RU2585537C1 - STRAIN Rhodococcus wratislaviensis - DESTRUCTOR OF PERSISTENT TOXIC ORGANOCHLORINE COMPOUNDS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain Rhodococcus wratislaviensis Ch625, having the ability to efficiently decompose complex organochlorine compounds: polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane), dichlorodiphenyltrichloroethane, deposited in the Russian National Collection of Microorganisms of the Federal State budget institution Science Institute of Biochemistry and Physiology of Microorganisms named G.K. Scriabin, Russian Academy of Sciences (RAS IBPM) under accession number VKM Ac-2631D.

EFFECT: invention improves the efficiency of destruction of polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane), dichlorodiphenyltrichloroethane.

1 cl, 3 tbl, 4 ex

Description

The invention relates to the field of biotechnology and is a new aerobic strain of bacteria capable of efficiently decomposing a complex of organochlorine compounds: polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane), dichlorodiphenyl trichloroethane.

These substances are included in the group of persistent organic pollutants (see Federal Law of the Russian Federation dated 27.06.2011 N 164-ФЗ "On Ratification of the Stockholm Convention on Persistent Organic Pollutants".

However, when implementing the provisions of the Stockholm Convention, the problem of destroying compounds of the group of persistent organic pollutants arises, since hundreds of tons of these substances have been accumulated over a long period of production and use (see, for example, Treger Yu.A. Persistent organic pollutants. Problems and solutions // Vestnik MITT, 2011, v. 6, No. 5), a significant part of which has entered the environment.

Studies of recent decades have shown that the most environmentally safe way to utilize polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane), dichlorodiphenyltrichloroethane is their biological destruction using bacteria (see, for example, Vasilyeva G.K., Strizhakova E.P. Bioremediation of soils and sediments contaminated with polychlorinated biphenyls // Microbiology. 2007. T. 76. No. 6. S.725-741; Beyer A., Biziuk M. Environmental fate and global distribution of polychlorinated biphenyls // Rev. Environ. Contamin. Toxic. 2009. V. 201. P. 137-158). There is evidence of bacterial strains of the genera Pseudomonas, Burkcholderia and Rhodococcus that fully decompose individual congeners of low chlorinated biphenyls (see, for example, the description of the invention "Bacterial degradation of 4-biphenil" to US patent 4999300 A, publication date 12.03.1991; description of the invention Compositions and methods useful in bioremediation of polychlorinated biphenyls "to patent US 6537797 (B1), publication date 03.25.2003; description of the invention" The strain of the bacterium Rhodococcus ruber - destructor of polychlorinated biphenyls "to patent RU 2262531 C2, publication date 10/20/2005). The bacterial strains decomposing dichlorodiphenyl trichloroethane, lindane and α (β) -hexachlorocyclohexane were isolated and described (see, for example, Xie H., Zhu L., Xu Q., Wang J., Liu W., Jiang J., Meng Y .Isolation and degradation ability of the DDT-degrading bacterial strain KK // Environ. Earth Sci. 2011. V. 62. P. 93 - 99; Ortiz I., Velasco A., Le Borgne S., Revah S. Biodegradation of DDT by stimulation of indigenous microbial populations in soil with cosubstrates // Biodegradation. 2013. V. 24. P. 215-225; Kumar M., Gupta SK, Garg SK, Kumar A. Biodegradation of hexachlorocyclohexaneisomers in contaminated soils // Soil Biology Biochemistry. 2006. V. 38. P. 2318 - 2327; Camacho-Perez B., Rios-Leal E., Rinderknecht-Seijas N., Poggi-Varaldo HM Enzymes involved in the biodegradation of hexachlorocyclohexane: A mini review // J Environmental Management. V. 95. P. S 306-S318; Abhilash P.C., Srivastava S., Singh N. Comparative bioremediation potential of four rhizospheric microbial species against lindane // Chemosphere. 2011. V. 82. P. 56-63; Benimeli C. S., Castro G. R., Chaile A. P., Amoroso MJ. Lindane uptake and degradation by aquatic Streptomyces sp.strain M7 // Int. Biodeterior Biodegrad. 2007. V. 59. P. 148-155).

Limited information is available on the anaerobic decomposition of hexachlorobenzene by microorganisms (see, e.g., Reineke W. Aerobic and anaerobic biodegradation potentials of microorganisms // The handbook of environmental chemistry Vol.2, Part K / Biodegradation and persistence (ed. By B. Beek), Springer-Verlag: Berlin, Heidelberg. 2001. 161 p .; Brahushi F., Dorfler U., Schroll R., Munch J.Ch. Stimulation of reductive dechlorination of hexachlorobenzene in soil by inducing the native microbial activity // Chemosphere. 2004 V. 55. P. 1477-1484).

