Strain of bacteria desulfovibrio sp. for wastewater treatment from heavy metal ions

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Desulfovibrio sp. A 4/1 is deposited in the Russian Collection of Microorganisms (RCM) under the registration number of RCM B-2820D, and is resistant to heavy metal ions.

EFFECT: increase in degree of wastewater treatment.

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The strain of bacteria Desulfovibrio sp. isolated from sludge deposits sump water from the cooling system of the furnace Chelyabinsk metallurgical plant. The strain is characterized by high resistance to elevated concentrations of copper ions (up to 600 mg/l) and other divalent metal cations.

The invention relates to industrial Microbiology and the receipt of a new strain of bacteria that can be used in biotechnology for clean industrial wastewater engineering, instrument engineering, electrical engineering enterprises from elevated concentrations of copper ions and other heavy metals.

Aware of the use of sulphate-reducing bacteria for wastewater treatment from heavy metal ions. Biogenic sulfide during biochemical purification binds heavy metal ions into insoluble sulfides.

A known strain of bacteria Desulfovibrio oxamicus BKM-2465 D (EN 2355756), characterized by a high degree of wastewater purification from heavy metals at elevated concentrations of sulfates. This strain has the ability to clean waste water from chromium (YI). The disadvantage of this strain is its ability to work at total concentrations of heavy metals up to 190 mg/L.

The closest to the essence and the achieved result to the claimed invention is one the SJ strain of bacteria Desulfovibrio sp. SSC is-2 (EN 2269571), used for treatment of wastewater from heavy metal ions. The proposed strain isolated from industrial effluent electroplating plants, containing heavy metal ions. However, the aforesaid patent, the total concentration of heavy metals used in the treatment waters is 197,9 mg/l, and copper is 8.9 mg/l In the wastewater metallurgical enterprises, the concentration of copper ions can reach 300 mg/L.

The lack of strain SSC is-2 is not sufficiently high resistance to heavy metal ions, in particular copper ions, which significantly limits its use in biotechnology.

The objective of the invention is obtaining resistant metals, highly active strains of sulphate-reducing bacteria used in wastewater treatment for removing heavy metal ions.

The technical result - obtaining strain of bacteria Desulfovibrio sp. VKM B-2820D can purify waste water from heavy metal ions.

Strain Desulfovibrio sp. A 4/1 deposited by the all-Russian collection of microorganisms (VKM) Institute of biochemistry and physiology of microorganisms them. G. K. Skryabin RAS (ibfm) under registration number VKM B-2820D. The proposed strain Desulfovibrio sp. VKM B-2820D not known in science and technology, therefore, the properties that he manifests, are new. Analysis of the nucleotide sequence of the 16S rRNA gene of strain VKM is-2820D showed the proposed strain is a new strain belongs to the genus Desulfovibrio.

The resulting strain is characterized by the following features.

Cultural and morphological features of patentable determine strain when cultivated on standard freshwater environment Videla (Widdel, Bak, 1992). The temperature of the cultivation of the strain was +28 ºC. The cultivation is carried out in the next 3-4 days without copper ion, 7-8 days with the addition of copper ions at a concentration of 200 mg/l and more.

Generic and species name of the strain is a strain of bacteria Desulfovibrio sp.

The origin is isolated from sludge deposits sump water from the cooling system of the furnace Chelyabinsk metallurgical plant. Morphological characteristics - form cells cholerae, a size of 0.5-1.0×3,0×5.0 µm.

The sporulation - no.

Response to gram - negative.

The mobility of the cells are motile.

The physiological characteristic is the ratio of the oxygen - gone anaerobic.

As a substrate for the growth of the use of lactate, ethanol, glycerol, malate, citrate, oxalacetate, fumarate, formate, succinate, simple sugars. Lactate is metabolized by way of incomplete oxidation with the formation of acetate. The highest growth rate and the lowest doubling time shows on the medium with lactate. Most products of protein and sulfide recorded in the medium with E. what anolon. The strain is resistant to copper ions (up to 600 mg/l), cobalt (up to 450 mg/l), Nickel (up to 225 mg/l), cadmium (up to 225 mg/l), chromium (up to 25 mg/l). Under cultivation of strain with ions of Fe²⁺form a black precipitate of sulphide of iron. The optimum pH of 7.2.

A useful property in connection with which the culture is deposited, the deposition of ions of heavy metals in concentrations greater than 200 mg/l hydrogen sulfide.

The cultivation conditions - freshwater environment Widely.

Storage conditions - saved by re-seeding on the environment of the cultivation or liofilizirovannam.

Example 1. The study of the ability of a strain of bacteria Desulfovibrio sp. VKM B-2820D to the deposition of copper ions.

Pure cultures of SRB Desulfovibrio sp. VKM B-2820D cultivated in synthetic medium containing copper ions at a concentration of 350 mg/L.

Sowing was performed in a sterile laminar flow Cabinet that before disinfected with ultraviolet light for 30 minutes. Before sowing synthetic environment (table 1) was brought to boil and then quickly cooled under running cold water to remove dissolved oxygen. Cooled to room temperature, the medium was made nutrients (table 2) (per 1 l) in the following sequence: vitamins (2 ml) salt solution (10 ml), a solution of cofactors (1 ml), the organic substrate - lactate (1.6 ml), NaHCO₃(when making the pH is brought up to 7.2 to 7.4), H₂S (2 ml). Before making hydrogen sulfide was added to the mother solution of copper in the amount of 4 ml per 1 liter of synthetic environment.

