RU2174553C2 - Method of biodestruction of heptyl, nonsymmetric dimethyl-hydrazine - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: method is based on biodestruction of heptyl (nonsymmetric dimethylhydrazine) by specially selected associative culture consisting of strains of bacteria Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3. Biopreparation prepared on the basis of this culture provides biodecomposition of heptyl. In use of associative culture or biopreparation the content of heptyl in water is decreased from 1000 to 0.3 mg/l and from 1000 to 3.2 mg/l at 30 C for 20 days. Content of formaldehyde and dimethylamine that are toxic products of heptyl decomposition is decreased simultaneously. In biodestruction of heptyl in soil its content is decreased from 11.3 to 0.35 g/kg at 18-20 C for 30 days. Proposed method of biodestruction can be used with success in treatment of water and soil from heptyl and products of its decomposition. Method is simple in practice realization and exhibits ecological purity. EFFECT: enhanced effectiveness of method, simplified procedure, improved ecology. 2 tbl, 5 ex

Description

 The invention relates to the field of ecology and biotechnology and is devoted to the problem of the disposal of toxic and toxic substances using microorganisms.

Это вещество относится к числу наиболее вредных и опасных химических соединений. Гептил представляет собой бесцветную жидкость, хорошо растворимую в воде, сильный яд. По химическим свойствам несимметричный диметилгидразин - сильное основание, сильный восстановитель, подвергающийся активному окислению (Справочник по токсикологии и гигиеническим нормативам (ПДК) потенциально опасных химических веществ, ред. В.С.Кушнева и Р.Б.Горшкова, Москва "ИздаТ", 1999; Е. Schmidt. Hyd-razine and Its Derivatines: Preparation, Properties, Applications, 1984).Liquid rocket fuel, known as heptyl, by its chemical structure is an asymmetric (secondary) dimethylhydrazine:

This substance is one of the most harmful and dangerous chemical compounds. Heptyl is a colorless liquid, soluble in water, a strong poison. By its chemical properties, asymmetric dimethylhydrazine is a strong base, a strong reducing agent that undergoes active oxidation (Handbook of Toxicology and Hygienic Standards (MPC) of potentially hazardous chemicals, edited by V. S. Kushnev and R. B. Gorshkov, Moscow, IzdaT, 1999 ; E. Schmidt. Hydrazine and Its Derivatines: Preparation, Properties, Applications, 1984).

 The problem of cleaning environmental objects, metal structures and products contaminated with toxic and toxic substances, as well as the disposal of excess quantities of these substances in warehouses, is extremely important for modern ecology.

 Of all the known methods for the decontamination of harmful organic compounds, the most promising are biotechnological methods.

 The study of patent and periodic scientific material showed a lack of data on the problem of the biological utilization of asymmetric dimelhydrazine. There are only a few reports of bacterial cultures capable of utilizing hydrazine, the derivative of which is asymmetric dimethylhydrazine. In these publications, the ability to utilize it as a source of nitrogen was chosen as a key property for screening microorganisms consuming hydrazine, while the carbon source was carbon dioxide. Aerobic chemolithotrophic bacteria of the genera Nitromonas and Achromobacter were discovered, which were reported as organisms capable of using hydrazine in the nitrogen cycle (A. McLiner a. J. Street. Decomposition of Hydrazine in Aqueous Solutions. 1989; D. Kuch. Bioremediation of Hydrazine. 1994). This approach significantly narrows the scope of the search for potential heptyl biodestructors, does not take into account that this substance can be a carbon source for heterotrophic microflora, and does not give a complete picture of the potential possibilities of natural microbiocenoses to deactivate harmful and toxic compounds.

 The most justified for solving the problems of heptyl decontamination is the application of the ecological and physiological approach, which consists in taking into account the features of the natural environment, subjected to pollution and used to decontaminate harmful and toxic compounds.

