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

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

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

Biologics, based on the associations of the oil destructive microorganisms capable of oxidizing a wide range of petroleum hydrocarbons from long-chain alkanes to aromatic compounds, have been used successfully in the technology of remediation (cleanup) of oil-contaminated objects. Biological products are designed to clean from oil and petroleum products soil and waters, to restore the function of self-cleaning soil and water, wastewater treatment industrial plants, wastewater treatment car washes, service stations, gas stations, oil companies and oil refining, dewaxing wells, technical tanks.

Known commercial preparations on the basis of oil-oxidizing microorganisms. The medicines are monocultures (drugs "Poutical", "ekoyl", "Destroil" and others), or Association with two or more than twenty different types of oil-oxidizing microorganisms: bacteria, fungi, yeast ("Devoroil", "Simbinal", "Rotatory") (table 1).

In recent times more likely to use biological products, consisting of 2 or more strains, as the use of monocultures may not completely solve the problem of cleaning. Oil is a complex mnogo nonentry substrate, containing hundreds of different chemical compounds (Baryshnikov L.M., Grishankov VG, Arinbasarova MU, Shkidchenko A.N., Voronin A.M. Biodegradation of petroleum strains destructors and their associations in the liquid environment // go active. biochem. and microbiol. - 2001. - V.37. No. 5. - S-548), and one strain is not capable of the full range of enzymes required for biodegradation. The use of multiple strains that differ in the spectrum of the consumed substrate, can lead to complete destruction of the oil (Kobzev E.N., Petricevic D.B, Shkidchenko A.N. Investigation of the stability of the Association of the oil destructive microorganisms in an open system // go active. biochem. and microbiol. - 2001. - V.37. No. 4. - S-418).

In terms of natural microflora observed simultaneous assimilation of different oil fractions of different groups of microorganisms (Shkidchenko A.N., Arinbasarova MU Study neftedestruktivnoi activity of microflora of the coastal zone of the Caspian sea // go active. biochem. and microbiol. - 2001. - V.38. No. 5. - S-512).

When several strains of destructors in their consortium septemtrionalis effect is enhanced. The reasons for this are:

different priority to the use of components of oil of different strains;

different speed of growth of microorganisms;

- produced starmometer, which may be growth factors for other strains of the consortium.

The conditions of application of most biological products require the access of oxygen in the soil, keep the soil moisture at least 40% of regular sprinkling and rolling. Work pH biologics typically from 5 to 9, operating temperature from +10 to +40°C. At low ambient temperature to +5°C, bacterial growth slows down to a complete stop biological activity. During the subsequent rise in temperature again microorganisms begin to multiply.

When choosing a strain for the development of the drug usually should take into account such criteria as high oxidizing activity, resistance to salts of heavy metals, epatajnosti and non-toxic strains for humans and animals (there are regional laws T.V. Principles and methods of intensification of the biological destruction of hydrocarbons in the environment // go active. biochem. and microbiol. - 1996. - 32. No. 6. - S-585). Due to the fact that technology microbiological purification of contaminated soils provide aerobic conditions, it is necessary to select a microorganism-destructor among aerobic and facultative anaerobic strains. In addition, cells of strain should have a high resilience to be able to grow and disposal of petroleum products under conditions of wide range is as temperatures, pH, humidity, lack of nutrients (Stabnikova E.V., Selezneva M.V., Reva O., Ivanov, C. N. The choice of active microorganisms-destructors of hydrocarbons for cleaning contaminated soils. // Go active. biochem. and microbiol. - 1995. Volume 31. No. 5. - S-539).

In Russia there are a variety of preparations that were developed for cleanup of soils from oil pollution (commercial names of drugs: avalon, Basicity, Valentis, Devoroil, Finished, Naftoks, Nicole, Petrolane, Poutical, Roder, Wagon, etc.) (Patent RF №2077397); (Dyadechko NR. Tolstokorova LE the Gashev S.N. etc. On biological remediation of contaminated sandy soils of the Middle Ob basin //soil science, 1990, N 9, p.148-151); (RF patents №№2114071, 2053205).

The most famous of them and has long been used in practice are the biologics Puteal (Dyadechko NR. Tolstokorova LE the Gashev S.N. etc. On biological recultivation neftezagryaznennyh sandy soils of the Middle Ob basin // soil science, 1990, N 9, p.148-151) and Devoroil (patent No. 2114071 from 22.05.97).

