RU2764434C1 - Method for cleaning environmental objects from oil and petroleum products contamination - Google Patents

Abstract

FIELD: environment protection.

SUBSTANCE: invention relates to the field of environmental protection, in particular to eco-biotechnology and ecology. The invention relates to a method for water purification from oil and petroleum products contamination, providing for the introduction into contaminated water of a suspension of a biological product containing the oil-oxidizing microorganisms Pseudomonas putida PI Ko-1 and Micrococcus sp. PI Ku-1, while the aqueous suspension of the biopreparation is prepared at a concentration of 0.5-10.0 g/l with a titer of 1⋅10⁸-1⋅10¹² cells/ml, after which oil-oxidizing microorganisms are activated by bubbling the aqueous suspension of the biopreparation with air at a flow rate of 3-5 m³ per 1 m³ of suspension for 1.5-2.5 hours. The biopreparation additionally contains Rhodococcus erythropolis AS-1269 with a weight ratio of 3-12, wt. %, of each microorganism, while Vitaplan biopreparation is injected into the suspension of the biopreparation in an amount of 0.05-1.0 g per 1 liter of suspension and mixed to obtain a homogeneous suspension, and a homogeneous suspension of the biopreparation is injected into contaminated water in an amount of 1 g per 1 liter of contaminated water.

EFFECT: purification from oil and oil products contamination with a high rate of destruction, including heavy oil fractions, efficient in a wide range of pH and temperature of the medium being cleaned.

1 cl, 1 tbl, 1 ex

Description

The invention relates to the field of environmental protection, in particular to ecobiotechnology and ecology, and in particular to methods for cleaning a polluted environment, in particular water, from contamination by hydrocarbons of oil and oil products using microorganisms.

A known method of cleaning a polluted environment from pollution by oil and oil products, involving the introduction of microorganisms into the contaminated environment in the form of a suspension in a nutrient medium containing sources of nitrogen, phosphorus, potassium and water; as microorganisms, a consortium of bacterial strains Acenitobacter oleovorum TsMPM B-1878 and Acenitobacter oleovorum VKPM U-4091 is used in a ratio of 10:1-1:10 by cell titer, RU 2038333, publ. 06/27/1995.

Adaptation of practically all oil-oxidizing microorganisms after their introduction into the medium being cleaned requires a considerable time, usually 3-6 months. This is due to a sharp change in the conditions of their life, both biotic and abiotic. Under the changed conditions, the enzyme systems of microorganisms should be adequately activated, providing the cell with nutrition and energy. The process of adaptation of microorganisms to new living conditions significantly increases the total time required for the effective purification of a polluted environment from oil and oil products.

The same disadvantages are inherent in the method of cleaning a polluted environment from pollution by oil and oil products, providing for the introduction of microorganisms Acenitobacter valentis subsp into the cleaned environment. paraffinium VKPM B-6728 and Acenitobacter valentis subsp. paraffinium VKPM B-6726, or Acenitobacter valentis subsp. paraffinium VKPM B-6727 at an ambient temperature of 10-50°C and pH 5.5-8.5, RU2053204C, publ. 1995.

There is also known a method of cleaning a polluted environment, in particular, soil from oil and oil products pollution, involving the introduction into the polluted environment of an aqueous suspension of a biological product containing oil-oxidizing microorganisms, in which an aqueous suspension is prepared at a concentration of 0.5-10.0 g/l with a titer of 1 ⋅10 ⁸ -1⋅10 ¹² cells/ml, after which oil-oxidizing microorganisms are activated by bubbling an aqueous suspension of the biological product with air at a flow rate of 3-5 m ³ per 1 m ^{3 of the} suspension for 1.5-2.5 hours, RU2108426C, publ. 04/10/1998. During bubbling, the enzyme systems of microorganisms are activated due to the action of atmospheric oxygen. Due to the activation of the enzymes of oil-oxidizing microorganisms, the period of their adaptation is reduced by 1-2 months when introduced into the medium being cleaned.

