RU2600868C2 - Preparation for cleaning soil from oil contaminants - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention refers to microbiology and biotechnology, particularly to technology for production of preparations for cleaning soil from oil contaminants. Preparation contains a biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on substrate surface carrier. Natural zeolite deposit Honguruu is used as a substrate-carrier.

EFFECT: invention allows to accelerate the process of destruction of oil contaminants and reduce the length of recovery of soil.

1 cl, 9 tbl, 2 ex

Description

The invention relates to microbiology and biotechnology, namely the technology for the production of preparations intended for the purification of soils from oil pollution.

Essence: a preparation is presented for cleaning soil from oil pollution, containing the biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on the natural zeolite of the Honguruu deposit at a ratio of 1:10.

The drug allows for a relatively short time to effectively clean the soil from oil pollution.

Known biological product “Putidul” (1. USSR author's certificate No. 1076446, Strain Pseudomonas putida 36, used to clean water and soil from oil and oil products. A1, C02F 3/34, B09C 1/10, C12N 1/20, C02F 3 / 34, C02F 101: 32, C12N 1/20, C12R 1:40 Application: 3474745, 07/22/1982 Published: 02/28/1984 Applicant: West Siberian Oil and Gas Research Institute Authors: Dyadechko VN , Tolstokorova L.E., Morozova T.N.), used for cleaning the soil from oil pollution. The biological product is based on a monoculture of bacteria Pseudomonas putida 36 (2. Dyadechko V.N., Tolstokorova L.E., Gashev S.N. et al. On the biological reclamation of oil-contaminated sandy soils of the Middle Ob region // Soil Science. - 1990, No. 9. - S. 148-151). Its disadvantage is the complicated technology of preparation of the drug, which provides for spray drying of a live culture of bacteria, which causes injury to bacteria and, as a result, their death or loss of necessary activity due to inactivation of cells under high temperatures (+ 60 ° C). To restore the vital activity of bacteria, the authors apply a complex set of measures: heating a large amount of water (5 m ³ ) to + 18 + 28 ° C, mixing, aeration and all this for a long time (16-24 hours), which is quite enough to perform in the field complicated.

A known method of cleaning soils from oil and oil products contaminants, in which a consortium of oil-oxidizing microorganisms Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ky-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt. % of each microorganism, while together with a suspension of a biological product, rhizotorfin is introduced into the contaminated medium in an amount of 30-120 g / m ² (3. Patent RU 2191643. A method for cleaning soils from contamination by oil and oil products. C1, B09C 1/10, C12N 1 / 20, C12N 1/20, C12R 1:01. Application: 2001119562/13, 07/09/2001. Published: 10/27/2002. Applicant: Closed Joint-Stock Company Polyinform Author (s): Saxon VM, Kuznetsov S .A., Boykova I.V., Novikova I.I. Patentee: Closed Joint-Stock Company Polyinform), where rizotorfin is a fertilizer of nitrogen-fixing microorganisms.

The disadvantage is that when preparing a working suspension of microorganisms, the temperature is maintained at + 18 + 22 ° C, i.e. microorganisms that are part of the consortium are not capable of degradation of oil products at low positive temperatures (+ 4 + 8 ° C).

A known method of cleaning the soil using a biological product "Devoroil" containing a consortium of hydrocarbon-oxidizing microorganisms Rhodococcus maris, Rhodococcus erythropolis, Pseudomonas stutzeri, Yarrowia lipolytica. This method involves the storage of a suspension of microorganisms using NaCl, biologically active substances (vitamins) and osmoprotectors (betaine) (4. Patent RU 2114071. A method for cleaning soil, natural and waste water contaminated with oil and oil products, using biological products. C02F 3/34 , B09C 1/10, C12N 1/20, C12N 1/26, C12Q 1/02. Application: 97107716/13, 05.22.1997. Published: 06/27/1998. Applicant: Borzenkov Igor Anatolevich Authors: Borzenkov I.A. , Belyaev S.S., Ibatullin PP, Pospelov M.E., Svitnev A.I. Patentee: Igor Anatolyevich Borzenkov). The disadvantage is that despite the addition of protective substances to the bacterial suspension, the number of microorganisms decreases regardless of the storage temperature.

