RU2650864C1 - Biological destructor of non-symmetric dimethylhydrazine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: ecobiotic "Centrum-MMS" is proposed, which is an association of microorganisms Pseudomonas fluorescens VKM B-6847 and Rhodococcus erythropolis VKM As-1769, which is proposed for biodegradation of asymmetric dimethylhydrazine.

EFFECT: invention allows to reduce the amount of asymmetric dimethylhydrazine in aqueous solutions by more than 50 times.

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Description

FIELD OF THE INVENTION

The invention relates to biotechnology for environmental protection and can be used in biological treatment processes when contaminated with asymmetric dimethylhydrazine.

State of the art

In technologies for environmental bio-purification from various xenobiotics, a significant amount of biological preparations of environmental purpose is known, containing a set of bacterial strains with aerobic respiration, intended for biochemical destruction and disposal of pollutants mainly of hydrocarbon nature (oil and products of its industrial processing).

Ecobiological preparations used for biological purification of the environment have both certain advantages and disadvantages, among which we note: a limited range of utilized substrates, non-optimal microbial composition, complexity of biomass production, in particular for multicomponent preparations, violation of natural relationships in natural biocenoses after introduction microorganisms, as well as the poor effectiveness of drugs in natural conditions, especially at relatively high concentrations of contaminants Iteli, the negative impact of various impurities, insufficient aeration, low temperatures, etc.

The neutralization of environmental objects from liquid jet fuels, known as heptyl (asymmetric dimethylhydrazine - UDMH), is an urgent and unsolved problem of modern ecology.

Heptyl is considered an extremely dangerous ecotoxicant and belongs to substances of the first hazard class. It is known that the use of UDMH in rocket and space technology is due to its “special operational properties, and it is not expected to replace it, as fuel, in the future. From the point of view of the environmental consequences arising from this, associated with spills of hydrazine fuels into the Earth’s atmosphere, this is a global environmental problem ”[Kolesnikov, S.V. Oxidation of asymmetric dimethylhydrazine (heptyl) and identification of the products of its transformation during straits: - Monograph. Novosibirsk: Publishing House. SibAK, 2014. - 110 p.].

When tanks with UDMH residues fall, as well as during unsuccessful launches of various missiles, significant areas of territories and water surfaces are contaminated by components of rocket fuels along the entire flight path of missiles, and the formation of infected areas, the number of which increases with each new launch, which are currently being carried out with Cosmodromes Baikonur (Kazakhstan), Plesetsk, Svobodny and Kapustin Yar.

Given the properties of UDMH, such as toxicity, high volatility, unlimited solubility in water and aqueous solutions of acids, etc., there is a significant risk of heptyl entering the biosphere and, in particular, into the human body, with all the ensuing negative consequences. A study of the environmental consequences of the fall of the separating parts of launch vehicles shows that UDMH can enter the environment up to several tons per year [Environmental monitoring of space rocket activity. Principles and Methods / Ed. N.S. Kasimova, O.A. Shpiguna. - M .: Restart, 2011. - 472 p.].

Effective and safe technologies for neutralizing heptyl, including biological ones, have remained insufficiently developed to date. We believe that one of the promising directions for solving this problem may be the use of ecobiological preparations intended for microbiological destruction of asymmetric dimethylhydrazine, although we do not know the presence of such commercial preparations in Russia or abroad.

Currently, there are few reports of species and associations of microorganisms capable of utilizing UDMH.

Thus, a method is known (Patent for invention No. 2174553 dated February 11, 1998, the Applicant is Biotech-Japan CJSC) for biodegradation of asymmetric dimethylhydrazine based on the use of an associative culture including the following bacteria strains: Acinetobacter sp. H-1, Rhodococcus sp. H-2, Arthrobacter sp. H-3 in a ratio of 1: 3: 2, respectively.

In model experiments during cultivation in flasks at temperatures of 20 and 30 ° C, a decrease in the initial concentration of heptyl in water from 100 mg⋅ ^-1 to 0.3 mg⋅ ^{-1 was} shown, in the soil - from 11.3 g⋅kg ^{- 1} to 0.35 g⋅kg ^-1 .

The patent is terminated on February 12, 2003.

The disadvantages of this method:

in fact, the authors for biodegradation of heptyl do not use a biopreparation not properly designed, but an associative culture;

specific biotechnological parameters that are optimal for the implementation of the biological destruction of UDMH are not given;

maximum values determining the concentrations of asymmetric dimethylhydrazine do not exceed 0.1% for water and 1% for soil;

the natural strains included in the association are not identified by species and since they are a “specially selected culture”, there are no indications of genetic fixation and resistance of their specific biodestructive properties (the ability to biodegrade UDMH), that is, the main feature by which they were selected .

