RU2453508C2 - Method for biological treatment of water from trinitrotoluene - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: contaminated natural and waste water is treated using cells of a yeast strain Geotrichum sp. VKPM F-1037 in series-connected bioreactors fitted with suitable equipment with a continuous stream of the contaminated and purified medium. Optimum temperature from +24°C to + 32°C, medium pH from 3.0 to 7.0 and rate of mixing biomass and medium from 100 to 250 rpm are maintained in the bioreactors.

EFFECT: method enables destruction of TNT and its metabolites in a short period of time when treating water contaminated with high concentrations of TNT.

5 cl, 1 dwg

Description

The invention relates to the field of biological water treatment and can be used for the treatment of natural and industrial wastewater, contaminated mainly by nitroaromatic xenobiotics.

The production of chemical synthesis products and their use in various fields of human activity is accompanied by pollution of soil, surface and groundwater with environmentally hazardous compounds, in particular 2,4,6-trinitrotoluene (hereinafter referred to as TNT) and its derivatives. Various methods are known for cleaning TNT-contaminated objects. At the same time, cleaning processes using microorganisms are more economical and environmentally friendly.

A known method [1] of transformation of TNT by free and immobilized cells of Arthrobacter sp. 8929.

The disadvantage of the method [1] is that when it is carried out in a cleaned medium, 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT) and 2,4 are accumulated -diamino-6-nitrotoluene (2,4-DANT), not further destroyed by cells of Arthrobacter sp. 8929. In addition, the potential of TNT transformation by the method [1] is significantly limited by the permissible concentration of xenobiotic and does not exceed 60 mg / l, whereas in practice the wastewater contains much higher concentrations of TNT. All this limits the possibility of applying the known method [1] in environmental activities.

A known method [2] of removing TNT from contaminated water with immobilized cells of Phanerochaete chrysosporium ATCC 24725 under various cultivation conditions.

The disadvantage of this method [2] is that the removal of TNT is not accompanied by its (TNT) destruction (destruction, degradation). As a result of the method [2], unidentified metabolites are formed, which subsequently do not undergo deep destruction by cells of the Ph strain. chrysosporium ATCC 24725 and accumulate, which limits the application of the known method on an industrial scale.

The known method [3] of transformation of TNT immobilized on filters by cells of the community of microorganisms. The method is carried out by a sequential two-stage purification process under anaerobic and aerobic conditions.

The disadvantage of this method [3] is the slowness of the biological treatment process. So, for adaptation of the microorganism community to TNT, it takes 8 days at an initial concentration of the initial xenobiotic of 5 mg / l, and the total duration (time) of the process takes 150 days. In addition, during the transformation of TNT, metabolites such as 2-ADNT, 4-ADNT, 2,4-DANT and 2,6-diamino-4-nitrotoluene (2,6-DANT) are formed, which retain toxicity and are characterized by increased resistance to destruction by a well-known community of microorganisms. This disadvantage also limits the application of the known method [3] in practice.

A known method [4] of purification of TNT-containing wastewater, carried out by continuous cultivation of the immobilized strain of Pseudomonas fluorescens B-3468 under anaerobic and aerobic conditions.

The disadvantage of this method [4] is that during its implementation amine derivatives of TNT resistant to further biological effects accumulate, while TNT biodegradation is absent, which limits the possibility of practical application of the known method [4].

The closest in technical solution to the proposed invention is the prototype, is a method of removing TNT from the cleaned environment by cells of the cyanobacteria Anabaena sp. [5].

The disadvantage of the prototype [5] is that high concentrations of TNT have a toxic effect on the cyanobacteria Anabaena sp., Therefore, require a long period of adaptation of cyanobacteria to the original xenobiotic by gradually increasing its (xenobiotic) concentration (from 1 mg / l to 58 mg / l) . This prolongs and increases the cost of the biotechnological process. In addition, the transformation of TNT by cyanobacteria Anabaena sp. It is accompanied by the accumulation in the medium to be purified of final and biodegradation resistant metabolites, for example, azoxy-tetranitrotoluene. Failure of Anabaena sp. to destroy the aromatic ring of TNT with the removal of nitro groups significantly limits the possibility of using the known method [5] in environmental protection activities.

