RU2535978C1 - Biopreparation for cleaning environmental objects from hydrocarbon pollution, method of obtaining and application thereof - Google Patents

Biopreparation for cleaning environmental objects from hydrocarbon pollution, method of obtaining and application thereof Download PDF

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RU2535978C1
RU2535978C1 RU2013112839/10A RU2013112839A RU2535978C1 RU 2535978 C1 RU2535978 C1 RU 2535978C1 RU 2013112839/10 A RU2013112839/10 A RU 2013112839/10A RU 2013112839 A RU2013112839 A RU 2013112839A RU 2535978 C1 RU2535978 C1 RU 2535978C1
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pah
vkpm
bak
consortium
biological
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Ирина Анатольевна Афти
Марина Ивановна Янкевич
Виктория Владимировна Хадеева
Илья Валерьевич Пивоваров
Михаил Юрьевич Королев
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Ирина Анатольевна Афти
Марина Ивановна Янкевич
Виктория Владимировна Хадеева
Илья Валерьевич Пивоваров
Михаил Юрьевич Королев
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Abstract

FIELD: chemistry.
SUBSTANCE: invention relates to biotechnology and ecology, namely to protection of the environment. Biopreparation for cleaning environmental objects from petroleum pollution and PAH represents consortium of microorganisms, consisting of the following strains of bacteria: Rhodococcus qingshengii BAC-PAH-1 VKPM AS-1946, Pusillimonas ginsegisoli BAC-PAH-2 VKPM V-11370, Shinella granuli BAC-PAH-3 VKPM V-11371, taken in equal ratios Biopreparation is applied in liquid form of immobilised cell biomass To obtain liquid form submerged cultivation of strains of bacteria, included into consortium, is realised with obtaining cell suspension with its further processing in protective medium To obtain immobilised form of biopreparation submerged cultivation of consortium on nutritional medium is performed with obtaining biomass and further immobilisation of cell biomass on mineral or organic carrier or stabilisation of cell suspension Biopreparation is introduced in amount 0.5-1 l/m2 in concentration 1×106-1×107 CFU/g of biopreparation and complex mineral fertiliser - Azophosca, in concentration 20-200 g/m2.
EFFECT: invention makes it possible to increase efficiency of cleaning soil from pollution with petroleum, petroleum products and polycyclic aromatic hydrocarbons.
13 cl, 2 tbl, 8 ex

Description

The invention relates to biotechnology and ecology. It can be used to protect the environment during bioremediation of industrial and natural objects technologically contaminated with oil and polycyclic aromatic hydrocarbons (PAHs).
In addition to petroleum hydrocarbons, polyaromatic hydrocarbons and, especially, the most toxic of them, benz (a) pyrene, which, according to the WHO (World Health Organization), is a provocator of oncological diseases, are widespread, highly toxic environmental pollutants. Benz (a) pyrene and other PAHs are formed during the incomplete combustion of solid and liquid fuels in power plants, in internal combustion engines and are concentrated in the surface soil layer. With inhaled dust, water, and food products, the source of which was contaminated with PAH soils, man-made toxicants penetrate the human body through trophic chains, which leads to devastating health consequences. Therefore, in the sanitary rules governing the content of toxicants in soils of populated areas, farmland, benzo (a) pyrene is classified as toxicants of the first hazard class, the concentration of which in the soil is limited by the MPC of 20 μg / kg of soil (maximum permissible concentration).
In addition to the hygienic significance of pollution, PAHs also have a negative ecological aspect - under the influence of PAHs, soil biota is inhibited, which is recorded by suppression of the biochemical activity of soils, for example, dehydrogenase, catalase activity.
Urban, to a large extent, roadside territories and industrial zones, which are especially associated with parking lots, with the storage and use of petroleum products — the fuel bases of various industrial enterprises, railway and motor vehicle parks — are largely subject to technogenic pollution by PAHs. Currently, due to the saturation of the urban environment with motor vehicles, the flow of pollution by these highly toxic substances is significantly increasing. The behavior of PAHs in soils depends on climatic conditions and their interaction with organic and mineral components of the soil. In the absence of strong oxidizing agents and UV radiation, biological systems play a leading role in PAH degradation processes. The ability of microorganisms to utilize PAHs by incorporating them into their metabolism has been observed in many types of aerobic bacteria that use PAHs as a carbon-containing substrate to provide themselves with energy.
