcs. 3 K. was deposited at the State Scientific Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences and Scientific and Technical Center “Nika” LLC, 2003 (filling date 11/25/03).

Strain Bacillus cereus pcs. 3 K. deposited as a production strain for the manufacture of a biological product-oil destructor under the registration number "VNIISKHM D143" dated December 24, 2003

The bacterial culture of the strain of Bacillus cereus 3 K. isolated from technogenic soil contaminated with diesel fuel (St. Petersburg).

Samples were taken from the technogenic soil on the territory of the Kirovsky plant in the district of the TPP (St. Petersburg). Isolation was carried out by inoculation (serial dilution method) on a solid nutrient medium Pridham-Gottlieb, in which crude oil was used as the only carbon source.

Cultivation conditions - growth is plentiful, on a nutrient medium with pancreatic sprat hydrolysis (SPA), temperature 25-27 ° С, aerob.

Pigmentation of the culture is absent.

The shape of the colonies is amoeboid, irregular in shape, the profile is flat, the edge is wavy, folded, whitish.

The shape and size of vegetative cells - sticks, sticks with rounded ends, size 0.6-0.8 × 1.9-2.6 microns.

Spore formation - ellipsoidal spores, central location, do not change the shape of the cell, size 0.6-0.8 × 1.2-2.1 microns.

Mobility of cells - mobility in a vegetative state.

Flagellation is peretrichial.

Gram staining - Gram ⁺ .

Relation to a carbon source - utilizes maltose, sucrose, mannose, glucose, lactose, inosan, racimose, sorbitol, xylose, arabinose, glycerin, mannitol, galactose, rhamnose.

Attitude to the source of nitrogen - good growth on media with mineral and organic sources of nitrogen (NH ₃ · NO ₃ ), peptone, asparagine).

Other diagnostic signs - forms catalase, lycetinase; the Voges-Proskauer reaction is positive, it does not form indole, glucose is used poorly without gas formation, there is no growth at 60 ° C, rhizoid colonies and parasporal inclusions.

The bacterial culture of the strain of Bacillus cereus 3 K. has a high hydrocarbon-oxidizing activity: it is able to utilize crude oil in the soil. The strain of Bacillus cereus 3 K. is used in a biological product-oil destructor based on microbial fertilizers Bamil and Omug.

As an analogue, the biological product Roder is used. Biological product Roder is introduced into the oil-contaminated soil twice a month. Roder biological product consists of oil-oxidizing microorganisms Rhodococcus ryber AC-1513 D Rhodococcus erythropolis AC-1514 D in the form of a concentrated suspension of bacterial cells with a titer of 10 ¹² / ml, diluted with water at a ratio of 1:40, respectively, at t = 25-30 ° С adding 0.2% mineral fertilizer (N, P, K).

The method of biological reclamation is carried out on a swampy peaty area of an emergency oil spill in the vicinity of the town of Usinsk, located in the Usinsk-Kolvinsky spruce-forest-tundra region in the subzone of the forest-tundra and extreme north taiga of the Pechersk-Ural sub-province.

The laying of a field experiment with oil is carried out in a pilot plot with an area of 2000 m ² (contamination dose in the range of 17 - 110 g / kg) and after applying mineral fertilizer (N, P, K), they determine: a control section (without introducing an oil-destructive biological product), for introducing the biological product Roder, a plot for introducing a biological product-oil degrader based on the Omug microbial fertilizer and a plot for introducing a biological product-oil degrader based on the microbial fertilizer Bamil.

The invention is illustrated by examples.

EXAMPLE 1

Determine the oil destruction activity of oil-contaminated soil in the experimental plots.

An analysis is made of the oil-destructive activity of soil bacteria by fluorescence spectroscopy.

The dynamics of oil-destructive activity in oil-contaminated soil, depending on the introduction of a biological product-oil destructor based on the Bamil microbial fertilizer, is presented in table 1.

Table 1 shows that the bacteria of microorganisms of biological preparations disposed of oil at the level of natural microorganisms - alkanotrophs at a dose of oil of 17 g / kg, and at a dose of 110 g / kg, oil was destroyed most efficiently when using a biological preparation-oil degrader based on the Bamil microbial fertilizer.

EXAMPLE 2

The number of alkanotroph bacteria in oil-contaminated soil of the experimental plots is determined by the Koch method: sowing soils on the Pridham-Gottlieb nutrient medium. Oil is used as the only carbon source - 1%.