The disadvantage of this method is the long time required to effectively clean the contaminated environment from oil and oil products under conditions of high concentration of hydrocarbons and in the presence of heavy metal salts in a wide range of pH and temperature of the medium being cleaned, due to the low rate of destruction of polyaromatic hydrocarbons.

Known a method for cleaning a polluted environment, in particular soil, from oil and oil products pollution, which involves introducing into the polluted environment a suspension of a biological product containing oil-oxidizing bacteria. An aqueous suspension of the biological product is prepared at a concentration of 0.5-10.0 g/l with a titer of 1⋅10^eight-1⋅10¹²cells / ml, after which oil-oxidizing microorganisms are activated by bubbling an aqueous suspension of a biological product with air at a flow rate of 3-5 m³per 1 m³suspension for 1.5-2.5 hours. As a biological product, a 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 at a weight ratio of 3-12 wt.% of each microorganism. Together with a suspension of a biological product, rhizotorfin is introduced into a contaminated environment in an amount of 30-120 g/m², RU 2191643С, publ. October 21, 2002.

This technical solution is taken as a prototype of the present invention.

The disadvantage of this method is the long time required to effectively clean the contaminated environment from oil and oil products under conditions of high concentration of hydrocarbons and in the presence of heavy metal salts in a wide range of pH and temperature of the medium being cleaned, due to the low rate of destruction of polyaromatic hydrocarbons.

The present invention is based on the solution of the problem of creating a highly efficient method for cleaning polluted water from oil and oil products with a high rate of destruction, including heavy oil fractions, effective in a wide range of pH and temperature of the medium to be cleaned.

The problem is solved due to the fact that in the method of water purification from pollution by oil and oil products, involving the introduction into the polluted water of a suspension of a biological product containing oil-oxidizing microorganisms Pseudomonas putida PI Ko-1 and Micrococcus sp. PI Ku-1, while an aqueous suspension of the biological product is prepared at a concentration of 0.5-10.0 g/l with a titer of 1⋅10 ⁸ -1⋅10 ¹² cells/ml, after which oil-oxidizing microorganisms are activated by bubbling the aqueous suspension of the biological product with air and consumption of 3-5 m ³ per 1 m ³ suspension for 1.5-2.5 h, according to the invention, the biological product additionally contains the oil-oxidizing microorganism Rhodococcus erythropolis AC-1269, while the weight ratio of each microorganism is 3-12 wt.%, moreover, the biological product Vitaplan is introduced into the suspension of the biological product in the amount of 0.05-1.0 g per 1 liter of suspension and mixed to obtain a homogeneous suspension, while the homogeneous suspension of the biological product is introduced into the contaminated water in the amount of 1 g per 1 liter of contaminated water.

Increasing the cell concentration (more than 10.0 g/l with a titer of 1⋅10 ⁸ -1⋅10 ¹² cells/ml) does not lead to a significant increase in the rate of destruction of the pollutant, however, it unreasonably increases the cost of cleaning work, since the increase in the rate of biodegradation will be insignificant compared to the increase in the cost of the drug. If the concentration of microorganism cells is less (0.5 g/l with a titer of 1⋅10 ⁸ -1⋅10 ¹² cells/ml) in the biological product, the technical result is not achieved due to a decrease in the required rate of biodegradation of the pollutant.

Due to the implementation of the distinctive features of the invention, the claimed method acquires an important new property, which consists in creating a highly efficient method for purifying water from oil and oil products pollution at a high concentration of polyaromatic hydrocarbons and in the presence of heavy metal salts with a high degradation rate, effective in a wide range of pH and temperature of the medium to be cleaned .

Each of the microorganisms: Pseudomonas putida PI Ko-1, Micrococcus sp. PI Ku-1 and Rhodococcus erythropolis AC-1269, as well as Vitaplan, which is a source of gram-positive spore-forming facultative aerobic bacteria, being active destructors of hydrocarbon pollution under conditions of high concentrations of salts of heavy metals and polyaromatic hydrocarbons, contribute to the activation of the destructive activity of other microorganisms that are part of the consortium, which provides a significant increase in the biological activity of both natural destructor microorganisms located in a polluted environment, and microorganisms included in the consortium.