The biological product for cleaning environmental objects from oil pollution and PAHs contains the biomass of the following bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAU-3 11371 in equal proportions.
The biological product is used in the form of a stabilized liquid suspension, to obtain an immobilized form of the biological product, the biomass of bacterial cells is immobilized on a mineral or organic carrier.
A known consortium of oil-oxidizing microorganisms Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1, which along with the destruction of oil and oil products utilize polycyclic aromatic hydrocarbons, namely anthracene, phenanthrene and pyrene derivatives, in soils with a high content of soluble forms of manganese and copper salts (RU No. 2191643 B09C 1/10, 2001.07 / 09).
The disadvantage of this method is that the destruction of polyaromatic compounds is limited to tri- and tetra-nuclear polyaromatic compounds, there is no information on the biodegradation of benzo (a) pyrene (5-core PAH), the most highly toxic compound, first hazard class.
A known method of cleaning the soil from oil and polycyclic aromatic hydrocarbons in conditions of increased mineralization of the environment, which is a consortium of bacteria
Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34, Halomonas sp.SMB31, Salinicola socius SMB3 (RU No. 2388816 C12R1 / 00, 2008.04 / 07).
The consortium can be used to clean oil, petroleum products and polycyclic aromatic hydrocarbons, namely naphthalene, biphenyl, acenaphthene, phenanthrene, anthracene under saline conditions.
The disadvantage of this method is the narrow spectrum of biodegradable polycyclic aromatic hydrocarbons, which does not represent the most toxic 5-core aromatic hydrocarbons, including benz (a) pyrene.
Closest to the present invention is a bacterial strain Pseudomonas alcaligenes MEV, which utilizes oil, fuel oil, diesel fuel, polycyclic aromatic hydrocarbons containing from 2 to 4 benzene rings - naphthalene, phenanthrene, anthracene, fluorantene, phenol, it is also resistant to heavy metal ions - Pb, Zn, Mo, Fe, Cr (RU 2228953, C12N 1/20, C02F 3/34, B09C 1/10).
The disadvantage of this purification method is that it carries out the destruction of PAHs containing only 2, 3 and 4 aromatic rings, and also contains only one strain of bacteria of the genus Pseudomonas, although it is known that biological products consisting of microorganisms of different taxonomic groups more efficiently utilize organic toxicants.
The objective of this invention is to create an effective method for cleaning environmental objects from contamination with oil and polycyclic aromatic hydrocarbons using a new biological product, consisting of a consortium of degradation microorganisms capable of deep destruction of oil and polycyclic hydrocarbons.
The technical result that can be achieved by using the present invention is to increase the efficiency of cleaning environmental objects from contamination with oil and polycyclic aromatic hydrocarbons due to the fact that a consortium of microorganisms that are part of the biological product is capable of degrading oil and polycyclic hydrocarbons in a wide range ambient temperatures, as well as in conditions of soil pollution by heavy metal ions and with a high salt background.
The problem is solved due to the fact that in a biological product for cleaning environmental objects from oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including a consortium of microorganism strains and a protective medium for liquid form or an inert carrier for dry form, it is proposed to use it as a consortium microorganism strains bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions. Additional differences of the proposed biological product are:
- the liquid form contains as a protective medium a solution of 5% sodium chloride,
- the dry form contains a mineral or organic carrier as an inert carrier on which a consortium of bacterial strains is immobilized: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli-BA 3 VKPM B-11371 in the ratio of 1:20,
- as a mineral or organic carrier may be, for example, horse peat, expanded perlite, vermiculite, buckwheat husk, polypropylene fiber, etc.