It is known that the drug Poutical consists of a single strain of the bacterium Pseudomonas putida 36. The drug is produced by deep cultivation of bacteria in a nutrient medium at 30°C, under aerobic conditions, followed by spray drying or lyophilization obtained culture of fluid (RF patent 2053205). Because the drug Putida which is a monoculture, it has less potential and more narrow spectrum of activity for hydrocarbons than drugs 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.) (patent RF №2114071 from 22.05.97). In accordance with the patent Devoroil also get deep cultivation in a nutrient medium of the constituent microorganisms and subsequent drying of the obtained biomass. However Devoroil consists of microorganisms of different taxonomic and species requiring separate cultivation, which greatly complicates and increases the cost of the process of manufacturing of the drug. On the other hand, by co-cultivation of microorganisms belonging to different taxonomic groups and different growth rate, substrate specificity, temperature optimum growth and so, there is competition for food sources, and as a result in this drug not survive all kinds of claimed, and, accordingly, decreases the effectiveness of this drug.

The lack of a biological product "Devoroil", in addition to the need for separate growing component of the microorganisms is the use of expensive carbohydrate environment to obtain biomass the s of the drug and the higher the temperature of the cultivation of 20-40°C, that limits the scope of application only in the middle and South of Russia.

Known consortium of bacterial strains Bacillus brevis and Arthrobacter species used for purification of water and soil from oil and oil products (patent RF №2232806). The disadvantage of this method should recognize its low efficiency in case of high degree of contamination of oil.

Natural microbial community of the soil, including a large number of bacteria-destructors, in small doses, contamination cope with the degradation of petroleum hydrocarbons, so when small particles (less than 10%) in an average strip of Russia it is better to create suitable conditions for the activity of indigenous bacteria than introductionat new species. However, in the case of the Northern ecosystems depleted species diversity and limiting amounts of nutrients, even when the dirt about 10% of the appropriate use of biologics. The introduction of adapted microorganisms, destroying oil is also recommended in case of physico-chemical characteristics of the place of pollution makes it impossible to increase the natural microbiota (high concentrations of heavy metals, soil salinity, extreme pH values, and others).

The objective of the invention is the creation of the Association of bacteria, oxidizing oil and oil products, and how is imediasee contaminated objects North of Siberian using this Association, components which are indigenous strains do not show antagonism against endogenous microorganisms able to grow at low temperatures (4-10°C), low pH values (4.5 to 6), elevated levels of NaCl (1-4%), to dispose of a wide range of oil components at temperatures of 10-15°C.

The technical result of the invention is to improve the effectiveness of cleaning contaminated objects at low ambient temperature (3-15°C).

The problem is solved by the creation of the Association isolated from oil-contaminated soil Siberian North bacteria, deponirovanie in the collection of cultures of microorganisms of the State research center of Virology and biotechnology "Vector":

Acinetobacter species, the registration number In-1037; Pseudomonas species, the registration number In-989; Bacillus species, registration number B-1040 taken in equal amounts. For remediation of oil-contaminated objects use aqueous suspension Leoville dried bacteria of the Association at the rate of 10⁹cells on m².

The sample was collected. In order not to disturb the biodiversity of the ecosystem, for each region needs its own characterized the composition of microorganisms.

As substrates for the selection of strains with a wide range of enzymes that destroy the complex organic substances, COI is litovali oil-contaminated soil in areas of oil production (Khanty-Mansi Autonomous Okrug, Yamalo-Nenets Autonomous Okrug). Samples of water and sludge deposits were placed in sterile plastic tubes with a volume of 50 ml, samples of soil were placed in sterile paper bags and in sterile plastic tubes with a volume of 50 ml.

Selection of microorganisms-destructors of oil. Examples of natural substrates were incubated in liquid selective medium 8E (g/l): (NH₄)₂HPO₄- 1.5; KH₂PO₄- 0.7; MgSO₄×7N₂About - 0.8; NaCl - 0.5; pH 7.2 with the addition of oil by weight (0.5 - 2%) on the rocking chair at temperatures 10°C; then the cumulative culture were transferred to agar medium 8E. Microorganisms with the ability to active growth on selective nutrient media with oil, were isolated in pure culture. In total there are 34 strain, of which 3 were selected strain with high neverthelessa capacity, relatively high growth rate at 10°C, acidic pH values and high salt content in the environment. The strains were identified by biochemical and physiological characteristics. Primary biodegradation of oil in the tubes was evaluated on the following parameters: breaking the surface of the oil film, the turbidity of the nutrient medium for the growth of biomass, the formation of a homogeneous emulsion of oil in the environment, gas.

Morph the logical characteristics of the obtained isolates-destructors studied using light microscopy of the living and the stained cells using a microscope Carl Zeiss Axioskop 40 (Carl Zeiss, Germany). The study of the physiological, biochemical properties, were performed by standard methods (Methods for General bacteriology, 1983; Determinant of bacteria ..., 2001).

