RU1792925C - Strain of bacterium pseudomonas putida - a destructor of aromatic compounds and mezithyl oxide - Google Patents

Abstract

Usage: utilization of aromatic compounds and mesityl oxide in phenolic wastewater. Essence: the bacterial strain Pseudomonas putlda GFS-8 deposited under the number VKPM B-5248. The strain has a high destructive activity against aromatic compounds, as well as mesityl oxide: completely utilizes 1000 mg / l of phenol in 96 hours. 1 table.

Description

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Arunakumarl A. of al. Utilization of omatlc substances by Pseudomonas anacearum. // Ind.I.Exp. Biol., 1984, v. 22, 10p.32-36.

Masque C. et al. Selection and adaptation a phenoldegrading strain of Pseudomonas. Blotechn.Lett. 1987, v.9, No. 9, p.655-660.

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The invention relates to applied microbiology and biotechnology and consists in isolating a new bacterial strain that can be used to treat wastewater, both phenolic and other, containing analogous compounds.

. | The cumene method includes phenol, acetophenone, α-methylstyrene, mesityl oxide, in the composition of wastewater from the production of phenol and acetone. dimethylphenylcarbinol, isopropylbenzene hydroperoxide (hyperis) and other components. The environmental harmfulness of these wastewater is due to the high toxicity of most of the components and increases when mixed.

Phenol is the target product of this production and therefore is a part of wastewater as a main component. In addition, it is a part of effluents for the production of phenol-formaldehyde resins, nitro- and chlorophenols, phenylsulfonic acids, caprolactan salicylic and picric acids and other aromatic compounds. Extremely permissible

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the concentration of phenol in water is 0.001 mg / l.

Acetophenone during the production of phenol and acetone is formed during acid hydrolysis of hyperysis. It can also be found in the wastewater of the perfume industry, where it is used as an aromatic substance, and in the wastewater of pharmaceutical industries. MAC in water 0.1 mg / L. In addition, when disinfecting water by chlorination (in particular in sewage treatment plants), acetophenone can turn into chloroacetophenone - a poisonous substance - a lacrimator with an intolerable concentration of 0.005 mg / l / 2 min.

a-Methylstyrene is obtained by acid hydrolysis of hyperysis, by dehydration of dimethylphenylcarbinols, by dehydrogenation of isopropylbenzene, by the production of phenol and acetone, a-methylstyrene is a part of sewage from the production of butadiene methyl styrene rubber, organic synthesis plants and petrochemicals. MAC in water 0.1 mg / L.

In the process of producing phenol from acetone by the cumene method, acetone, resulting from the decomposition of hyperysis, in the presence of sulfuric acid is converted to mesityl oxide. Methyl oxide is also used as a solvent for cellulose ethers, polyvinyl chloride, natural resins, and is an insecticide.

Strains capable of utilizing phenol are known. For example, French researchers have selected a strain of Nocardla sp. B 87, capable of destroying phenol at a concentration of 2000 mg / L. However, the destruction process required the presence of a source of nitrogen and carbon.

Known strains capable of utilizing other xenobiotics in addition to phenol. For example, the strain Pseudomonas putida CB-173 (300 mg / l phenol, for 48 h, at 30 ° С) can destroy nonionic surfactants, but it implements these processes on a complete medium.

A known strain that destroys a-methylstyrene (Bacillus cereus Kk 3.2000 mg / l, for 72 hours). However, there is no evidence of its ability to use other phenolic wastewater components as nutrient substrates.

Known strains capable of destroying many aromatic compounds. For example, the strain Pseudomonas sotanacearum is able to use 46 aromatic substrates as the sole carbon source. However, neither phenol nor other components of phenolic wastewater are included in its spectrum

destructive activity.

Closest to the claimed is Pseudomonas sp. QT31, destroying phenol at a concentration of 1000 mg / l in 36-95 hours, depending on the time of adaptation. The disadvantage of all these strains is their lack of biodegradability of a wide range of substrates, which are components of wastewater

phenol production .:

An object of the invention is to provide a bacterial strain capable of utilizing wastewater components for the production of phenol and acetone (such as phenol, acetophenone, α-methylstyrene, mesityl oxide) and retaining its destructive activity in wastewater.

The proposed strain is deposited in the All-Union collection of industrial

microorganisms VNII genetics under registration number VKPM B-5248.