 So, for the deactivation of pesticides used in agriculture (pesticides and herbicides), a method for the decomposition of organophosphorus pesticides using bacteria Bacillus megaterium, described in ed. testimonial. N 1735359. In this method, Bacillus megaterium bacteria are pre-grown on a nutrient medium containing pesticides, and then introduced into the soil contaminated with the same pesticide. The disadvantage of this method is the orientation on the allocation and use of a pure culture of natural microorganisms, which are characterized by a weak ability to utilize xenobiotics. This is unacceptable for solving practical problems associated with the cleaning of soils and reservoirs contaminated with heptyl, and for the disposal of stocks of substandard heptyl accumulated in warehouses.

 A method for the decontamination of pesticides is described, which consists in adapting the spontaneous microflora of farm animal manure or bird droppings during their accelerated composting in the presence of increasing concentrations of pesticides. The resulting mature compost can be used as a “sourdough” because it contains microflora capable of deactivating pesticides (Pat. RF N 2077398). However, this method cannot be used for the decomposition of heptyl entering water bodies, or for the disposal of substandard heptyl stored in warehouses.

 The purpose of the invention was to develop an environmentally friendly method for the destruction of heptyl using biological products based on microorganisms isolated from natural environments. This goal is achieved by the fact that for the destruction of heptyl and its decomposition products, an associative culture is used isolated from soils saturated with heptyl or oil products, as well as from soils into which organic fertilizers were applied (manure or composts based on plant waste), as well as a biological product created based on this culture.

 Bacterial biological products and technologies developed on the basis of the above principles were used to decompose harmful and toxic compounds such as oil and oil products (Pat. RF N 2053204; Pat. RF N 2067993; Pat. RF N 2076150; Patent US N: 5496723; Patent N: US 5759800; European Patent N 0668246).

 In practical terms, for the destruction of heptyl in water and soil, an associative culture was used, including the following bacterial strains: Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3.

 This associative culture for the utilization of heptyl was obtained on the basis of bacterial strains that were isolated from accumulative cultures with their subsequent selection based on the activity of destruction of toxic and toxic substances.

 Characterization of strains.

 The bacterial strain Acinetobacter species H-1.

One-two-day culture of bacteria on meat-peptic agar (MPA) is a short-oval bacillus, single and double, with a size of 1.2-1.5 x 1.3-1.5 microns. The colonies on the MPA are rounded, smooth, evenly convex with a flat edge. Gram-negative. The dispute does not form. Hemoorganotroph. Aerobe. Assimilates: glycerin, D-glucose, lactose, sucrose, mannitol, maltose, xylose, D-galactose, rhamnose, cellobiose, asparagine, urea, ammonium sulfate, potassium nitrate. Growth temperature 10-40 ^o C. Non-pathogenic.

 The bacterial strain Rhodococcus species H-2.

One-two-day bacterial culture on MPA is a short-oval bacillus with a size of 1.4 x 1.5 microns. The colonies on the MPA are rounded, with a smooth edge, shiny, uniformly convex, whitish. Gram-positive. The dispute does not form. Hemoorganotroph. Aerobe. Assimilates: glycerin, D-glucose, lactose, sucrose, raffinose, mannose, fructose, acetate, pyruvate, lactate, glutamate, asparagine, urea, ammonium sulfate, potassium nitrate. Growth temperature 15 - 43 ^o C. Non-pathogenic.

 The bacterial strain Arthrobacter species H-3.

One-two-day bacterial culture on MPA is an irregular stick, varying in size 1.1-1.5 x 1.3-1.8 microns. Cells are often located at an angle to each other, forming V-shaped. Gram-positive. The dispute does not form. The colonies on the MPA are rounded with a smooth edge, whitish, shiny, uniformly convex. Hemoorganotroph. Assimilates: D-glucose, D-xylose, sucrose, maltose, lactose, D-gluconate, glycine, uric acid, hydroxybenzoate, inositol, citrate, raffinose, asparagine, urea, ammonium sulfate, potassium nitrate. Growth temperature 10 - 40 ^o C. Non-pathogenic.

 Preparation of a biological product.

