RU2503511C1 - Method of removal oil products from contaminants - Google Patents

Abstract

FIELD: process engineering.

SUBSTANCE: invention relates to microbiological cleaning of environments. Proposed method comprises introduction of consortium of oil oxidant microorganisms along with introduction of mineral nutrients. Said oil oxidant microorganisms represent strains of bacteria Rhodococcus qingchengii All Russian Collection of Industrial Microorganisms AC-1877, strain of bacteria Rhodococcus erythopolis All Russian Collection of Industrial Microorganisms AC-1878 and strain of yeasts Rhodotorula mucilaginosa All Russian Collection of Industrial Microorganisms Y-3604. Consortium of said oil oxidants is introduced into contaminated medium in amount of 0.2-0.5 wt % of contaminant mass. Note here that along with oil oxidant microorganisms and mineral nutrient introduced in contaminated medium, introduced is surfactant in amount of 0.2-0.5 % of contaminant mass.

EFFECT: higher efficiency of oil products removal.

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Description

The invention relates to biotechnology, in particular to microbiological methods of environmental cleaning, and can be used to clean the earth from oil pollution using a consortium of microorganism strains.

For the processes of cleaning the soil from oil pollution, various microorganisms (bacteria, fungi, yeast) are used individually or their combinations with each other:

- strains of Rhodococcus erythropolis IEGM 708 and Rhodococcus ruber IEGM 327 (RF patent No. 2180276, B09C 1/10, 03/10/2002);

- strain Fusarium sp.N 56 (RF patent No. 2126041, C12N 1/14, 02/10/1999);

- strain Pseudomonas putida 9 (RF patent No. 2134722, C12N 1/20, 08.20.1999).

Known microorganisms differ in the rate of disposal of petroleum products and in terms of use, ensuring their greatest effectiveness (temperature, pH, sources of phosphorus and nitrogen).

However, biopreparations based on consortia from various types of microorganisms have higher efficiency and stability in the natural ecosystem:

- a consortium of strains of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ky-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 (RF patent No. 2191643, B09C 1/10, 10.27.2002);

- a consortium of strains of Rhodocuccus sp.367-2 or Rhodococcus maris 367-5 or Rhodococcus eruythropolis 367-6, strain Pseudomonas stutzeri 367-1 and strain Yarrowia lipolytica 367-3 (RF patent No. 2104249, C02F 3/34, 02/10/1998) ;

- a consortium of strains of Rhodococcus sp., Rhodococcus maris, Rhodococcus erythropolis, Pseudomonas stutzeri, Candida sp. (RF patent No. 2023686, C02F 3/34, 11/30/1994).

In addition, the cleaning of soil and contaminated surfaces can be done with detergents based on surface-active substances (surfactants):

- a mixture of nonionic and ionic surfactants with a ratio of (2-8): 1, respectively (RF patent No. 2169175, C11D 1/83, 06/20/2001);

- a composition containing 85% fatty acid alkyl ester, surfactant and acid, while the concentration of surfactant is from about 0.1 to 10% (RF patent No. 2305121, C09K 8/524, 08/27/2007).

The most acceptable surfactant for cleaning soil from oil pollution is syntanol, which is a mixture of polyethylene glycol ethers and synthetic primary alcohols, since it has high biodegradability (RF patent No. 2412985, C11D 1/02, 02/27/2011).

However, the high operating costs when cleaning the soil with detergent solutions and the problem of disposing of spent detergent solution containing a large amount of oil products make this method unattractive for widespread use, despite the significantly shorter time spent in comparison with cleaning biological products.