The proposed strain is characterized by the following morphological-cultural and physiological-biochemical properties.

Morphological signs. The cells of the strain are short rods with blunt ends located singly and in pairs, gram-negative, spores and capsules do not form.

Cultural properties. On a synthetic mineral medium M9 of the following composition, mg / l: No. 2НР04 6000; KH2P04 3000 mg / l; NaCI 500; NH C11000; agar agar 15000 containing aromatic compounds in

after 72 hours of incubation at 30 ° C, it forms colonies with a diameter of 1.5-2 mm of regular round shape, smooth, shiny, and crown as the sole source of carbon and energy.

Physiological and biochemical properties. Aerobic, motile, catalase- and oxidase-positive, forms arginine dihydrolase and urease; gelatinase, amylase, phenylalanine deaminase does not form. On a Hugh-Leifson medium, it forms acids from L-arabinose, L-xylose, glucose, maltose, sucrose and galactose. On MPA and Kipg, B forms a lemon yellow fluorescent pigment.

It can use acetate, L-arabinose, sucrose, malonate, ethanol, sorbitol, L-alanine, L-glucamine, mannitol, succinate, glycerin, L-xylose, glucose, sucrose, L-rhamnose as the only source of carbon and energy and lectose.

Optimum culturing conditions on complete nutrient media: pH 7.0 ± 0.5; 30 ± 1 ° C.

Storage conditions. Sterile columns (M9 medium, agar-agar 7500 mg / l, acetophenone or phenol at a concentration of 500 mg / l), incubated at 30 ° C for 72-96 hours, pour 1 ml of sterile paraffin oil and store for 4 ° C, subcultured 2 times a year.

Sensitivity to antibacterial drugs. The strain is resistant to lincomycin, ristomycin, benzylpenicillin, oleadomycin; the strain is sensitive to streptomycin, polymyxin, rifampicin, tetracycline, chloramphenicol, erythromycin, carbenicillin, gentamicin.

 Pathogenic properties. During intraperitoneal infection of outbred white mice (10 animals per sample) with a suspension of the daily culture of the strain at a concentration of 1 billion microbial bodies-ml in a volume of 0.5 ml per animal, no disease was found. The observation period is 7 days. This gave grounds to consider the culture of the strain non-pathogenic.

-: Based on a complex of morphological-cultural and physiological-biochemical properties, the strain was identified as Pseudomonas putida.

 PRI me R 1. Obtaining strain GFS8.

f The strain Pseudomonas putida GFS8 was taken from a soil sample taken from a plant producing fensl and acetone by direct sowing of a soil suspension onto an aggated

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M9 of the following composition, mg / l: M 42YP04 6000; KH2RSch 3000; NHUCI 1000; NaCl 500, phenol 500, agar-agar 15000; pH 7.0 ± 0.2.

: The isolated culture was passaged on solid media of the same mineral composition containing gradually increasing phenol concentrations (from 500 to 1125 mg / L), and the largest colonies were selected for further studies.

 On M9 medium containing 1-125 mg / L phenol as the sole carbon source, growth of this culture was not observed. Clones selected from a dense synthetic mineral medium M9 with a phenol content of 100 mg / L were tested; the ability to utilize this substrate1 upon cultivation in a liquid medium of the same composition in 250 ml flasks containing 100 ml of medium. Content Change | phenol in the culture medium was monitored spectrophotometrically (270 nm). .

Further studies have shown

almost this strain is capable of besides pheno

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It also metabolized α-metistyrene, mesityl oxide (500 mg / L), 4-chlorobenzoic acid (750 mg / L) and benz mid (500 mg / L) as the sole carbon source when cultured on M9 mineral medium at 30 ° C. As a result of passage through dense mineral media, as described above, it was possible to obtain clones capable of utilizing higher concentrations of acetophenone 1250 mg / l, mesityl oxide 1000 mg / l.

Example 2. The use of strain GFS8 as the only phenol destructor under conditions of periodic cultivation on a circular shaker.

The daily culture of strain GFS8 grown on an agarized complete nutrient medium was washed three times by centrifugation at 6000 rpm in a 0.9% sodium chloride solution. 1 ml of suspension (10 cells / ml) was added to a 250 ml flask containing 100 ml of non-agarized medium | I9 (medium composition see Example 1) and 1000 mg / l phenol as the sole carbon source of m energy. Cultivation was carried out on a circular shaker (120-135 rpm) at 28-30 ° C. The change in the phenol content in the culture medium was monitored by UV spectrophotometry (maximum absorption of this substance 270 nm) and photocolorimetry (reaction with 4-aminoantipyrine).