The aim of the invention is to improve the quality and efficiency of treatment (neutralization, bioremediation) of natural and wastewater contaminated with toxic 2,4,6-trinitrotoluene and its derivatives, reducing treatment time, increasing the productivity of treatment facilities, expanding the scope of biotechnology for cleaning contaminated objects, pollution prevention.

The goals are achieved in that the treatment of TNT-contaminated natural and waste water is carried out with free and / or immobilized cells of the yeast strain Geotrichum sp. VKPM F-1037 in bioreactors connected in series and equipped with appropriate equipment with a continuous flow of contaminated and cleaned media, ensuring (in bioreactors) optimal temperatures, pH of the medium, aeration, mixing speed of biomass and medium.

The proposed method is carried out in a device consisting of a container with a contaminated medium, for example, a tank and bioreactors, connected in series by pipelines with shut-off and control equipment. Bioreactors are also equipped with samplers, air-venting channels, mixers for mixing biomass and medium, pumps for pumping the medium, and equipment for ensuring aeration of the medium.

The essence of the proposed method is that the neutralization of TNT-contaminated water is carried out using a yeast strain Geotrichum sp. VKPM F-1037. The method is carried out by continuous cultivation of the strain in the medium to be cleaned in a device consisting of at least three bioreactors in the form of tanks connected in series to each other, equipped with the necessary operational and control equipment. In another embodiment, the cleaning is carried out in series-connected reservoirs, for example flowing ponds of treatment facilities equipped with the necessary operational and control equipment. Yeast strain Geotrichum sp. cultivated in an optimal temperature range from + 24 ° C to + 32 ° C, pH from 3.0 to 7.0 and at a speed of mixing the medium from 100 to 250 rpm; when deviating from the optimal values of the parameters, the process of purification of polluted waters slows down.

The essence of the proposed method is illustrated by examples.

Example 1. Purification of natural and waste water from TNT by free cells of the strain of the yeast Geotrichum sp. VKPM F-1037.

The method is carried out in a device consisting of a tank and bioreactors connected in series with each other by pipelines with shut-off and control equipment. Bioreactors are equipped with samplers, air-venting channels, mixers, for example, magnetic mixers for uniform distribution of yeast and oxygen biomass in the thickness of the medium to be cleaned, pumps for pumping the medium to be cleaned, and an aeration system.

Cleaning is carried out in a plant for continuous cultivation of yeast (Fig.), Where: 1 - the first bioreactor; 2 - second bioreactor; 3 - the third bioreactor; 4 - a reservoir containing a liquid medium with TNT; 5, 6, 7 - samplers; 8, 9, 10 - air vents equipped with filters; 11, 12, 13 - magnetic stirrers with magnets; 14, 15, 16, 17 - piping system with locking and regulating equipment; 18, 19, 20, 21 - pumps; 22, 23, 24 - aeration system.

The installation consists of a tank 4 containing a liquid, purified synthetic medium with TNT, and three bioreactors 1, 2, 3, connected in series by a system of pipelines 14, 15, 16, 17 and equipped with pumps, for example, peristaltic pumps 18, 19, 20, 21 for pumping cleaned environment. All bioreactors are equipped with an aeration system 22, 23, 24, which supplies sterile air (to the bioreactors), as well as air-venting channels with 0.22 µm filters 8, 9, 10. In addition, bioreactors are equipped with samplers 5, 6, 7. The mixing process The cleaned medium inoculated with yeast cells (with introduced cells) of Geotrichum sp. is carried out with magnetic stirrers 11, 12, 13. The cleaning process is carried out in the temperature range from + 24 ° C to + 32 ° C.

As a cleaning medium, for example, a synthetic medium of the following composition is used: glucose - 11.2 mm, (NH ₄ ) ₂ SO ₄ - 7.6 mm, MgSO ₄ -2.0 mm, Na ₂ HPO ₄ - 9.8 mm KH ₂ PO ₄ - 6.2 mmol (pH 7.0). TNT is added to the synthetic medium at a concentration of 100 mg / L. The medium for maintaining the strain of Geotrichum sp. and biomass (strain) accumulation is Saburo agar medium containing glucose - 10.0 g / l, peptone - 10.0 g / l, yeast extract - 5.0 g / l, NaCl - 0.25 g / l, agar - 20.0 g / l. Distilled water is used to prepare all media.

The process of biological destruction (biological destruction) of TNT and its metabolites is carried out in two stages.

