SU1652335A1 - Actinomyces strain streptomyces rochei, carrying out complete degradation of 2,4,6-trichlorophenol, or 2,4-dichlorophenol, or 2,6- dichlorophenol, or 2-chlorophenol - Google Patents

Abstract

The invention relates to biotechnology and can be used to purify wastewater and soil from chlorinated phenols. The purpose of the invention is to obtain a new strain capable of decomposing elevated concentrations of a wide range of chlorinated phenols. Streptomyces rochel strain BKM Ac-1284D was obtained by extracting cotton fields from a mixed sample of soil. . The strain completely decomposes 2,4,6-trichlorophenol at a concentration of up to 400 mg / l, as well as 2,4-dichlorophenol, 2,6-dichlorophenol, 2-chlorophenol at a concentration in the medium up to 50 mg / l as in growth conditions x, as well as at rest washed cells.

Description

J

The invention relates to biotechnology and can be used to purify wastewater and soil from chlorinated phenols.

The purpose of the invention is to obtain a new strain with the ability to decompose elevated concentrations of a wide range of chlorinated phenols.

The invention consists of isolating the strain of actinomycetes Streptomyces rochei BKM Ac-1284D, degrading 2-chloro- or 2,4-or 2,6-dichloro, or 2,4,6-trichlorophenol. Strain completely decomposes 2-chlorophenol, 2,4-, 2,6-dichlorophenols at a concentration of 50 mg / l; 2,4,6-trichlorophenol (2,4,6-TCP) at a concentration of 10-400 mg / l, using them as the sole carbon source. In the process of chlorophenols utilization, a stoichiometric amount of chlorine ions is released into the medium.

The strain Streptomyces rochei 303 was isolated by the method of cumulative culture from a mixed sample of the soils of cotton fields of the Uzbek SSR, which were treated with high doses of chloroaromatic pesticides for 15–20 years, deposited in the All-Union Collection of Microorganisms No. Ac-12840.

To obtain a pure culture, the spore suspension is plated on a selective medium, a stand-alone colony is transferred to a full-fledged agar medium coated with a membrane filter (Milllpore. USA) with a pore size of 0.12 µm. Air mycelium sprouted through the filter is subcultured on selective agar medium and iso

ate yu

CJ

coke

used as a seed in experiments.

Characteristics of the strain.

Morphological and cultural characteristics.

Sporosum spirals, the surface of the spores is smooth. LL-diaminopimelic acid is present in the cell wall. The differentiating sugars are not contained. The hyphae are long, well branchy. The spores are round, 1-1.5 microns in diameter. Gram-positive. non-acid-resistant.

On oats and mineral agar, aerial mycelium is gray, substrate colorless, sometimes pale pink, soluble pigment is not formed. On organic agar, aerial mycelium is white or gray-white, the substrate mycelium is colorless or slightly yellow, and there is no soluble pigment. No organic melanoid pigment is formed on organic tyrosine agar. The diameter of the colonies 2-8 mm. Has the smell of geosmin. Substrate mycelium, as a rule, is not fragmented.

Physiological signs.

As a carbon source uses cellobiose. fructose, inositol, sucrose, galactose, lactose, maltose, mannitol, L-rhamnose, xylose, inulin. Does not utilize raffinose, adonite, erythritol, D-arabinose. As a source of nitrogen, L-histidine, hydroxyproline, and slightly a-aminobutyric acid are used.

Hydrolyzes pectin, lysitin, arbutin.

Forms H2S, possesses nitrate reduction activity. It grows on media with 7% NaCI: 0.01% NaNa: 0.1% phenol at 45 ° C. Resistant to neomycin at a concentration of 50 µg / ml. Sensitive to rifampicin at a concentration of 50 µg / ml.

Does not possess antibiotic activity against Asperglllus nlger, Bacillus sobtllls, Streptomyces murlnus.

It grows in the temperature range 10-37 ° C, the optimum is 27 ° C.Aerob.

Strain is not pathogenic.

Storage conditions: in lyophilized form - in ampoules at 4 ° C and under liquid nitrogen - at 95-9b ° C. The strain can be maintained by regular transfers (1 time in 14 days) on the mineral medium of the composition, g / l: NasSCM 0.1; MgS04 7H20 0.8: KH2PO 0.7; (MND) 2NP041.5; 2,4,6-trichlorophenol 0,2: agar 15.0 (2.4,6-trichlorophenol is added as sodium phenol).

EXAMPLE 1. Streptomyces rochel BKM Bc-1284D is grown for 5 days on barbeque with mineral agar medium composition, g / l: NaSOo 0.1; MgS04 7H O

0.8; KH2P04 0.7; (1.5; 2,4.6-TCP (in the form of sodium salt) 0.05; agar 15.0. The grown culture is introduced into a liquid medium of similar composition, poured into 100 ml in

Medical flasks with a volume of 700 ml, 2,4,6-TCP, are introduced at a concentration of 10 mg / l, cultivated at 29 ° C in the dark on a rocking chair at 180-200 rpm.

