RU2482076C2 - Method for bioremediation of water contaminated with trinitrotoluene - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a method for bioremediatio of water contaminated with 2,4,6-trinitrotoluene (TNT). The method is carried out in three series-connected bioreactors with continuous flow of the purified medium. At the first step, bioreactors are filled by a third with a liquid TNT-containing medium and mixers and the aeration system are turned on. A day old culture of yeast Yarrowia lipolytica VKPM Y-3492 is then added to the first bioreactor. The Y. lipolytica is cultured until full conversion of TNT to a S-3 monohydride Meisenheimer complex of a deep red colour. A day old culture of Y. lipolytica is added to the second bioreactor at the same time as the beginning of operation of the first bioreactor and then cultured until accumulation of yellow-orange S3, S5 dihydride Meisenheimer complexes. A day old culture of Y. lipolytica is added to the third bioreactor at the same time as the beginning of operation of the first and second bioreactors and then cultured until full decomposition of TNT-dihydride complexes with decolouration of the purified medium. At the second step, the biodegradation of TNT is carried out in continuous mode of culturing Y. lipolytica. The process of culturing Yarrowia lipolytica VKPM Y-3492 is carried out at temperature ranging from +24°C to +31°C, pH from 3.0 to 7.0 and rate of mixing the medium of 100-250 rpm. The source of carbon and energy for growth of the strain and degradation of TNT used is monosaccharides or triatomic alcohols or aliphatic hydrocarbons.

EFFECT: method increases efficiency of the process of decontaminating water contaminated with TNT and cuts the bioremediation time: provides 85% decomposition of TNT in concentration of 100 ml/g a day.

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Description

The invention relates to the field of water treatment and can be used for bioremediation (bio-treatment) technologies of natural and wastewater contaminated mainly with nitroaromatic compounds.

It is known that actions associated with the production, storage, use and disposal of nitroaromatic compounds, for example, 2,4,6-trinitrotoluene (hereinafter referred to as TNT) and its derivatives, lead to their widespread distribution in the environment (compounds). As a result of synthesis, as well as the burial of these compounds, environmental pollution occurs, for example, groundwater, which is used by the population.

Operation and depreciation of equipment of industrial enterprises using nitroaromatic compounds in technological processes lead to an increase in the quantity and accumulation of waste. These wastes are characterized by high toxicity and cumulative ability, and may also have a mutagenic effect. The dumping of toxic wastes of industrial enterprises into water bodies and streams significantly worsens their general sanitary condition, while having a negative effect on living organisms both by toxicity and by a change in the regime of nutrients. To reduce the anthropogenic impact of nitroaromatic compounds on environmental objects, it is necessary to use effective technologies for the treatment of contaminated wastewater.

A known method [1] of remediation of TNT-contaminated territories, based on covalent binding of TNT and its nitro derivatives with an organic soil matrix.

The disadvantage of this method [1] is the slow process of remediation and its low efficiency. So, after a 28-day treatment of TNT-contaminated soil under anaerobic and aerobic conditions, the percentage of binding of TNT and its metabolites to the organic soil matrix was 40%. In addition, there is a risk of re-contamination with these compounds as a result of their cleavage from the organic matrix of the soil with the depletion of fertility. In this regard, the application of the method [1] is very limited.

A known method [2] of washing and removing TNT from objects contaminated with explosive mixtures.

The disadvantage of this method [2] is that its use is not accompanied by the destruction and mineralization of TNT. This leads to the conservation and circulation of TNT and its metabolites in the environment.

A known method [3] non-biological treatment of groundwater contaminated with explosives, for example, TNT, carried out by dithionite-reduced sediments.

The disadvantage of the method [3] is that the transformation potential of TNT according to the method is significantly limited and does not exceed 65 mg / l for 72 hours (3 days). In addition, in the process of transformation, such metabolites are formed as 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6- nitrotoluene (2,4-DANT) and 2,6-diamino-4-nitrotoluene (2,6-DANT), which retain residual toxicity and are characterized by increased resistance to subsequent exposure.

A known method [4] of removing TNT from bioreactors by continuous cultivation of microbial communities in a liquid TNT-containing medium under aerobic, microaerophilic and anaerobic conditions.