Described (Patent for invention No. 2236453 dated September 18, 2002, the Applicant is a Research Center for Toxicology and Hygienic Regulation of Biological Preparations) a bacterial strain Rhodococcus globerulus 19ph, when a culture fluid (10 ⁷ klml ^-1 ) is introduced into contaminated heptyl ( 200 mg-kg ^-1 ) soil, its concentration decreased by 39% during the month of observation.

The patent is terminated on September 19, 2010.

The disadvantage when using this strain is that the development is not brought to the creation of a finished biological product, as well as the duration and relatively low level of biodegradation of UDMH.

There is also a patent for the invention of the Republic of Kazakhstan No. 19817 dated December 15, 2010, which describes a plant for biochemical treatment and reclamation of industrial wastewater from hydrazine, heptyl and its metabolites, which is a three-section concrete biopond with continuous aeration of wastewater and the use of natural association for biodegradation microorganisms belonging to the three genera of Rhodococcus and two genera of pseudomonas. As stimulants of bacterial growth, molasses, plant proteins, ammophos and potassium chloride are introduced.

According to the authors, the installation allows you to achieve 100% biochemical treatment of wastewater and soil from heptyl and its metabolites at their initial concentration of 100 mg kg ^-1 .

The disadvantages of the proposed method are:

the technical impossibility of using a stationary installation for remediation of contaminated soils and water surfaces in natural conditions on large areas corresponding in linear parameters to the length of missile spans and dispersion zones of UDMH;

biotechnological problems associated with the complexity of producing a six-component bacterial product, especially since the simultaneous cultivation is technologically unacceptable due to the different nutritional needs of the strains (two of which are not identified at all);

the declared titer of microorganisms, recommended for biodegradation, equal to 10 ¹² -10 ¹⁴ cells · cm ^-3 , in our opinion, is unattainable;

complete biodegradation of heptyl is achieved with its relatively low initial level of 0.01%;

The claimed technical solution is very close to the prototype "Installation for microbiological wastewater treatment" (AS USSR No. 1542916, 1990).

Disclosure of invention

Earlier (2010), we proposed for the biological purification of water and soil from oil and oil products the ecological product Centrum - MMS (RF Patent No. 2428471 of June 13, 2010). The proposed biological product for environmental purposes "Centrum - MMS" is characterized by:

the component composition represented by two types of microorganisms with a pronounced ability to utilize hydrocarbons when they are combined;

high biodegradation activity even with a single application to crude oil and various types of hydrocarbon fuels;

activity in the disposal of "heavy" as well as aromatic hydrocarbons;

the possibility of a single use for remediation of soils and water surfaces at relatively low ambient temperatures (8-12 ° C), which is especially important for the northern oil producing regions of the Russian Federation.

Subsequently, at the stages of implementation and implementation (TU-92917-005-2010 and Industrial Regulation No. 01-MP) of the ecological product “Centrum-MMS”, a more complete range of its biodestructive capabilities was established. So, the drug utilized not only a wide range of aliphatic, aromatic and naphthenic hydrocarbons, such as: crude oil, fuel oil, gasoline, diesel fuel, but also engine, engine and turbine oils. The possibilities of decomposition and disposal by the Centrum MMS MMS of other xenobiotics, including UDMH, have not been studied before.

Thus, taking into account the accumulated theoretical and experimental data, in order to solve the environmental problems of cleaning territories and water surfaces contaminated with asymmetric dimethylhydrazine, the authors were faced with the task of establishing the biodestructive activity of the Centrum-MMS ecological product in relation to UDMH and assessing the level of its biological decomposition .

The implementation of the invention

To solve the problem used:

Ecobiological product “Centrum-MMS” (batch 01-17), prepared in accordance with the applicable specifications and industrial regulations;

synthetic nutrient medium (CaCl ₂ ⋅ 6H ₂ O - 0.1 g; MgO ₄ ⋅ 5H ₂ O -0.02 g; FeSO ₄ ⋅ 7H ₂ O - 0.01 g; Na ₂ HPO ₄ - 3.0 g; Na ₂ CO ₃ - 0.1 g; NH ₄ Cl - 2.0 g; NaCl - 5.0 g; KH ₂ PO ₄ - 2.0 g; MgSO ₄ ⋅ 7H ₂ O - 0.2 g; glucose - 5 g; distilled water to 1 dm ³ );

granular phosphate fertilizer - superphosphate (GOST 5956-78), potassium chloride (GOST 4568-95);

technical sample of asymmetric dimethylhydrazine;

shaker incubator KS 4000i with flasks with a capacity of 250 cm ³ ;

laboratory bioreactor with a capacity of 5 dm ³ with forced air supply.

Test conditions:

the temperature of the reaction medium is 18-20 ° C;

observation time - 7 days;

the concentration of UDMH in the shaker-incubator in the reaction mixture is 0.01-1.0%;

the concentration of UDMH in the laboratory bioreactor in the reaction mixture is up to 2.0%.