Known bacterial preparation consisting of highly active live aerobic oil-oxidizing bacteria (Mycobacterium, Pseudomonas, etc.) grown on solid carrier substrates with titers of 2.5-7.0 · 10 ⁹ cells / g. As a carrier substrate for the immobilization of microorganisms, gamma-sterile peat with a pH of 6.8-7.0 in an amount of 40-45 wt. % and water in the amount of 58.95-53.25 wt. % In order to maintain oil-oxidizing activity during storage, nutrient substrates are additionally introduced into the preparation: ammonium oxalate (0.05-1.0 wt.%) And normal paraffins (1.0-1.5 wt.%). The shelf life of a biological product is 6 months at a temperature of 10-15 ° C from the moment of its manufacture (5. Patent RU 2053205. Biological product for cleaning soil and water from oil and oil products. C1, C02F 3/34, C09K 3/32, B09C 1 / 10, B09C 101: 00. Application: 94034274/13, 09/29/1994 Published: 01/27/1996 Applicant: All-Russian Petroleum Research and Development Institute of Geology, Authors: Belonin MD, Rogozina EA, Svechina PM, Khotyanovich A.V., Orlova N.A. Patentee: All-Russian Petroleum Research and Exploration Institute). The biological product contains oil-oxidizing bacteria, which require a pH of 6.8-7.0 and a large amount of water (about 60% water) to maintain their vital functions, which reduces its effectiveness over time.

A known method of cleaning soils and soils contaminated with oil and oil products using an oleophilic biological product based on the association of oil-oxidizing microorganisms - Rhodococcus erythropolis IEGM 708 and Rhodococcus ruber IEGM 327 and biosurfactant-Rhodococcus, characterized in that the soil is thoroughly cultivated to reach the initial level residual oil 5-10 weight. % and the subsequent addition of an organic baking powder, an oleophilic biological product is applied in an amount of at least 10 liters per 0.5-1.0 m ^{3 of} soil / soil at least once a week for the first month and subsequently at least once a month before the end of the bioremediation cycle, in combination with periodic loosening and moistening, and additionally produce phytoremediation - sowing with perennial grasses (6. Patent RU 2193464. Method for bioremediation of soils and soils contaminated with oil and oil products. C1, B09C 1/10, C12N 1/26, C12N 1/26, C12R 1:01. Application: 2001130686/13, 11/14/2001. flashed: 11/27/2002 Applicants: Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences, PermNIPIneft Limited Liability Company Authors: Ivshina IB, Kostarev SM, Kuyukina MS, Zakshevskaya LV Patent holders: Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences, Limited Liability Company PermNIPIneft). However, the above method involves the use of solid-liquid phase bioreactor and / or aerated soil pads as the initial treatment of highly contaminated soil. This method of reducing the level of pollution is time-consuming and involves disruption of the fertile soil layer (humus part of the soil profile), which is not applicable for accidents and oil spills in the Far North (including in the tundra zone). Since a prerequisite for cleaning and restoring oil-contaminated permafrost soils is the preservation of the fertile soil layer. This is explained by the fact that at the place of removing the soil cover under permafrost conditions, the formation of thermokarst defrost with the formation of funnels, dips or alas is possible, which in turn leads to the development of thermoerosion and causes great damage to the vulnerable and difficult to restore soil ecosystem of the Far North.

A non-laborious and closest to the proposed technical solution is a method of cleaning soils from petroleum hydrocarbons, which provides for the immobilization of hydrocarbon-oxidizing microorganisms extracted from specific contaminated objects on the natural zeolite of the Honguruu deposit and its introduction into soil contaminated with petroleum hydrocarbons. Moreover, the degree of destruction is carried out in 4 months (7. Erofeevskaya L.A., Burmistrova T.I., Alekseeva T.P., Tereshchenko N.N. // Zeolite as an indispensable component in the rehabilitation of disturbed lands. Biological sciences. New word in science and practice: hypotheses, approbation of research results. 2013, No. 3. - S. 7-9).