In bottles of 120 ml was made about 50 ml of synthetic medium amended additives and 10 ml of inoculum (bacteria cultures), followed by adding medium to the top. Rubber stoppers were displayed to the edges of the vials using a sterile needle, which reduced the likelihood of penetration of oxygen. At the end of sowing the vials were closed with aluminum caps, sealed vial capping machine and put thermostat at a temperature of 28 OC.

On medium containing copper ions at a concentration of 350 mg/l for 15 days in the environment copper is precipitated as sulphide. The concentration of copper ions in the liquid phase remains 0.26 mg/L.

Example 2. The study of the ability of a strain of bacteria Desulfovibrio sp. VKM B-2820D to the deposition of Nickel ions.

Pure cultures of SRB Desulfovibrio sp. VKM B-2820D cultivated in synthetic medium containing divalent Nickel at a concentration of 250 Ni/L.

Sowing was performed in a sterile laminar flow Cabinet that before disinfected with ultraviolet light for 30 minutes. Before sowing synthetic environment (table 1) was brought to boil and then quickly cooled under running cold water to remove dissolved oxygen. Cooled to room temperature, the medium was made nutrients (table 2) (1 is) in the following sequence: vitamins (2 ml), salt solution (10 ml), a solution of cofactors (1 ml), the organic substrate is glycerol (10 ml), lactate (0.8 ml), a solution of NaHCO₃(pH was brought to 7.0 - 7,8), sodium sulfide solution (2 ml). Stock solution of Nickel was added in the amount of 25 ml per 1 liter of synthetic environment (thus was achieved the concentration of Nickel in the environment 250 mg/l).

In bottles of 120 ml was made about 50 ml of synthetic medium amended additives and 10 ml of inoculum (bacteria cultures), followed by adding medium to the top. Rubber stoppers were displayed to the edges of the vials using a sterile needle, which reduced the likelihood of penetration of oxygen. At the end of sowing the vials were closed with aluminum caps, sealed vial capping machine and put thermostat at a temperature of 28 OC.

Beginning of formation of crystalline Nickel sulfide occurs within 7 days, and a stable crystalline phase sulfide is formed within 20 days.

Example 3. The study of the ability of a strain of bacteria Desulfovibrio sp. VKM B-2820D to the deposition of cobalt ions.

Pure cultures of SRB Desulfovibrio sp. VKM B-2820D cultivated in synthetic medium containing divalent cobalt in a concentration of 400 MgSO(II)/l

Sowing was performed in a sterile laminar flow Cabinet that before disinfected with ultraviolet light for 30 minutes. Before sowing synthetic environment (t the blitz 1) brought to the boil and then quickly cooled under running cold water to remove dissolved oxygen. Cooled to room temperature, the medium was made nutrients (table 2) (per 1 l) in the following sequence: vitamins (2 ml) salt solution (10 ml), a solution of cofactors (1 ml), the organic substrate is glycerol (10 ml), lactate (0.8 ml), a solution of NaHCO₃(pH was brought to 7.0 - 7,8), sodium sulfide solution (2 ml). Stock solution of cobalt was added in the amount of 40 ml per 1 liter of synthetic environment (thus was achieved the concentration of cobalt in the environment 400 mg/l).

Vials were made about 50 ml of synthetic medium amended additives and 10 ml of inoculum (bacteria cultures), followed by adding medium to the top. Rubber stoppers were displayed to the edges of the vials using a sterile needle, which reduced the likelihood of penetration of oxygen. At the end of sowing the vials were closed with aluminum caps, sealed vial capping machine and put thermostat at a temperature of 28 OC.

Beginning of formation of the crystalline cobalt sulfide occurs within 12 days, and a stable crystalline phase sulfide formed during 25 days.

Example 4. The study of the ability of a strain of bacteria Desulfovibrio sp. VKM B-2820D to the deposition of cadmium ions.

Pure cultures of SRB Desulfovibrio sp. VKM B-2820D cultivated in synthetic medium containing divalent cadmium at a concentration of 75 Cd(II)/l

the Sowing was performed in a sterile laminar flow Cabinet, you disinfected with ultraviolet light for 30 minutes. Before sowing synthetic environment (table 1) was brought to boil and then quickly cooled under running cold water to remove dissolved oxygen. Cooled to room temperature, the medium was made nutrients (table 2) (per 1 l) in the following sequence: vitamins (2 ml) salt solution (10 ml), a solution of cofactors (1 ml), the organic substrate is glycerol (10 ml), lactate (0.8 ml), a solution of NaHCO₃(pH was brought to 7.0 - 7,8), sodium sulfide solution (2 ml). Stock solution of cadmium was added in the amount of 7.5 ml per 1 liter of synthetic environment (thus was achieved the concentration of cadmium in the environment of 75 mg/l).

Vials with a volume of 120 ml was made about 50 ml of synthetic medium amended additives and 10 ml of inoculum (bacteria cultures), followed by adding medium to the top. Rubber stoppers were displayed to the edges of the vials using a sterile needle, which reduced the likelihood of penetration of oxygen. At the end of sowing the vials were closed with aluminum caps, sealed vial capping machine and put thermostat at a temperature of 28 OC.

Crystallization of cadmium sulfide begins after 10 days of cultivation, the cultivation of 18 days the cadmium sulfide crystallizes completely.

The strain of bacteria Desulfovibrio sp. for about isdi waste water from heavy metal ions, deposited by the all-Russian collection of microorganisms, Institute of biochemistry and physiology of microorganisms, Russian Academy of Sciences (ibfm) under registration number VKM B-2820D.