Analytical studies of experimental samples on the content of UDMH were carried out by two independent methods: chromato-mass spectrometric and gas chromatographic. The combination of the capabilities of the two selected analytical methods allowed us to identify and quantify the main parameters of the biological destruction of UDMH.

Chromatography-mass spectrometric analysis was performed on a Trace GC Ultra / DSQII chromatography-mass spectrometer under the following conditions:

TR 5 MS capillary column 60 m long, 0.25 mm diameter;

injector temperature - 200 ° C;

the temperature of the ion source is 200 ° C;

carrier gas - helium M 60, feed rate 1.2 ml min ^-1 ;

the temperature of the chromatographic column during analysis increased from 40 to 200 ° C at a rate of 10 deg граmin ^-1 .

Mass chromatograms were recorded in the range of mass numbers 35-300. Identification was carried out using the electronic library of mass spectra "NIST 2005".

Gas chromatographic analysis was carried out under the following optimal conditions:

chromatographic column temperature  80 ° C evaporator temperature 120 ° C detector temperature 320 ° C carrier gas feed rate (nitrogen) 28 ml⋅min ^-1 hydrogen feed rate 16 ml⋅min ^-1 air flow rate 150 ml⋅min ^-1

The volume of each sample of the working solutions introduced into the column of the chromatograph was 0.2 μl.

Detector signals and chromatograms were recorded using the TsvetKhrom software package. The calibration dependence for the quantitative assessment of the components was evaluated according to the results of the analysis of calibration samples.

Each sample was analyzed three times. For this, 2.0 ml of the test solution was introduced into a penicillin vial with 10.0 ± 0.5 g of potassium hydroxide. Then the bottle was closed with a rubber stopper with a gasket from a PTFE film, an aluminum cap was put on and crimped by a press (or a sealing device was used). The bottle was shaken several times, placed in a thermostat and kept for 15 min at a temperature of 80 ° C. 1.0 cm of the gas phase was taken from the vial for analysis using a syringe preheated to 80 ° C.

The results of the experimental studies are presented in the table.

Notes:

1. Patent for the invention of the Russian Federation No. 2174553 dated February 11, 1998

2. Patent for the invention of the Republic of Kazakhstan No. 19817 dated December 15, 2010

Thus, as a result of experimental studies, for the first time, the Centrum-MMS ecobiological product was found to have a previously unknown property - the ability to biodegrade such an ecotoxicant of hazard class I as asymmetric dimethylhydrazine.

The obtained experimental results suggest that the aerobic bacteria that are the basis of the Centrum-MMS ecobiological product, Pseudomonas fluorescens VKM B-6847 and Rhodococcus erythropolis AC-1769 species, have the ability to use UDMH as the sole source of carbon and nitrogen. We also indicate that, according to analytical studies, the most common products of the biological decomposition of heptyl were di- and trimethylamines, azomethane, tetramethylmethanediamide, which were determined only on the 1st – 6th day of observation. Nitrosodimethylamine was not found in the analyzed samples, which indirectly indicates the occurrence of UDMH conversion processes precisely in the biodestructive type.

It was found that this biodestructor effectively decomposes UDMH in water at concentrations from 0.5% and above (up to 1.58%), which significantly exceeds the same parameters for existing prototypes. Significant differences are the higher decomposition rate of the ecotoxicant, as evidenced by the relatively short biodegradation time, as well as the lower temperature of the heptyl decomposition processes when using the Centrum-MMS preparation than the known prototypes: 6-7 days at 18-20 ° С and 20-25 days at temperatures up to 30-35 ° C, respectively.

A list has also been established and technological parameters optimized in the course of experimental studies that allow achieving maximum biodegradation of asymmetric dimethylhydrazine in the reaction medium, which were equal to:

concentration of hydrogen ions, units pH 6.2-7.8 temperature, ° С 20-25 weight of applied ecobiological product (up to concentration biocomponent), cm ^-3 10 ⁸ -10 ⁹ duration of exposure, days 6-7 the frequency of treatment with an ecobiological product, times one level of aerability, mg⋅dm ^-3 15-20

amount of additives, g⋅dm ^-3 :

granular superphosphate 5,0 potassium chloride 2.5

Thus, for the first time, experimental data have been obtained confirming the ability of the association of bacterial microorganisms of the species Pseudomonas fluorescens VKM-6847 and Rhodococcus erythropolis Ac-1769, which are part of the Centrum-MMS eco-preparation, to biologically destroy UDMH in aqueous solutions. The decomposition efficiency of asymmetric dimethylhydrazine was significantly higher than that of other types of microorganisms and their associations described previously. The Centrum-MMS ecobiological product has been proposed for use for the biological destruction of asymmetric dimethylhydrazine, an organic compound of hazard class I used as rocket fuel.

Claims (1)

The use of Centrum-MMS ecobiological product for oil and oil products purification as a biological destructor of asymmetric dimethylhydrazine aqueous solutions.