A biological product was obtained by culturing the listed bacteria cultures individually on a mineral medium with 2% (by volume) liquid paraffin and subsequent mixing of individual bacterial biomasses in a ratio of 1: 3: 2. The composition of the medium for the cultivation of bacteria, g / l: NH ₄ H ₂ PO ₄ - 10, K ₂ HPO ₄ - 10, MgSO ₄ • 7H ₂ O, ZnSO ₄ • 7H ₂ O, FeSO ₄ • 7H ₂ O, MnSO ₄ • 7H ₂ O - at 0.0125. Cultivation was carried out in an apparatus with intensive stirring (1200 rpm) and air supply of 600 l / h at 30 - 34 ^o C, pH 7.0. The resulting biomass was subjected to separation and drying. Drying mode: temperature at the inlet is 106-110 ^o C, at the outlet 45-50 ^o C. The resulting biological product was a beige powder, without a specific odor, with a moisture content of 8 - 10%, a culture titer of the producer 10 ⁸ -10 ¹⁰ cells / g .

 Heptyl destruction by the association of cultures and biological product.

The process of destruction of heptyl in water was carried out in model experiments in flasks on a rocking chair (220 rpm) on a mineral medium of the above composition, both using a liquid associative culture and with a powdered biological product. The heptyl content in the medium was 100 and 1000 mg / L. An associative culture or biological product was introduced in an amount of 1-2% of absolutely dry biomass to a given concentration of heptyl. The cultivation temperature of 20 and 30 ^o C, pH 7.0. The concentration of heptyl and its decomposition products (formaldehyde and dimethylamine) was determined after 1.5, 5, 7, and 20 days. In the table. 1 presents data on the dynamics of the destruction of heptyl in water.

As can be seen from the data presented, the destruction of heptyl actively occurred when using both an associative bacterial culture and a biological product at 20 ^o C and 30 ^o C: at 20 ^o C after 1.5 days of cultivation when using an associative culture decreased from 100 mg / l to 4.5 - 7.0 mg / l, when using a biological product - up to 3.8 - 8.1 mg / l. At 30 ^o C after 1.5 days, the concentration of heptyl decreased from 100 mg / l to 2.3 - 2.6 mg / l when using an associative culture and to 3.2 - 5.7 mg / l when using a biological product. At the same time, a decrease in the content of intermediate decomposition products of heptyl formaldehyde and dimethylhydrazine was observed from 65 and 47 mg / l (in control) to 0.17 and 1.2 mg / l when using an associative culture and to 0.5 and 1.5 mg / l when using a biological product, respectively. After 20 days of cultivation, using both an associative culture and a biological product, the residual concentration of heptyl was 0.3 - 0.4 mg / L, that is, the heptyl content decreased by almost 300 times.

 The possibility of biodegradation of high (up to 1000 mg / l) heptyl concentrations in water using a biological product has been shown. According to the table. 1 after 1.5 days, a decrease in the concentration of heptyl from 1000 mg / L to 44 mg / L was noted; after 20 days of cultivation, the concentration of heptyl decreased to 3.2 mg / L.

 The process of destruction of heptyl in the soil was carried out by solid-phase fermentation. The concentration of heptyl in the soil was 11.3 g / kg of soil. The amount of biological product introduced into the soil was 1 - 2% of a given amount of heptyl. From the table. Figure 2 shows that when a biological product is introduced into the soil, intensive destruction of heptyl occurs: the concentration of heptyl after 30 days decreased from 11.3 g / kg to 0.35 g / kg of soil. In control 1 (without soil mixing), heptyl destruction was practically absent. In control 2 (with soil mixing) and in control 3 (with soil mixing and the addition of mineral salts) the decrease in heptyl concentration was also insignificant.

 Our invention is illustrated by the following examples.

