RU2617941C2 - Thermotolerant bacterial strains consortium for oil and oil products degradation in soil and waters in hot climates - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention can be used as a basis of a biological product for soil and water remediation, including saline ones, in hot climates. The consortium contains Gordonia amicalis, Rhodococcus erythropolis and Rhodococcus pyridinivorans bacteria, deposited in the Russian National Collection of Microorganisms IBPM named after G.K. Skryabin RAS under accession number VKM Ac-D 2720, VKM Ac-D 2722 and VKM Ac-D 2721, respectively. At that, the number of microorganisms for the consortium on the treated area should be at least 1×10⁴-1×10⁵ CFU/g of soil (1×10⁴-1×10⁵ CFU/ml), depending on the contamination level.

EFFECT: invention improves the efficiency of soil and water purification at temperatures up to 50 degrees, soil and water salinity to 7 percent and 10 percent soil humidity.

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Description

The invention relates to the field of environmental biotechnology and biotechnology of environmental protection and can be used when carrying out activities to clean oil-contaminated soils and water in a hot climate. The use of thermo-tolerant bacteria allows the treatment of soils and water at temperatures from 20 ° C to 50 ° C, the consortium remains viable up to 53 ° C.

The invention is a consortium of thermotolerant non-pathogenic bacteria capable of efficiently disposing of oil and oil products at elevated temperatures. The consortium includes three bacterial strains - Gordonia amicalis VKM Ac-2720 D, Rhodococcus erythropolis VKM Ac-2722 D, Rhodococcus pyridinivorans VKM Ac-2721 D. It is possible to use the consortium in saline (up to 7% salt) soils and waters, in the pH range 6-8, as well as with low soil moisture (about 10%). The drug is effective at a level of soil contamination up to 10% (100 g of oil per 1 kg of soil), in case of a higher oil content, it is necessary to reduce its concentration by available methods. For effective cleaning, the number of microorganisms of the consortium in the treated area should be at least 1 × 10 ⁴ -1 × 10 ⁵ CFU / g of soil (1 × 10 ⁴ -1 × 10 ⁵ CFU / ml) depending on the level of contamination.

Evaluation of the effectiveness of cleaning soil or water using a consortium is carried out by measuring the residual hydrocarbon content in comparison with the control to assess abiotic loss. The technical result consists in reducing the concentration of oil hydrocarbons in soil and water at elevated temperatures. The implementation of the method of cleaning soils and waters by a consortium of thermo-tolerant bacteria is an environmentally safe and promising approach to eliminating the effects of oil spills in hot climates and with increased salinity of the soil and water.

Technical field

The invention relates to the field of environmental biotechnology and can be used in the aftermath of oil spills and the cleaning of oil-contaminated soils and waters in situ and ex situ in hot climates.

The main risk due to environmental pollution by oil and oil products are exposed to the regions where the fields are located and oil is actively extracted and transported. More than half of the world's oil reserves are located in areas with a hot climate, where the specifics of natural, in particular temperature conditions, forces us to more carefully approach the choice of an adequate remediation method for contaminated lands.

One of the main problems of remediation of territories in hot climates is the fact that high temperatures reduce the viscosity of oil and, thus, accelerate its diffusion deep into the soil. Also, the high evaporation rates of light oil fractions at elevated temperatures lead to air pollution with toxic products, while the remaining non-volatile components with a high molecular weight form films that are poorly biodegradable. High rates of water evaporation at temperatures above 40 ° C lead to salinization of soils and waters. Drilling fluids that enter the soil during oil production also contribute to salinization of the territories. The use of a consortium of thermotolerant bacteria-oil destructors makes it possible to remove hydrocarbons from soil and water in such conditions. The bacteria that make up the consortium are able to dispose of oil at temperatures up to 50 ° C, salinity up to 7%, pH 6-8 and soil moisture 10%.

State of the art

A significant part of oil-contaminated sites in Russia and the CIS countries is located in regions with a hot climate. However, in a patent search, no commercial biological products intended for cleaning oil-contaminated areas in hot climates were found. However, there are drugs based on both consortia and monocultures, with the upper limit of the working temperature range within 40-50 ° C.

