RU2603277C1 - Acidophile strain desulfosporosinus sp._ for contaminated ecosystems with extremely acidic values cleaning of heavy metal ions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used to purify industrial waste water from mining, machine building, instrument making industry, electroplating facilities, as well as for cleaning mine, quarry, drainage water from heavy metals ions. Strain of Desulfosporosinus sp. bacteria, resistant to high concentrations of heavy metals ions, is deposited in Russian National Collection of Microorganisms of Microbial Biochemistry And Physiology Institute (IBFM RAS) under registration number VKM B-3021 D.

EFFECT: invention increases degree of waste water purification, having extremely acidic pH values (< 3).

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Description

The invention relates to biotechnology and biological methods for the treatment of contaminated industrial wastewater with extremely acidic pH values (<3) and is a new strain of acidophilic sulfate-reducing bacteria (CRP) Desulfosporosinus sp. OL VKM B-3021 D. The strain can be used in biotechnological schemes for the purification of extremely acidic wastewater from mining, machine-building, instrument-making, galvanic industries, as well as for the treatment of mine, quarry, and drainage waters having extremely acidic pH values from sulfates and ions heavy metals.

A known bacterial strain Streptomyces chromogenes s.g. 0832 used for wastewater treatment from heavy metal ions (RU 2312073). The proposed method for wastewater treatment from heavy metal ions allows the most complete removal of heavy metal ions from the wastewater of food industry enterprises, providing a high degree of extraction in a short period of time. The disadvantage of the proposed method is the impossibility of its implementation in an extremely acidic environment.

The most promising for the purification of contaminated ecosystems are sulfate-reducing bacteria. Biotechnologies for the purification of contaminated ecosystems are based on the ability of sulfate-reducing bacteria to produce hydrogen sulfide during its life, which binds heavy metal ions to insoluble sulfides.

A known strain of bacteria Desulfovibrio oxamicus VKM B-2465 D (RU 2355756), capable of treating wastewater from heavy metal ions at elevated sulfate concentrations. The disadvantage of this strain is its ability to work with a total concentration of heavy metals up to 190 mg / l, while it is known that in the wastewater of industrial enterprises, the total concentration of heavy metal ions is most often more than 200 mg / l.

A known strain of bacteria Desulfovibrio sp. SVB-2 (RU 2269571). The strain is intended for wastewater treatment from heavy metal ions at a total concentration of heavy metals in the used wastewater of 197.9 mg / l, and copper - 8.9 mg / l. The disadvantages of the strain SVB-2 include insufficiently high resistance to heavy metal ions, in particular copper ions, and its inability to grow in extremely acidic pH values - the strain grows at pH 6.5 ... 8.5.

A known strain of the bacterium Desulfovibrio sp. for the treatment of wastewater from heavy metal ions (RU 2542402). Bacterial strain Desulfovibrio sp. A 4/1 was isolated from the sludge sedimentation of a water sump from the cooling system of melting furnaces of the Chelyabinsk Metallurgical Plant. The strain is characterized by high resistance to increased concentrations of copper ions (up to 600 mg / l), nickel ions (up to 250 mg / l), cobalt ions (up to 400 mg / l), cadmium ions (up to 75 mg / l). The disadvantage of the strain Desulfovibrio sp. is its inability to grow at extremely acidic pH values, the optimal pH of the medium for strain A 4/1 is 7.2.

The closest in essence and the achieved result to the claimed invention is the bacterial strain Desulfotomaculum halophilum RETECH-SRB-II (CN 101434919), deposited in the Chinese type culture collection center (CCTS) (Wuhan University) with deposit number M207061, for the purification of acid mine drainage water . The strain is able to precipitate sulfides of heavy metals in various concentrations (Cu ²⁺ - 0.04-0.6 g / l; Zn ²⁺ - 0.5-1.0 g / l; As ³⁺ - 0.07-0, 6 g / l; Fe ²⁺ - 1.0-2.0 g / l; Fe ³⁺ - 0.5 g / l) at acidic pH values (2.0-4.5). The disadvantage of the strain is the need for its use together with fermented rice straw (fermentation time 10-30 days), which lengthens the duration of the biotechnological process.