One of the disadvantages of this method is its relatively low speed (4 months) for cleaning contaminated soils from oil, which is not always acceptable in the conditions of a short northern summer. In addition, the preparation of a drug based on hydrocarbon-oxidizing microorganisms isolated from specific contaminated sites is not always convenient with a quick response to eliminate an emergency oil spill in the field and requires additional time and additional research: going to the emergency spill site, taking soil samples to isolate microorganisms, cultivation and isolation of microorganisms, control of the oil-oxidizing ability of selected cultures of microorganisms, the accumulation of biomass, immobil zatsiyu microorganisms to the sorbent carrier, drying the resulting biological preparation, control of the obtained preparation, etc. All these stages extend the preparation of soils for self-healing. It should also be emphasized that in the process of oil oxidation, the practical use of microbial degradation of oil is determined by the composition of the biological product, which determines the viability of bacteria, their number and the technology of using drugs for practical purposes.

Another disadvantage of this method is that it is applicable for cleaning soils from fresh oil pollution and is not applicable for cleaning soils with chronic oil pollution (up to 10 years).

The objective of the invention is to accelerate the degradation processes of both fresh and old oil pollution and reduce the timing of soil preparation for self-healing.

The technical result consists in reducing the level of oil pollution in soils to 83% in 80 days in soils with a long-term oil pollution (10 years) and to 53-55% in 60 days in soils with fresh oil pollution.

The task is carried out, and the technical result is achieved by creating a preparation for cleaning soils from oil pollution, containing the biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on the natural zeolite of the Honguruu deposit at a ratio of 1:10.

Characterization of strains.

1) Rhodococcus sp. VKM Ac-2626D

Strain Rhodococcus sp. VKM Ac-2626D isolated from permafrost soil (Yakutsk, Central Yakutia).

The strain is characterized by the following symptoms: gram-positive, fixed sticks. In a one-day culture on the mineral environment, Müns forms short thick sticks. During division, a characteristic arrangement of cells at an angle to each other is observed.

On the MPA it forms smooth colonies, creamy pink in color, with a diameter of 1-5 mm.

The strain grows at a temperature of + 4 + 30 ± 1 ° C under aerobic conditions and weakly under anaerobic conditions. Growth optimum + 4 + 20 ± 1 ° С.

Oxidase negative, does not decarboxylate lysine, decarboxes ornithine, is not able to break down phenylalanine. Indolenegative. Hydrogen sulfide does not produce. Not active against inositol. Urease is negative. The β-galactosidase test is negative. Does not ferment lactose, glucose, sodium citrate, sodium malonate. Uses petroleum hydrocarbons as an energy source.

Resistant to amoxiclav, ampicillin, kaotima, amoxicillin, pefloxacin, amosin. Low resistance to chloramphenicol. It is sensitive to benzylpenicillin, mozivar, oxacillin, cefatoxime, ceftriaxone, polymyxin, furazolidone, metranidazole.

When sequencing the variable regions of 16SpPHK, the following assembled nucleotide sequence was obtained for the studied strain:

According to the results of the analysis of sequencing of variable regions of 16SpPHK, the test strain is classified as Rhodococcus sp.

The results of processing sequences are presented in table 1.

Screening using the GenBank and RDP-II database showed that the studied strain is closest to the genus Rhodococcus, and the homology with the species R. qingshengii and R. jialingiae is 100%.

The strain is not virulent, not toxic, not toxigenic, not phytotoxic (the test was carried out on white mice and seeds of higher plants (oats cultivar Skakun and wheat cultivar Prilenskaya-19)).

Storage conditions:

1. In lyophilized form in ampoules at + 4 ° C.

2. The strain can be maintained by regular reseeding (1 time in 2 weeks) on the slanted MPA.

The strain was identified and deposited by the All-Russian Collection of Microorganisms (VKM) of the IBPM RAS (Pushchino, Moscow Region, Nauki Ave., Building 5). Collector number VKM Ac-2626D.

2) Serratia plymuthica VKM B-2819D

The strain Serratia plymuthica VKM B-2819D was isolated from soil contaminated with Arctic diesel fuel (Chapchylgan, Central Yakutia).

The strain is characterized by the following symptoms: gram-negative, rod-shaped, peritrichial, asporogenous, hemorrhotrophic, facultative anaerobic bacteria.

It grows at a temperature of + 4 + 25 ± 1 ° C, the optimum growth is + 4 + 15 ± ° C under aerobic conditions, but can grow at a temperature of + 30 ° C and under anaerobic conditions, however, it loses pigment.