The disadvantage of this method [4] is that during continuous cultivation of microbial communities in the medium to be purified, the following TNT transformation metabolites are formed: hydroxylamino-dinitrotoluene (GADNT), amino-dinitrotoluene (ADNT), diamino-nitrotoluene (DANT) and azoxynitrotoluene. These metabolites are not biodegradable and accumulate in the environment.

The known microbiological method [5] for the neutralization of objects contaminated with TNT (up to 100 mg / l) with a strain of the fungus Penicillium sp. I-2081.

The disadvantage of the method [5] is that the purification method using a strain of Penicillium sp. I-2081 is time-consuming (optimally 10 days). To implement the method, it is necessary to have a high concentration of glucose (optimally 15 g / l) and an increased concentration of fungal biomass (optimally 150 g / l) in the neutralized medium, which complicates and increases the cost of the bioremediation process by the Penicillium sp. I-2081.

Known microbiological method [6] for the treatment of wastewater containing TNT. The method is carried out by two-stage continuous cultivation of a strain of Pseudomonas fluorescens B-3468, immobilized on a fiberglass carrier.

The disadvantage of the method [6] is that in wastewater there is an accumulation of 2-ADNT, 4-ADNT and 2,4-DANT that are not degradable by the Ps strain. fluorescens B-3468. This limits the possibility of implementing the known method [6] in environmental activities.

Closest to the technical solution to the proposed invention, the prototype is a method of neutralizing TNT-contaminated liquid with immobilized cells of the fungus Phanerochaete chrysosporium BKM-F-1767 in a bioreactor under continuous cultivation [7].

The disadvantage of the prototype [7] is that the bioremediation of TNT-contaminated liquid is carried out in a single bioreactor equipped with appropriate equipment for conducting and monitoring the TNT biotransformation process. The use of a single bioreactor is dictated by the feature of the TNT biotransformation process carried out by Ph. chrysosporium BKM-F-1767. This process is characterized by unsatisfactory (low) for practice effectiveness, limiting the application of the method. Another disadvantage is the implementation of the biological process by the culture of the fungus Ph. chiysosporium BKM-F-1767 with an age of at least 7 days. This prolongs and increases the cost of the biotechnological process. In addition, the low resistance of the known fungus to the toxic effect of TNT does not allow it (the fungus) to be used for bioremediation of objects contaminated with high concentrations of TNT. In addition, the transformation of TNT fungus Ph. chiysosporium BKM-F-1767 is accompanied by the accumulation of stable metabolites in the medium to be purified (2-ADNT, 4-ADNT, 2,4-DANT and 2,6-DANT), which reduces the effectiveness of bioremediation with the implementation of the prototype. At the same time, the average level of TNT mineralization by a known fungus remains at a low level (15.3% of the initial TNT content (50 mg / L) for 41 days).

The aim of the invention is to improve the quality and efficiency of the process of bioremediation (neutralization, treatment) of water contaminated with toxic nitroaromatic compounds, reducing the time of bioremediation, expanding the scope of biotechnology for cleaning contaminated objects, preventing environmental pollution.

The goals are achieved by the fact that bioremediation of waters contaminated with nitroaromatic compounds is carried out using a yeast strain Yarrowia lipolytica VKPM Y-3492 in three bioreactors connected in series and equipped with appropriate equipment with a continuous flow of contaminated and purified medium, ensuring optimal temperature values (in bioreactors), pH of the medium, aeration, mixing speed of biomass and medium. Monosaccharides, trihydric alcohols, aliphatic hydrocarbons are used as a source of carbon and energy for strain growth and degradation of TNT.

The method is carried out in a device consisting of a tank and three bioreactors connected in series by pipelines with shut-off and control equipment, equipped with samplers, air-venting channels, mixers for mixing biomass and medium, pumps for pumping the medium, and an aeration system.

The essence of the proposed method lies in the fact that the bioremediation of water contaminated with TNT is carried out using a yeast strain Y. lipolytica VKPM Y-3492.

The method is carried out by continuous cultivation of the strain in the medium to be cleaned in a device consisting of 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 biotechnological process is carried out in three bodies of water connected in series, for example, flow treatment plants, equipped with the necessary operational and control equipment. The strain of Y. lipolytica yeast is cultivated in the optimal temperature range from +24 to + 31 ° C and pH from 3.0 to 7.0; when deviating from the optimal parameter values, the process of bioremediation of polluted waters slows down.