Mineral medium Pridham-Gottlieb, g / l: (NH ₄ ) ₂ SO ₄ - 2.64; K ₂ HPO ₄ - 5.65; KH ₂ PO ₄ - 2.38; MgSO ₄ · 7H ₂ O - 1.0; CuSO ₄ - 0.064; FeSO ₄ - 0.01; MnCl ₂ - 0.079; ZnSO ₄ - 0.015.

The dynamics of the number of bacteria-alkanotrophs in oil-contaminated soil depending on the introduction of biological products-oil destructors based on the microbial fertilizers Bamil and Omug are presented in table 2.

From table 2 it is obvious that in the oil-contaminated soil with added biological products, the number of bacteria increased by an average of 3 orders of magnitude compared with the control.

EXAMPLE 3

The biological activity of the experimental plots is evaluated by the intensity of CO ₂ emission.

СО _{2 is} determined a month after the introduction of oil-degrading biological products based on microbial fertilizers Bamil and Omug into the oil-contaminated soil by gas chromatography.

A sample of soil weighing 20 g (calculated on an absolutely dry weight) is placed in 120 ml flasks, closed with a cotton stopper and incubated in a humid chamber at a temperature of 27 ° C. The intensity of CO ₂ emission is determined after 3 days. For this, the flasks are “washed” with compressed air for 3 minutes and hermetically closed with plastic caps with an elastic rubber gasket. Incubated for 24 hours in a thermostat at a temperature of 27 ° C. After that, a 0.5 ml gas sample is taken with a medical syringe and introduced into the receiver of the Chrom-4 gas chromatograph, and then calibrated. For chromatography, sorption columns of stainless steel 120 cm long with an inner diameter of 2 mm filled with Polysorb-1 sorbent are used. Helium is used as the gas, which is passed at a speed of 20 mm / min. The temperature of the detector thermostat is 100 ° C. The temperature of the chamber and columns is 20-22 ° C. The output of CO ₂ occurs at the 48th second, a clear peak appears on the chromatograph, fixing the yield of CO ₂ .

The intensity of CO ₂ emission in oil-contaminated soil, depending on the application of biological preparations-oil destructors based on the microbial fertilizers Bamil and Omug, is presented in table 3.

Table 3 shows that the treatment of oil-contaminated soil with biological preparations-oil destructors based on microbial fertilizers Bamil and Omug activated the emission of CO ₂ by 20-35% compared with the control, which characterizes the rate of oxidation of carbon compounds in the soil, but most successfully activated their biological preparation-oil destructor based on microbial fertilizer Bamil.

The above examples confirm that bioremediation using oil-based biodegradation products based on microbial fertilizers Bamil and Omug increases the number of bacteria that decompose oil, intensifies the oxidation of carbon-containing compounds in the soil and shortens the recovery time.

The proposed method of biological recirculation was carried out at oil spill sites in the Far North, and based on the results of processing oil-contaminated soils with different degrees of pollution, monitoring and studying periods of activation of soil restoration, it can be concluded that the proposed method meets the criterion of "industrial applicability".

Biological reclamation using effective biological products-oil destructors for cleaning oil-contaminated soils allows you to:

- increase the manufacturability of biological reclamation through the use of biological products, oil destructors;

- improve soil restoration processes and reduce the time for reclamation due to the high oil-destructive ability of the used biological products-oil destructors;

- use a promising technology for the production of dry, bulk biological products-oil destructors due to the immobilization of bacterial cultures of the strain Bacillus cereus 3 K. on microbial fertilizers carriers;

- to simplify the technology of biological reclamation through the use of a simple method of applying biological products, oil destructors by scattering when placing seeds or in front of him;

- to improve the process of biological reclamation due to liming of the soil and the introduction of mineral fertilizers;

- increase oil destruction activity due to harrowing after applying the biological product-oil destruction to a depth of 5-10 cm;

- carry out the cleaning of oil-contaminated soils in areas with a short thermal period.

Claims (1)

The method of biological reclamation of oil-contaminated soil in the natural conditions of the Far North, including liming, mineral fertilizer application, treatment of contaminated areas with a biological product-oil destructor containing microbial fertilizer - a carrier in the form of fermented agricultural waste and a bacterial culture immobilized on a carrier, seed application and harrowing, different the fact that the treatment is carried out with a dry biological product-oil destructor once a season during a warm period in at a dose of 100 g / m ² , Bacillus cereus 3 K. strain with a titer of 10 ¹² cells / g of carrier is used as a bacterial culture, while the biological product-oil destructor is scattered when seeds are placed or before it, followed by harrowing to a depth of 5-10 cm