RU2509150C2 - Association of strains of bacteria-oil decomposers, and remediation method of oil-contaminated objects - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes an association of strains of bacteria-oil decomposers, which have been extracted from oil-contaminated soil, Acinetobacter species B-1037, Pseudomonas species B-989, Bacillus species B-1040, deposited at The State Research Centre of Virology and Biotechnology VECTOR. Besides, at least 30% bacteria of each strain is contained in the association. Remediation of oil-contaminated soils includes water suspension of lyophilic dried biomass of the strain association based on 10⁹ cells per square metre. Strains of the association can utilise a wide range of oil components at the temperature of 10-15°C.

EFFECT: improving cleaning efficiency of oil-contaminated soils.

2 cl, 5 dwg, 2 tbl, 4 ex

Description

The invention relates to biotechnology for environmental protection, in particular for remediation of soils contaminated with oil products.

Biological products created on the basis of associations of microorganisms-oil destructors capable of oxidizing a wide range of oil hydrocarbons - from long-chain alkanes to polyaromatic compounds, are successfully used in the technology of remediation (purification) of oil-contaminated objects. Biological products are intended for cleaning soil and water areas from oil and oil products, for restoring the functions of soil and water self-cleaning, wastewater treatment of industrial enterprises, wastewater treatment of car washes, service stations, gas stations, territories of oil and oil refining industry enterprises, dewaxing wells, technical tanks.

Known commercial preparations based on oil-oxidizing microorganisms. The composition of the preparations includes monocultures (Putidul, Ekoil, Destroil, etc.) or associations with two or more than twenty types of various oil-oxidizing microorganisms: bacteria, fungi, yeast (Devoroil, Simbinal, " Rodotorin ") (table 1).

Recently, biologics consisting of 2 or more strains are increasingly used, since the use of monoculture cannot completely solve the problem of purification. Oil is a complex multicomponent substrate containing several hundred different chemical compounds (Baryshnikova L.M., Grischenkov V.G., Arinbasarov M.U., Shkidchenko A.N., Voronin AM Biodegradation of oil products by destructive strains and their associations in a liquid medium // Prikl. Biochemical and microbiol. - 2001. - T.37. - No. 5. - S.542-548), and one strain is not able to possess the entire spectrum of enzymes necessary for biodegradation. The use of several strains that differ in the spectrum of the consumed substrates can lead to complete destruction of oil (Kobzev E.N., Petrikevich S.B., Shkidchenko A.N. Study of the stability of the association of microorganisms-oil destructors in an open system // Prikl. Biochem. and microbiol. - 2001. - T.37. - No. 4. - S.413-418).

Under the conditions of natural microbiocenosis, the simultaneous assimilation of different oil fractions by various groups of microorganisms is observed (Shkidchenko A.N., Arinbasarov M.U. Study of oil destructive activity of microflora of the coastal zone of the Caspian Sea // Prikl. Biochem. And microbiol. - 2001. - T. 38. - No. 5. - S. 509-512).

With the joint use of several strains of destructors in the consortium, their oil-utilizing effect is enhanced. The reasons for this are:

- different priority use of oil components by different strains;

- different growth rates of microorganisms;

- metabolites produced by the strain, which may be growth factors for other strains of the consortium.

The conditions for the use of most biological products require the organization of oxygen access to the soil, maintaining soil moisture of at least 40% by regular sprinkling and turning over. The working pH of biological products is usually from 5 to 9, the working temperature is from +10 to + 40 ° C. When the ambient temperature drops to + 5 ° С, the growth of bacteria slows down, until the biological activity is completely stopped. With a subsequent increase in temperature, microorganisms begin to multiply again.

When choosing a strain for drug development, one should usually take into account criteria such as high oil oxidizing activity, resistance to heavy metal salts, non-pathogenicity and non-toxicity of the strain for humans and animals (Koronelli T.V. Principles and methods of intensifying the biological destruction of hydrocarbons in the environment // Prikl. Biochem. and Microbiol. - 1996. - T.32. - No. 6. - S.579-585). Due to the fact that the microbiological treatment technology for contaminated soils involves aerobic conditions, it is necessary to choose a microorganism-destructor among aerobic and facultative anaerobic strains. In addition, the cells of the strain must have high viability in order to be able to grow and utilize the oil product in a wide range of temperatures, pH, humidity, lack of nutrients (Stabnikova E.V., Selezneva M.V., Reva O.N. , Ivanov V.N. Choice of an active microorganism-degrader of hydrocarbons for cleaning oil-contaminated soils // Prikl. Biochemical and microbiol. - 1995. - Volume 31. - No. 5. - S.534-539).