Known biological product for cleaning soil and water from oil (RF patent No. 2365438, Karaseva E.V. et al., KubSU, 2006). The composition of the drug includes microorganisms Arthrobacter sp. BKM Ac-2272 D and Rhodococcus sp. BKM Ac-2045 D; and also (wt.%): glycerol - 8-10, KNO ₃ - 0.360-0.367, KH ₂ PO ₄ - 0.054-0.055, Na ₂ HPO ₄ ⋅12H ₂ O - 0.126-0.128. The number of viable cells in the preparation is assumed to be at least 3-10 ¹⁰ cells / ml. Cells of microorganisms of strains of Arthrobacter sp. BKM Ac-2272 D and Rhodococcus sp. BKM Ac-2045 D was grown together under conditions of deep cultivation in a liquid mineral medium with diesel fuel as a carbon source.

The disadvantage of the drug is the inability of the bacteria that make up its composition to dispose of oil and its products at temperatures above 40 ° C. In addition, the drug is introduced in the form of a culture fluid, which requires a large flow of water during the remediation of large areas.

Known biological product "Destroyl" and the method of its use in wastewater treatment plants for the disposal of petroleum products (application 2008129964/13, Nadein AF). The Destroil preparation contains a monoculture of Acinetobacter sp., The number of viable cells is 1 × 10 ⁸ CFU / ml. The working conditions of the drug: pH 4.5-8.5 and temperatures - from +5 to + 38 ° C, viable to + 42 ° C. In the method of remediation, based on the joint use of the drug and activated sludge in aeration tanks, the temperature of the wastewater should not be higher than 22 ° C. The consumption of the drug is 2 g per 1 m ³ water-sludge mixture located in the aeration tank.

The disadvantage of the drug is the use of monoculture, which narrows the spectrum of compounds oxidized in the process of remediation. Also, the use of a gram-negative strain as the main agent of bioremediation limits the maximum temperature of use of the drug.

Known biological product "Biooil-Yugra" (RF patent No. 2337069, Alekseev A.Yu. et al., 2007), based on the association of yeast and bacterial strains: Saccharomyces sp. KKM SSC WB "Vector" No. Y-1036, Bacillus sp. KKM SSC WB "Vector" No. B-1040, Bacillus sp. KKM SSC WB "Vector" No. B-1039, Enterobacter sp. KKM SSC WB "Vector" No. В-1038. The composition also includes protectors (sucrose, agarose and gelatin). The working range of the drug: temperature - from + 1 ° C to + 41 ° C (optimum 7-27 C); pH 4.5-9.0, salinity up to 7%.

The microbial association is selected for the disposal of oil in temperate and cold climates. The disadvantage of the drug when used in hot climates is its low efficiency with increasing temperature.

The known drug "Oleovorin" (Order of the Goskomekologiya RF dated 04/02/97 No. 139 on the approval of the conclusion of the expert commission of the state environmental review of materials for environmental substantiation of technical conditions (TU) on the bacterial drug "Oleovorin"). The drug "Oleovorin" was developed on the basis of bacterial strains of the genus Acinetobacter oleovorum isolated from a natural biocenosis (strains VSB-712 and VSB-568), which have oxidative activity in relation to oil and oil products. The technical specifications of the preparation indicate that Oleovorin is active in the disposal of mono- and polyaromatic hydrocarbons. There is no patent for Oleovorin.

The disadvantage of a biological product is its low activity against alkanes, which make up up to 70% of all oil hydrocarbons. In addition, the use of two closely related strains in a preparation brings its properties closer to monoculture based preparations.

In the patent for invention No. 2014286 (Bitteeva MB et al., 1994) it is proposed to use the thermotolerant associative strains of bacteria Mycococcus lactis VSB-574 and VSB-D-5 for cleaning the water surface and tanks from oil products, as well as wastewater containing petroleum products. The strains were cultured at 40-42 ° C, pH 6.8-7.0, on mineral medium N9 (composition, 1 l: NH ₄ H ₂ PO ₄ - 10 g, K ₂ HPO ₄ - 10 g, MgSO ₄ - 0.7 g, trace elements Fe ₂ (SO ₄ ) ₃ - 12.5 mg, ZnSO ₄ - 12.5 mg, MnSO ₄ - 12.5 mg, CuSO ₄ - 3 mg). As a carbon source used engine oil, oil with tarred sediment in a concentration of 0.5 vol. %, dewaxing of diesel fuel at a concentration of 10 vol. %

The use of closely related strains brings the properties of the drug closer to monoculture. The low used concentrations of pollutants in the tests suggest that the effectiveness of such a drug may decrease with an increase in the content of petroleum products in the culture medium.

Known biological product "Devoroil" (RF patent No. 2023686, C02F 3/34, 1994.11.30), developed on the basis of a consortium of microorganism strains: Rhodococcus erytropolis 367-2, Rhodococcus maris 367-5, Rhodococcus erytropolis 367-6, Pseudomonas stutzeri 367-6 1, Candida lipolytica 367-3. The microorganisms included in the preparation are capable of growth and destruction of hydrocarbons in a wide range of salinity (from 0.05% to 15%). The operating temperature range is + 5 ° C to + 40 ° C.