The essence of the proposed method is illustrated by examples.

Example 1. The neutralization of waters containing 2,4,6-trinitrotoluene, a strain of yeast Y. lipolytica VKPM Y-3492 in the presence of monosaccharides, for example, glucose as a source of carbon and energy.

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

The neutralization is carried out in the installation for continuous cultivation of yeast (Figure 1), where 1 is 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 reservoir 4 containing a liquid purified (neutralized) 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 the cleaned medium. All bioreactors are equipped with an aeration system 22, 23, 24, which supplies sterile air (to the bioreactors), as well as air-venting channels 8, 9, 10, equipped with 0.22 μm filters. In addition, bioreactors are equipped with 5,6,7 samplers. The process of mixing the cleaned medium inoculated with Y. lipolytica yeast cells (with introduced cells) is carried out with magnetic stirrers 11,12,13. The biotechnological process is carried out in the temperature range from +24 to + 31 ° C.

As a cleaned 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 mM (pH 7.0). TNT is added at a rate of 100 mg / l before autoclaving the medium. The medium for maintaining the Y. lipolytica strain and biomass accumulation is Saburo agar medium containing glucose 10.0 g / l, peptone 10.0 g / l, yeast extract 5.0 g / l, NaCl 0.3 g / l, agar - 20.0 g / l. Distilled water is used to prepare all media.

The process of 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.

In bioreactor 1 containing a liquid synthetic medium with TNT, a daily culture of U. lipolytica is introduced through line 14 to a final A _{600 of} 0.2 and cultivated (Y. lipolytica) until TNT is completely converted into C-3 Meisenheimer monohydride complex (3-Н ^- -THT) dark red. As a result of the transformation of TNT with a yeast strain, the major metabolite - 3-H ^- -THT (up to 85%) accumulates in bioreactor 1. At the same time maintain a neutral pH value of the cleaned medium.

Simultaneously with the beginning of the operation of bioreactor 1, bioreactor 2 containing a liquid synthetic medium with TNT is introduced through line 15 by daily culture of Y. lipolytica to the final A ₆₀₀ 0.4 and cultivated (Y. lipolytica) until yellow-orange C3, C5 dihydride complexes are accumulated Meisenheimer (3.5-2H ^- -THT, isomers 3.5-2H ^- -THT · H ⁺ , 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 4.8-5.4 due to the intense production of organic acids by the yeast.

Simultaneously with the beginning of the work of bioreactors 1 and 2, a daily culture of Y. lipolytica to the final A ₆₀₀ 1,0 is introduced into the bioreactor 3, containing a liquid synthetic medium with TNT, through a pipe 16 and cultivated (Y. lipolytica) until the TNT dihydride complexes are completely destroyed. Bioremediation in bioreactor 3 is accompanied by discoloration of the medium being cleaned and the accumulation of nitrate ion, which is the final nitrogen-containing inorganic degradation product 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.

The second stage includes pumps 18, 19, 20, 21, and the biodegradation of TNT and its metabolites is carried out under conditions of continuous cultivation of Y. lipolytica yeast cells with the provision (operation of pumps and shut-off and control equipment) of a complete renewal of the bioreactor medium in the period from 18 to 24 hours.

In each of the bioreactors 1, 2, 3, the optimal concentration of Y. lipolytica cells (from A ₆₀₀ 0.2 to A ₆₀₀ 6.0) is maintained by regulating the rate of liquid synthetic medium from TNT from reservoir 4 to bioreactor 1, the medium to be cleaned from the bioreactor 1 to the bioreactor 2, the cleaned medium from the bioreactor 2 to the bioreactor 3, and the cleaned medium from the bioreactor 3, for example, to a system for collecting and removing the purified medium (not shown in FIG. 1 as not directly related to the bioremediation method). The flow rate of the medium to be cleaned through bioreactors is established while maintaining the stability of biological processes and ensuring the 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 the medium (from 10 to 20 ml / h), the intensity of mixing (100-250 rpm).