Pre-tank 4 is filled with liquid TNT-containing synthetic medium.

At the first stage, bioreactors 1, 2, 3 are filled with one third of liquid TNT-containing synthetic medium from tank 4; include magnetic stirrers 11, 12, 13 and aeration system 22, 23, 24.

The daily culture of Geotrichum sp. Is introduced into the bioreactor 1 containing a liquid synthetic medium with TNT. to a final optical density of A ₆₀₀ 0.2 cells are cultured and (Geotrichum sp.) to the fullest possible conversion of TNT-3 monohydride complex Meyzenheymera (3-H ^- -TNT) dark red. As a result of the transformation of TNT with a yeast strain, bioreactor 1 accumulates a major metabolite - 3-H ^- -THT (up to 82%). At the same time, a neutral or slightly acidic pH value of the medium being cleaned is maintained.

Simultaneously with the start of the bioreactor 1 operation, a diurnal culture of Geotrichum sp. Is introduced into the bioreactor 2 containing a liquid synthetic medium with TNT. to the final A ₆₀₀ 0.4 and cultivated (Geotrichum sp.) until the yellow-orange C3, C5 Meisenheimer dihydride complexes (3.5-2H ^- -THT; isomers 3.5-2H ^- -THT · H ⁺ ) are accumulated, which are the main metabolites of the conversion of the initial xenobiotic in bioreactor 2. In bioreactor 2, the pH decreases to the optimal level of 4.7-5.4 due to the intensive production of organic acids by the yeast.

Simultaneously with the start of the operation of bioreactors 1 and 2, a diurnal culture of Geotrichum sp. Is introduced into bioreactor 3 containing a liquid synthetic medium with TNT. to the final A ₆₀₀ 1.0 and cultured (Geotrichum sp.) until the formation and complete destruction of TNT-dihydride complexes. Purification in the bioreactor 3 is accompanied by discoloration of the medium being cleaned and the accumulation of nitrate ion, which is the major final nitrogen-containing inorganic product of the destruction of toxic TNT. In bioreactor 3, the pH decreases to optimal values from 3.0 to 3.8 due to intensive synthesis and excretion of organic acids by yeast. This completes the first stage.

In the second stage, pumps 18, 19, 20, 21 are turned on, and the biodegradation of TNT and its metabolites is carried out under continuous cultivation of Geotrichum yeast cells. sp. with provision (operation of pumps and shut-off and control equipment) of a complete renewal of the bioreactor environment over a period of time from 18 to 24 hours.

In each of the bioreactors 1, 2, 3, the optimal optical density of the cells of Geotrichum sp. (from A ₆₀₀ 0.2 to A ₆₀₀ 5.0) by controlling the rate of liquid synthetic medium from TNT from the tank 4 to the bioreactor 1, the medium to be cleaned from the bioreactor 1 to the bioreactor 2, the medium to be cleaned from the bioreactor 2 to the bioreactor 3 and the purified medium from a bioreactor 3, for example, to a system for collecting and removing the purified medium (not shown in FIG. as not directly related to the cleaning method). The flow rate and flow rate of the cleaned medium through bioreactors is established while maintaining the stability of biological processes and ensuring spatial separation of TNT transformation metabolites in all three bioreactors 1, 2, 3. The process is controlled by sampling through samplers 5, 6, 7 and their (samples) subsequent analysis and correct, for example, by changing the speed of receipt of the medium, the intensity of mixing.

As a result of the implementation of the proposed cleaning method according to Example 1, the biodegradation of TNT through the destruction of intermediate TNT hydride complexes reaches 82% (of the initial concentration) for 1 day. Such a deep destruction of TNT by the proposed method significantly exceeds the capabilities of the prototype and ensures its (the proposed method) application in an industrial environment.

Another advantage of the proposed method for the biological destruction of TNT and its metabolites in the device is the ability to neutralize TNT-contaminated water using cells immobilized on various carriers, as shown in the following examples.

As carriers for the immobilization of cells of the strain of the yeast Geotrichum sp. various carriers are used, for example: solid (chitin, cellulose, silica, ceramics, fiberglass) and gel (alginate, polyacrylamide and agarose gels, polyvinyl alcohol cryogel).

Example 2. Purification of natural and waste water from TNT by cells of the yeast strain Geotrichum sp. VKPM F-1037.