On MPA it forms wet colonies with a diameter of 1-3 mm from raspberry to orange. On the mineral environment, Muntz with oil grows in the form of pasty colonies of raspberry color.

Oxidase-positive, does not decarboxylate lysine and ornithine, is not able to break down phenylalanine. Indolenegative. Hydrogen sulfide does not produce. Active against inositol. Urease positive. The test with β-galactosidase is positive. Ferments sucrose, mannitol, sorbitol, maltose. Does not ferment lactose, sodium acetate, sodium citrate, sodium malonate. Uses petroleum hydrocarbons as an energy source.

Resistant to caotima, amoxicillin, pefloxacin, amoxiclav, chloramphenicol, cefatoxime, ceftriaxone, polymyxin, furazolidone, metranidazole, amosin. Weakly resistant to benzylpenicillin, mozivar, oxacillin, ampicillin.

When sequencing the variable regions of 16SpPHK, the following assembled nucleotide sequence was obtained for the studied strain:

According to the results of the analysis of sequencing of variable regions of 16SpPHK, the test strain is assigned to the species Serratia plymuthica. The results of processing sequences are presented in table 2.

Screening using the GenBank and RDP-II database showed that the studied strain is closest to the species Serratia plymuthica (99.77%).

The strain is not virulent, not toxic, not toxigenic, not phytotoxic (the test was carried out on white mice and seeds of higher plants (oats cultivar Skakun and wheat cultivar Prilenskaya-19)).

Storage conditions:

1. In lyophilized form in ampoules at + 4 ° C.

2. The strain can be maintained by regular reseeding (1 time in 2 weeks) on the slanted MPA.

The strain was identified and deposited by the All-Russian Collection of Microorganisms (VKM) of the IBPM RAS (Pushchino, Moscow Region, Nauki Ave., Building 5). Collector Number VKM B-2819D.

Characterization of the substrate carrier.

According to many authors, natural mineral and organic materials are preferable for immobilization of UOM. At the same time, the sorbent carrier used to immobilize UOM cells should have the following basic properties: permeability, oil capacity, non-caking, porosity, and flowability.

The natural zeolite of the Khonguruu deposit (Western Yakutia) is the most fully compliant - a light-green frame aluminosilicate, in the structure of which there are communicating cavities occupied by large ions of various elements and water molecules that have significant freedom of movement, due to which ion exchange and reversible dehydration (8. Kolodeznikov KE, Novgorodov PG, Stepanov VV Types of zeolite raw materials of the Honguruu deposit // Prospects for the use of zeolite rocks of the Honguruu deposit: Sat. Tr. - Yakutsk: NSC SB RAS, 1993. - S. 5-13).

The zeolite crystal lattice is formed by tetraids with a well-developed inner surface, which ensures the sorption ability of the mineral and good adhesion to microbial cells, allowing oil destructors to be fixed not only on the surface of the carrier, but also inside, which allows its use as a sorbent carrier for immobilization of UOM.

It was established that the zeolite of the Honguruu deposit is saturated with oil almost immediately after the sample is immersed in oil. After 1 minute, the oil capacity of the sample reaches 0.4-0.5 g / g and does not change for six hours, which corresponds to the literature of other researchers (9. Arens V.Zh., Saushin A.Z., Gridin O.M. , Gridin AO Purification of the environment from hydrocarbon pollution. - M., Publishing house "Interbook", 1999. - 371 p .; 10. Novgorodov P.G., Erofeevskaya L.A., Alekseeva T.P. , Burmistrova TI Influence of zeolite on the destruction of petroleum hydrocarbons // Chemistry of oil and gas. - Abstracts of the VII International Conference. - Tomsk - 2009. - P. - 843-846).

As a catalyst for the destruction of petroleum hydrocarbons, zeolite, when introduced into oil-contaminated soils, also contributes to the formation of centers of active destruction of petroleum hydrocarbons in contaminated soil due to the sorption of oil-absorbing cultures of microorganisms on its surface (11. Tereshchenko NN, Lushnikov SV, Pysheva E .V. Reclamation of oil-contaminated soils // Ecology and Industry of Russia. - 2002. - No. 10. - P. 17-20).

