RU2131852C1 - Strain of bacterium desulfomicrobium apsheronum vkm v-2012 d egast-3 used for sewage treatment from heavy metal ions - Google Patents

Abstract

FIELD: technical microbiology, ecology. SUBSTANCE: invention proposes the highly active strain of sulfate-reducing bacteria providing biomass increment in sewage treatment from heavy metal ions - Desulfomicrobium apsheronum VKM V-2012 D EGAST-3. Strain is isolated from industrial glycerol-containing sewers. Strain is the stringent anaerobe, pH range is from 6.0 to 9.0, it grows at the temperature range from 0 to 41 C. Effectiveness of sewage waters from galvanic production using the proposed strain is 99.9%. EFFECT: enhanced effectiveness of sewage treatment. 1 tbl, 2 ex

Description

 The invention relates to industrial microbiology and relates to the production of a new strain of bacteria producing sulfide, which can be used in biotechnology for the treatment of industrial wastewater from engineering, instrument-making, and electrical enterprises.

 It is known about the use of sulfate reducing bacteria (SBB) for wastewater treatment from heavy metal ions. In the process of biochemical purification, heavy metal ions are bound by biogenic hydrogen sulfide to insoluble sulfides, as well as the reduction of certain metals and precipitation in the form of hydroxides. To increase the efficiency of biochemical purification methods using SVB, a high level of bacterial growth activity is required.

 To create the necessary concentration of microorganisms in the process of wastewater treatment from heavy metal ions, various methods are used or they add easily digestible organic food for SVB / 1 /, or use specially selected biocenoses of microorganisms / 2 /.

 However, the microorganisms used do not provide special activity for the growth of SFS in industrial wastewater, characterized by varying degrees of pollution and different composition of polluting components.

 The bacterial strain DesulfovibrioVulgaris VKM B-1970 D (UV-3) used for wastewater treatment from heavy metal ions was adopted as a prototype. This strain is a highly active strain of sulfate-reducing bacteria, providing a stable increase in biomass during wastewater treatment from heavy metal ions / 3 /.

 The strain SBB DesulfovibrioVulgaris VKM B-1970 D was obtained by isolating from the wastewater a galvanic shop with a high content of dissolved metals and subsequent selection by standard methods on elective media. The basic composition of the medium is shown in the example.

 The resulting bacterial strain was deposited in the All-Union Collection of Microorganisms (VKM) under the number VKM B-1970 D.

 The definition of culture is given by Bergey's determinant ("Bergey's Manual of Determinative Bacteriology", v. 1, 909, Baltimore William, Wilkins, 1984).

 Example. Cultural and morphological characteristics of the strain. The characteristic of vegetative cells - vibrios, cells of 0.4-0.7; 1.5 to 4 microns; gram negative; acid resistance - unstable, do not form spores. The type of reproduction and divergence of cells during division is direct uniform division, the type of flagellation is mobile, polar flagging; there are no inclusions. The type of colony — spherical and disk-shaped colonies in the thickness of agar, growth features on a nutrient medium — reduce sulfates to form sulfides.

 Physiological and biochemical characteristics of the strain.

The cultivation conditions are anaerobic, the optimum temperature is 30 ^o C. (range 10-35 ^o C), the optimal pH is 7.2-7.4 (range 5.5-8.5), the pH change with growth is absent or slightly alkalized . The smell is hydrogen sulfide.

 The relation to pH, temperature and oxygen is indicated under cultivation conditions, it is suppressed by streptomycin, ampicillin and other antibiotics that inhibit gram-negative microflora. Cells do not contain phages, are not lysogenic, withstand salt content up to 20-30 g / l, gelatin does not thin, starch does not hydrolyze.

 This strain was used for wastewater treatment of galvanic production.

 The disadvantage of this strain is the insufficiently high efficiency of the degree of purification of wastewater from galvanic plants from heavy metal ions.

