RU2401298C1 - BACTERIA STRAIN Achromobacter sp - ORGANOPHOSPHONATE DESTRUCTOR AND METHOD OF APPLICATION THEREOF FOR SOILS BIOREMEDIATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a new bacterial strain Achromobacter sp., VKM B-2534 D, which can be used for soil and fluid cleanup, e.g. organophosphonate-contaminated soil and surface water. Bacteria strain Achromobacter sp. Kg 16 has been recovered by the enrichment culture method from glyphosate-contaminated soil, it is deposited in the Russian National Collection of microorganisms, No. VKM B-2534 D. Said strain utilises organophosphonates: glyphosate, metylphosphonate, aminometylphosphonate, phosphonoacetate, 2-aminoethylphosphonate, N-(phosphonomethyl)iminodiacetate. Strain stability to high glyphosate concentration is shown among other factors that expand the application range, including in emergencies.

EFFECT: invention allows improving efficiency of soils and fluid cleanup, eg organophosphonate-contaminated soil and surface water.

2 cl, 6 tbl, 5 ex

Description

The invention relates to microbiology and biotechnology and is a new bacterial strain that can be used to clean soil and liquid media, such as ground and surface waters, contaminated with organophosphonates.

Organophosphonates are part of a large number of xenobiotics, including herbicides. Although the use of chemical plant protection products is strictly regulated, pollution of the agroecosystem with these substances most often occurs due to high consumption rates and poorly tuned equipment. Organophosphonates are characterized by the presence of a chemically stable carbon-phosphorus bond, resistant to photolysis, chemical hydrolysis, and thermal destruction. However, the carbon-phosphorus bond can be broken due to the enzymatic action of microorganisms with the release of inorganic phosphorus, which is used by cells as a biogenic element.

For remediation of contaminated areas, physical, chemical and biological methods are used. Biological technologies, including the use of strains of microorganisms, are most preferable due to their environmental safety, low cost of work and high enough efficiency, which has been repeatedly demonstrated in solving various environmental problems.

The organophosphonate, glyphosate [N- (phosphonomethyl) lycine], which is difficult to degrade and toxic to nature and humans, is an active ingredient in a number of herbicides, such as Gound Bio and Roundup, and is widely used throughout the world.

Glyphosate undergoes rapid detoxification in the soil as a result of soil binding and microbial degradation [1, 2]. However, glyphosate sorption by the soil matrix reduces the degree of mineralization and makes the herbicide more persistent. It can accumulate in the arable layer of the soil, from where, through the root system of plants, it enters leaves, berries, fruits, and, migrating along food chains, into the body of warm-blooded animals and humans.

An analysis of the papers published to date indicates the presence of organophosphonate destructive microorganisms, including glyphosate, in nature.

Bacterial strains Pseudomonas sp. LBr [3] and Flavobacterium sp. [4], isolated from glyphosate-contaminated sources, are capable of utilizing the herbicide as the sole source of phosphorus under batch cultivation conditions. The bacterial strain Pseudomonas sp. LBr is resistant to glyphosate concentrations in the medium of up to 20 mM (about 3 g / L) glyphosate, but its main part (up to 90%) is not utilized, but undergoes transformation to aminomethylphosphonate, which contains a stable carbon-phosphorus bond in its structure. Bacterial strain Flavobacterium sp. also transforms glyphosate (150 mg / L) to aminomethylphosphonate, with about 60% of this compound remaining in the culture fluid.

Thus, the disadvantage of these strains is the incomplete degradation of glyphosate and the accumulation in the environment of the product of its degradation.

Known bacterial strain Arthrobacter sp. GLP-1, isolated in an accumulative culture with glyphosate, has a broad substrate specificity for organophosphonates. It uses glyphosate as the sole source of phosphorus with a C-P bond cleavage and remains viable at glyphosate concentrations in the medium of up to 850 mg / L. The glyphosate biodegradation efficiency calculated on the basis of the data presented in the article is 13 mg / g biomass [5].

Known bacterial strain Ochrobactrum sp. GPK 3, isolated from glyphosate contaminated soils, has a high destructive potential under conditions of periodic cultivation. It decomposes up to 220 mg / l of glyphosate when the glyphosate content in the medium is 500 mg / l with an efficiency of 78 mg / g of biomass [6].