The introduction of zeolite into oil-contaminated soil can also enhance its nitrogen-fixing ability due to the presence of microelements such as Mo and Fe in the zeolite, which activate the nitrogenase enzyme complex of nitrogen-fixing microorganisms (in particular, bacteria of the genus Azotobacter and cyanobacteria) (12. Tereshchenko NN, Lushnikov S.V., Pysheva E.V. Biological nitrogen fixation as a factor in accelerating the microbial destruction of petroleum hydrocarbons in the soil and methods of its stimulation // Biotechnology. - 2004. - No. 5. - P. 41-45).

The chemical composition of the zeolite of the Honguruu deposit allows it to be used as a source of macro- and microelements necessary for activation and nutrition of oil-oxidizing soil microorganisms (13. Novgorodov P.G., Erofeevskaya L.A., Alekseeva T.P., Burmistrova T.I. zeolite for the destruction of petroleum hydrocarbons // Chemistry of oil and gas. - Abstracts of the VII International Conference. - Tomsk - 2009. - S. - 843-846) (table 3).

The physical characteristics of the zeolite of the Honguruu deposit correspond to the values of table 4.

Thus, natural zeolite is a promising carrier sorbent used for immobilization of UOM cells, as it possesses the basic properties listed above, namely: permeability, oil capacity, non-caking, porosity and flowability.

Based on the above stated research results, a preparation was prepared for cleaning soil from oil pollution, containing the biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on the natural zeolite of the Honguruu deposit at a ratio of 1:10.

The parameters and characteristics of the biological product must correspond to the values of table 5.

The technology of preparation of the drug for cleaning soils from oil pollution

- Obtaining uterine culture

To obtain uterine cultures, agar cultures of strains of Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D. These crops are seeds used for sowing in fermenters.

For each bacterial isolate, which is at this stage a production strain, a separate seed is prepared and cultivation is carried out in a separate fermenter. In this case, individual species and strain features of the bacterial destructor are taken into account. To obtain a uterine culture, a mineral medium is used (14. Kersten D.K. Morphological and cultural properties of indicator microorganisms in oil and gas surveys // Microbiology, 1963, No. 5, P. 1024-1030) of the following composition (wt.%): KNO ₃ - 0 ,four; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; distilled water - the rest; pH is 7.2. The amount of seed should be 5-10% of the volume of the nutrient medium.

Isolate cultures grown on chamfered timing agar, the basis of which is a fish meal hydrolyzate or industrial meat-peptone agar (MPA), are washed off with physiological saline and a suspension is prepared. The concentration of cells in suspension should be 1 × 10 ⁹ cells / cm ^-3 according to the optical standard GISK them. AM Tarasevich; volume - not less than 25-50 cm ³ .

- Cultivation of seed in flasks

Nutrient mineral medium of the same composition is poured into 150 cm ³ into flasks with a capacity of 500 cm ³ , 15-25 cm ^{3 of} bacterial suspension prepared by flushing are inoculated into the medium. 1.5 cm ^{3 of} sterile oil is added there. Crops in flasks are incubated in UVMT for 72 hours at 200 rpm, a cultivation temperature of 29 + 1 ° C.

- Obtaining seed for immobilization

The cultivation of the bacteria that make up the basis of the drug is carried out on the same medium; sterile crude oil is used as the sole carbon source; the concentration of hydrocarbon-containing products in the fermentation liquid is at least 1% (by volume). The fermentation process is carried out with vigorous stirring at a temperature of 29 + 1 ° C for 36-48 hours.

At the end of the cultivation process, the produced fluid is poured into sterile containers and used to immobilize the carrier sorbent.

- Immobilization of strains on a sorbent carrier

The seeds for immobilization are mixed in an apparatus with a stirrer with a zeolite crumb of a fraction of 1-3 mm in a ratio of 1:10 (1 part of a bacterial suspension: Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D per 10 parts of zeolite raw materials). After impregnation of the zeolite with seed for immobilization, it is dried by contact at a temperature of 37 ° C to constant weight or at room temperature for 2 days. The drug is ready to use. Dry biological product is placed in kegs or two-layer plastic bags and sealed. The drug is applied in an amount of 100 g per 10 kg of soil.

The parameters and characteristics of the drug should correspond to the values of table 5.