 The objective of the invention is to obtain a highly active strain of bacteria Desllifomicrobium apsheronum VKM V-2012 D EGAST-Z, which provides a stable increase in biomass when treating wastewater from heavy metal ions and thereby expanding the range of bacterial strains used to treat wastewater from heavy metal ions.

 The essence of the invention lies in the fact that a new strain of bacteria Desulfomicrobium apsheronum VKM V-2012 D EGAST-3 is proposed, used for wastewater treatment from heavy metal ions.

 The strain is isolated from industrial effluents containing glycerin and is an active producer of hydrogen sulfide for the precipitation of heavy metal ions.

 The resulting strain of bacteria Desulfomicrobium apsheronum VKM B-2012 D EGAST-3 was deposited in the All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms RAS under the number VKM B-2012 D. The strain Desulfomicrobium apsheronum VKM B-2012 D EGAST-3 is characterized by the following cultural-morphological and physiological - biochemical properties.

1. Cultural and morphological characteristics of the obtained strain are determined on standard media, the recipes of which are taken from the catalog of the All-Russian collection of microorganisms N 92 and 109 and are given below in the publication "All-Russian collection of microorganisms. Catalog". Pushchino - Moscow, VNITI, 1992, 362 p. Cultivation is carried out under anaerobic conditions at 30 ^o C for two days.

 2. Cultural signs: with the growth of the strain, the liquid medium opalesces, a black precipitate of iron sulfide accumulates at the bottom of the cultivation vessel, and the smell of hydrogen sulfide is present.

 3. Morphological signs: under microscopy, cells are rod-shaped cells with a size of (0.7-0.9) • (1.4-2.9) microns. Cells are mobile by electron microscopy; one polar flagellum (monotrichi) has been identified; reproduction by division with a uniform divergence of cells. According to the structure of the wall - gracile, which is confirmed by negative gram stain. Not acid resistant. They do not form a dispute. Intracellular inclusions during cultivation on these media are absent.

 Features of growth: reduction of sulfate to sulfide (hydrogen sulfide) with the accumulation of black ferrous sulfide in the standard medium, as well as (after exhaustion of iron) hydrogen sulfide.

 4. Physiological and biochemical characteristics of the proposed strain.

Strict anaerobic. It is capable of sulfate respiration: it uses sulfate or sulfur compounds of intermediate oxidation (sulfite, thiosulfate) as an electron acceptor, and uses molecular hydrogen, lactate, pyruvate, formate, ethanol as hydrogen donors. Accordingly, the above-mentioned lactate, pyruvate, formate, and ethanol are capable of using carbon sources. Ammonium and amino acids of yeast extract are able to use nitrogen sources. Gelatin does not dilute, starch does not hydrolyze. Does not need growth stimulants. PH range 6–9 with optimum at 7.2–7.4, 63DSM medium used. With growth, a slight alkalization of the medium is observed, which correlates with the replacement of sulfate by sulfide. The temperature range for strain growth is from 0 to 41 ^o C, optimum at 3 ^o C. Cells contain cytochrome C. The growth of the strain is suppressed by streptomycin, ampicillin and other antibiotics that inhibit gram-negative microflora. Storage conditions: it is saved by transfers on the cultivation medium. It can be stored in the refrigerator at 4-10 ^o C for 3-6 months.

N 92. Medium with lactate.
1 g solution
K ₂ HPO ₄ - 0.5
NH ₄ Cl - 1.0
CaCl ₂ • 6H ₂ O - 0.1
MgSO ₄ • 7H ₂ O - 2.0
Na ₂ SO ₄ - 1.0
Lactate Na - 5.0
Yeast Extract - 1.0
Resazurin - 0.001
Cysteine - 0.5
Distilled water, ml - 950.0
Solution 2, g:
NaHCO ₃ - 4.0
Distilled Water - 40.0
Solution 3:
Na ₂ S • 9H ₂ O, g - 0.3
Distilled water, ml - 6.0
Solution 4:
FeSO ₄ • 7H ₂ O, g - 0.4
Distilled water, ml - 10.0
Preparation: bring solution 1 to a boil while blowing through a solution of an oxygen-free gas mixture consisting of 97% N ₂ and 3% H ₂ . Sterilize these gases in the atmosphere. Solution 3 to sterilize in an atmosphere of N ₂ . After sterilization, connect the solutions. After mixing, the pH of the medium is adjusted to 6.8.