Pre cells of the yeast strain Geotrichum sp. immobilized on a carrier, for example on a fiberglass cloth (fiberglass). For this, Saburo liquid medium is inoculated with the yeast cells of Geotrichum sp. and cultured for 18 hours. Then, pieces of fiberglass are introduced into the Saburo liquid medium with cells and cultured (cells) in the presence of a carrier for 4 hours. Yeast cells are adsorbed onto the carrier. Then pieces of fiberglass with immobilized yeast cells are transferred to bioreactors 1, 2, 3 with the medium to be cleaned.

The next stage, the stage of biological destruction of TNT, is carried out according to Example 1. By the implementation of the process according to Example 2, the destruction of TNT is achieved within 75-80%.

Example 3. Purification of natural and wastewater from TNT by cells of the yeast strain Geotrichum sp. VKPM F-1037.

Pre cells of the yeast strain Geotrichum sp. include in the structure of the gel, for example alginate gel, by known methods [6, 7].

Then the carrier with immobilized yeast cells is transferred into bioreactors 1, 2, 3 with the medium to be purified.

Next, the process of biological destruction of TNT is carried out according to Example 1. The implementation of the process according to Example 3 achieve the destruction of TNT in the range of 75-80%.

The effectiveness of the cleaning process of the proposed method significantly exceeds the efficiency of the prototype, which improves the performance of treatment facilities. In addition, the proposed method allows the use of both free and immobilized cells of the yeast strain Geotrichum sp. VKPM F-1037 for the implementation of the biotechnological process for the treatment of contaminated waters. The universality of the application of the TNT purification method is ensured by the fact that, depending on the conditions for the implementation of the neutralization process, Geotrichum sp. Cells immobilized on various carriers are used. The possibility of immobilization of the strain Geotrichum sp. allows you to increase the resistance of immobilized cells to high concentrations of TNT. This expands the scope of biotechnology for the detoxification of natural and wastewater contaminated with nitroaromatic xenobiotics.

The dynamics of the transformation of TNT and its metabolites, accompanied by a change in the color of the medium being cleaned in bioreactors, allows visual (without the use of analytical equipment) control of the cleaning process in an industrial environment. This greatly simplifies the monitoring of the treatment facilities. If necessary, the dynamics of the bioremediation process is monitored by analyzing samples taken from bioreactors 1, 2, 3 using samplers 5, 6, 7. In the practice of treating natural and waste waters contaminated with TNT and its metabolites, visual control of the bioremediation process is sufficient.

TNT transformation products are determined by various physicochemical methods, for example, UV-visible spectrophotometry, high-performance liquid and ion chromatography.

Spectrophotometric measurements are performed on a spectrophotometer, for example, Lambda 35 (Perkin Elmer, USA). Biomass is estimated by the change in the optical density of the medium with cells at a wavelength of 600 nm. The control is a cell-free medium. 3-H ^- -THT is detected by the absorption peak at 476 nm, the accumulation of 3.5-2H ^- -THT and the sum of the isomers 3.5-2H ^- -THT · H ⁺ - by spectral shifts in the region of 440-445 nm.

TNT and its transformation products are analyzed by high performance liquid chromatography, for example, on a Series 200 chromatograph (Perkin Elmer, USA), in the reverse phase version using a Supelcosil octyl C-8 column (150 × 4.6 mm; 5 μM), with detection at 254 and 476 nm. Initially, the mobile phase consists of 99% Na-phosphate buffer (pH 7.0; 25 mM) and 1% methanol. Over 2.0 minutes, the amount of methanol is increased to 30%, over the next 13.0 minutes - up to 43%. In the next 12.5 minutes, the methanol content was increased from 43% to 100%. This gradient is left unchanged for 0.5 min. Subsequently, over 1.0 min, the ratio of the mobile phase is returned to its original level and remains constant over the next 5.0 min. The flow rate of 1.0 ml / min, temperature plus 50 ° C.

Nitrite and nitrate ions are determined using an ion chromatograph, for example, 761 Compact IC (Metrohm AG, Switzerland) equipped with a Metrosep A Supp 5 - 150 separation column (6.1006.520). The elution is carried out with solutions of 1.0 mm NaHCO ₃ and 3.2 mm Na ₂ CO ₃ at a rate of 0.7 ml / min.

