RU2603249C1 - Strain of bacteria desulfovibrio species vk-9 for purification of acidic wastewater from heavy metal ions - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to industrial microbiology. Bacterial strain Desulfovibrio sp. VK-9 has high resistance to high concentrations of copper ions (up to 125 mg/l), nickel ions (up to 250 mg/l), cobalt ions (up to 350 mg/l) and cadmium ions (up to 60 mg/l) and can be used for purification of acidic wastewater from machine building, instrument-making, electrical, mining enterprises, electroplating industries, as well as for acidic mine water purification from high concentrations of copper ions and other heavy metals. Strain Desulfovibrio sp., is deposited in Russian National Collection of microorganisms of Institute of biochemistry and physiology of microorganisms RAS IBFM under registration number VKM V-3000D.

EFFECT: invention allows to increase degree of wastewater purification from heavy metal ions.

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Description

Bacterial strain Desulfovibrio sp. VK-9 is isolated from laminated mats with a wooden structure flooded with water at the exit of the adit, the Akatuyskoe deposit of polymetallic ores (Trans-Baikal Territory). The strain is characterized by high resistance to increased concentrations of copper ions (up to 125 mg / l), nickel ions (up to 250 mg / l), cobalt ions (up to 350 mg / l) and cadmium ions (up to 60 mg / l). The strain is capable of growth at wide pH values from 2.7 to 7.5.

The invention relates to industrial microbiology and relates to the production of a new strain of bacteria that can be used in biotechnology for the treatment of acidic industrial wastewater from machine-building, instrument-making, electrical, mining and processing enterprises, galvanic industries, and also for the purification of acidic mine water from elevated concentrations of copper ions and other heavy metals.

A known method of biosorption water purification from heavy metal ions using yeast Saccharomyces cerevisiae (RU 2509734). Brewing yeast biomass is capable of utilizing wastewater containing zinc (20 mg / dm ³ ), nickel (100 mg / dm ³ ), and copper (40 mg / dm ³ ). The disadvantage of the proposed method is the inability to implement it in acidic wastewater, as the used yeast culture is able to grow at a pH of 5.5-8.0.

It is known about the use of sulfate-reducing bacteria for wastewater treatment from heavy metal ions. Hydrogen sulfide formed by sulfate-reducing bacteria in the process of biochemical purification binds heavy metal ions to insoluble sulfides.

A known strain of bacteria Desulfovibrio sp. SVB-2 (RU 2269571), used for wastewater treatment from heavy metal ions. The proposed strain isolated from industrial runoff galvanic industries containing heavy metal ions. However, the total concentration of heavy metals indicated in the patent in the used wastewater is 197.9 mg / l, and copper - 8.9 mg / l. In the wastewater of metallurgical enterprises, the concentration of copper ions can reach 300 mg / l. Strain SVB-2 grows at a pH of 6.5-8.5, an optimum of 7.2.

The disadvantage of strain SVB-2 is not sufficiently high resistance to heavy metal ions, in particular copper ions, which significantly limits its use in biotechnology. It is also impossible to use it in acidic wastewater having a pH <5.0.

The known strain Desulfovibrio halophilus RETECH-SRB-I (CN 101434916), deposited in the Chinese type culture collection center (CCTCC) (Wuhan University) with the deposit number M207060, used to clean acid mine wastewater from heavy metals. The strain is able to purify acid mine water with a pH value of from 1.5 to 6.0 from metal ions in various concentrations: Cu ²⁺ - 0.07-0.6 g / l; Zn ²⁺ - 0.5-1.0 g / l; As ³⁺ - 1.2-2.8 g / l; Fe ²⁺ - 1.0-2.0 g / l; Fe ³⁺ - 0.3-3 g / l. The disadvantages of the proposed strain include its inability to grow on highly toxic metals such as cadmium and nickel, which limits the use of the strain in wastewater of galvanic plants and wastewater of other industries containing one of the most toxic metals - cadmium. In addition, the strain has a less wide range of pH values at which it can be used in biotechnology for wastewater treatment.

The closest in essence and the achieved result to the claimed invention is a bacterial strain Desulfovibrio sp. for the treatment of wastewater from heavy metal ions (RU 2542402). Bacterial strain Desulfovibrio sp. A 4/1 is isolated from silt 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 high concentrations of copper ions (up to 600 mg / l) and other divalent metal cations. The disadvantage of the strain Desulfovibrio sp. is its inability to grow at acidic pH values, the optimal pH of the medium for strain A 4/1 is 7.2.

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

The technical result is the use of a strain of bacteria Desulfovibrio sp. VK-9 for the treatment of acidic wastewater, including mine, from heavy metal ions.