In Russia there are various biological products designed to clean soils from oil pollution (commercial names of the preparations are Avalon, Batsispetsin, Valentis, Devoroil, Dostroil, Naftoks, Nikoil, Petrolan, Putidul, Roder, Universal, etc.) (RF Patent No. 2077397); (Uncle 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, N 9, p.148-151); (RF patents No. 2114071, 2053205).

The most famous of them and have long been used in practice are Putidul biologics (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, N 9, p. 148-151) and Devoroil (patent No. 2114071 from 05.22.97).

It is known that the drug Putidul consists of one strain of the bacterium Pseudomonas putida 36. The drug is obtained by deep cultivation of the bacteria in a nutrient medium at 30 ° C, under aerobic conditions, followed by spray drying or lyophilization of the obtained culture fluid (RF patent 2053205). Since Putidul is a monoculture, it has less potential and a narrower spectrum of action on hydrocarbons than preparations consisting of two or more strains of microorganisms, for example, Devoroil.

It is known that the composition of the drug Devoroil includes strains of Pseudomonas stutzeri, Rhodococcus erythropolis, Rhodococcus maris, Rhodococcus sp., Yarrowia lipolytica (formerly Candida sp.) (RF patent No. 2114071 from 05.22.97). In accordance with the patent, Devoroil is also obtained by deep cultivation in the nutrient medium of its microorganisms and subsequent drying of the resulting biomass. However, Devoroil consists of microorganisms of various taxonomic and species belonging, requiring separate cultivation, which significantly complicates and increases the cost of the drug production process. On the other hand, during the joint cultivation of microorganisms belonging to different taxonomic groups and differing in growth rate, substrate specificity, temperature optimum of growth, etc., there is a competition for food sources, and as a result, not all of the declared species survive in such a preparation and, accordingly, the effectiveness of such a drug decreases.

The disadvantage of the Devoroil biological product, in addition to the need for separate cultivation of the microorganisms included in it, is the use of an expensive carbohydrate medium for biomass preparation and a higher cultivation temperature of 20-40 ° C, which limits the scope to only in central and southern Russia.

A known consortium of strains of microorganisms Bacillus brevis and Arthrobacter species, used to purify water and soil from oil and oil products (RF patent No. 2232806). The disadvantage of this method should be recognized as its low efficiency in cases of a high degree of oil pollution.

The natural microbial community of the soil, which includes a large number of bacteria-destructors, at low doses of pollution copes with the decomposition of petroleum hydrocarbons, so with small pollution (less than 10%) in central Russia it is more rational to create suitable conditions for the activity of native bacteria than to introduce new species. However, in the case of northern ecosystems with depleted species diversity and limiting amounts of nutrients, even with pollution of about 10% it is more advisable to use biological products. The introduction of adapted microorganisms that destroy petroleum products is also recommended if the physicochemical characteristics of the pollution site make it impossible to grow a natural microbiota (high concentrations of heavy metals, salinity of the soil, extreme pH values, etc.).

The objective of the invention is the creation of an association of bacteria that oxidize oil and oil products, and a method for the remediation of oil-contaminated objects in the Siberian North using this association, the components of which are indigenous strains, do not show antagonism towards endogenous microorganisms, are able to grow at low temperatures (4-10 ° C) , low pH (4.5-6), high NaCl (1-4%), dispose of a wide range of oil components at temperatures of 10-15 ° C.

The technical result of the invention is to increase the efficiency of cleaning oil-contaminated objects at low ambient temperatures (3-15 ° C).

The problem was solved by creating an association of bacteria isolated from oil-contaminated soil of the Siberian North, deposited in the collection of microorganism cultures of the State Scientific Center for Virology and Biotechnology "Vector":

Acinetobacter species, registration number B-1037; Pseudomonas species, registration number B-989; Bacillus species, registration number B-1040, taken in approximately equal amounts. For remediation of oil-contaminated objects, an aqueous suspension of leophilically dried association bacteria is used at a rate of 10 ⁹ cells per m ² .