No data on the destructive activity of the strain under the conditions of soil experiments.

A known method of purification of liquid effluents containing glyphosate using an immobilized mixed culture of microorganisms ATCC 55050, which includes the bacterial strain Moraxella anatipestifer ATCC 55051. The mixed culture has a high ability to biodegradation of glyphosate in aqueous systems. When contaminated water containing 1400 mg / l of glyphosate is passed through a column with immobilized biomass of microorganisms making up the ATCC 55050 association, the destruction efficiency is 85% in 5 days [7].

The literature sources that we studied allow us to conclude that in studies conducted with organophosphonate destructive strains, the main attention was paid to studying the properties of these microorganisms, their substrate specificity, and the mechanisms of destruction of such compounds.

The process of glyphosate destruction in soil was studied only under the influence of native microflora [1, 2, 8]. The maximum degree of degradation of the toxicant was no more than 11% [8] with a dose of herbicide of 100 mg / kg of soil.

We did not find any documents in the literature that would describe the methods of bioremediation of soils contaminated with organophosphonates using introduced microorganisms-destructors.

No data were also found on the study of integral toxicity and harmlessness for warm-blooded animals of isolated organophosphonate destructive microorganisms.

The closest to the proposed strain in terms of glyphosate biodegradation efficiency and broad substrate specificity is the bacterial strain Pseudomonas sp. PG2982. This strain utilizes 1 mM (up to 170 mg / l) glyphosate under conditions of periodic cultivation [9]. However, there is no information on its use for bioremediation of soils contaminated with glyphosate.

The objective of the invention is to obtain an active strain-destructor of a wide range of organophosphonates, which has a high utilization ability in environmental conditions, is resistant to high glyphosate concentrations, does not have pathogenicity and can be used to clean the soil and liquid media from contamination with glyphosate and other organophosphonates.

The proposed bacterial strain Achromobacter sp.Kg 16 was isolated by the accumulative culture method from the soil of the Krasnodar Territory, which has been treated with Roundup herbicide for a long time, identified in accordance with the analysis of the primary nucleotide sequence of 16 S ribosomal RNA genes and deposited in the All-Russian Collection of Microorganisms (CM) under registration number VKM B-2534 D.

In a pure culture, this strain was isolated from an accumulation culture on MS1 agar medium (g / l): NH ₄ Cl - 2.0; MgSO ₄ · 7H ₂ O - 0.2; K ₂ SO ₄ - 0.5; agar-agar - 18.0; trace elements (mg / l): FeSO ₄ · 7H ₂ O - 2.5; CaCl ₂ · 6H ₂ O - 10.0; CuSO ₄ · 5H ₂ O - 2.0; H ₃ VO ₃ - 0.06; ZnSO ₄ · 7H ₂ O - 20.0; MnSO ₄ · H ₂ O - 1.0; NiCl ₂ · 6H ₂ O - 0.05; Na ₂ MoO ₄ · 2H ₂ O - 0.3, pH 7.0-7.2.

The carbon source is monosodium glutamate at a concentration of 10 g / l. As a source of phosphorus, 0.5 g / l of glyphosate is used as part of the Grass Bio herbicide (manufacturer - Tekhnoexport, Russia), which contains 360 g / l of glyphosate in the form of isopropylamine salt.

The proposed bacterial strain Achromobacter sp. VKM V-2534 D is characterized by the following features.

Cultural and morphological characters.

When growing in Luria-Bertani (LB) liquid media, meat-peptone broth and fish meal enzymatic hydrolyzate (FGRM) after 24-48 hours, rod-shaped cells are short, mobile with polar flagging, cell length 1.4-2.5 microns, width 0.6 microns ; do not form a dispute, gram-negative. The colonies on agarized media are round, up to 3 mm in diameter, convex with a cone in the center, cream-colored, shiny, oily, translucent on the periphery, the edge is even.

Physiological and biochemical characteristics.