The disadvantage of Devoroil is the inability to utilize hydrocarbons by microorganisms at temperatures above 40 ° C.

The closest analogue of the proposed microbial consortium for remediation of soils and water in hot climates is the biological product "Mikrozim Petrotrit", developed by RSE Trading (Moscow), sanitary and epidemiological conclusion No. 77.99.02.515.D.001102.03.05 of 03/11/2005. However, there is no patent for the drug. The composition of "Microzyme" includes a consortium of microorganisms of the genera Bacillus sp., Atherobacter sp., Nocardia sp., Rhodococcus sp., Pseudomonas sp., Salts, carrier; the number of viable cells in the preparation is assumed to be 10 ¹² CFU / g. Corn flour is used as a carrier. “Microzyme” can be used with Nutrizim top dressing. Working range of the drug: temperature from + 5 ° C to + 50 ° C (optimum from + 10 ° C to + 40 ° C), pH 4-10.

The disadvantage of the drug “Microzym Petrotrit” is a large number (12 strains) of microorganisms used, which significantly complicates the technology for its production and increases the cost of its production and biomass production.

Despite the presence on the Russian market of biological products with a working temperature limit of about 50 ° C, according to publicly available information, their use is mainly designed for moderate or even cold climates. There is no information on testing these drugs at elevated temperatures.

SUMMARY OF THE INVENTION

The development task is to create an effective microbial consortium for cleaning soil and water from oil both at moderate and elevated (up to 50 ° C) temperatures. The solution to the problem is achieved through the use of thermo-tolerant bacteria capable of utilizing oil in the temperature range of 20-50 ° C, which makes them promising agents for the remediation of oil-contaminated soils and waters in regions with a hot climate.

Taking into account the high rates of water evaporation at elevated temperatures and, as a consequence, the salinity of soils and aquatic ecosystems, the consortium was developed using microorganisms capable of utilizing hydrocarbons in the presence of salt in the medium (up to 7%) and at low humidity (about 10% )

The consortium includes bacteria-destructors of both aliphatic and aromatic fractions of oil. The use of bacteria capable of degradation of a wide range of substrates provides a more efficient removal of oil from soils and aquatic ecosystems. The effectiveness of the developed consortium of bacteria in the removal of oil hydrocarbons in soil and water was confirmed using model experiments both at normal (24 ° C) and elevated (45 ° C) temperatures. When cultured in a liquid medium with 2% oil and 3% salt, the consortium provides the destruction of 68% hydrocarbons at 24 ° C and 38% hydrocarbons at 45 ° C for 14 days. In the process of oil destruction in soil, the consortium utilized 70% of oil at 24 ° C and 59% of oil at 45 ° C (initial oil concentration of 2%, salinity of 3%, soil moisture of 10%) for 21 days.

The developed microbial consortium can be used in the course of restoration of oil-contaminated soils and water, and the elimination of the consequences of emergency oil and oil products spills in regions with a hot climate.

Information confirming the possibility of carrying out the invention

The invention is presented by a consortium of thermotolerant microorganisms-oil destructors and can be used in remediation of oil-contaminated soils and waters in regions with a hot climate. The consortium is composed of one strain of bacteria of the genus Gordonia and two strains of bacteria of the genus Rhodococcus, is effective in the temperature range of 20-50 ° C, the maximum working temperature of the consortium is 53 ° C. The consortium can also be used in saline (up to 7%) soils and waters.

The effectiveness of the invention in the removal of oil hydrocarbons in soil and water was confirmed by model experiments both at normal (24 ° C) and at elevated (45 ° C) temperatures.

When modeling water and ground media, it was shown that a consortium of three strains utilized oil more efficiently than individual crops. The possibility of use and effectiveness of the invention is confirmed by the following examples.

Example 1. Monitoring abiotic loss of oil in aqueous systems.

Sea salt to a final concentration of 3% and oil to a final concentration of 2% are added to the Evans liquid mineral medium or sterile tap water. Model systems are closed to prevent the ingress of extraneous microflora, however, they are continuously leaked continuously to ensure evaporation, during the whole experiment. The experiment is carried out both at 24 ° C and at 45 ° C. At the end of the 14th day of the experiment, the residual concentration of hydrocarbons in the samples is measured by IR spectroscopy using an AN-2 oil analyzer in accordance with RD 52.24.476-2007 “Mass concentration of oil products in waters. The measurement technique using the IR-photometric method ", after which they evaluate abiotic decrease in systems without microorganisms.