A significant difference of the proposed method for multistage cultivation of the yeast strain Geotrichum sp. BKPM F-1037 in the presence of TNT is a high (up to 82%) biodegradation efficiency of TNT and its metabolites in a short period of time (1 day), whereas the transformation of TNT with the prototype only leads to the accumulation of metabolites of the initial xenobiotic in the absence of their decomposition. The accumulation of stable TNT transformation products that occurs when using the prototype does not solve the problem of cleaning TNT-contaminated objects, but only contributes to secondary environmental pollution by formed metabolites.

Another advantage of the proposed method using a strain of Geotrichum sp. VKPM F-1037 is the possibility of its use for the purification of waters contaminated with high concentrations of TNT, since the Geotrichum sp. possesses increased (compared with analogues and prototype) resistance to the toxic effect of TNT (up to 200 mg / l).

Possibility of carrying out the purification process using immobilized cells of the strain Geotrichum sp. VKPM F-1037 can significantly expand the scope of biotechnology for environmental protection.

The application of the proposed method improves the quality and simplifies the process of biological treatment of natural and wastewater contaminated with toxic nitroaromatic compounds (exemplified as particularly resistant to biological destruction of TNT and its derivatives), shortens cleaning time, expands the scope of biotechnology for cleaning environmentally hazardous objects, and prevents pollution of natural Wednesday.

The present invention satisfies the criteria of novelty, since when determining the prior art methods are not found that are inherent in signs identical (that is, coinciding in the function performed by them and the form of these signs) to all the signs listed in the claims, including the purpose of the application.

The claimed method has an inventive step, because it has not been identified technical solutions that have features that match the distinguishing features of this invention, and the popularity of the influence of distinctive features on the specified technical result is not established.

The claimed technical solution can be implemented at the treatment facilities of industrial enterprises synthesizing nitroaromatic compounds, as well as in the activities of environmental organizations using well-known standard technical devices and equipment. This meets the criterion of "industrial applicability" of the invention.

Information sources

1. Tore A.M., K. Jamil, T. R. Baggi. Transformation of 2,4,6-trinitrotoluene (TNT) by immobilized and resting cells of Arthrobacter sp. // J. Hazard. Subst. Res. - 1999. - V. 2. - P. 3-1-3-9.

2. Sublette, K. L., E. V. Ganapathy, S. Schwartz. Degradation of munition wastes by Phanerochaete chrysosporium // Appl. Biochem. Biotechnol. - 1992. - V.34 / 35. - P.709-723.

3. Wang Zh., Zh. Ye, M. Zhang, X. Bai. Degradation of 2,4,6-trinitrotoluene (TNT) by immobilized microorganism-biological filter // Process Biochem. - 2010 .-- V.45. - P.993-1001.

4. RF patent "Method for biological wastewater treatment" RU No. 1471493, IPC ⁶ C02F 3/34, priority dated 12/08/1986. Description text dated 09/10/1995.

5. Pavlostathis S.G., G. H. Jackson. Biotransformation of 2,4,6-trinitrotoluene in a continuous-flow Anabaena sp. system // Water Res. - 2002. - V. 36. - P.1699-1706.

6. Flink J.M., A. Johansen. A novel method for immobilization of yeast cells in alginate gels of various shapes by internal liberation of Ca-ions // Biotechnol. Lett. - 1985. - V.7. - P.765-768.

7. Stormo K.E., R.L. Crawford. Preparation of encapsulated microbial cells for environmental applications // Appl. Environ. Microbiol. - 1992. - V.58. - P.727-730.

Claims (5)

1. The method of biological treatment of water from trinitrotoluene, which consists in the fact that the treatment of contaminated natural and waste water is performed using a yeast strain Geotrichum sp. VKPM F-1037 in bioreactors connected in series and equipped with appropriate equipment with a continuous flow of contaminated and cleaned media with optimal temperature values in the bioreactors from 24 to 32 ° C, pH of the medium from 3.0 to 7.0, mixing rates of biomass and medium from 100 to 250 rpm 2. The method according to claim 1, characterized in that the use of free yeast cells. 3. The method according to claim 1, characterized in that immobilized yeast cells are used. 4. The method according to claim 3, characterized in that the yeast cells are immobilized on a solid carrier. 5. The method according to claim 3, characterized in that the yeast cells are immobilized in a gel.