Sample collection. In order not to violate the biodiversity of the ecosystem, each region needs its own characterized composition of microorganisms.

Oil-contaminated soils in oil production areas (Khanty-Mansi Autonomous Okrug, Yamalo-Nenets Autonomous Okrug) were used as substrates for isolating strains having a wide range of enzymes that destroy complex organic substances. Samples of water and sludge were placed in 50 ml sterile plastic tubes, soil and soil samples were placed in sterile paper bags and in 50 ml sterile plastic tubes.

Isolation of oil-degrading microorganisms. Samples of natural substrates were incubated in a liquid selective nutrient medium 8E (g / l): (NH ₄ ) ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 0.7; MgSO ₄ × 7H ₂ O - 0.8; NaCl - 0.5; pH 7.2 with the addition of oil by weight (up to 0.5 - 2%) on a rocking chair at temperatures of 10 ° C; then, the resulting accumulation cultures were transferred to 8E agar medium. Microorganisms with the ability to actively grow on selective nutrient media with oil were isolated in pure cultures. A total of 34 strains were isolated, of which 3 strains were selected, which possess high oil utilization ability, a relatively high growth rate at a temperature of 10 ° C, acidic pH values and an increased salt content in the medium. Strains were identified by biochemical and physiological characteristics. Primary biodegradation of oil in test tubes was evaluated by the following parameters: breaking the surface film of oil, clouding of the nutrient medium due to the growth of biomass, the formation of a homogeneous emulsion of oil in the medium, gas formation.

The morphological features of the obtained destructor isolates were studied using light microscopy of living and stained cells using a Carl Zeiss Axioskop 40 microscope (Carl Zeiss, Germany). The study of physiological, biochemical properties was carried out by standard methods (Methods of General Bacteriology, 1983; Determinant of bacteria ..., 2001).

Selected strains have the following morphological, physiological and biochemical properties.

The strain Acinetobacter species B-1037 on dense nutrient media forms whitish, translucent, shiny, smooth colonies with a smooth edge; the cells of the strain are gram-negative, motile rods, 0.8 × 1.7-2 microns in size. The strain Acinetobacter species B 1037 is an aerobic, grows in the temperature range from 4-6 to 35 ° C; does not hydrolyze starch, casein, does not have proteolytic activity, is catalase-positive, ferments glucose, lactose, mannitol, maltose, sucrose, sorbitol with gas formation, does not dilute gelatin. It has citrate activity, grows on RPA with a NaCl concentration of 5%; does not have hemolytic properties.

The strain Bacillus species 1040 on dense nutrient media forms whitish, translucent, shiny, smooth colonies with a smooth edge; the cells of the strain are gram-positive bacilli, 1 × 1.2-1.5 μm in size. The strain Bacillus species 1040 is an aerobic, growing in the temperature range from 4-6 to 35 ° C; does not hydrolyze starch, does not thin gelatin, does not form acetoin, does not utilize glucose, galactose, xylose, ramnose, dulcite, mannitol, maltose, arabinose, sucrose, sorbitol, inositol, lactose. It has citrate, catalytic, fibrinolytic activity, does not have lipase, plasmocoagulase, oxidase, lecithinase, urease, caseinolytic activity on milk agar, hemolytic properties.

The strain Pseudomonas species 989 on dense nutrient media forms whitish, translucent, shiny, smooth colonies with a smooth edge; cells of the strain are gram-negative shortened rods of 1-2 and in short chains, 0.9-1.0 × 1.5-1.8 μm in size. The strain Pseudomonas species 989 is an aerobic strain, grows in the temperature range from 4-6 to 35 ° C; does not hydrolyze starch, does not thin gelatin, does not form acetoin, does not utilize glucose, lactose, galactose, xylose, rhamnose, dulcite, mannitol, maltose, arabinose, sucrose, sorbitol, inositol. It has catalytic, oxidase activity, caseinolytic activity on milk agar, does not have citrate, lecithinase, lipase, urease activity, hemolytic, fibrinolytic, plasma-coagulase properties. Pseudomonas species is resistant to penicillin, ampicillin, oleandomycin, oxacillin, ristomycin, carbenicillin, lincomycin, rifampicin, chloramphenicol; sensitive to erythromycin, monomycin, tetracycline, streptomycin, kanamycin, neomycin, gentamicin, polymyxin.