After 96 hours of cultivation of the claimed strain under the indicated conditions, phenol was not detected in the culture medium by the above method.

Example 3. Use of strain GFS-8 as an acetophenone destructor under conditions of periodic cultivation on a circular rocking chair.

The study of the utilization of aceto1 phenon of the claimed strain was carried out according to a scheme similar to that described in Example 2, with the difference that instead of phenol, acetophenone was used at a concentration of 1250 mg / L. The change in the acetophenone content in the culture medium was monitored by UV spectrophotometry (maximum absorption 245 nm) and gas chromatography.

After 96 hours of cultivation of the claimed strain under the indicated conditions, acetophenone in the medium by the above methods was not determined.

Example 4. Use of strain GFS-8 as an a-methylst-α-roll destructor under conditions of periodic cultivation on a circular rocking chair.

The study of the utilization of a-methylstyrene according to the inventive strain was carried out according to a scheme similar to that described in Example 2, with the difference that 500 mg / L a-methylstyrene was used as the sole carbon source instead of phenol. Change in the content of a-methylstyrene The cultivation was monitored by UV spectrometry (maximum absorbance 244 nm) and gas chromatography.

After 24 hours of cultivation of the claimed strain under the indicated conditions, α-methylstyrene in the medium by the above-mentioned methods hell was determined,.

Example 5. The use of strain GFS-8 as a destructor of mesityl oxide under conditions of periodic cultivation on a circular rocking chair.

The study of the process of utilization of mesityl oxide after the claimed strain was carried out according to the scheme similar to that described in Example 2, with the difference that mesityl oxide at a concentration of 1000 mg / L was used instead of phenol. The change in the mesityl oxide content in the culture medium was monitored by UV spectrophotometry (maximum absorption 243 nm) and gas chromatography.

After 96 hours of cultivation of the claimed strain under the indicated conditions, mesityl oxide was not determined by the above methods in the medium.

Example 6. The use of strain GFS-8 as a destructor of phenol and acetophenone when they are combined in the medium under conditions of periodic cultivation on a circular rocking chair.

The study of the utilization of phenol and acetophenone when they are combined in the culture medium was carried out according to the scheme similar to that described in example 2, with the difference that in addition to phenol 150 mg / l, the non-agarized mineral synthetic medium M9 (composition see example 1) contained acetophenone in such the same concentration of 150 mg / l, Change in the content of both substrates in the culture medium

Claims (1)

SUMMARY OF THE INVENTION Bacterial strain Pseudomonas putida GFS-8 VKPM-B-5248 — the destructor was controlled by UV spectrophotometry (maximum absorption; phenol 270 nm, acetophenone 245 nm) and gas chromatography, After 15 hours of cultivation of the claimed strain under the indicated conditions, acetophenone was not determined by the above methods. After 19 hours, analysis revealed the absence of phenol in the medium. The test results of the biodestructive activity of the claimed strain, as well as the prototype strain are shown in the table. Comparison of the biodestructive characteristics of the claimed strain with the prototype shows that the strain Pseudomonas sp QT 31 after prolonged adaptation capable completely dispose of 1000 mg / l phenol per hour, and the claimed strain destroys the same phenol concentration in 96 hours. However, it differs favorably from the prototype by the ability to degrade acetophenone (1250 mg / L in 96 hours), a-methylstyrene (500 mg / L in 24 hours), mesityl oxide (1000 mg / L in 96 hours ), Which are components of phenolic wastewater. Testing the strain in samples of real wastewater containing phenol, acetophenone, mesityl oxide, 2-methylstyrene showed its viability and destructive activity in relation to these components. The presence of sufficiently high (350 mg / L) concentrations of dimethylphenylcarbinol and minor admixtures of hyperis, acetone do not reduce the biodestructive activity of the claimed strain with respect to the main components of wastewater. Thus, the proposed strain .has a high destructive activity, has been tested in samples of real industrial wastewater and can be recommended for local treatment of phenolic wastewater from components such as phenol, acetophenone, a-methylstyrene, mesityl oxide, both individually and in combination in wastewater fifty tical compounds a. and mesi oxide