The problem is also solved due to the fact that in the method of obtaining a biological product for cleaning environmental objects from pollution by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including deep cultivation of a consortium of microorganisms on a nutrient medium to obtain a cell suspension and its further processing in a protective environment to obtain a liquid form, it is proposed to use bacterial strains as a consortium of microorganism strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimo nas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions.
An additional difference of the proposed method was that a solution of 5% sodium chloride was added as a protective medium.
The problem is also solved due to the fact that in the method of obtaining a biological product for cleaning environmental objects from pollution by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including deep cultivation of a consortium of microorganisms on a nutrient medium to obtain a cell suspension and its further immobilization on an inert carrier to obtain a dry form, it is proposed to use bacterial strains as a consortium of microorganism strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC- 1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions.
Additional differences of the proposed method are:
- as a protective medium make a solution of 5% sodium chloride,
- a consortium of bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 are immobilized on an inert carrier, in a ratio of 1:20, in a carrier of 1:20
- as an inert carrier, mineral or
organic carrier, e.g. horse peat, expanded perlite, vermiculite, buckwheat husk, polypropylene fiber, etc.
The problem is also solved due to the fact that in the method of purification of environmental objects from pollution by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including the introduction of a biological product into a polluted medium, consisting of a consortium of microorganisms and a protective medium for liquid form or an inert carrier for solid forms, it is proposed to use the following bacterial strains as a consortium of microorganisms: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH -3 VKPM B-11371 in equal proportions. The introduction of a biological product is proposed to be carried out at the rate of 0.5-1 l / m2. in a concentration of 1 × 10 6 -1 × 10 7 CFU / g of a biological product together with complex mineral fertilizer - azofoska, in a concentration of 20-200 g / m 2 .
Additional differences of the proposed method are:
- the liquid form of the biological product contains as a protective medium a solution of 5% sodium chloride,
The dry form of the biological product contains a mineral or organic carrier as an inert carrier, on which a consortium of bacterial strains is immobilized: Rhodococcus qingshengii BAK-PAH-1 VKPM AS-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shaella gran 3 VKPM B-11371 in the ratio of 1:20,
- as a mineral or organic carrier may be, for example, horse peat, expanded perlite, vermiculite, buckwheat husk, polypropylene fiber, etc.
The proposed microbial community is a natural association derived from soil contaminated with creosote and petroleum products. The composition of the consortium is stable over time, the ratio of strains may vary depending on the cultivation conditions of the microbial community.
The bacterial consortium is non-pathogenic (non-virulent, non-toxic, non-toxic), its advantage is the high rate of destruction of polycyclic hydrocarbons.
The composition of the biological product includes a consortium of destruction bacteria of petroleum and polyaromatic hydrocarbons: bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AS-1946, Pusillimonas ginsegisoli BAK-PAU-2 VKPM B-11370, Shinella granuli-BAK-3K BAK 11371, which were identified to the species by analysis of genes encoding 16S rRNA of VKMP, FSUE GosNIIGenetika, and deposited in the All-Russian collection of industrial microorganism strains (VKPM).
The bacterial strain Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, isolated from soil contaminated with creosote.
Cultural and morphological signs: gram-positive irregular rods with pointed ends 0.5 × 2.0-4.0 microns. Bushy, primary branching and mycelium are not characteristic, the cycle of bacillus-coccus is absent. The colonies are quite large - 4-7 mm in diameter, roundish, convex, cream, dull (not shiny), on the 7-10th day the colonies are dirty orange with a denser center, rather dry.
Physiological and biochemical signs: aerob, chemoorganotroph, catalase-positive. Halotolerance (8% NaCl) is not characteristic, hydrogen sulfide does not form.
The bacterial strain Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370 was isolated from soil contaminated with creosote.
Cultural and morphological characteristics: gram-negative small thin regular bacilli 0.3 x 1.0-3.0 microns, motile. The colonies are brilliant 4-7 mm in diameter, rounded orange-beige, slightly convex, with a lighter translucent edge.
Physiological and biochemical characteristics: aerob, O / F - test - "+/-", extracellular pigments are not characteristic, oxidase - and catalase-positive. Able to use glucose, valine and arginine as the sole carbon source.