The nature of the relationship of the association strains with endogenous soil microbiota was studied experimentally. The absence of antagonism is illustrated by the photographs shown in Fig. 1, where the horizontal strip is the sowing of indigenous microorganisms isolated from samples of oil-contaminated soil of the Yamal-Nenets Autonomous Okrug, the vertical strip is the sowing of the association of microorganisms Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989. As can be seen, the association of microorganisms Ac species 1037, Bacillus species 1040 and Pseudomonas species 989 does not inhibit the growth of indigenous microorganisms isolated from oil-contaminated soil of the Yamalo-Nenets Autonomous Okrug, because endogenous bacteria grow equally intensively both in the presence of association (photo on the left) and in the control (photo on the right). Therefore, when introducing large amounts of biomass of the strains, inhibition of local microbiota and crowding out of endogenous microorganisms will not be observed.

Example 1. Getting Association

An association was composed of bacteria Acinetobacter species 1037, Bacillus species 1040, and Pseudomonas species 989. For this, bacteria grown in meat-peptone broth to a concentration of 1-3 × 10 ⁹ cells / ml were mixed so that the number of bacteria of each strain was not less than 30% in the mixture. After that, the bacterial mass was poured into 3 ml ampoules, frozen at 70 ° C for a day, then transferred to a freeze drying apparatus, the vacuum was turned on and dried for 2 days. Sealed ampoules were stored at 4 ° C.

After storage for a month, the concentration of bacteria of each strain in the ampoule was determined. For this, the ampoule was opened, 3 ml of a sterile 0.9% NaCl solution (physiological solution) was introduced into it, the contents of the ampoule were transferred into a volumetric flask, the contents were brought to 100 ml with physiological saline. The suspension was held for 12 hours at 18-20 ° C. Then, ten-fold dilutions were made in physiological saline and plated on Petri dishes. After culturing for 2 days at room temperature, the grown colonies were counted and the concentration of each strain in the association was determined. The results are presented in table 1.

Table 1 The concentration of bacteria Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 in association after freeze drying and storage for a month at 4 ° C Acinetobacter species 1037 Bacillus species 1040 Pseudomonas species 989 1.20 × 10 ⁹ 1.05 × 10 ⁹ 1.15 × 10 ⁹ 35.29% 30.88% 33.82%

The association was tested for the ability to utilize oil components and for the ability to bioremediation of oil-contaminated objects.

Example 2. The growth rate of the association at 10 ° C and 30 ° C

The growth rate of bacterial associations was studied in 8E liquid medium with the addition of 1% glucose at a temperature of 10 and 30 ° C. Microorganisms were quantified by the Koch plate method by counting the grown colonies (Methods of General Bacteriology, 1983).

The number and ratio of strain cells was determined by microscopy of suspensions at different stages of cultivation using a phase contrast microscope, plating on agarized media to obtain isolated colonies with their further counting and identification (Labinskaya, 1978). When assessing the activity of strains in the association, the change in the optical density of the culture fluid during incubation of the strains was taken into account. The results are presented in Fig. 2, where the growth dynamics of the association of Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 at t = 30 ° C and 10 ° C (mineral medium, without aeration) is shown.

As can be seen from Fig. 2, the bacteria association Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 grows well at 10 ° C, reaching 10 ^8.5 cells / ml by the 12th day, while at 30 ° C the maximum concentration there was only 10 ^7.5 cells / ml.

After 12 days of cultivation, the suspension was tested for a percentage in the environment of bacteria of different strains. It amounted to approximately Acinetobacter species 1037: Bacillus species 1040: Pseudomonas species 989 as 1.2: 0.9: 1. Thus, the growth rate of association bacteria is approximately the same, and when the Acinetobacter species 1037, Bacillus species 1040, and Pseudomonas species 989 strains are jointly cultured, some bacteria are not replaced by others.

Example 3. Oxidation by bacteria of the association of oil components

Biodegradation of oil by microorganism strains was carried out under conditions of periodic cultivation in 8E liquid mineral medium without additional aeration and with aeration using a temperature-controlled rocking chair (Sanyo, Japan; Orbi-safe, Great Britain) with a rotation speed of 200 rpm. The initial concentration of strain cells in the flasks averaged 1 × 10 ⁷ cells / ml.

