RU2128219C1 - Strain of bacterium desulfovibrio vulgaris egast-1 used for treatment of sewage from heavy metal ions - Google Patents

Abstract

FIELD: industrial microbiology, biotechnology, ecology. SUBSTANCE: invention proposes a new strain of bacterium Desulfovibrio vulgaris EGAST-1 for treatment of sewage from heavy metal ions. Strain shows high resistance during culturing on media polluted with metal ions and provides stable mass increment. Strain binds and removes ions of heavy metals. EFFECT: enhanced effectiveness of sewage treatment. 1 tbl, 1 ex

Description

 The invention relates to industrial microbiology and relates to a new strain of bacteria - sulfide producers, 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 Desulfovibrio Vulgaris BKM B-1970 D (UV-3) used for the treatment of wastewater 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 Desulfovibrio Vulgaris BKM 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 (BKM) under the number BKM 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 0.4-0.7 in size; 1.5-4 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 (limits 10-35 ^o C), the optimal pH is 7.2-7.4 (limits 5.5-8.5), the pH change with growth is absent or weak alkalization . 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 strain of bacteria Desulfovibrio Vulgaris BKM B-1020 D used for wastewater treatment from heavy metal ions and thereby expanding the range of bacterial strains used for wastewater treatment. The invention is achieved by the use of a strain of bacteria Desulfovibrio Vulgaris BKM B-1020 D for wastewater treatment of galvanic production. The bacterial strain isolated from the cutting fluid (coolant) of the metalworking shop.

 The proposed strain is deposited in the All-Union Collection of Microorganisms (BKM) under registration number - BKM B-2010 D.

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

1. Cultural and morphological characteristics of the patented strain were determined during its cultivation on standard media, the recipes of which are taken from the catalog of the All-Russian collection of microorganisms 92 and 109 and are given below in the publication "All-Russian collection of microorganisms. Catalog", Pushchino-Moscow, VNITI, 1992, 362 pp. The cultivation was carried out under anaerobic conditions at 30 ^o C for 2 days.

 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; there is a smell of hydrogen sulfide.

 Morphological signs: under microscopy, the cells are represented by single vibrios with a size of 0.5-0.7 x 1.5-3 microns; cells are motile, one polar flagellum (monotrichi) was detected by electron microscopy; 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.

2. Physiological and biochemical characteristics of the patented strain
Strict anaerobic. It is capable of sulfate breathing: it uses sulfate or sulfur compounds of intermediate oxidation (sulfite, thiosulfate) as an electron acceptor, while it uses hydrogen, lactate, pyruvate, formate, ethanol as hydrogen donors. Accordingly, the above-mentioned lactate, pyruvate, formate, and ethanol are capable of using carbon sources. In addition, it is able to use industrial coolant as a carbon source (growth substrate). 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. The pH range is from 6-9 with the optimum at 7.2-7.4. With growth, a slight alkalization of the medium is observed, which correlates with the replacement of sulfate by sulfide. The temperature range for the growth of strain 0-41 ^o C with optimum at 3 ^o C. Cells contain cytochrome C and desulfoviridine. The growth of the strain is suppressed by streptomycin, ampicillin and other antibiotics that inhibit Gram-negative microflora.

 3. Prescriptions of media from the catalog of the All-Russian collection of microorganisms.

N 92. Medium with lactate for DESULFOVIBRIO
Solution 1, g:
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.

NaHCO ₃ , g - 4
Distilled water, ml - 40
Solution 3.

Na ₂ S • 9H ₂ O, g - 0.3
Distilled Water - 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. Medium for DESULFOVIBRIO.

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.

Thioglycolate Na, g - 0.1
Ascorbic acid, g - 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, g - 1.5
ZnCl ₂ - 70.0
MnCl ₂ • 4H ₂ O - 100.0
H ₃ BO 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, the pH of the medium after mixing is adjusted to 7.2-7.4.

Example 1. Obtaining a strain of Desulfovibrio Vulgaris BKM B-2010 D
The deposited strain was obtained by sampling industrial effluents and isolating from them an accumulative culture of bacteria by growing under anaerobic conditions at 30 ^o C on media, the prescriptions of which are given in the text, with the change in the media that production coolant is used as the only carbon source for growth . Then, strain selection is continued at increasing concentrations of heavy metals in the culture medium. The final selection of the pure culture of the patented 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 the use of a strain of bacteria Desulfovibrio Vulgaris BKM B-1020 D 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 Desulfovibrio Vulgaris BKM B-1020 D microorganisms, which are fixed on a pumice stone. The cultivation temperature in the biotank is maintained at 28-30 ^° C. The wastewater of galvanic production is passed through the biotank. 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, wastewater from galvanic plants can be effectively cleaned of heavy metal ions using a strain - Desulfovibrio Vulgaris BKM B-1020 D (EGAST-1).

LITERATURE
1. Copyright certificate of the USSR 857013, cl. C 02 F 3/28, 1979

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

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

Claims (1)

 Bacterial strain Desulfovibrio Vulgaris VKMB-2010 D, used for wastewater treatment from heavy metal ions.

Images