 N 109. Wednesday.

Solution 1, g:
K ₂ HPO ₄ - 0.5
NH ₄ Cl - 1.0
Na ₂ SO ₄ - 1.0
CaCl ₂ • 6H ₂ O - 0.1
MgSO ₄ • 7H ₂ O - 2.0
Lactate Na - 2.0
Yeast Extract - 1.0
Resazurin - 1.0
Distilled water, ml - 980.0
Solution 2:
FeSO ₄ • 7H ₂ O, g - 0.5
Distilled water, ml - 10.0
Solution 3, g
Thioglycolate Na - 0.1
Ascorbic acid - 0.1
Distilled water, ml - 10.0
Solution 4 (trace element solution SL-10), mg:
HCl (25%; 7.7 M), ml - 10.0
FeCl ₂ • 4H ₂ O - 1.5
ZnCl ₂ - 70.0
MnCl ₂ • 4H ₂ O - 100.0
H ₃ VO ₃ - 6.0
CoCl ₂ • 6H ₂ O - 190.0
CuCl ₂ • 2H ₂ O - 2.0
NiCl ₂ • 6H ₂ O - 24.0
Na ₂ MoO ₄ • 2H ₂ O - 36.0
Distilled water, ml - 990.0
Preparation: all solutions are sterilized separately by autoclaving at 121 ^o C for 15 min in a nitrogen atmosphere. Solution 1 is boiled immediately before autoclaving, flushing with nitrogen. To prepare the medium, solutions 1, 2 and 3 are mixed, and 1 ml is added from solution 4. After mixing, the pH of the medium is 7.2-7.4.

 An example of obtaining a strain of Desulfomicrobium apsheronum VKM B-2012 D EGAST-3.

The deposited strain is obtained by selecting production effluents and isolating from them an accumulative culture of bacteria by growing under anaerobic conditions at 30 ^o C.

 As the sole carbon source for growth, industrial effluents containing glycerin are used. Then, strain selection is continued at increasing concentrations of heavy metals in the culture medium. The final selection of the pure culture of the obtained strain is produced by the method of serial dilutions, followed by seeding in an agarized version of the nutrient medium and reseeding of single colonies.

 An example of using the bacterial strain Desulfomicrobium apsheronum VKM V-2012 D EGAST-3 for wastewater treatment from heavy metal ions.

The process is carried out in bioten with loading from pumice with an upward flow of liquid. Seeds are microbial strains grown in a liquid medium in flasks. Biotenes are inoculated with strains of Desulfomicrobium apsheronum VKM B-2012 D EGAST-3 microorganisms, which are fixed on a pumice stone. The cultivation temperature in the biotank is maintained in the range of 28-30 ^o C. Through the biotenk wastewater of galvanic production is passed. As a result of the life of the SRB, hydrogen sulfide is released, which combines with heavy metal ions, forming metal sulfides, which precipitate.

 The effectiveness of the biotench is shown in the table.

 The table shows that the efficiency of wastewater treatment of galvanic production using the proposed strain is 99.9%.

 Thus, the wastewater of galvanic plants can be effectively cleaned of heavy metal ions using a strain of Desulfomicrobium apsheronum VKM V-2012 D EGAST-3.

 Literature.

 1. USSR author's certificate N 857013, C 02 F 3/28, 1979.

 2. Copyright certificate of the USSR N 941319, C 02 F 3/34, 1980.

 3. RF patent N 2055817, cl. C 02 F 3/34, 1996.

Claims (1)

 The bacterial strain Desulfomicrobium apsheronum VKM V-2012 D, used for wastewater treatment from heavy metal ions.

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