The closest analogue to the proposed method (prototype) is a method for cleaning the soil from contamination with oil and oil products (RF patent No. 2191643, B09C 1/10, 10.27.2002), which provides for the introduction into the contaminated environment of a suspension of a biological product containing oil-oxidizing bacteria. An aqueous suspension of a biological product is prepared in 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 sparging an aqueous suspension of a biological product with air at a flow rate of 3-5 m ³ per 1 m ³ suspension for 1.5-2.5 hours. 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.% Each microorganism, while risotorfin is introduced into the contaminated medium in an amount of 30-120 g / m. The cleaning efficiency in this method is 82% - 90% for three months at a daily temperature of 12 ° C to 24 ° C. The disadvantage of this method is the low cleaning efficiency due to the low oxidation rate of oil hydrocarbons and, as a result, the impossibility of cleaning in one summer season. In addition, the use of a strain of soil bacteria Pseudomonas putida and Pseudomonas fluorescens for oil utilization is ineffective, because these types of bacteria poorly utilize hydrocarbons, and they are mainly used for humus formation and to ensure the protective functions of plants. The vital products of these bacteria are siderophores, antibiotic substances and auxin-gibberrelin-like substances that stimulate root formation and plant growth.

The problem to which the invention is directed is the development of a method for cleaning the earth from oil pollution using a consortium of microorganisms (the commercial name of the biological product is Romiger), which ensures a high rate of utilization of oil hydrocarbons.

The technical result, the achievement of which the invention is directed, is to increase the efficiency of cleaning the earth from oil pollution.

To achieve the specified technical result in the proposed method, a consortium of oil-oxidizing microorganisms is introduced into the contaminated environment with the simultaneous introduction of a mineral nutrient medium. The bacterial strain Rhodococcus qingchengii VKPM AC-1877, the bacterial strain Rhodococcus erythopolis VKPM AC-1878 and the yeast strain Rhodotorula mucilaginosa VKPM Y-3604 are used as oil-oxidizing microorganisms. A consortium of oil-oxidizing organisms contribute to the polluted environment in the amount of 0.2% ÷ 0.5% by weight of the pollutant. In this case, together with oil-oxidizing microorganisms, a surfactant is introduced into the contaminated medium in an amount of 0.2% ÷ 0.5% by weight of the pollutant.

The strains used in the proposed method were obtained by breeding from samples of soil samples from the Bovanenkovo oil and gas condensate field of the Yamal Peninsula.

The bacterial strain Rhodococcus qingchengii VKPM AC-1877.

The method of propagation of the strain is fermentation. The optimal composition of the mineral medium for the propagation of the strain: KNO ₃ - 4.0 g / l, KH ₂ PO ₄ - 0.4 g / l, Na ₂ HPO ₄ · 12H ₂ O - 1.4 g / l, MgSO ₄ - 0 , 8 g / l, FeSO ₄ · 7H ₂ O - 0.045 g / l, ZnSO ₄ · 7H ₂ O - 0.031 g / l, MnSO ₄ · 7H ₂ O - 0.031 g / l, CuSO ₄ - 0.004 g / l at pH 7.0-7.2. As a carbon source used liquid paraffin fraction C ₁₃ -C ₁₇ . The cultivation was carried out in a fermenter with a volume of 40 l in the cumulative mode at a rotational speed of the paddle mixer 600 rpm, air supply of 3.5 m ³ / h and a temperature of 32 ° C-34 ° C.

The morphological features of the strain were evaluated using phase contrast microscopy.

The cells of this strain are sticks, cocci, on a solid medium, whitish-colored colonies, matte, with a fine-grained surface.

The strain is non-pathogenic and non-toxic. The culture is stored in test tubes on mown L-agar at a temperature of 15 ° C ÷ 20 ° C. The viability of the strain is maintained by regular reseeding (2-3 times a year) on L-agar medium and on a liquid mineral medium containing nitrogen, phosphorus, potassium, magnesium, trace elements, with n-paraffins as the sole source of carbon and energy.

The bacterial strain Rhodococcus erythopolis VKPM AC-1878.

The method of propagation of the strain is fermentation. The optimal composition of the mineral medium for the propagation of the strain: KNO ₃ - 4.0 g / l, KH ₃ PO ₄ - 0.4 g / l, Na ₂ HPO ₄ · 12H ₂ O - 1.4 g / l, MgSO ₄ - 0 , 8 g / l, FeSO ₄ · 7H ₂ O - 0.045 g / l, ZnSO ₄ · 7H ₂ O - 0.031 g / l, MnSO ₄ · 7H ₂ O - 0.031 g / l, CuSO ₄ - 0.004 g / l at pH 7.0-7.2. As a carbon source used liquid paraffin fraction C ₁₃ -C ₁₇ . The cultivation was carried out in a fermenter with a volume of 40 l in the cumulative mode at a rotational speed of the paddle mixer 600 rpm, air supply of 3.5 m ³ / h and a temperature of 32 ° C ÷ 34 ° C.