The objective of the invention is to obtain a metal-resistant, highly active acid-tolerant strain of sulfate-reducing bacteria used for the treatment of wastewater from heavy metal ions.

EFFECT: isolation of an acidophilic strain of bacteria Desulfosporosinus sp. OL VKM VKM B-3021 D, resistant to low pH and high metal concentrations and intended for the purification of contaminated ecosystems with extremely acidic pH values and its deposition in the All-Russian collection of microorganisms.

Strain Desulfovibrio sp. OL deposited by the All-Russian Collection of Microorganisms (VKM) of the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS (IBPM) under registration number VKM B-3021 D.

The proposed strain of Desulfosporosinus sp. OL is not known in science and technology, therefore, the properties that it exhibits are new, and therefore, the claimed solution has an inventive step.

The origin of the strain was isolated from fouling from the bottom of the vetland, the Komsomolsky tailing site, site KU-2, Kemerovo region. Russia.

Analysis of the nucleotide sequence of the 16S rRNA gene of strain OL showed that the proposed strain is a new strain and belongs to the genus Desulfosporosinus.

Cultural and morphological characteristics of the patented strain were determined determined by its cultivation in a standard freshwater medium Widdel [Widdel F. and. Bak F. Gram-negative mesophilic sulfate-reducing bacteria. N.y .: Springer. 1992].

The resulting strain is characterized by the following features.

The temperature of the cultivation of the strain is + 28 ° C.

The generic and species name of the strain is a strain of bacteria Desulfosporosinus sp. OL.

Morphological characteristic - the form of cells - sticks.

Spore formation - spore-forming.

Gram reaction is positive.

Cell motility is motile.

Physiological characteristic - attitude to oxygen - anaerobic.

Alcohols (ethanol, glycerol), sugars (fructose, glucose, sucrose) and organic acids (lactate, propionate, acetate), as well as peptone and tryptone are used as a substrate for growth.

Resistance to heavy metals. The strain is resistant to copper ions (up to 450 mg / l), cobalt (up to 200 mg / l), nickel (up to 700 mg / l), cadmium (up to 20 mg / l). When cultivating a strain with Fe ^{2+ ions} , a black precipitate of iron sulfides is formed.

The optimal pH of the medium is 4.0.

A useful property, in connection with which the culture is deposited, is resistance to high concentrations of heavy metal ions, acidotolerate properties.

Cultivation conditions - Widdel's freshwater environment.

Storage conditions - stored by reseeding on cultivation medium or lyophilized.

The ability of the strain to reduce the content of the main polluting heavy metal ions present in wastewater was studied in laboratory experiments. The results are shown in the examples below.

Example 1. The study of the ability of the bacterial strain Desulfosporosinus sp. OL to the deposition of copper ions.

Pure CRP culture Desulfosporosinus sp. OL was cultured on a synthetic medium containing copper ions at a concentration of 450 mg / L.

Sowing was carried out in a sterile laminar box. To remove dissolved oxygen in the medium before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water. Nutrients were added to the medium cooled to room temperature (table 2) (per 1 l) in the following sequence: vitamins (2 ml), salt solution (10 ml), cofactor solution (1 ml), organic substrate - lactate (1 , 6 ml), H ₂ SO ₄ (when making pH is adjusted to 3,0), H ₂ S (2 mL) was added. Before introducing hydrogen sulfide, a mother liquor of copper (CuSO ₄ · 5H ₂ O (10 g per 50 ml of distilled water)) was added in an amount of 4.5 ml per 1 liter of synthetic medium.

About 10 ml of synthetic medium with additives and 1 ml of inoculum were added to 10 ml vials. Anaerobic conditions were created by filling the vials with medium to the top. To prevent air oxygen from entering the vial, rubber stoppers were rubbed to the edges of the vials with a sterile needle. The vials were closed with aluminum caps, sealed with a seaming machine, and the thermostat was placed at a temperature of 28 ° C.

On a medium with copper ions in a concentration of 450 mg / l for 10-15 days, copper is precipitated in the form of sulfide.

Example 2. The study of the ability of the bacterial strain Desulfosporosinus sp. OL to the deposition of nickel ions.