The applicant is not aware of any sources of information that would contain information about a technical solution that is adequate to the declared differences and provides for the achievement of the new properties described above (technical result). These circumstances allow, according to the applicant, to conclude that the claimed technical solution meets the condition of "inventive step".

The proposed method is carried out as follows. The parameters of the method and data on its effectiveness are shown in the table.

Apply a biological product containing a consortium of oil-oxidizing microorganisms Pseudomonas putida PI Ko-1, Micrococcus sp. PI Ku-1, Rhodococcus erythropolis AC-1269 at a weight ratio of 3-12 wt.% of each microorganism.

Strains Pseudomonas putida PI Ko-1, Micrococcus sp. PI Ku-1 and Rhodococcus erythropolis AS-1269 were deposited in the State Collection of Microorganisms of the All-Russian Research Institute of Plant Protection.

The biopreparation is filled with sterile peat. As a mineral additive, diammophos, 1 wt.%, and carbamide, 0.5 wt.% are used.

Strain Pseudomonas putida PI Ko-1 - small short sticks, sizes (0.2-0.3)⋅(0.5-0.8) microns; colonies of the strain are round, smooth, with a shiny surface, slightly convex, translucent, colorless, 3-5 mm in diameter.

Micrococcus sp.PI Ku-1 strain - cocci, 0.6-1.0 µm in diameter; colonies of the strain are round, with a smooth edge, yellow, opaque, smooth, shiny, 2-5 mm in diameter.

Strain Rhodococcus erythropolis AC-1269 - straight or curved rods, decomposing into coccal forms, sometimes forming filaments with elementary branching. The strain is non-spore-forming, Gram-positive, immobile. On agar media forms rough light-beige colonies with uneven edges. Aerobe. Oxidase test is negative. The catalase test is positive. Partially acid resistant. Breaks down β-alanine, pyroglutamate, threonine-7-AMA, glucosides. Growth due to the use as a single carbon source: maltose, andosterone, benzoic acid, butane-2,3diol, pimelic, citraconic and D-mandelic acids, hydrocarbons (decane, hexadecane, diesel fuel, oil, naphthenic and polyaromatic hydrocarbons). Relation to pH: 5-9. Relation to NaCl: 3-5%. Relation to temperature: +4-30°С. Optimum cultivation temperature: 28°C. It has a pronounced ability to form biosurfactants - it synthesizes natural biosurfactants of the glycolipid group (trigasolipids) in response to the presence of n-alkanes in the medium, which contribute to a decrease in viscosity.

Vitaplan is a biological fungicide developed on the basis of beneficial microflora - Bacillus subtilis bacteria, intended for use as a therapeutic and prophylactic agent for protecting plants by retaining moisture in the seed germination zone, creating a favorable background of microorganisms in the root zone, exudate metabolism, metabolism of phenol-formaldehyde residues pitches. Preparative form - wettable powder, manufacturer - AgroBioTechnology LLC, Russia. The active substance is a mixture of Bacillus subtilis strains (titer 10 ¹⁰ CFU/g Bacillus subtilis, strain VKM B-2604D + 10 ¹⁰ CFU/g Bacillus subtilis, strain VKM B-2605 D) - gram-positive spore-forming facultative aerobic bacteria, https://bioprotection .ru/new/preparations/vitaplan-sp-100-g.html.

In a stainless steel container with a capacity of 2 m ³ containing water with mineral additives, the biological product is diluted at a concentration of 0.5-10 g/l, and a suspension with a titer of 1⋅10 ⁸ -1⋅10 ¹² cells/ml is obtained. Then the resulting suspension is bubbling by supplying air from a compressor at a pressure of 2 atm for 1.5-2.5 hours. The air consumption is 3-5 m ³ per 1 m ^{3 of the} biological product suspension. While maintaining the temperature of the suspension 18-22°C.