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

The strain of Acinetobacter species In-a dense nutrient medium forms a whitish, translucent, shiny, smooth colonies with smooth edge; cells of strain represent gram-negative, motile bacilli, size 0.8×1.7 to 2 μm. The strain of Acinetobacter species In 1037 is an aerobe, grows in the temperature range from 4-6°to 35 ° C; no hydrolyzes starch, casein, does not possess proteolytic activity, catalogoplantillas, sprayway glucose, lactose, mannitol, maltose, sucrose, sorbitol with gas, not liquefies gelatin. Possesses citrate activity grows on RPA with the concentration of NaCl 5%; does not have hemolytic properties.

The strain Bacillus species 1040 on solid nutrient media forms a whitish, translucent, shiny, smooth colonies with smooth edge; cells of strain represent gram-positive bacilli, 1×1.2-1.5 mm. The strain Bacillus species 1040 is an aerobe, grows in the temperature range from 4-6°to 35 ° C; no hydrolyzes starch, not liquefies gelatin, does not form acetoin not utilize glucose, galactose, xylose, rhamnose, dulcet, mannitol, Malta is, the arabinose, sucrose, sorbitol, Inositol, lactose. Possesses citrate, catalytic, fibrinolytic activity, does not have lipase, plasmalogens, oxidase, lecithinase, urease, caseinolytic activity on milk agar, hemolytic properties.

The strain Pseudomonas species 989 on solid nutrient media forms a whitish, translucent, shiny, smooth colonies with smooth edge; cells of strain represent gram-negative short Bacillus 1-2 and in short chains, the size of 0.9-1.0×1.5 to 1.8 microns. The strain Pseudomonas species all the aerobe, grows in the temperature range from 4-6°to 35 ° C; no hydrolyzes starch, not liquefies gelatin, does not form acetoin not utilize glucose, lactose, galactose, xylose, rhamnose, dulcet, mannitol, maltose, arabinose, saccharose, sorbitol, Inositol. Possesses catalytic oxidase activity, caseinolytic on milk agar, does not possess citrate, lecithinase, lipase, urease activity, hemolytic, fibrinolytic, plasmamembrane properties. Pseudomonas species are resistant to penicillin, ampicillin, oleandomycin, oxacillin, ristomycin, carbenicillin, lincomycin, rifampin, chloramphenicol; sensitive to erythromycin, monomitsina, tetracycline, streptomycin, kanamycin, neomycin, gentamycin, polymyxin.

The nature of waimao the relations strains Association with the endogenous microbiota of the soils studied in the experiment. The lack of antagonism illustrated by the images shown in figure 1, where the horizontal strip - seeding of native microorganisms isolated from samples of oil-contaminated soils in the area, a vertical strip - seeding of the Association of microorganisms Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989. As can be seen, the Association of microorganisms Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 does not inhibit the growth of indigenous microorganisms isolated from oil-contaminated soil YANAO, because endogenous bacteria grow equally intensely as in the presence of Association (photo left)and control (right photo). Therefore, when making large quantities of biomass strains will not experience oppression of the local microbiota and displace endogenous species of microorganisms.

Example 1. Receiving Association

Bacteria Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 was compiled Association. This bacteria grown in meat-peptone broth to a concentration of 1-3×10⁹cells/ml, were mixed so that the number of bacteria of each strain was a mixture of not less than 30%. After that, the bacterial mass was poured into ampoules of 3 ml were frozen at 70°C during the day, then transferred to the office of freeze drying, consisted of vacuum and dried for 2 days. Sealed vials were stored at 4°C.

After storage in accordance with the s of the month was determined by the concentration of bacteria of each strain in the ampoule. For this purpose, the ampoule was opened, was made in 3 ml of sterile 0.9% NaCl solution (saline), transferred the contents of the ampoule in a volumetric flask, saline brought the contents to 100 ml of the Suspension is kept for 12 hours at 18-20°C. Then did a ten-fold dilution in saline solution and were sown on Petri dishes. After culturing for 2 days at room temperature was calculated grown colonies and determined the concentration of each strain in the Association. The results are presented in table 1.

The Association was tested on the ability to dispose of oil components and the ability to bioremediation of oil-contaminated objects.

When is EP 2. The rate of growth of the Association at 10°C and 30°C

The growth rate of the Association of bacteria was investigated in liquid medium 8E with the addition of 1% glucose at a temperature of 10 and 30°C. the quantification of microorganisms was performed by a Cup method Koch by counting the grown colonies (Methods of General bacteriology, 1983).