Strain Desulfovibrio sp. VK-9 is deposited with the All-Russian Collection of Microorganisms (VKM) of the Institute of Biochemistry and Physiology of Microorganisms named after G.K.Skryabin RAS (IBPM) under registration number VKM V-3000D.

The proposed strain of Desulfovibrio sp. VK-9 is not known in science and technology, therefore, the properties that it exhibits are new, and therefore, the claimed solution has an inventive step.

Analysis of the nucleotide sequence of the 16S rRNA gene of strain VK-9 showed that the proposed strain is a new strain and belongs to the genus Desulfovibrio.

The resulting strain is characterized by the following features.

Cultural and morphological characteristics of the patented strain are determined during its cultivation in Widdel's standard freshwater medium (Widdel, Bak, 1992). The temperature of cultivation of the strain is +28 ºС. Cultivation is carried out for 3-4 days without metal ions, 7-8 days with the addition of metal ions in a concentration of 100 mg / l or more.

The generic and species name of the strain is the bacterial strain Desulfovibrio sp. VK-9.

Origin - Akatuyskoe deposit of polymetallic ores (Trans-Baikal Territory) isolated from laminated mats with a wooden structure flooded with water at the exit from the adit.

Morphological characteristic - the shape of the cells is a vibrio, 0.5-1.0 × 3.0 × 5.0 microns in size.

Spore formation - no.

Gram reaction is negative.

Cell motility is motile.

Physiological characteristics - attitude to oxygen - anaerobic.

Lactate is used as a substrate for growth. Lactate is metabolized along the path of incomplete oxidation to form acetate. The strain is resistant to copper ions (up to 125 mg / l), cobalt (up to 350 mg / l), nickel (up to 250 mg / l), cadmium (up to 60 mg / l). When cultivating a strain with Fe ^{2+ ions} , a black precipitate of iron sulfides is formed. The strain is capable of growth at pH values from 3 to 7.5. The optimal pH of the medium is 5.0-5.5.

A useful property in connection with which the culture is deposited is the precipitation of heavy metal ions in concentrations exceeding 100 mg / l by hydrogen sulfide.

Cultivation conditions - Widdel's freshwater environment.

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

Example 1. The study of the ability of the bacterial strain Desulfovibrio sp. VK-9 VKM B-3000D to the deposition of copper ions.

Pure CRP culture Desulfovibrio sp. VK-9 VKM B-3000D was cultured on a synthetic medium containing copper ions at a concentration of 125 mg / L.

Sowing was carried out in a sterile laminar cabinet, which was then disinfected with ultraviolet light for 30 minutes. Before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water to remove dissolved oxygen. 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 4.5), H ₂ S (2 ml). Before introducing hydrogen sulfide, a mother liquor of copper was added in an amount of 2 ml per 1 liter of synthetic medium.

About 120 ml of synthetic medium with added additives and 10 ml of inoculum (bacterial culture) were added to 120 ml vials, after which they were added to the top with medium. Rubber plugs were rubbed to the edges of the vials using a sterile needle, which reduced the likelihood of air oxygen entering. At the end of sowing, the bottles were closed with aluminum caps, the bottle was sealed with a seaming machine and the thermostat was placed at a temperature of 28 ºС.

Copper is precipitated as sulfide at the bottom of the vial.

Table 1 & The composition of the synthetic medium

Reagent Concentration, mg / L Na ₂ SO ₄ 4000 MgCl ₂ 6H ₂ O 400 NaCl (25%) 0.0125 ^* FeSO ₄ * 7H ₂ O 2.1 H ₃ BO ₃ 0,03 MnCl ₂ * 4H ₂ O 0.1 CoCl ₂ * 6H ₂ O 0.19 NiCl ₂ * 6H ₂ O 0.024 CuCl ₂ * 2H ₂ O 0.002 ZnSO ₄ * 7H ₂ O 0.144 Na ₂ MoO ₄ * 2H ₂ O 0,036 CuSO ₄ * 7H ₂ O 750 H ₂ O 1 liter   * - ml / l