The bacterial strain Shinella granuli BAK-PAH-3 VKPM B-11371 isolated from soil contaminated with creosote.
Cultural and morphological signs: gram-negative, small thin thin regular bacilli, 0.3 h 1.0-3.0 μm, motile. The colonies are brilliant, 5-8 mm in diameter, roundish, off-white, slightly convex, translucent, with a lighter thin edge.
Physiological and biochemical characteristics: aerob, O / F - test - "+/-", intracellular and extracellular pigments are not characteristic, oxidase - and catalase-positive. Able to use glucose, but not valine and arginine as the sole carbon source.
To obtain immobilized forms of a biological product, a suspension of the biomass of a bacterial consortium is applied to a mineral or organic inert carrier such as expanded vermiculite, expanded perlite, and peat, followed by drying.
The bacterial consortium is stored on an agarized enzymatic hydrolyzate of fishmeal containing 50 μg of creazote per Petri dish, at + 4 ° C, with regular reseeding.
The biological product obtained on the basis of this consortium was proposed for the first time to clean the soil of hydrocarbons, in connection with which we can conclude that the proposed consortium meets the criteria of the invention of "novelty" and "inventive step".
The invention is confirmed by the following examples.
Example 1: Obtaining a liquid form.
The proposed bacterial consortium was isolated from technologically contaminated soil. To obtain the biomass of the drug, co-cultivation of the strains that make up the biological product is carried out. For the cultivation of microorganisms of the destructors Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli, fermenters with a capacity of 100-250 liters with a fill factor of 0.6-0.7 are used. Fermenters are equipped with fine filters, sensors for temperature, pH, mixing speed and air flow for aeration. The introduction of seed into the fermenter is made from a ratio of 3 l of microbial suspension with a concentration of 109 cells / ml per 60 l of nutrient medium. Cultivation of microorganism cultures is carried out in a fermenter on a nutrient medium of the following composition (g / l):
- Acid hydrolyzate of casein or peptone - 10
- Yeast autolysate - 0.08 L
- K 2 HPO 4 - 2
- MqSO 4 - 0.3
- MnSO 4 - 0.05
- (NH 4 ) 2 SO 4 - 6
- NaCl - 0.5
- Glucose - 20
- Diesel fuel - 0.5
- Sofexil - 1 ml
- Tap water up to 60 l
pH of the medium - 6.8-7.2
We support the following values of the technological parameters of the cultivation process:
- Temperature 28 ± 1 C °
- Mixing - 200 rpm
- Aeration - 1 rpm
- pH - 6.9 ± 0.1 units pH
The pH value is maintained automatically by feeding a 15% solution of NH 4 OH, with continuous stirring.
The duration of the cultivation process is up to 22-26 hours. The resulting suspension has a high cell titer of 10 10 CFU / ml. The biological product is obtained by stabilizing the resulting suspension with a suitable protector. The biological product is stored in suspension at a temperature of 4-6 ° C for up to 60 days.
Example 2: Obtaining an immobilized (dry) form.
The following carriers can be used as a carrier for the immobilized form:
- organic (peat, sawdust, agricultural grain waste: buckwheat and barley husks, rice husks, polymer fibers),
- mineral (expanded vermiculite, perlite, kaolin, glauconite).
To obtain the immobilized form of the microbial preparation, a suspension of cells obtained as described in example 1 is applied to the surface of the carrier in a ratio of 1:20. The preparation obtained after immobilization has a stable stable form with a high titer of oil-oxidizing cultures 10 6 CFU / g of the preparation. The shelf life of the biological product in immobilized form without loss of activity is 1 year.
The surface or part of the surface of the cell held by the carrier is freely “washed” by the external environment (liquid or gaseous); while the consumption of substrates and the release of waste products of the microorganism are determined mainly by biological factors.
The advantage of the dry form of the immobilized preparation is that it is supplied in a form ready for application to oil-contaminated soil, without preliminary preparation at the place of use.
Immobilized cells of the drug are more resistant to adverse environmental effects and more effectively oxidize hydrocarbon contaminants at high concentrations.