After completion of the bubbling process, Vitaplan biopreparation is introduced into the container at the rate of 0.05-1.0 g/l of the suspension and mixed to obtain a homogeneous suspension. The drug thus obtained is introduced into a polluted aqueous medium containing petroleum products, including a significant amount of polyaromatic hydrocarbons and salts of heavy metals. The effectiveness of the method is judged by the loss of oil after 60 days.

Example. The parameters of the method and data on its effectiveness are shown in the table.

The experiments were carried out according to the following procedure. Erlenmeyer flasks with a volume of 750 ml were filled with 100 ml of water with different pH (ranging from 4.5 to 8.2) and an oil content of 12 mg/l, including a significant amount of polyaromatic hydrocarbons. The sum of polyaromatic compounds was 17.9 wt.%, including pyrene derivatives with isomers - 0.6 wt.%. The content of soluble lead salts was 340 µg/kg (MPC = 6.0 µg/kg), copper - 240 µg/kg (MPC = 3.0 µg/kg). The amount of the applied drug is 1 g per 1 liter of water. The flasks were kept on a thermostatically controlled shaker in the temperature range from 3°C to 40°C for 60 days. The total mass content of residual oil products in the flasks was determined on an AN-2 IR spectrophotometer in accordance with PND F 14.1:2:4.168-2000.

9 experiments were carried out with the parameters indicated in columns 2-5 of the table, while:

- experiment 1 corresponds to the maximum concentrations of the biological product and Vitaplan,

- experiments 2-5 correspond to intermediate concentrations of the biological product and Vitaplan,

- experiment 6 corresponds to the minimum concentrations of the biological product and Vitaplan,

- experiment 7 corresponds to the values of the concentration of the biological product and Vitaplan, less than stated in the claims,

- experiment 8 corresponds to the values of the concentration of the biological product and Vitaplan, exceeding those stated in the claims,

- experiment 9 corresponds to the concentration values of the biological product, which does not contain the Rhodococcus erythropolis strain and the biological product Vitaplan.

After 60 days, the total concentration of petroleum products at maximum concentrations of the biological product and Vitaplan decreased by 95-97%, polyaromatic hydrocarbons - by 93-95%, including pyrene derivatives with isomers - by 0.2-0.3 wt.%. The total concentration of oil products at the minimum concentrations of the biological product and Vitaplan decreased by 79-81%, polyaromatic hydrocarbons - by 72-74%, including pyrene derivatives with isomers - by 0.2-0.3 wt.%. The total concentration of petroleum products at concentration values of the biological product and Vitaplan, less than stated in the claims, decreased by 62-63%, polyaromatic hydrocarbons - by 46-48%, including pyrene derivatives with isomers - by 0.1 wt.%, which says about the insufficient efficiency of the cleaning method. The total concentration of petroleum products at concentrations of the biological product and Vitaplan, exceeding those stated in the claims, decreased by 97-99%, polyaromatic hydrocarbons - by 90-93%, including pyrene derivatives with isomers - by 0.6-0.8 wt. %, did not lead to a significant increase in the rate of destruction of the pollutant, but unreasonably increases the cost of cleaning work, since the increase in the rate of biodegradation will be insignificant compared to the increase in the cost of the drug.

Each of the microorganisms: Pseudomonas putida PI Ko-1, Micrococcus sp. PI Ku-1 and Rhodococcus erythropolis AC-1269, as well as Vitaplan, which is a source of gram-positive spore-forming facultative aerobic bacteria, being active destructors of hydrocarbon pollution under conditions of high concentrations of salts of heavy metals and polyaromatic hydrocarbons, contribute to the activation of the destructive activity of other microorganisms that are part of the consortium, which provides a significant increase in the biological activity of both natural destructor microorganisms located in a polluted environment, and microorganisms included in the consortium.

Due to the bubbling of an aqueous suspension of a biological product with air, the enzyme systems of microorganisms are activated due to the action of air oxygen, the enzymes of oil-oxidizing microorganisms are activated and the period of their adaptation is reduced when introduced into the medium being cleaned.