 Example 1

The destruction of heptyl in water was carried out by cultivation of an associative culture, including bacteria strains Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3. The cultivation was carried out in rocking flasks with valves that prevent the escape of heptyl, the stirrer speed is 180 rpm. / min., temperature 20 ^o C. Mineral salts of the composition were added to the water, g / l: NH ₄ H ₂ PO ₄ - 10, K ₂ HPO ₄ - 10, MgSO ₄ • 7H ₂ O, ZnSO ₄ • 7H ₂ O, FeSO ₄ • 7H ₂ O, MnSO ₄ • 7H ₂ O - 0.0125 each; pH 7.0. The concentration of heptyl was 100 mg / L. After 1.5 days, the heptyl content decreased to 4.5 mg / L.

 Example 2

The destruction of heptyl in water was carried out according to example 1, but at a cultivation temperature of 30 ^o C. After 1.5 days, the heptyl content decreased to 2.35 mg / l, and after 20 days to 0.32 mg / l, while the formaldehyde content decreased from 65 mg / l to 0.9 mg / l, the content of dimethylamine - to 0.25 mg / l.

 Example 3

The destruction of heptyl in water was carried out as in example 2, but instead of an associative culture, a biological product was used, obtained by periodically cultivating separately three strains of bacteria: Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3. Cultivation was carried out on a mineral medium (composition according to example 1) with the addition of 2% (by volume) liquid paraffin in an Abitex apparatus with stirring (1800 rpm) and aeration (600 l / h); pH 7.0; temperature 34 ^o C. After cultivation, the biomass was separated by separation and then dried. Drying mode: inlet temperature 105-110 ^o C, outlet 45 - 50 ^o C, which ensured the preservation of cells in a viable state. The obtained dry biomass was a powder without a specific odor with a moisture content of 10% and a producer culture titer of 10 ⁸ cells / g of powder. To obtain a biological product, the dry biomass of three cultures was mixed in equal amounts. The finished biological product was introduced in an amount of 1% (by weight) relative to the concentration of heptyl. After 1.5 days, the heptyl content decreased to 3.2 mg / L, after 20 days - to 0.3 mg / L.

 Example 4

 The destruction of heptyl in water was carried out as in example 3, but the concentration of heptyl was 1000 mg / L. After 1.5 days, the heptyl content decreased to 44.3 mg / L, the formaldehyde content was 1.8 mg / L, and dimethylamine - 15.0 mg / L. After 20 days of cultivation, the heptyl content decreased to 3.2 mg / L.

 Example 5

The destruction of heptyl in the soil was carried out using samples of sandy soil in an amount of 400 g, which was placed in porcelain glasses. The soil moisture was maintained at a level of 20 - 25% by feeding a solution of mineral salts (according to example 1). The initial concentration of heptyl in the soil was 11.3 g / kg. Biological product was added to glasses with soil and heptyl in an amount of 2% (by weight) relative to the concentration of heptyl contained and closed with glass lids to prevent heptyl escaping. The cultivation temperature was 18 - 20 ^o C. The soil during the experiment was thoroughly mixed daily with a spatula to the entire depth. After 7 days of cultivation, the concentration of heptyl decreased to 7.5 g / kg, after 30 days to 0.35 g / kg.

Thus, a method of biodegradation of heptyl - asymmetric dimethylhydrazine, the novelty of which is as follows:
- in the use for the biodegradation of heptyl of a specially selected associative bacterial culture Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3, providing fast and deep decomposition of heptyl;
- in obtaining a biological product based on an associative culture, which is convenient for the practical implementation of the proposed method, in particular for the destruction of heptyl in natural conditions;
- biodegradation of heptyl is accompanied simultaneously by the destruction of intermediate products of its decomposition - formaldehyde and dimethylamine;
- in environmental cleanliness, since the bacterial strains and biological product used for biodegradation of heptyl are non-pathogenic and, in their practical use, environmental pollution is eliminated and the water and soil are cleaned of heptyl and its decomposition products.

Claims (1)

 A heptyl biodegradation method, characterized in that the heptyl decomposition process is carried out using a biological product based on an associative bacterial culture Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp.H-3 under aeration conditions when sources of nitrogen, phosphorus and trace elements are added to an object contaminated with heptyl (water, soil).

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