2,4,6-TCP and its metabolic products

extracted from acidified to pH 2.0 culture liquid with ether. Qualitative analysis is carried out using thin layer chromatography on Sllufol UV-254 plates. The plates are developed in

solvent system benzene: dioxane: acetic acid 90: 10: 2. Detection was performed in UV light and spraying with a reagent on chlorine-containing substances (0.1 g AgNOa, dissolved in a mixture of 1 ml distilled water, 190 ml acetone and 10 ml 2-phenoxyethanol, 1 drop of 30% H202 was added to the solution) . After exposure for 15 minutes in long-wave ultraviolet light, chlorine-containing

the substances appear as gray spots on a white background. Compounds having phenolic hydroxyl are detected after spraying with diazotized benzidine in the form of yellow brown spots. Metabolism products are analyzed using chromatography-mass spectrometry using an LKB-2091 instrument and high-performance liquid chromatography (HPLC) system. Analysis conditions in the HPLC system:

LKB-2134 column 4 × 250 mm in size, filled with Spherisorb ODS-2 particles 5 pt, solvent - 70% methanol, 30% water, flow rate 0.7 ml / min, UV detector with variable wavelength LKB- 2151.

Measurements are performed at a wavelength of 280 and 311 nm.

Quantitative determination of 2,4,6-TCP is carried out spectrophotometrically by ultraviolet absorption with

I am 311 nm on a Specord M-40 instrument (Karl-Zeiss, lena, DDR), gas chromatographic on a Chuy model 304 from the company Rue Unlearn (Phillips) on a 1.5 m long column with an internal diameter of 2 mm with 7% PEGA and 3% H3P04 on Chromosorb W / AW 30/100

mesh, inlet and column temperatures of 150 ° C, detector of 160 ° C, nitrogen carrier gas, 30 ml / min.

The concentration of chlorine ions in the culture fluid was measured after cell separation by centrifugation. The analysis is carried out using an Orion 94-17 chlorine selective electrode on an Ion Analyzer EA940 ion analyzer (Orion). Electrode calibration is carried out according to standards prepared on culture medium.

The culture is capable of utilizing 2,4,6-TCP in a concentration of 10 mg / l. The time of complete decomposition is 1 day. In this case, a stoichiometric amount of chlorine ions is detected in the culture fluid.

EXAMPLE 2 The process is carried out analogously to Example 1, but 100 mg / l of 2,4,6-TPC in the form of sodium salt are introduced into the culture medium. At this concentration, 2,4,6-TCP disappears completely in 6 days. the stoichiometric amount of chlorine ions is determined in the culture fluid; the optical density of the cell suspension increases by about 5 times from 0.012 to 0.067.

Froze The process is carried out analogously to Example 1, but 400 mg / l of 2,4,6-TCP in the form of sodium salt is introduced into the culture medium. 2.4,6-ThPh disappears in 10 days; a stoichiometric amount of chlorine ions is detected in the culture fluid.

EXAMPLE 4 After growing the original strain on the scythes with selective intensity (as in Example 1), the culture is introduced into a liquid medium of the composition, g / l: bactopepton 5.0; yeast extract 3.0; glucose 5.0; K2HP04 0.2, poured into 100 ml in medical flasks with a volume of 700 ml, and grown for 20 h at 28-30 ° C with stirring. The cells are then separated by centrifugation and washed three times with 0.05 M phosphate buffer, pH 7.0. Cells are applied in an amount of 40 mg / ml in crude weight into flasks with 100 ml of 0.05 M phosphate buffer, pH 7.0. 2-Chlorophenol is added as an aqueous solution of sodium salt to a final concentration of 50 mg / L. Incubated at 29 ° C in the dark on a rocking chair at 180-200 rpm. After 0, 6, 15, 18, 21, 24 hours, samples are taken for analysis. The quantitative determination of chlorophenols, mineral chlorine and the identification of metabolites is carried out using the methods described in Example 1.

The remaining Streptomyces rochei cells are capable of decomposing 2-chlorophenol at a concentration of 50 mg / l during the day. In this case, a stoichiometric amount of chlorine ions is observed, and chlorohydroquinone is detected as a metabolite.

PRI me R 5. The process is carried out analogously to example 4, but on medium for incubation

2.4-dichlorophenol is added as sodium salt to a final concentration of 50 mg / l. The resting cells of the strain decompose 2,4-DCP for 1 day with the release of a stoichiometric amount of chlorine ions; chlorine hydroquinone is found as a metabolite.

EXAMPLE 6 The process is carried out analogously to Example 4, but 2,6-dichlorophenol is added as an sodium salt to the incubation medium for a final concentration of 50 mg / l.

The resting cells of the strain decompose 2,6-DCP for 1 day with the release of a stoichiometric amount of chlorine ions. Dichlorohydroquinone, chlorohydroquinone and chlorine-containing product of the splitting of the aromatic ring with a mass of 133 are found as metabolites.

Example. The process is carried out analogously to Example 4, but 2,4,6-dichlorophenol as the sodium salt is introduced into the incubation medium to a final concentration of 50 mg / l.

The remaining cells of the strain decompose 2,4,6-trichlorophenol during the day with the release of a stoichiometric amount of chlorine ions, and dichlorohydroquinone, chlorohydroquinone and chlorine-containing aromatic ring decomposition product with a mass of 133 are found as metabolites.

Thus, the strain of Streptomyces rochei completely decomposes 2-chloro, 2,4-dichloro-. 2,6-dichloro-, 2,4,6-trichlophenols. The decomposition of all four isomers is accompanied by stoichiometric emission of chlorine ion. The strain assimilates a wider range of chlorophenols, decomposes them both in growth conditions and at rest washed cells, which is of great importance since the strain can be used not only for the purification of effluent, where technology with immobilized non-multiplying cells is possible, but also for cleaning local contamination sites where it is required that the strain grows on these substrates.

Claims (1)

Invention Formula The actinomycete strain Steptomyces rochei BKM Ac-1284D, which carries out the complete decomposition of 2,4,6-trichlorfenol or 2,4-dichlorophenol, or 2,6-dichlorophenol, or 2-chlorophenol.