Information on bacterial strains that efficiently utilize several compounds of the group of persistent organic pollutants is not available in publicly available information sources.

The objective of the invention is the isolation and description of an aerobic bacterial strain that effectively decomposes several compounds of the group of persistent organic pollutants, namely polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane) and dichlorodiphenyl trichloroethane.

The technical result is expressed in the high efficiency of decomposition (destruction) of several compounds of the group of persistent organic pollutants, namely polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane) and dichlorodiphenyl trichloroethane.

To achieve a technical result, it is proposed to isolate from a soil contaminated with chemical compounds a new aerobic bacterial strain, which, as a result of evolutionary processes caused by prolonged pollution, has acquired the ability to destroy a number of compounds of the group of persistent organic pollutants.

The strain Rhodococcus wratislaviensis Ch625 was isolated from soil contaminated with compounds of the group of persistent organic pollutants and selected in the territory of the former Open Joint-Stock Company Sredne-Volzhsky Chemicals Plant (Chapaevsk, Samara Region, RF). Morphological and physiological characteristics of the strain: when grown for 3 days on an agarized rich medium, it forms round, with a curved profile (biomass accumulation in the center of the colony), matte, light pink colonies 2-3 mm in size; characterized by the life cycle of cocci-coli-cocci, capable of growth in a wide temperature range - from 8 to 42 ° C, with an optimum of 25-30 ° C; active growth of the culture occurs at a pH of 6-9. The strain Rhodococcus wratislaviensis Ch625 was deposited on 05/07/2013 by the All-Russian Collection of Microorganisms of the Federal State Budgetary Institution of Science, Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin of the Russian Academy of Sciences (IBPM RAS) at 5, Nauki Avenue, Pushchino, Moscow Region, 142290 under registration number VKM AC-2631D.

Genetic traits: according to the nucleotide sequences of the 16S rRNA gene, it is closest (100% homology) to the strain Rhodococcus wratislaviensis NCIMB 13082 ^T.

Based on morpho-physiological and molecular genetic characteristics, the strain was identified as Rhodococcus wratislaviensis.

An advantage of this invention is the ability of the strain Rhodococcus wratislaviensis Ch625 to decompose several compounds of the group of persistent organic pollutants, namely polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane) and dichlorodiphenyl trichloroethane without accumulation of toxic decomposition products. The destructive activity of the strain is manifested in standard environmental conditions and does not require serious economic costs for their creation.

A strain with such properties is described for the first time, in connection with which we can conclude that the proposed invention meets the criteria of "novelty" and "inventive step".

Example 1

Isolation of strain from contaminated soil.

To isolate the strain-destructor, the method of accumulative cultures was used: 1 g of soil contaminated with compounds of the POPs group and taken on the territory of the former Open Joint-Stock Company “Middle Volga Chemical Plant” (Chapaevsk, Samara Region, RF), was placed in flasks (volume 250 ml) containing 100 ml of K1 mineral medium (composition, g / l: K ₂ НРО ₄ × 3Н ₂ O-4.0; NaH ₂ PO ₄ × 2Н ₂ O-0.4; (NH ₄ ) ₂ SO ₄ -0.5;

MgSO ₄ × 7H ₂ O-0.15; Ca (NO ₃ ) 2 × 4H ₂ O-0.01; NaMoO ₄ × 2H ₂ O-0.18, FeSO ₄ × 7H ₂ O-1.98) and biphenyl as a carbon source in an amount of 1 g / L. Incubation was carried out for 1 month at 28 ° C.

Pure cultures of destructive bacteria were isolated by seeding of accumulative culture on Petri dishes with agarized K1 mineral medium (agar-agar concentration 15 g / l) and biphenyl as the sole source of carbon and energy with subsequent selection of single colonies. The purity of the cultures was controlled by plating on LB medium (composition, g / l: tryptone - 10, yeast extract - 5, NaCl - 10, agar -15).

Strains obtained from single colonies were tested for the degradability of polychlorinated biphenyls, dichlorodiphenyl trichloroethane, hexachlorocyclohexane (lindane) and hexachlorobenzene.

According to the results of the analysis of degradation activity, a strain of Rhodococcus wratislaviensis Ch625 was selected that effectively decomposes the entire spectrum of the presented compounds.

Example 2

Decomposition of the Rhodococcus wratislaviensis Ch625 strain of polychlorinated biphenyls (individual congeners and commercial mixtures).