The Rhodococcus erythropolis AC-1269 strain, in addition, has a pronounced ability to form biosurfactants - it synthesizes natural biosurfactants of the glycolipid group in response to the presence of n-alkanes in the medium, which contribute to a decrease in viscosity.

Vitaplan, due to the retention of moisture, creates a favorable background for microorganisms, for the metabolism of exudates, the metabolism of phenol-formaldehyde resin residues.

The proposed method, due to the synergy of the properties of all used microorganisms, can significantly reduce the total time required to purify the aquatic environment in a wide range of pH and ambient temperature (pH 4.5-8.2, T = 3-40°C) from oil and oil products pollution for by increasing the rate of destruction of polyaromatic hydrocarbons.

To implement the method used conventional equipment, known strains of microorganisms and biological product Vitaplan, which are produced industrially.

Method parameters and data on its effectiveness by example

at a consumption rate of 1 g/l of contaminated water

table

No. Quantity of Vita-plan, g/l * The ratio of strains in the biological product, wt.%; Biopreparation concentration, g/l;
Titer, CFU/ml Parameters of water suspension bubbling: air consumption per 1 m ^{2 of} suspension, m ³ ;
time, h Total loss of oil products, %,
loss of polyaromatic hydrocarbons, %,
including pyrene derivatives with isomers, wt.%,
at Ph environment: 4.5 6.8 8.2 one 2 3 4 5 6 7 eight one one 12; 12; 12 10; 1⋅10 ¹² 4; 2.5 97;95;0.2 97;94;0.3 95;93;0.2 2 0.2 5; 5; 5 one; 1⋅10 ⁸ 5; 2 83;77;0.3 83;76;0.3 82;75;0.3 3 0.8 12; 6; 3 2; 1⋅10 ⁹ 4; 2 88;81;0.4 88;80;0.4 86;80;0.4 4 0.9 6; 12; eight eight; 1⋅10 ¹⁰ 4; 2 89;83;0.5 89;83;0.5 87;82;0.4 5 one 7; 10; 12 9; 1⋅10 ¹¹ 4; 2 91;87;0.6 90;86;0.6 89;85;0.5 6 0.06 3; 3; 3 0.5; 1⋅10 ⁸ 5; 1.5 81;74;0.2 80;74;0.3 79;72;0.2 7 0.05 2; 2; 2 0.5; 1⋅10 ⁸ 5; 2.5 63;48;0.1 63;47;0.1 62;46;0.1 eight 1.1 15;15;15 one; 1⋅10 ⁸ 5; 2.5 98;91;0.7 99;93;0.7 97;90;0.6 9 0 12; 12; 0 6; 1⋅10 ⁹ 5; 2.5 55;22;0 54;20;0 53;20;0

* The ratio of strains: Pseudomonas putida PI Ko-1, Micrococcus sp. PI Ku-1, Rhodococcus erythropolis AC-1269, wt.%.

Claims (1)

SUBSTANCE: method of water purification from oil and oil product pollution, which involves introducing into polluted water a suspension of a biological product containing oil-oxidizing microorganisms Pseudomonas putida PI Ko-1 and Micrococcus sp. PI Ku-1, while an aqueous suspension of the biological product is prepared at a concentration of 0.5-10.0 g/l with a titer of 1⋅10 ⁸ -1⋅10 ¹² cells/ml, after which oil-oxidizing microorganisms are activated by bubbling the aqueous suspension of the biological product with air and consumption of 3-5 m ³ per 1 m ^{3 of} suspension for 1.5-2.5 h, characterized in that the biological product additionally contains Rhodococcus erythropolis AC-1269 at a weight ratio of 3-12 wt.% of each microorganism, while in suspension of the biological product, the biological product Vitaplan is introduced in the amount of 0.05-1.0 g per 1 liter of suspension and mixed to obtain a homogeneous suspension, and the homogeneous suspension of the biological product is introduced into the contaminated water in the amount of 1 g per 1 liter of contaminated water.