Requirements for production and safety conditions

Premises for working with microorganisms must comply with the Sanitary and Epidemiological Rules SP 1.3.2322-08 "Safety in Working with Microorganisms of the III-IV Pathogenicity (Hazard) Groups and Parasitic Pathogens".

Work on equipment when working with microorganisms and the manufacture of a sorbent carrier should be allowed to persons who have reached the age of 18 years who have been trained.

All employees must undergo periodic medical examinations in the manner prescribed by the health authorities (15. Order of the Ministry of Health of Russia dated 14.03.96 No. 90 “On the procedure for preliminary and periodic medical examinations of employees and medical regulations for admission to the profession”; Order of the Ministry of Health and Social Development of Russia of 08.16.04 No. 83 “On approval of lists of harmful and (or) hazardous production factors and work, during the implementation of which preliminary and periodic medical examinations (examinations) are carried out, and the procedure and conducting these examinations (examinations) ”(as amended on May 16, 2005).

Compliance with safety requirements must be ensured by complying with the relevant approved safety instructions and rules when carrying out appropriate work.

Bioremediation technology of oil-contaminated soils using the drug.

The drug is applied to the soil without preliminary preparation, in the form in which it entered the object, under surface treatment at the rate of 100 g of the drug per 10 kg of soil.

The drug is recommended to be applied at average daily soil temperatures above + 4 ° C. Permissible acidity is pH 5.0-8.0. Soil moisture in the range of 40-60%. The recommended time interval between introducing a biological product into the soil is 30-45 days.

After 30-45 days, re-sampling of soil samples for the content of residual amounts of oil and oil products is carried out. This analysis will determine the need for re-processing.

The quality of soil cleaning from oil pollution is controlled by the gravimetric method in accordance with the instructions for monitoring the state of soils at the facilities of the Minneftegazprom enterprises (16. RD 39-0147098-90. Environmental protection instructions for the construction of oil and gas wells on land, 1990).

Acceleration of terms of restoration (soil remediation) with the use of a drug for cleaning soils from oil pollution

The self-healing process of oil-contaminated soil ecosystems in regions with favorable climatic conditions takes 10-25 years, while the destruction of oil and its derivatives in the North is at least 50 years (17. Oborin A.A., Kalachnikova I.G., Maslivets T .A. Et al. Biological reclamation of oil-contaminated lands in the taiga zone // Restoration of oil-contaminated soil ecosystems: Collection of scientific papers, Moscow: Nauka, 1988. - P. 140-159). Subject to the requirements for storage, transportation and use of the proposed drug for cleaning soils from oil pollution, the concentration of oil in the soil is not more than 10%; oil penetration depth up to 20 cm; temperature not lower than + 4 ° С; pH of the medium is 5.0-8.0; humidity not less than 40-60%; soil aeration by loosening, since the preparation includes aerobic bacteria that work with the access of atmospheric oxygen, introducing the drug into oil-contaminated soil helps to create optimal conditions for the reproduction and functioning of indigenous hydrocarbon-oxidizing microorganisms, plant resistance to adverse factors, which leads to the stimulation of oil decomposition in the soil and leads to to reduce the terms of restoration of disturbed lands (up to 3-5 years). At low concentrations up to 3%, this period can be reduced to 1 year.

To accelerate the rehabilitation of oil-contaminated soils, the following measures must be observed:

1) oil-contaminated soils must be mixed with a clean fertile soil layer or peat;

2) according to the results of agrochemical studies (if necessary) - introduction of mineral components in the form of fertilizers that are missing in the soil in calculated doses. Usually, in agriculture, for basic, pre-sowing and local application, when sowing or for feeding plants, nitrogen-phosphorus-potassium fertilizers are used (nitroammophores, ammofoska or azofoska). These fertilizers are universal and are used on all types of soils and under all agricultural crops. With the main application on chernozem and heavy clay soils, it is advisable to apply fertilizers in the fall, and on light soils in the spring. Application rates depend on soil fertility and average 35–45 kg per hectare;

3) increase in the frequency of soil treatment with the drug up to 3-4 times for 1 growing season;

4) loosening the soil at least 1 time per week;

5) maintaining optimal moisture and soil pH;

6) sowing of perennial oil-tolerant grasses (fescue, creeping wheatgrass, Siberian wheatgrass, meadow timothy grass, field sow thistle, beechless campfire, meadow clover).