The morphological features of the strain were evaluated using phase contrast microscopy.

The cells are large, oval in shape, on a solid medium of a pink colony, with a matte flat surface.

The strain is non-pathogenic and non-toxic. The culture is stored in test tubes on mown L-agar at a temperature of 15 ° C ÷ 20 ° C. The viability of the strain is maintained by regular reseeding (2-3 times a year) on L-agar and on a liquid mineral medium containing nitrogen, phosphorus, potassium, magnesium, trace elements, with n-paraffins as the sole source of carbon and energy.

The yeast strain Rhodotorula mucilaginosa VKPM Y-3604.

The method of propagation of the strain is fermentation. The optimal composition of the mineral medium for the propagation of the strain: H ₃ PO ₄ (70%) - 2.6 g / l, KCl - 1.14 g / l, MgSO ₄ - 0.55 g / l, FeSO ₄ · 7H ₂ O - 0.045 g / l, ZnSO ₄ · 7H ₂ O - 0.031 g / l, MnSO ₄ · 7Н ₂ O - 0.031 g / l, CuSO ₄ - 0.004 g / l at pH 4.0-4.2. As a carbon source used liquid paraffin fraction C ₁₃ -C ₁₇ . The cultivation was carried out in a fermenter with a volume of 40 l in the cumulative mode at a rotational speed of the paddle mixer 600 rpm, air supply of 3.5 m ³ / h and a temperature of 32 ° C ÷ 34 ° C.

The morphological features of the strain were evaluated using phase contrast microscopy.

The cells are rod-shaped, on a solid medium, the colonies are pink in color, matte, with a flat surface.

The strain is non-pathogenic and non-toxic. The culture is stored in test tubes on a mowed agarized wort at a temperature of 15 ° C ÷ 20 ° C. The viability of the strain is maintained by regular reseeding (2-3 times per year) for agarized wort and liquid mineral medium containing nitrogen, phosphorus, potassium, magnesium, trace elements, with n-paraffins as the sole source of carbon and energy.

The introduction of surfactants into a contaminated environment contributes to the destruction of the film of liquid hydrocarbons and water-oil emulsions into fine particles. The resulting dispersed liquid hydrocarbons are much better oxidized microbiologically.

The method is as follows.

Cultures of bacterial strains of Rhodococcus qingchengii VKPM AC-1877 and Rhodococcus erythopolis VKPM AC-1878, which are part of a consortium of microorganisms, are grown together in a liquid nutrient medium. If necessary, the resulting biomass is concentrated, for example, in a centrifuge and dried in a freeze dryer to obtain a powder form of the biological product. Similarly, a powder form of the yeast strain Rhodotorula mucilaginosa VKPM Y-3604 is obtained. Then a consortium of these strains is taken, taken in the ratio of bacteria: yeast, as 2: 1. Determine the amount of oil in the cleaned environment. The required amount of biological product is calculated depending on the type of pollution: 0.2% for light hydrocarbons, 0.5% by weight of pollutants for dark hydrocarbons, respectively. Next, the required amount of surfactant, in particular syntanol, is calculated, depending on the conditions of pollution: 0.2% for purification of light hydrocarbons from oil, 0.5% of the pollutant mass for dark ones. Before introducing into the contaminated medium, the required amount of the powdered form of a biological product is diluted with an aqueous solution of a mineral nutrient medium, including nitrogen, phosphorus, potassium, magnesium, and trace elements, after which the resulting solution is mixed with constant aeration for 3 h ÷ 4 h to activate microorganisms. 2-3 minutes before the end of mixing, aeration is stopped and the required amount of surfactant is added. Then, a solution containing a suspension of a biological product and a surfactant is introduced into a contaminated medium and mixed uniformly.

The following are examples confirming the high efficiency of the proposed method.