The oil-utilizing properties of the bacterial association are tested by gas chromatography. The content of n-alkanes in the medium was determined after cultivation of the association for 21 days in the medium with oil at 10 ° C as compared with the control (medium with oil without the association of microorganisms). In Fig. 3. The gas chromatogram of the chloroform oil extract without the addition of destructors (A) and after biodegradation by the association of the strains Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 (B) for 21 days of incubation at a temperature of 10 ° C and a pH of 7.0 is shown.

The studied strains had a degree of oil destruction in the range from 69 to 84% for 21 days of cultivation in a liquid medium with oil at 10 ° C.

Under different experimental conditions (adding NaCl to the nutrient medium, changing pH, temperature, oil from different deposits), the utilization of n-alkanes by the association of strains remained high.

Example 4. The method of remediation of oil-contaminated objects

The association’s ability to utilize petroleum products in the soil was investigated in a model experiment. For this, an experimental training ground was laid out, divided into sections. Each plot was 1.5 m × 2 m in size, a buffer zone of 2 m × 2 m was left between the plots. Before the experiment, the plots were dug up so that the grass did not absorb pollutants. In addition, 2 positive control sections were prepared, one of them was dug up: 2 m × 2 m sections located at a distance from the experimental sites in order to exclude the influence of pollutants.

The plots are treated with diesel fuel or used automobile oil to a final concentration of 10% in the upper soil layer. For this, 40 liters of used automobile oil or diesel fuel were poured onto the experimental plot.

After 2 days, the experimental plots were treated with an aqueous suspension of freeze-dried microorganisms-oil destructors Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp 1040, taken in equal quantities. Bacteria introduced in the amount of 10 ⁶ cells / cm ² . Control plots were watered with the same amount of water without microorganisms.

The treatment of experimental plots with the association of microorganisms-oil destructors in the amount of 10 ⁶ cells / cm ² was carried out three times with an interval of 10 days. Observations were carried out for 70 days. The results are presented in Fig. 4, 5. The results of seeding bacteria from soil samples are presented in Table 2.

In Fig. 4. - Restoration of the soil cover during the experimental 10% contamination with used automobile oil (from top to bottom, 2nd, 20th 70th day of the experiment).

In Fig. 5. - Restoration of the soil cover within 70 days after the experimental 10% contamination with diesel fuel (from top to bottom the 2nd, 20th 70th day of the experiment).

table 2 Sowing Results Pollutant Plot CFU in 1 g of soil 2nd day of the experiment (before making the association) 10th day of the experiment 20th day of the experiment Used car oil the control 1.8 × 10 ⁶ 4.8 × 10 ⁶ 1.3 × 10 ⁷ experience 1.5 × 10 ⁶ 1.5 × 10 ⁷ 7.5 × 10 ⁷ Diesel fuel the control 6.6 × 10 ⁵ 8.5 × 10 ⁵ 3 × 10 ⁶ experience 6.8 × 10 ⁵ 8.4 × 10 ⁶ 1.9 × 10 ⁷ Soil control 4.3 × 10 ⁷ 2.7 × 10 ⁷

As can be seen from Figures 3 and 4 and Table 2, the use of the association of strains Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 accelerated the reduction of soil toxicity after contamination with used automobile oil and diesel fuel and contributed to the restoration of microbiocenosis and grass cover.

It can be seen from the above that the association of the Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 strains grows well at low temperatures (10 ° С), is able to utilize a wide range of oil components, in the model experiment, the introduction of the soil into the soil accelerated the reduction of toxicity after contamination with used automobile oil and diesel fuel and contributed to the restoration of microbiocenosis and grass cover, the bacteria included in the association do not show antagonism towards endogenous microbiota.

Claims (2)

1. The Association of strains of bacteria-oil destructors Acinetobacter species B-1037, Bacillus species B-1040, Pseudomonas species B-989, deposited in the Federal State Budget Scientific Center, VB Vektor, for the remediation of oil-contaminated soils, and the bacteria of each strain in the association contain at least 30% . 2. The method of remediation of oil-contaminated soils by introducing a biological product, characterized in that an aqueous suspension of freeze-dried biomass of the association of strains of bacteria-oil destructors Acinetobacter species B-1037, Bacillus species B-1040, Pseudomonas species B-989, deposited in ФЦ WB "Vector", at the rate of 10 ⁹ cells per m ² .

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