Example 3:
A consortium of microorganisms was grown on Raymond's mineral medium. Oil, fuel oil, diesel fuel was added to the nutrient medium in an amount of 1% by weight of the nutrient medium as the sole source of carbon. The experiment was carried out in triplicate. 200 cm 3 of mineral medium and 2000 mg of oil, fuel oil and diesel fuel were introduced into flasks with a volume of 750 cm 3 . The flasks were seeded with Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli cells in equal proportions to a concentration of 10 6 CFU / cm. As controls, flasks with a mineral medium with oil products were used. The flasks were cultured on a shaker at 200 rpm and 22 ° C for 14 days. The measurement of the concentration of petroleum products was performed by the fluorimetric method according to PND F 16.1.21-98. The results showed that within 14 days at 22 ° C this consortium utilizes oil, fuel oil and diesel fuel at: 76.3; 54.2 and 96.6%, respectively.
Example 4:
A consortium of bacterial strains: Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli was grown on Raymond's mineral medium; 1% oil was used as the sole carbon source. The grown cell suspension was introduced into flasks with a 200 cm 3 mineral medium containing 1% oil and various degrees of salinity (3%, 5%, and 7% NaCl). As controls, flasks with a mineral medium of various salinity with oil were used. The flasks were cultured on a shaker at 200 rpm and 22 ° C for 14 days. The measurement of the concentration of petroleum products was performed by the fluorimetric method according to PND F 16.1.21-98. The results showed that within 14 days at 22 ° C this consortium utilizes oil in the presence of high salinity.
The degree of salinity of the mineral medium NaCl 3% NaCl 5% NaCl 7%
The degree of destruction,% with consortium 74.6 71.6 66.8
without consortium 0 0 0
Example 5:
A consortium of bacteria: Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli, which were grown on Raymond's mineral medium with the addition of diesel fuel in an amount of 1%. The grown cell suspension was introduced into soil contaminated with crude oil (3% hydrocarbon content in the soil) of different degrees of salinization (3%, 5% and 7% NaCL) at the rate of 10 7 cells per 1 g of soil. After 21 days of research, the content of petroleum products in the soil was evaluated by IR spectrometry. In the soil after treatment by a consortium of microorganisms, a decrease in the concentration of petroleum products is noted. Significant inhibition of the process of destruction of hydrocarbons is observed with a mineralization of 7%, the concentration of oil products in the variants without introducing microorganisms remained at the same level, in the experimental variants it decreased by 28.2%, 20.5% and 15.4%, respectively
Thus, the proposed consortium can be used to clean the soil from oil in saline conditions.
Example 6:
One of the most toxic environmental pollutants, which according to the World Health Organization (WHO) has a significant impact on human health, is the group of polycyclic aromatic hydrocarbons (PAHs), which includes compounds such as naphthalene, anthracene, phenanthrene, 3,4-benz (a) pyrene, (benz (a) pyrene) and others.
A cultured cell suspension of Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli (see example 1) was used to treat soil contaminated with polycyclic aromatic hydrocarbons (PAHs). During 3 months of research, the content of PAHs was evaluated by the KHA method (MUK 4.1.1062-01). The results of the neutralization of contaminated PAH soil proposed by the consortium are presented in the table. In the soil after treatment with a biological product, there is a decrease in the concentration of PAHs throughout the study period. 3 months after the application of the biological product, the total concentration of PAHs decreased by 31.6 times and amounted to 67.6 μg / kg of soil. The concentration of the most toxic benz [a] pyrene (hazard class 1) decreased by 24 times and amounted to 3.31 μg / kg.