Bacterial strain Achromobacter sp. BKM B-2534 D is a strict aerobic grows at temperatures from +4 to + 34 ° C in the range of pH 6.0-8.0. Temperature optimum 28 ± 1 ° С, optimum pH 6.5-7.5. Glutamate, pyruvate, succinate, fumarate, citrate, and malate are consumed as a carbon source. Does not use sucrose, glucose, maltose, arabinose, xylose, lactose, trehalose. Prototroph, does not need additional growth factors. Has a negative anaerobic and aerobic Hugh-Leifson test.

It has oxidase and catalase activity, it does not form pigment, it does not thin out gelatin, and starch does not hydrolyze. The culture is resistant to antibiotics: streptomycin, erythromycin, lincomycin, nalidixic acid.

In the presence of organic acids, it breaks down the carbon-phosphorus bond of glyphosate as well as other organophosphonates, such as methylphosphonate, 2-aminoethylphosphonate, aminomethylphosphonate, phosphonomycin, phosphonoacetate, N- (phosphonomethyl) iminodiacetate, using the released inorganic phosphorus as a carbon source.

The strain is not pathogenic, does not have virulence, toxicity, toxigenicity and dissemination in the internal organs of experimental animals.

The strain is stored in test tubes on the jambs of solid medium MS1 (agar-agar 18 g / l) under a layer of sterile liquid paraffin at a temperature of + (2-4) ° C and in a freeze-dried state.

Table 1 shows data that show that the proposed bacterial strain has a high destructive potential compared to previously described microorganisms.

The advantages of the strain include the ability to grow in a medium with a glyphosate concentration of 10 g / l and destruction efficiency of 345 mg / g of biomass. In addition, the strain actively decomposes glyphosate in the soil, where its content exceeds 10 times the recommended norms for the use of the herbicide.

The advantages of the strain also include the ability to decompose other organophosphonates, such as methylphosphonate, 2-aminoethylphosphonate, aminomethylphosphonate, phosphonomycin, phosphonoacetate, N- (phosphonomethyl) iminodiacetate.

The proposed bacterial strain does not have pathogenicity, as a result of which it can be safely used for biotechnological purposes, in particular for cleaning soils contaminated with organophosphonates.

A bioremediation method, comprising introducing a strain of bacteria Achromobacter sp. VKM V-2534 D in the soil contaminated with glyphosate provides high cleaning efficiency. After 28 days, the degree of glyphosate destruction in the soil with the introduced bacterial strain was 2.5 times higher than with only natural microflora. In this case, there is a decrease in integrated toxicity and phytotoxicity to indicators of unpolluted soil.

The invention is illustrated by the following examples, but is not limited to.

Example 1. The destruction of glyphosate, depending on the duration of fasting on phosphorus seed.

Cultivation of the bacterial strain Achromobacter sp. VKM B-2534 D is carried out in 750 ml Erlenmeyer flasks with 100 ml of medium on a shaker (180 rpm) at 28 ± 1 ° С.

Seed is grown for 3 days on MS1 agar medium with glyphosate.

Cells are washed into a sterile flask with MS1 liquid medium without a phosphorus source and incubated on a rocking chair 180 rpm at 28 ± 1 ° C for phosphorus starvation.

After 0, 36, 48 and 72 hours, part of the suspension was taken and used for inoculation of experimental flasks (750 ml volume) with 100 ml of MS1 medium with 0.5 g / l glyphosate as part of the Grass Biot herbicide and 10 g / l sodium glutamate with the initial optical with a density of 0.1-0.2 units. Cultivation is carried out on a rocking chair from 180 rpm, the pH of the medium is maintained at 6.5-7.5 by introducing a sterile 20% solution of H ₂ SO ₄ . Samples of the culture fluid were taken daily, the cells were separated by centrifugation, and the glyphosate content was determined in the supernatant by high performance liquid chromatography. Cultivation is stopped after 120 hours upon the transition of the culture to the stationary phase of growth.

The results presented in table 2 indicate that the strain has a maximum destructive activity when the inoculum is starved for phosphorus for 36 hours. The efficiency of glyphosate destruction is 56 mg / g biomass, that is, it increases by 1.4 times compared with non-starving cells.