Pure CRP culture Desulfosporosinus sp. OL was cultured on a synthetic medium containing divalent nickel at a concentration of 700 mg / L.

Sowing was carried out in a sterile laminar box. To remove dissolved oxygen in the medium before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water. Nutrients were added to the medium cooled to room temperature (table 2) (per 1 l) in the following sequence: vitamins (2 ml), salt solution (10 ml), cofactor solution (1 ml), organic substrate - lactate (1 , 6 ml), a solution of H ₂ SO ₄ (when pH is adjusted to 3.0), H ₂ S (2 ml). Before the introduction of hydrogen sulfide, a mother liquor of nickel (NiCl ₂ (2.2 g per 100 ml of distilled water)) was added in an amount of 70 ml per 1 liter of synthetic medium.

About 10 ml of synthetic medium with additives and 1 ml of inoculum were added to 10 ml vials. Anaerobic conditions were created by filling the vials with medium to the top. To prevent air oxygen from entering the vial, rubber stoppers were rubbed to the edges of the vials with a sterile needle. The vials were closed with aluminum caps, sealed with a seaming machine, and the thermostat was placed at a temperature of 28 ° C.

Nickel sulfide precipitates as a precipitate on the walls and bottom of the bottle.

Example 3. The study of the ability of the bacterial strain Desulfosporosinus sp. OL to the deposition of cobalt ions.

Pure CRP culture Desulfosporosinus sp. OL was cultured on a synthetic medium containing cobalt divalent at a concentration of 200 mg / L.

Sowing was carried out in a sterile laminar box. To remove dissolved oxygen in the medium before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water. Nutrients were added to the medium cooled to room temperature (table 2) (per 1 l) in the following sequence: vitamins (2 ml), salt solution (10 ml), cofactor solution (1 ml), organic substrate - lactate (1 , 6 ml), a solution of H2SO4 (when pH is adjusted to 3.0), H2S (2 ml). Before the introduction of hydrogen sulfide, a mother liquor of nickel (CoCl ₂ × 6H ₂ O (2 g per 50 ml of distilled water)) was added in an amount of 20 ml per 1 liter of synthetic medium.

About 10 ml of synthetic medium with additives and 1 ml of inoculum were added to 10 ml vials. Anaerobic conditions were created by filling the vials with medium to the top. To prevent air oxygen from entering the vial, rubber stoppers were rubbed to the edges of the vials with a sterile needle. The vials were closed with aluminum caps, sealed with a seaming machine, and the thermostat was placed at a temperature of 28 ° C.

The formation of cobalt sulfide begins within 10-12 days.

Example 4. The study of the ability of the bacterial strain Desulfosporosinus sp. OL to the deposition of cadmium ions.

Pure CRP culture Desulfovibrio sp. A 4/1 VKM B-2820D was cultured on a synthetic medium containing divalent cadmium at a concentration of 20 mg / L.

Sowing was carried out in a sterile laminar box. To remove dissolved oxygen in the medium before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water. Nutrients were added to the medium cooled to room temperature (table 2) (per 1 l) in the following sequence: vitamins (2 ml), salt solution (10 ml), cofactor solution (1 ml), organic substrate - lactate (1 , 6 ml), a solution of H ₂ SO ₄ (when pH is adjusted to 3.0), H ₂ S (2 ml). Before the introduction of hydrogen sulfide, a mother liquor of nickel (CdCl ₂ × 2.5H ₂ O (1 g per 50 ml of distilled water)) was added in an amount of 2 ml per 1 liter of synthetic medium.

About 10 ml of synthetic medium with additives and 1 ml of inoculum were added to 10 ml vials. Anaerobic conditions were created by filling the vials with medium to the top. To prevent air oxygen from entering the vial, rubber stoppers were rubbed to the edges of the vials with a sterile needle. The vials were closed with aluminum caps, sealed with a seaming machine, and the thermostat was placed at a temperature of 28 ° C.

Cadmium sulfide is deposited on the walls and bottom of the bottle.

Claims (1)

The bacterial strain Desulfosporosinus sp., Deposited in the All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBPM) under registration number VKM B-3021 D, designed to clean contaminated ecosystems with extremely acidic pH values from heavy metal ions.