The abiotic loss of oil in the experiment at 24 ° C and 45 ° C was 6% and 11%, respectively, after 14 days.

Example 2. The destruction of oil by microorganisms in aqueous systems.

The systems are prepared according to the same scheme as in Example 1. The experiment is carried out both with monocultures of microorganisms and with a consortium:

a) a cell suspension of a strain of Gordonia amicalis VKM Ac-2720 D;

b) a cell suspension of a strain of Rhodococcus erythropolis VKM Ac-2722 D;

c) a cell suspension of a strain of Rhodococcus pyridinivorans VKM Ac-2721 D;

d) a consortium of three strains in a ratio of 1: 1: 1.

The number of microorganisms in the experiment should be at least 1 × 10 ⁴ CFU / g of soil (1 × 10 ⁴ CFU / ml of water). To obtain a consortium, monocultures are grown separately, then monoculture suspensions are prepared with a cell concentration of at least 1 × 10 ⁴ CFU / ml and mixed in equal proportions.

The experiment is carried out in parallel at 24 ° C and 45 ° C. At the end of the experiment, the residual concentration of hydrocarbons in the samples is measured and abiotic loss is estimated in systems without microorganisms.

In FIG. 1 shows the results of purification - the degree of microbial degradation of oil by G. amicalis VKM As-2720 D, R. erythropolis VKM As-2722 D, R. pyridinivorans VKM As-2721 D strains and a consortium composed of these three strains in water model systems is shown net of abiotic attrition. Abiotic oil loss after 14 days at 24 ° C and 45 ° C was 6% and 11%, respectively. In a liquid cultivation medium with 2% oil and 3% salt, the consortium provides 68% destruction at 24 ° C and 38% destruction at 45 ° C for 14 days.

Example 3. Monitoring abiotic loss of oil in soil systems.

In sterile sand, sea salt is added to a final concentration of 3%, oil to a final concentration of 2%, and sterile liquid mineral medium or tap water to a final concentration of 10%. Systems with sand are closed to prevent the ingress of extraneous microflora, however, it is leaky to ensure evaporation, and they are loosened every 3-4 days during the entire experiment. The experiment is carried out both at 24 ° C and at 45 ° C. At the end of the experiment, the residual concentration of hydrocarbons is measured by IR spectroscopy using an AN-2 oil analyzer in accordance with MUK 4.1.1956-05 "Determination of oil concentration in the soil by infrared spectrophotometry", after which the abiotic loss in systems without microorganisms is evaluated.

Example 4. The destruction of oil by microorganisms in soil systems

The preparation of the systems is carried out according to the same scheme as in Example 3. The experiment is carried out both with monocultures of microorganisms and with a consortium. The system options are described in Example 2. The number of microorganisms in the experiment should be at least 1 × 10 ⁴ CFU / g of soil (1 × 10 ⁴ CFU / ml). To obtain a consortium, monocultures are grown separately, then monoculture suspensions are prepared with a cell concentration of 1 × 10 ⁴ CFU / ml and mixed in equal proportions.

The experiment is carried out in parallel at 24 ° C and 45 ° C. At the end of the experiment, the residual concentration of hydrocarbons in the samples is measured and abiotic loss is estimated in systems without microorganisms.

In FIG. 2 shows the cleaning results - the degree of microbial degradation of oil by G. amicalis VKM As-2720 D, R. erythropolis VKM As-2722 D, R. pyridinivorans VKM As-2721 D strains and a consortium composed of these three strains in the soil minus abiotic attrition. Abiotic oil loss after 21 days at 24 ° C and 45 ° C was 20% and 33%, respectively. In the process of oil destruction in the soil, the consortium disposed of 70 and 59% of oil (at temperatures of 24 ° C and 45 ° C, respectively) at an initial oil concentration of 2%, salinity of 3%, and humidity of 10% in 21 days.

Claims (1)

A consortium of bacterial strains of Gordonia amicalis VKM Ac-2720 D, Rhodococcus erythropolis VKM Ac-2722 D, Rhodococcus pyridinivorans VKM Ac-2721 D for the destruction of oil and oil products in soils and waters in regions with a hot climate, characterized by the fact that it is able to utilize oil and in the temperature range of 20-50 ° C and with salinity of up to 7%, in the pH range of 6-8, with soil moisture of about 10% and oil concentration in soil / water up to 10%.

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