PAH name source 1 month 2 months 3 months
mcg / kg mcg / kg mcg / kg mcg / kg
Naphthalene 166.4 29.2 11.7 1,69
Acenaphthylene 77.9 54,2 16.9 8.04
Acenaften 11,4 <0.01 <0.01 <0.01
Fluoren 131.2 10.1 2.27 2.01
Phenanthrene 474.3 38.8 12.8 7.51
Anthracene 40 12,2 4,58 1.68
Fluorantent 416.2 34.3 12.1 8.31
Pyrene 466.1 40.6 17.4 14.96
Benz [a] anthracene 93.7 10.9 3.14 2.76
Chrysen 47.4 26.2 8.96 5.78
Benz [b] fluorantent 83.7 29.9 7.75 3,55
Benz [k] fluorantent 15.6 12.8 3.18 1.49
Benz [a] pyrene 79.6 42.1 10.5 3.31
Dibenz [a, h] anthracene 8.8 7.4 1,63 2.61
Benz [g, h, i] perylene 5.2 3,1 0.98 0.47
Indeno [1,2,3-cod] pyrene 21 11.8 2.71 3.43
Amount, mcg / kg 2138.5 363.6 116.6 67.6
The proposed consortium can be used to clean soils from oil, as well as polycyclic aromatic hydrocarbons.
Example 7: Field trials of a biological product in liquid form.
Testing the hydrocarbon oxidizing ability of a consortium of Rhodococcus qingshengii, Pusillimonas ginsegisoli, Shinella granuli cells on oil sludge and an oil-contaminated water surface.
Bioremediation (cleaning) of oil-contaminated soil was carried out in combination with agrotechnical measures - loosening the soil, applying complex azofoska fertilizer at the rate of 20-200 g / m 2 , and periodical moistening.
1. Place of the experiment: Leningrad Region, Volosovsky District, site for the disposal of soils and soils, 2011. Pollutant: oil sludge, neutralization time - 50 days, the number of treatments - 2 biological treatment. The initial concentration of oil products is 110.33 g / kg, the degree of purification is 78.58%, the hazard class is reduced from III to IV hazard class.
2. The place of the experiment: Bologoe, locomotive depot, 2012. Pollutant: oil sludge, neutralization time - 30 days, the number of treatments - 2 biological treatment. The initial concentration of petroleum products is 92.33 g / kg, the degree of purification is 62.23%. The initial concentration of benz (a) pyrene is 0.061 mg / kg, the degree of purification is 60.6%, the hazard class is reduced from III to IV hazard class.
3. Location of the experiment: Leningrad region, Vsevolozhsk district, the territory of the boiler room, 2011. Pollutant: oil sludge, neutralization time - 90 days, the number of treatments - 4 biological treatment. The initial concentration of petroleum products is 86.4 g / kg, the degree of purification is 81.6%. The initial concentration of benz (a) pyrene is 0.068 mg / kg, the degree of purification is 82.4%, the hazard class is reduced from III to IV hazard class.
In all cases, there was a decrease in the number of microorganisms on the first day, which may be due to the toxic effect of oil products and the adaptation of microorganisms to environmental conditions, there is a general tendency to a sharp increase in the number of hydrocarbon-oxidizing microorganisms in the subsequent oxidation period.
4. Location of the experiment: Leningrad region, Vsevolozhsk district, storm drains, 2012. Pollutant: petroleum products, the number of treatments - 5 biological treatments. The initial concentration of oil products is 0.35 g / l, the degree of purification is 85%.
Example 8: Field trials of a dry form of a biological product.
Testing a biological product on oil sludge and water surface contaminated with oil (immobilized form). Bioremediation (cleaning) of oil-contaminated soil was carried out in combination with agrotechnical measures - loosening the soil, applying complex azofoska fertilizer at the rate of 20-200 g / m 2 , and periodical moistening.
1. Biological product on an organic medium / peat /. Place of experiment: Leningrad Region, Volosovsky District, site of neutralization, 2012. Pollutant: oil sludge, neutralization time - 90 days, the number of treatments - 4 biological treatment. The initial concentration of petroleum products is 138 g / kg, the degree of purification is 82.36%, the hazard class is reduced from III to IV hazard class.
2. Biological product on a mineral carrier / vermiculite /. Place of experiment: Petrozavodsk, locomotive depot, 2012. Pollutant: oil sludge, neutralization time - 30 days, the number of treatments - 2 biological treatment. The initial concentration of petroleum products is 68.5 g / kg, the degree of purification is 71.64%. The initial concentration of benz (a) pyrene is 0.091 mg / kg, the degree of purification is 72.4%.
