RU2388816C2 - Agent for cleaning of contaminated soils from oil and polycyclic aromatic carbohydrates under conditions of high medium mineralisation - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: agent represents a consortium of the following bacteria - Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34, Halomonas sp.SMB31 and "Salinicola socius" SMB35, deposited in collection of Chemical mutagenesis laboratory of Institute of Ecology and Genetics of Microorganisms, Urals department of Russian Academy of Sciences under No. SMB3.

EFFECT: high speed of polycyclic carbohydrates destruction under conditions of high salt concentrations (NaCl to 7%).

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Description

The invention relates to the field of biotechnology and can be used to clean the soil from contamination with oil and polycyclic aromatic hydrocarbons in saline conditions by introducing a microbial consortium.

The most dangerous components of oil are polycyclic aromatic hydrocarbons, which are characterized by the least degradation potential, high toxicity, mutagenic and carcinogenic effects [1, 2]. Moreover, oil pollution of the soil is often accompanied by salinization due to the influx of highly mineralized formation water into it, which leads to inhibition of the activity of soil microorganisms [3].

Known strains of the microorganisms Pseudomonas alcaligenes MEV B-8278 [4] and Pseudomonas stutzeri MEV-S1 B-8277 [5], which, along with the destruction of oil and oil products, utilize polycyclic aromatic hydrocarbons. However, these bacteria are not capable of growth and destruction of hydrocarbons in saline conditions.

Known consortia of microorganisms Rhodococcus sp. Bkmac-1500D, Rh. maris AC-1501D, Rh. erythropolis AC-1502D, Pseudomonas stutzeri BKM B-1972 D, Candida sp. BKM-y-2778 D [6]; Rhodococcus ruber BKM Ac-1513D, Rhodococcus erythropolis BKM Ac-1514D [7]; Acinetobacter calcoaceticus Baumann et al. strain 279 VKPM-B-7179, Pseudomonas fluorescens Stanier et al. strain 325 VKPM-B-7152, Alcaligenes faecalis Castellani et Chalmers strain 404 VKPM-B-7180 [8]; Candida maltosa VKPM U-2256, Candida maltosa VKPM U-2257 [9], which can degrade oil and aliphatic hydrocarbons in the presence of salinization. However, these microbial consortia are not capable of degradation of polycyclic aromatic hydrocarbons.

A known consortium is Sacharomyces sp., Pseudomonas sp. [10], capable of growth and destruction of oil under saline conditions, as well as degradation of polycyclic aromatic hydrocarbons - anthracene and naphthalene, but the utilization of anthracene and naphthalene and its rate during salinization of the medium have not been verified.

The closest in technical essence to the proposed solution is a consortium of microorganisms Rhodococcus sp.BKM Ac-2532D, Rhodococcus sp.BKM Ac-2533D, Pseudomonas sp.BKM Ac-2387D, Pseudomonas putida BKM Ac-2380D [11], which destroys oil and polycyclic aromas hydrocarbons - naphthalene and phenanthrene in the presence of salt (NaCl), however, its disadvantage is the narrow salinity range of the biological product (up to 3% NaCl) and the unknown rate of destruction of polycyclic aromatic hydrocarbons.

The objective of the invention is to create a tool - an oil-oxidizing consortium of microorganisms with a high rate of destruction of a wide range of polycyclic aromatic hydrocarbons and the ability to grow at elevated salt concentrations (NaCl up to 7%).

The above problem is solved by a means for purifying contaminated soils from oil and polycyclic aromatic hydrocarbons under conditions of increased mineralization, which is a consortium of bacteria Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34 Halomon sp.SMB31 and Salinicola socius SMB35.

This microbial community is a natural association, isolated from technologically saline soils contaminated with hydrocarbons. The bacteria of the consortium are connected by protocooperative relations, in connection with which joint industrial cultivation of microorganisms is possible. The composition of the consortium is stable over time. The ratio of the strains may vary depending on the cultivation conditions of the microbial community, but Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Salinicola socius SMB35 dominate in culture media. The main feature of the drug is that its microbial consortium can develop in a mineralized (NaCl up to 7%) environment and utilize oil and polycyclic aromatic hydrocarbons.

The resulting consortium of bacterial strains is stored in the collection of the Laboratory of Chemical Mutagenesis of the Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences under collector number SMB3.

Description of the bacterial strains that make up the consortium

Strain Rhodococcus sp.SMB37

Morphological and cultural characters

Gram-positive cells, motionless. With development, the bacterial culture is characterized by the life cycle of cocci-coli-cocci.

On a full-fledged agar medium, it forms opaque colonies with a pale pink color, convex with a rhizoid edge.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. Mycolic acids are part of the cell wall. Hydrolyzes starch. Not capable of hydrolysis of gelatin and urea. The oxidase test is negative, the catalase test is positive. Uses arabinose, xylose, maltose, mannitol with the formation of acid as a carbon source. It is capable of growth in the range of NaCl concentration from 0 to 10%. Growth is possible at pH 6-9. It does not need additional growth factors.