The number and ratio of cells of strains was determined by microscopy suspensions at different stages of cultivation using a phase-contrast microscope, by plating on agar medium to obtain isolated colonies from further counting and identification (Labinsky, 1978). When assessing the activity of the strains in Association took into account the change in optical density of the culture fluid during incubation of the strains. The results are presented in Fig.2, which shows 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 environment, without aeration).

As can be seen from Fig.2, the Association of the bacteria Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 grows well at 10°C, reaching up to 12 days the concentration of 10^8,5cells/ml, while at 30°C the maximum concentration was only 10^7,5cells/ml

After 12 days of cultivation, the suspension was tested for the percentage of bacteria of different strains. It was approximately Acinetobacter species 1037: Bacillus species 100: Pseudomonas species 989 as 1.2:0,9:1. Thus, the rate of bacterial growth Association is about the same and by co-cultivation of strains of Acinetobacter species 1037, Bacillus species 1040 and Pseudomonas species 989 is not the replacement of one of the other bacteria.

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

The biodegradation of oil by strains of microorganisms were carried out in conditions of periodic cultivation in liquid mineral medium 8E without additional aeration and aeration, using thermally rocker ("Sanyo", Japan; "Orbi-safe", UK) with a rotation speed of 200 rpm Initial concentration of cells of strains in flasks averaged 1×10⁷CL/ml

Septemtrionalis Association properties of the bacteria tested by gas chromatography. Determined the content of n-alkanes in the medium after cultivation of the Association within 21 days in an environment with oil at 10°C compared with control (medium with oil without the Association of microorganisms). 3. shows the gas chromatogram of the chloroform extract oil without adding destructors (a) and after biodegradation Association strains of Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 (B) within 21 days of incubation at 10°C and pH 7.0.

The studied strains had a degree of degradation of oil in the range from 69 to 84% 21 days of cultivation in liquid medium with oil at 1°C.

In different experimental conditions (addition of nutrient medium NaCl, pH change, temperature, oil from different fields) utilization of n-alkanes Association strains remained high.

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

The ability of the Association to recycling oil in the soil was investigated in a model experiment. For this was laid experimental polygon, divided into sections. Each plot had a size of 1.5 m×2 m between plots was left a buffer zone of 2 m×2 m Before the experiment plots were dug so that the grass is not sorbirovanna itself pollutants. In addition, there were prepared 2 plots the positive control, one of them dug: plots of 2 m×2 m, located at a distance from the experimental plots, in order to exclude the influence of pollutants.

Plots treated with oil or used motor oil to a final concentration in the upper soil layer of 10%. For this purpose, the experimental plot was poured 40 liters of waste motor oil or diesel fuel.

After 2 days experimental plots were treated with an aqueous suspension of lyophilized microorganisms-oil degrading Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp 1040, taken in equal quantities. Bacteria included in the amount of 10⁶cells/cm². Control plots were watered so W is the amount of water without microorganisms.

The processing of experimental plots by the Association of the oil destructive microorganisms in the amount of 10⁶cells/cm²conducted three times with an interval of 10 days. Observations were carried out over 70 days. The results are presented in Fig.4, 5. The results of the seeding of bacteria from soil samples is presented in table 2.

Figure 4. - Restoration of soil cover over after experimental 10% contamination of used motor oil (from top to bottom 2-th, 20-th of the 70-day experiment).

Figure 5. - Restoration of soil within 70 days after experimental 10% contamination with diesel fuel (top-down 2-th, 20-th of the 70-day experiment).

As can be seen from figures 3 and 4 and table 2, the use of the Association strains of Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 accelerated reduction of toxicity soil contaminated used motor oil and diesel fuel and contributed to the restoration of microflora and grass.

From the above it is evident that the Association strains of Acinetobacter sp.1037, Pseudomonas sp.989, Bacillus sp.1040 grows well at low temperatures (10°C), is capable of utilizing a wide range of oil components in the model experiment, the soil Association has accelerated the reduction of the toxicity of the settlement of the E. contamination of spent motor oil and diesel fuel and contributed to the restoration of microflora and grass, bacteria belonging to the Association, do not show antagonism against endogenous microbiota.

1. The Association of bacterial strains-oil degrading Acinetobacter species In-1037, Bacillus species In-1040, Pseudomonas species B-989 deposited in the FBSI SRC VB "Vector", for remediation of oil-contaminated soils and bacteria of each strain in Association contains at least 30%.

2. The method of remediation of oil-contaminated soils by introducing a biological product, characterized in that as a biological product used aqueous suspension of lyophilized biomass Association strains of bacteria-oil degrading Acinetobacter species B-1037, Bacillus species B-1040, Pseudomonas species In-989 deposited in the FBSI SRC VB "Vector", at the rate of 10⁹cells on m².