In all cases, after the introduction of the immobilized preparation, active growth of both heterotrophs and oil destructors was immediately observed. Immobilized cells of the drug are more resistant to adverse environmental effects and do not require a period of adaptation.
3. Biological product on a vegetable carrier / buckwheat husk /. Place of the experiment: Leningrad region, Tosnensky district, storm drains, 2012. Pollutant: petroleum products, the number of treatments - 8 biological treatments. The initial concentration of oil products is 0.15 g / l, the degree of purification is 60%.
4. Biopreparation on polypropylene fiber. St. Petersburg Metro, local sewage treatment facilities, outlet well. A loading from polypropylene fiber with an immobilized consortium was established. The concentration of oil products in water before loading 0.37 mg / L, after loading 0.01 mg / L.

Claims (13)

1. A biological product for cleaning environmental objects from contamination by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including a consortium of microorganism strains and a protective medium for liquid form or an inert carrier for dry form, characterized in that the consortium of microorganism strains consists of the following bacterial strains : Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions.
2. The biological product according to claim 1, characterized in that the liquid form contains a solution of 5% sodium chloride as a protective medium.
3. The biological product according to claim 1, characterized in that the dry form contains an inert carrier mineral or organic carrier on which a consortium of bacterial strains is immobilized: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAU-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in a ratio of 1:20.
4. The biological product according to claim 3, characterized in that as a mineral or organic carrier can be, for example, horse peat, expanded perlite, vermiculite, husk of buckwheat, polypropylene fiber.
5. A method of obtaining a biological product for cleaning environmental objects from pollution by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including the deep cultivation of a consortium of microorganisms in a nutrient medium to obtain a cell suspension and its further processing in a protective medium to obtain a liquid form, characterized in that a consortium of microorganism strains consists of the following bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKP M B-11371 in equal proportions.
6. The method according to claim 5, characterized in that as a protective medium contribute a solution of 5% sodium chloride.
7. A method of obtaining a biological product for cleaning environmental objects from contamination with oil, oil products, polycyclic aromatic hydrocarbons (PAHs), including the deep cultivation of a consortium of microorganisms on a nutrient medium to obtain a cell suspension and its further immobilization of the cell suspension on an inert carrier to obtain a solid form, characterized the fact that the consortium of microorganism strains consists of the following bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 B KPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions.
8. The method according to claim 7, characterized in that the consortium of bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-1 VKPM B- 11371 immobilized on an inert carrier in a ratio of 1:20.
9. The method according to claim 8, characterized in that as an inert carrier, a mineral or organic carrier is used, for example horse peat, expanded perlite, vermiculite, buckwheat husk, polypropylene fiber.
10. A method of cleaning environmental objects from contamination by oil, oil products, polycyclic aromatic hydrocarbons (PAHs), comprising introducing into the contaminated medium a biological product consisting of a consortium of microorganisms and a protective medium for a liquid form or an inert carrier for a solid form, characterized in that the biological product consists of the following bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in equal proportions, the introduction of a biological product a couple of 0.5-1 l / m2 in a concentration of 1 × 10 6 - 1 × 10 7 CFU / g of a biological product together with complex mineral fertilizer - azofoska, in a concentration of 20-200 g / m 2 .
11. The method according to claim 10, characterized in that the liquid form of the biological product contains a solution of 5% sodium chloride as a protective medium.
12. The method according to claim 10, characterized in that the solid form of the biological product contains as an inert carrier a mineral or organic carrier on which a consortium of bacterial strains is immobilized: Rhodococcus qingshengii BAK-PAH-1 VKPM AS-1946, Pusillimonas ginsegisoli BAK-PAH- 2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371 in a ratio of 1:20.
13. The method according to p. 12, characterized in that as a mineral or organic carrier can be, for example, horse peat, expanded perlite, vermiculite, buckwheat husk, polypropylene fiber.
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