By sequence 16S rDNA is closest to the species R. rhodochrous, however, it differs from it by its resistance to high salt concentrations and the ability to use naphthalene as the sole source of carbon and energy.

Strain Rhodococcus sp.SMB38

Morphological and cultural characters

Gram-positive cells, motionless. With development, the bacterial culture is characterized by the life cycle of cocci-coli-cocci.

On a full-fledged agar medium, it forms opaque pink colonies with a smooth edge.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. Mycolic acids are part of the cell wall. Hydrolyzes urea. Not capable of hydrolysis of starch. The oxidase test is negative, the catalase test is positive. Uses galactose, lactose, mannitol with the formation of acid as a carbon source. It is capable of growth in the range of NaCl concentration from O to 10%. It does not need additional growth factors.

By sequence 16S rDNA is closest to the species R. jostii, however, it differs from it by its resistance to high salt concentrations, the ability to use naphthalene as the sole source of carbon and energy, and hydrolyze urea.

Strain Arthrobacter sp.SMB32

Morphological and cultural characters

Gram-positive cells, motionless. In young cultures, sticks of irregular shape, with the development of culture break up into small cocci. They do not form a dispute.

On a full-fledged agar medium, it forms round, convex, with a smooth edge, white, yellowing colonies with age.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. Mycolic acids in the cell wall were not detected. The oxidase test is negative, the catalase test is positive.

By sequence 16S rDNA is closest to A. nicotianae.

Strain Microbacterium sp.SMB33

Morphological and cultural characters

The cells are rod-shaped, gram-positive, motile. With the development of a bacterial culture, the life cycle of coccus-bacillus-coccus is absent. They do not form a dispute.

On a full-fledged agar medium, it forms rounded, convex, light green colonies.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. Gelatin and starch do not hydrolyze. Forms acid from glucose. The oxidase test is negative, the catalase test is positive.

By sequence 16S rDNA is closest to the species M. maritypicum.

Strain Thalassospira sp.SMB34

Morphological and cultural characters

The cells are curved rod-shaped, gram-negative, motile due to the polar flagellum. They do not form a dispute.

On a full-fledged agar medium, it forms round, shiny, smooth, with a smooth edge, beige colonies.

Physiological and biochemical characteristics

Hemorganotroph. Oxidase and catalase tests are positive. Does not hydrolyze starch, gelatin and urea. Phenylalanine deaminase and β-galactosidase activity does not have, restores nitrate to nitrite. It is capable of growth in the presence of salt (NaCl up to 8%).

By sequence 16S rDNA is closest to the species Thalassospira lucentensis, but differs from it in its ability to reduce nitrate and resistance to high salt concentrations.

Strain Halomonas sp.SMB31

Morphological and cultural characters

The cells are rod-shaped, gram-negative, motile due to the polar flagellum. They do not form a dispute.

On a full-fledged agar medium, it forms round, convex, with an even edge, fawn-colored colonies.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. The oxidase test is negative. Catalase reaction is positive. Does not hydrolyze gelatin, starch and urea. It forms acid from glucose, inositol, mannitol, sucrose, sorbitol. It is capable of growth in the range of NaCl concentrations from 3 to 30%, with an optimum at 3-10%. Growth is possible at pH 6-7. It does not need additional growth factors.

By sequence 16S rDNA is closest to H. taeanensis, however, it differs from it in the range of sodium chloride concentrations and pH, in which culture growth is possible, the ability to hydrolyze tween 80 and urea, and the ability to reduce nitrate.

Strain Salinicola socius SMB35

Morphological and cultural characters

The cells are rod-shaped, gram-negative, motile due to the polar flagellum. They do not form a dispute.

On a full-fledged agar medium, it forms round, convex, with a smooth edge, yellow colonies.

Physiological and biochemical characteristics

Aerobe. Hemorganotroph. The catalase reaction is positive, the oxidase reaction is negative. Hydrolyzes starch, gelatin and urea. It forms acid from glucose, mannitol, sorbitol. It is capable of growth in the range of NaCl concentrations from 0.5 to 30%, with an optimum at 3-10%. The pH range of the growth medium is 6.0-8.0. It does not need additional growth factors.

By sequence 16S rDNA is closest to the species of the genera of the Halomonadaceae family (the genera Halomonas, Cobetia and Chromohalobacter), however, it differs significantly from them by the only, polar flagellum, a wide range of sodium chloride concentrations at which growth is possible, a negative oxidase reaction, and the lack of the ability to reduce nitrate to nitrite, the ability to hydrolyze gelatin, starch and tween-80.

An advantage of the consortium of bacteria Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34, Halomonas sp.SMB31 and Salinicola socius SMB35 is a high destruction rate of polycyclic hydrocarbons and salts (NaCl up to 7%).

The studied consortium was proposed for the first time to clean the soil of oil and polycyclic aromatic hydrocarbons, in connection with which we can conclude that the proposed consortium meets the criteria of the invention of "novelty" and "inventive step".