Example 1. Carried out the cleaning of the earth from diesel fuel. 10 kg of dry sandy soil were introduced into a rectangular container with a volume of 15 dm ³ . Diesel fuel in the amount of 0.5 kg was applied to the soil with a finely dispersed layer with constant mixing (the initial concentration of oil products in this way was 5%). Then 1 g of dry biological product was dissolved in 2 l of an aqueous solution of a mineral nutrient medium (composition: KNO ₃ - 4.0 g / l, KH ₂ PO ₄ - 0.4 g / l, Na ₂ HPO ₄ · 12H ₂ O - 1 , 4 g / l, MgSO ₄ - 0.8 g / l, FeSO ₄ · 7H ₂ O - 0,045 g / l, ZnSO ₄ · 7H ₂ O - 0,031 g / l, MnSO ₄ · 7H ₂ O - 0,031 g / l, CuSO ₄ - 0.004 g / l,) in an apparatus with a stirrer and bubbler. Aeration and stirring were carried out for 4 hours. 2 minutes before the end of stirring, the air supply was turned off and 1 g of syntanol ALM-10 was added to the suspension (TU 2483-003-71150986-2006). The resulting solution was added to contaminated soil with stirring. Soil moisture was maintained by periodic irrigation at 60% of the total soil moisture capacity. Aeration was carried out by periodically mixing the soil, while the soil temperature was 18 ° C ÷ 20 ° C. Sampling was carried out once every five days. The oil content in the soil was determined by IR spectrometry. As a result, over 60 days of cleaning, the concentration of oil products in the soil decreased from 5% to 0.42%, i.e. the degree of purification was 91.6%.

Example 2. Carried out the cleaning of the soil from fuel oil. 10 kg of dry sandy soil were introduced into a rectangular container with a volume of 15 dm ³ . Fuel oil in the amount of 0.5 kg was applied dropwise to the soil layer with constant mixing (initial concentration of oil products was 5%). Then, 2.5 g of dry biological product was dissolved in 2 l of an aqueous solution of a mineral nutrient medium (composition: KHO ₃ - 4.0 g / l, KH ₂ PO ₄ - 0.4 g / l, Na ₂ HPO ₄ · 12H ₂ O - 1.4 g / l, MgSO ₄ - 0.8 g / l, FeSO ₄ · 7H ₂ O - 0,045 g / l, ZnSO ₄ · 7Н ₂ O - 0,031 g / l, MnSO ₄ · 7Н ₂ O - 0,031 g / l, CuSO ₄ - 0.004 g / l,) in an apparatus with a stirrer and bubbler. Aeration and stirring were performed for 4 hours. 2 minutes before the end of the process, the air supply was turned off and 2.5 g of syntanol ALM-10 was added to the suspension (TU 2483-003-71150986-2006). The resulting solution was added to contaminated soil with stirring. Soil moisture was maintained by periodic irrigation at 60% of the total soil moisture capacity. Aeration was carried out by periodically mixing the soil, while the soil temperature was 18 ° C-20 ° C. Sampling was carried out once every five days. The oil content in the soil was determined by IR spectrometry. As a result, over 60 days of cleaning, the concentration of oil products in the soil decreased from 5% to 0.76%, i.e. the degree of purification was 84.8%.

The proposed method allows to increase the efficiency of land purification from pollution by oil products through the use of a consortium of microorganisms that provide a high rate of utilization of oil products.

Claims (1)

A method of cleaning the earth from oil product pollution, including introducing into the polluted environment a consortium of oil-oxidizing microorganisms with the simultaneous introduction of a mineral nutrient medium, characterized in that the bacterial strain Rhodococcus qingchengii VKPM AC-1877, the bacterial strain Rhodococcus erythopolis VKPM and VKPM are used as oil-oxidizing microorganisms yeast Rhodotorula mucilaginosa VKPM Y-3604, a consortium of oil-oxidizing organisms contribute to the polluted medium in the amount of 0.2% ÷ 0.5% by weight of the pollutant, while together with the oil-oxidizing mik By means of organisms, a surfactant is introduced into the contaminated medium in an amount of 0.2% ÷ 0.5% by weight of the pollutant.