Table 2 - Composition of nutrients added to the synthetic medium

Solution (insertion amount per 1 liter of synthetic medium) Reagent Concentration 1. Vitamins
(2 ml / l) 4-aminobenzoic acid 4 mg / l Biotin (Vitamin H) 1 mg / l Niacin (Vitamin B ₅ ) 10 mg / l Calcium Pantothenate (Vitamin B ₃ ) 5 mg / l Pyridoxine Dihydrochloride (Vitamin B ₆ ) 15 mg / l Cyanocobalamin (Vitamin B ₁₂ ) 5 mg / l Thiamine (Vitamin B ₁ ) 10 mg / l Riboflavin (Vitamin B ₂ ) 0.5 mg / l Folic acid 0.2 mg / l 2. Saline solution
(10 ml / l) KH ₂ PO ₄ 20 g / l NH ₄ Cl 25 g / l NaCl 100 g / l Kcl 50 g / l CaCl ₂ 11.3 g / l H ₂ O 1 liter 3. The solution of cofactors
(1 ml / l) Na OH 4 g / l Na ₂ SeO ₃ x 5H ₂ O 6 mg / l Na ₂ WO ₄ x2H ₂ O 8 mg / l 4. The solution of Na ₂ S
(2 ml / l) Na ₂ S x 9 H ₂ O 4.8 g

Example 2. The study of the ability of the bacterial strain Desulfovibrio sp. VK-9 VKM B-3000D for the deposition of nickel ions.

Pure CRP culture Desulfovibrio sp. VK-9 VKM B-3000D was cultured on a synthetic medium containing divalent nickel at a concentration of 250 mg Ni / L.

Sowing was carried out in a sterile laminar cabinet, which was then disinfected with ultraviolet light for 30 minutes. Before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water to remove dissolved oxygen. 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 4.5), a solution of sodium sulfide (2 ml). Nickel stock solution was added in an amount of 25 ml per 1 liter of synthetic medium (thus, a concentration of nickel in the medium of 250 mg / l was achieved).

About 120 ml of synthetic medium with added additives and 10 ml of inoculum (bacterial culture) were added to 120 ml vials, after which they were added to the top with medium. Rubber plugs were rubbed to the edges of the vials using a sterile needle, which reduced the likelihood of air oxygen entering. At the end of sowing, the bottles were closed with aluminum caps, the bottle was sealed with a seaming machine and the thermostat was placed at a temperature of 28 ºС.

Nickel is precipitated as sulfide at the bottom of the vial.

Example 3. The study of the ability of the bacterial strain Desulfovibrio sp. VK-9 VKM B-3000D for the deposition of cobalt ions.

Pure CRP culture Desulfovibrio sp. VK-9 VKM B-3000D was cultured on a synthetic medium containing bivalent cobalt at a concentration of 350 mgCo (II) / L.

Sowing was carried out in a sterile laminar cabinet, which was then disinfected with ultraviolet light for 30 minutes. Before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water to remove dissolved oxygen. 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 4.5), a solution of sodium sulfide (2 ml). Stock cobalt solution was added in an amount of 35 ml per 1 liter of synthetic medium (thus, the concentration of cobalt in the medium was 350 mg / l).

About 50 ml of synthetic medium with added additives and 10 ml of inoculum (bacterial culture) were added to the vials, after which they were added to the top with medium. Rubber plugs were rubbed to the edges of the vials using a sterile needle, which reduced the likelihood of air oxygen entering. At the end of sowing, the bottles were closed with aluminum caps, the bottle was sealed with a seaming machine and the thermostat was placed at a temperature of 28 ºС.

Cobalt is precipitated as sulfide at the bottom of the vial.

Example 4. The study of the ability of the bacterial strain Desulfovibrio sp. VK-9 VKM B-3000D for the deposition of cadmium ions.

Pure CRP culture Desulfovibrio sp. VK-9 VKM B-3000D was cultured on a synthetic medium containing divalent cadmium at a concentration of 60 mgCd (II) / L.

Sowing was carried out in a sterile laminar cabinet, which was then disinfected with ultraviolet light for 30 minutes. Before sowing, the synthetic medium (table 1) was brought to a boil and then quickly cooled under a stream of cold water to remove dissolved oxygen. 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 4.5), a solution of sodium sulfide (2 ml). Stock solution of cadmium was added in an amount of 6.0 ml per 1 liter of synthetic medium (thus, a concentration of cadmium in the medium of 75 mg / l was achieved).

About 120 ml of synthetic medium with added additives and 10 ml of inoculum (bacterial culture) were added to 120 ml vials, after which they were added to the top with medium. Rubber plugs were rubbed to the edges of the vials using a sterile needle, which reduced the likelihood of air oxygen entering. At the end of sowing, the bottles were closed with aluminum caps, the bottle was sealed with a seaming machine and the thermostat was placed at a temperature of 28 ºС.

Cadmium is precipitated as sulfide at the bottom of the vial.

Claims (1)

Bacterial strain Desulfovibrio sp. VK-9 for the treatment of acidic wastewater from heavy metal ions deposited in the All-Russian Collection of Microorganisms of the IBPM RAS under registration number VKM B-3000D.