Example 1

The proposed bacterial consortium was isolated from soil taken from a depth of 5-10 cm on the territory of industrial salt development of the potash plant OJSC Uralkali (Berezniki, Perm Territory), as follows:

1.0 g of soil was introduced into 250 ml of Raymond mineral medium of the following composition (g / l): NH ₄ NO ₃ - 2.0, MgSO ₄ × 7Н ₂ О - 0.2, K ₂ HPO ₄ - 2.0, Na ₂ HPO ₄ - 3.0, CaCl ₂ × 6H ₂ O - 0.01, Na ₂ CO ₃ - 0.1, supplemented with 1% solution of MnSO ₄ × 2H ₂ O - 2 ml / l of medium and 1% solution of FeSO ₄ × 7H ₂ O - 1 ml / l of medium, NaCl - 60 g / l of the medium; pH of the medium is 7.3. Naphthalene (1 g / l of medium) was used as the sole source of carbon and energy. Incubation was carried out for 2 weeks on a thermostatic rocker UVMT-12-250 at 100 rpm and 28 ° C. A consortium of bacteria was obtained by repeated reseeding (more than 10) of the accumulative culture and cultivation under the above conditions.

To establish the composition of the consortium, pure bacterial cultures were isolated on Raymond's complete agar medium (5 g / L tryptone, 2.5 g / L yeast extract as growth substrates, and 15 g / L Difco agar) in the presence of 3% NaCl.

Example 2

A consortium of bacteria Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34, Halomonas sp.SMB31 and Salinicola socius SMB35 grown on Raymond mineral medium with naphthalene (see. ), were introduced into the soil with varying degrees of salinization (3%, 5% and 7% NaCl), contaminated with oil at a dose of 1%, at the rate of 10 ⁷ cells per 1 gram of soil. After 20 days, the soil oil content was estimated by the gravimetric method and the concentration of polycyclic aromatic hydrocarbons was measured using an Agilent 6890N gas chromatography-mass spectrometer (Agilent Technology, USA).

In the soil with the introduction of a bacterial consortium, a large decrease in oil was noted (Table 1). The largest difference in the oil content between soils with and without the introduction of a consortium was observed with a mineralization of 7%, so if in the soil without a consortium the utilization of oil was 0%, then with a contribution of 26.8%.

The results of the impact of the proposed consortium on the destruction of polycyclic aromatic hydrocarbons are presented in Table 2. It is shown that over 20 days, the utilization of naphthalene ranged from 93.3% to 95.7%, anthracene - from 46.6% to 62.0%, during the same time the degree of degradation of naphthalene and anthracene by analog Andreeva et al., RU 2272071 C2, 03.20.2006) was 50%. The invention also differs from prototypes in a large range of recyclable polycyclic aromatic hydrocarbons.

Table 1 The degree of destruction of oil in the soil (%) Experience Options Soil salinity NaCl 3% NaCl 5% NaCl 7% Soil without consortium 9.0 6.6 0 Consortium soil 21.3 21.5 26.8

table 2 The degree of destruction of polycyclic hydrocarbons in oil-contaminated soil (%) Hydrocarbons Soil salinity NaCl 3% NaCl 5% NaCl 7% naphthalene 95.7 94.4 93.3 biphenyl 55.2 57.1 45.9 acenaphthene 57.5 55.7 50.5 phenanthrene 45.3 53.5 39.5 anthracene 53.0 62.0 46.6

Thus, the proposed consortium can be used to clean the soil of oil, as well as polycyclic aromatic hydrocarbons (naphthalene, biphenyl, acenaphthene, phenanthrene, anthracene) under salinization conditions.

References

1. Atlas R.M. Microbial degradation of petroleum hydrocarbons: an environmental perspective // Microbiolgical reviews. 1981. V. 45. P.180-209.

2. Pikovsky Yu.I. Natural and man-made hydrocarbon flows in the environment. M .: Publishing house of Moscow State University, 1993.207 p.

3. Solntseva N.P. General laws of soil transformation in areas of oil production (manifestation forms, basic processes, models) // Restoration of oil-contaminated soil ecosystems. M .: Nauka, 1988.S. 23-42.

4. RF patent No. 2228953 C2, 2004.

5. RF patent No. 2228952 C2, 2004.

6. RF patent No. 2023686 C1, 1994.

7. RF patent No. 2174496 C2,2001.

8. RF patent No. 2107722 C1, 1998.

9. RF patent No. 2114174 C1, 1998.

10. RF patent No. 2272071 C2, 2006.

11. RF patent No. 2312891 C1, 2007 - prototype.

Claims (1)

Means for cleaning contaminated soils from oil and polycyclic aromatic hydrocarbons in conditions of increased mineralization of the environment, which is a consortium of bacteria Rhodococcus sp.SMB37, Rhodococcus sp.SMB38, Arthrobacter sp.SMB32, Microbacterium sp.SMB33, Thalassospira sp.SMB34, Halomonas sp.SMB31 and Salinicola socius SMB35, deposited in the collection of the Laboratory of Chemical Mutagenesis of the Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences under No. 5 of the cost center.