The destruction of polychlorinated biphenyls by the strain Rhodococcus wratislaviensis Ch625 was carried out in experiments with “washed cells”. The strain was grown in K1 liquid medium with biphenyl (1 g / L) at 28 ° C to an OD of ₆₀₀ = 1-0. Cells washed twice in K1 medium (1 ml, OD ₆₀₀ = 2.0) were transferred to vials with Teflon caps.

An acetone solution of chlorobiphenyls was added as a substrate to a final concentration: monochlorobiphenyl - 100 mg / l, dichlorobiphenyl - 50 mg / l, trichlorobiphenyl - 50 mg / l, Delor 103 - 0.14 mg / l, Sovol - 0.53 mg / l .

To isolate chlorinated biphenyls, the culture fluid was extracted with a mixture of conc. H ₂ SO ₄ -12.5% dodecyl sulfate-Na-hexane (1:10:25) for 60 minutes at 30 ° C, stirring speed 200 rpm. The extracts were dehydrated with Na ₂ SO ₄ and analyzed on a GC6890N gas chromatograph (Agilent Technology, USA) with a MSD5973N mass selective detector (Agilent Technology, USA).

The quantitative content of the isolated chlorobiphenyls was evaluated based on a comparison of the values of the obtained peaks and standard compounds.

The degradation products of chlorobiphenyls were determined spectrophotometrically and by high performance liquid chromatography (HPLC).

To isolate and identify the biodegradation products of chlorinated biphenyls, the culture fluid was purified from bacterial cells by centrifugation (9660 g for 3 min in a miniSpin centrifuge (Eppendorf ', Germany). The supernatant was used for further analyzes.

The formation of meta-cleavage products of the aromatic ring of chlorobiphenyls - 2-hydroxy-6-oxo- (chlorophenyl) hexa-2,4-dienoic acids (HFCA) was determined by spectroscopy on a UV-Visible BioSpec-mini spectrophotometer (Shimadzu, Japan) at γ _max from 390 nm to 440 nm.

The presence in the supernatant of chlorobenzoic and hydroxybenzoic acids (CBC and (OH) BK), as well as catechols, was determined by high performance liquid chromatography - analysis on an LC-20A chromatograph (Shimadzu, Japan) with a Discovery C18 column (150 × 4.6 mm) ( Supelco, Sigma-Aldrich, USA) and a UV detector at 205 nm. The analysis was carried out in the system acetonitrile-0.1% H ₃ PO ₄ (70:30). Identification was carried out by comparing the retention time on the column of the studied and standard compounds. The number of products formed was estimated by the size of the area and the height of the peaks in the chromatogram relative to these values of standard compounds.

As a result of the studies, it was found that the strain Rhodococcus wratislaviensis Ch625 decomposes monochlorinated biphenyls (hereinafter in Tables 1, 2 PCB1, PCB3 (No. according to IUPAC classification) by 99.9%, dichlorinated biphenyls (further in Tables 1, 2 PCB8 (No. according to IUPAC classification ) by 97.8%, trichlorinated biphenyls (further in tables 1, 2 PCB17, PCB28 (IUPAC classification number) by 82.2% for 2 days, commercial mixtures of Delor 103 and Sovol polychlorinated biphenyls - by 99% for 8 days (tables 1, 2).

During the decomposition of polychlorinated biphenyls by the strain Rhodococcus wratislaviensis Ch625, insignificant amounts of intermediate compounds are fixed, and there is a tendency to their further utilization. The spectrum of detected metabolites indicates the decomposition of polychlorinated biphenyls to the main metabolic compounds of the cell.

Thus, the strain Rhodococcus wratislaviensis Ch625 is an active destructor of both individual congeners of polychlorinated biphenyls and their commercial mixtures.

Example 3

Decomposition of the Rhodococcus wratislaviensis Ch625 strain of pesticides of the group of persistent organic pollutants: γ-hexachlorocyclohexane (lindane), hexachlorobenzene, dichlorodiphenyl trichloroethane.

The destruction of pesticides by the strain Rhodococcus wratislaviensis Ch625 was carried out in experiments with "washed cells". The strain was grown in K1 liquid medium with biphenyl (1 g / L) at 28 ° С to an OD of ₆₀₀ ~ 1.0. Cells washed twice in K1 medium (1 ml, OD ₆₀₀ = 2.0) were transferred to vials with Teflon caps.

As a substrate, a solution of γ-hexachlorocyclohexane (lindane) (200 μg / ml), hexachlorobenzene (2.5 mg / ml), dichlorodiphenyl trichloroethane (200 μg / ml) was added. The analysis was performed by gas chromatography with mass spectrometric detection on a GC6890N gas chromatograph (Agilent Technology, USA) with a MSD5973N mass selective detector (Agilent Technology, USA) and a HP-5MS SN quartz capillary column US15189741-1 (30 m × 0.25 mm) (Agilent Technology, United States). The carrier gas is helium (1 ml / min), the temperature of the evaporator is 230 ° C. Sample volume 0.2 μl. Preliminary, the samples were extracted with chloroform for 3 hours.