As a result of the implementation of the proposed bioremediation method according to Example 1, the biodegradation of TNT through the destruction of intermediate TNT hydride complexes reaches 85% in 1 day. The remaining 15% of TNT are transformed into GADNT, which covalently bind to biological macromolecules and subsequently do not show toxicity.

Example 1 shows that the inventive method, providing 85% decomposition of TNT at a concentration of 100 mg / L for 1 day, compared with the prototype, providing 15.3% decomposition of TNT at a concentration of 50 mg / L in 41 days, can significantly improve the quality and the effectiveness of the bioremediation process of TNT-contaminated water.

Another advantage of the proposed method for the biological destruction of TNT and its derivatives in wastewater is the possibility of its (method) implementation in the presence of a wider (compared to the prototype) spectrum of carbon and energy sources, which is proved in the following examples.

Example 2. The neutralization of water containing TNT, a yeast strain Y. lipolytica VKPM Y-3492 using a trihydric alcohol, for example, glycerol as a source of carbon and energy.

The process of biological destruction of TNT is carried out according to Example 1, using another source of carbon and energy - glycerol, for example, at a concentration of 2.4 ml / l. The implementation of the process according to Example 2 achieve the degradation of TNT within 80%.

Example 3. Purification of waters contaminated with TNT, a strain of yeast Y. lipolytica VKPM Y-3492 in the presence of an aliphatic compound of hexadecane as a source of carbon and energy.

The process of biodegradation of TNT is carried out according to Example 1 using a different source of carbon and energy - hexadecane, for example, at a concentration of 3.0 ml / L. The implementation of the process according to Example 3 achieve 30% decomposition of TNT.

In Examples 2 and 3, the efficiency of the bioremediation process by the proposed method is inferior to the efficiency of the process according to Example 1, but significantly exceeds the efficiency. prototype. Thus, the proposed method allows the use of alternative, in comparison with the prototype, sources of carbon and energy for the implementation of the bioremediation process. This allows you to expand the scope of biotechnology for the detoxification of natural and waste water.

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 biotechnological process in production conditions, which greatly simplifies the process control and shortens their duration. If necessary, the dynamics of the bioremediation process is monitored by analyzing selected samples 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 scanning two-beam 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%. The subsequent 12.5 min of chromatography was associated with an increase in methanol content to 100%, this gradient was 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. Flow rate 1.0 ml / min, temperature + 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). Elution is carried out with solutions of 1.0 mM NaHCO ₃ and 3.2 mM Ma2CO3 at a rate of 0.7 ml / min.

A significant difference between the method of continuous multistage cultivation of the yeast strain Y. lipolytica VKPM Y-3492 in the presence of TNT is a higher (85%) compared with the prototype (15.3%), the biodegradation efficiency of TNT and its metabolites, the possibility of using the proposed biotechnological treatment scheme in various industries. In addition, the proposed method using strain Y. lipolytica VKPM Y-3492 allows the purification of waters contaminated with high concentrations of TNT without the use of dilution. Compared with the known analogues and prototype, the claimed method can significantly reduce bioremediation time (up to 1 day instead of 41 days for the prototype), while increasing the productivity and efficiency of biological treatment plants and contributing to the preservation of the environment.

The possibility of implementing the bioremediation process using alternative sources of carbon and energy for the growth of the Y. lipolytica strain VKPM Y-3492 can significantly expand (compared with the prototype) the scope of biotechnology for environmental protection.

The application of the proposed method helps to improve the quality and simplify the process of biological treatment of natural and wastewater contaminated with toxic nitroaromatic compounds (exemplified by TNT destruction resistant), reduce bioremediation time, expand the scope of biotechnology for cleaning environmentally hazardous objects, and prevent environmental pollution.

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 execution 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.

Used sources

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Claims (1)

The method of bioremediation of water contaminated with 2,4,6-trinitrotoluene, which consists in cultivating a yeast strain Yarrowia lipolytica VKPM Y-3492 in three bioreactors connected in series with a continuous flow of a purified medium using monosaccharides or trihydric alcohols, or aliphatic hydrocarbons as a source of carbon and energy , ensuring optimal temperature values from + 24 ° C to + 31 ° C, medium pH from 3.0 to 7.0, mixing speed of biomass and medium 100-250 rpm.