The use of the proposed drug for cleaning soils from oil pollution is confirmed by specific examples.

Example 1. Cleaning the soil from chronic oil pollution

The drug was tested to clean the loamy soil from chronic oil pollution in the tank farm located in Central Yakutia. The initial content of oil products (NP) in the soil before the introduction of the biological product was 50294 mg / kg. The duration of oil pollution is 10 years. The effectiveness of the drug was evaluated by the degree of degradation of oil pollution, the dynamics of the number of major groups of soil microorganisms and phytotoxicity of the soil.

The oil content in the soil was determined by the gravimetric method in accordance with the instructions for monitoring the condition of soils at the facilities of the Minneftegazprom enterprises (18. RD 39-0147098-90. Environmental protection instructions for the construction of oil and gas wells on land, 1990).

It was found that the degree of biodegradation of chronic oil pollution in the experimental plot 80 days after the drug was introduced into the soil was 83.58% (table 6).

The dynamics of oil pollution biodegradation was consistent with the dynamics of the accumulation of heterotrophic microorganisms, including oil destructors, the number of which 80 days after the introduction of a biological product into the soil increased by an average of 2 orders of magnitude (table 7).

Biotesting showed that phytotoxicity decreased in the process of cleaning up old oil pollution in the soil of the experimental plot, as evidenced by an increase in the percentage of germination of seeds of test plants from 30 to 90%.

Thus, the results indicate the effectiveness of the proposed drug for cleaning soils from chronic oil pollution.

Example 2. Cleaning the soil from fresh oil pollution

The drug was tested to clean the loamy soil from fresh oil pollution in a laboratory experiment. The effectiveness of the drug was evaluated by the degree of degradation of oil pollution and the dynamics of the number of main groups of soil microorganisms.

The oil content in the soil was determined by the gravimetric method analogously to example 1.

For the experiment, the soil was placed in special containers and the required volume of oil was added, after which it was thoroughly mixed for uniform distribution. Then, a drug was added to each container.

As a control, soil contaminated with oil was used without the addition of a biological product. Each experiment was carried out in triplicate. The exposure of the experiments was 60 days at room temperature. The scheme of the model experiment is presented in table 8.

It was found that the degree of biodegradation of fresh oil pollution for one application to the soil after 60 days was 53.42-55.32% (table 11).

The dynamics of biodegradation of oil pollution was consistent with the dynamics of the accumulation of heterotrophic microorganisms, including oil destructors, the number of which significantly increased 60 days after the introduction of the biological product into the soil (table 9).

The results obtained indicate the effectiveness of the drug for cleaning soils from fresh oil pollution.

Thus, the advantage of the proposed drug for cleaning soil from oil pollution is the presence in its composition of the biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM AC-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on the natural zeolite of the Khonguruu deposit at a ratio of 1:10, which allows stimulating in soils with both fresh and fresh soil in a relatively short period of time (60-80 days) with long-standing oil pollution (up to 10 years) microbiological degradation of oil products (NP) and reduce phytotoxicity in relation to higher plants.

The method of preparation and use of the proposed drug is economically viable, since it does not require complex technological equipment for its implementation. And the zeolite, which serves as a carrier for microorganisms in this method, is both affordable and cheap raw materials, since zeolites have a surface occurrence and are developed in an open way. The reserves of 11462 thousand tons were approved at the Honguruu field. At the cost of natural zeolites are 20-200 times cheaper than substitutes.

Purification of the soil from oil pollution by the proposed preparation allows to prevent the spread of pollutants to the associated landscapes and to eliminate stains of contaminated soil, which significantly improves the sanitary and environmental condition of the disturbed area.

The proposed drug for cleaning the soil from oil pollution is environmentally friendly, since the basis for its preparation are natural formations - not pathogenic microorganisms in the composition: Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D and natural zeolite of the Honguruu deposit (Western Yakutia).

Claims (1)

A preparation for cleaning soil from oil pollution, containing the biomass of hydrocarbon-oxidizing microorganisms Rhodococcus sp. VKM Ac-2626D and Serratia plymuthica VKM B-2819D, taken in equal proportions, immobilized on the natural zeolite of the Honguruu deposit at a ratio of 1:10.