The quantitative content of the isolated pesticides was evaluated based on a comparison of the values of the obtained peaks and standard compounds.

Pesticide degradation products were determined by high performance liquid chromatography (HPLC) as described above (Example 2).

The strain Rhodococcus wratislaviensis Ch625 carries out the destruction of γ-hexachlorocyclohexane (lindane) by 27%, hexachlorobenzene by 20% and dichlorodiphenyl trichloroethane by 31% (see table 3).

The accumulation of intermediate products (chlorobenzene, chlorcatechol, chlorobenzoic acids) formed during the decomposition of these pesticides by the strain Rhodococcus wratislaviensis Ch625 was insignificant, which indicates the efficient utilization of the initial substrate.

Example 4

Analysis of the degradation activity of the strain Rhodococcus wratislaviensis Ch625 after cultivation on various substrates.

The activity of the strain Rhodococcus wratislaviensis Ch625 was studied with respect to the model congener of polychlorinated biphenyl-2,4′-dichlorobiphenyl (PCB8) in experiments with “washed cells” after culturing the strain on various substrates.

The biomass of the strain Rhodococcus wratislaviensis Ch625 was grown by periodic cultivation in K1 liquid medium for 48 hours at 28 ° C to an OD of ₆₀₀ = 1.0. As a source of carbon and energy, the following was introduced: 1) sodium benzoate - 1 g / l, 2) sodium benzoate + biphenyl, 1 g / l and 0.2 g / l, respectively, 3) biphenyl - 1 g / l. Sodium benzoate is used as a substance that is an affordable substrate (freely produced, low price, widely used in industry). Cells washed twice in K1 medium (1 ml, OD ₆₀₀ = 2.0) were transferred to vials with Teflon caps. 2,4′-dichlorobiphenyl was added at a concentration of 90 mg / L. Analysis of destruction and metabolites was performed as described in example 2.

As a result of the study, it was found that the growth of the biomass of the strain Rhodococcus wratislaviensis Ch625 occurs at a high and almost the same rate during cultivation on all three substrates. The specific growth rate was: 1) on benzoate - 0.076 h ^-1 , 2) on benzoate + biphenyl - 0.078 h ^-1 , 3) on biphenyl - 0.077 h ^-1 . The differences lie within the confidence interval; therefore, it can be argued that the specific growth rate when using sodium benzoate as a source of carbon and energy varies insignificantly.

An analysis of the results showed that after cultivation on sodium benzoate, the strain Rhodococcus wratislaviensis Ch625 decomposes 99.6% of PCB8 in 4 days, after cultivation on a mixed substrate (sodium benzoate and biphenyl) - 99.8% PCB8, and after cultivation on biphenyl - 97.8% PCB8.

Analysis of the metabolic profile showed that the aromatic ring of chlorobiphenyls - 2-hydroxy-6-oxo- (chlorophenyl) hexa-2,4-dienoic acid only slightly accumulates during the first 24 hours of destruction in option 2 (after a mixed substrate) and then its concentration reduced to zero. The accumulation of chlorobenzoic acids and their metabolites of hydroxybenzoic acids was recorded in all three variants, however, after preliminary cultivation on benzoate, the rate of metabolite decrease was 20% higher than in the other two experimental variants.

It was proved that the strain Rhodococcus wratislaviensis Ch625 retains high degradation activity against polychlorinated biphenyls after cultivation on sodium benzoate, and this compound (sodium benzoate) can be recommended for increasing large volumes of the strain culture as readily available raw materials.

Thus, the strain Rhodococcus wratislaviensis Ch625 effectively decomposes several compounds of the group of persistent organic pollutants, namely, polychlorinated biphenyl, hexachlorobenzene, γ-hexachlorocyclohexane (lindane) and dichlorodiphenyl trichloroethane, and can be recommended for the development of a biopreparation compound based on it.

Claims (1)

The strain Rhodococcus wratislaviensis is a destructor of persistent toxic organochlorine compounds: polychlorinated biphenyls, hexachlorobenzene, γ-hexachlorocyclohexane (lindane), dichlorodiphenyl trichloroethane, deposited in the All-Russian Collection of Microorganisms of the Federal State Budgetary Institution of Physics and Microorganism Institute of Biochemistry. G.K. Scriabin, Russian Academy of Sciences under registration number VKM As-2631D.