Example 2. The destruction of glyphosate in a liquid medium with various concentrations.

Bacterial strain Achromobacter sp. VKM B-2534 D is grown in flasks in a liquid mineral medium MS1, as described in Example 1. Glyphosate at a concentration of 0.05 is used as a source of phosphorus; 0.25; 0.50; 1.0; 5.0 and 10.0 g / l. The inoculum is prepared as described in Example 1 and used after 36 hours of fasting.

The resulting culture fluid is analyzed to determine its glyphosate content. The data presented in table 3 show that the ability to decompose is preserved during the growth of the strain in an environment with a high concentration of glyphosate (up to 10 g / l), while achieving a maximum degree of destruction (345 mg / g biomass).

Example 3. The destruction of various organophosphonates.

Bacterial strain Achromobacter sp. BKM B-2534 D is grown as described in example 1.

A number of organophosphonates are used as a source of phosphorus: methylphosphonate, 2-aminoethylphosphonate, aminomethylphosphonate, phosphonomycin, phosphonoacetate, N- (phosphonomethyl) iminodiacetate, in amounts corresponding to phosphorus concentrations of 90-100 mg / l.

The inoculum is prepared as described in Example 1 and used after 36 hours of phosphorus starvation. After the cessation of bacterial growth in the culture fluid, the residual amount of organophosphonates is determined spectrophotometrically from the difference between total and inorganic phosphorus and the destruction efficiency is calculated.

The data in table 4 show that the proposed bacterial strain is able to degrade a wide range of organophosphonates under conditions of periodic cultivation.

Example 4. Biodegradation of glyphosate in the soil in laboratory experiments.

Glyphosate biodegradation in the soil is carried out using uncultured sod-podzolic soil. The sifted air-dry soil is placed in 500 g plastic cups and moistened to 60% of the full moisture capacity. 25 ml of a 0.03% solution of the Grad Biot preparation containing glyphosate is applied to the surface soil in a glass at the rate of 100 l / ha (80 ± 10 mg glyphosate / kg of absolutely dry soil). The soil is maintained for 3 days to undergo glyphosate sorption processes on soil particles.

To obtain a microbial suspension, the biomass of the bacterial strain Achromobacter sp. BKM B-2534 D was grown as described in Example 2 using 0.5 g / L glyphosate. The grown biomass is separated by centrifugation, washed twice with MS1 mineral medium without glyphosate and the cells are suspended in the same medium to a concentration of (1 ± 0.5) × 10 ⁸ cells / ml.

30 ml of cell suspension at a rate of 1 l / m ^{2 of} the soil surface are added to the test glasses with soil. Moreover, the cell titer is 10 ⁷ cells / g of absolutely dry soil.

In the control variant, soil contaminated with the Ground Bio preparation containing glyphosate is used without introducing microorganisms. The duration of the experiment is 28 days.

Glyphosate is recovered from the soil by extraction with 1 N NaOH. High-performance liquid chromatography is used to identify and quantify glyphosate.

Integral soil toxicity is investigated by biotesting on daphnia [10]. Samples are considered toxic in which the death of daphnia exceeds 50% in comparison with the control. Phytotoxicity of the soil is characterized by morphometric indicators of oat plants: the length of the root of plants [11].

The results shown in table 5 show that the glyphosate content in the experimental soil sample treated with the claimed strain is reduced by 57%, and in the control version by 24%. Integral toxicity and phytotoxicity in experimental samples are 5 and 24%, respectively, compared with 23 and 40% in control samples.

The results of microbiological studies show that the titer of introduced microorganisms during the observation period is reduced by an order of magnitude.

Example 5. Bioremediation of soil contaminated with glyphosate in a field experiment.

For bioremediation, two sections 1 × 2 meters each are used. 20 ml of Ground Bio per 2 m ² are applied to the soil at the rate of 100 l / ha (60 ± 10 mg glyphosate / kg of soil).

Microbial biomass of the bacterial strain Achromobacter sp. VKM B-2534 D is grown in the ANKUM-2 fermenter. Fermentation conditions: MS1 liquid mineral medium, glyphosate in the composition of the Grass Biot herbicide - 250 mg / l, sodium glutamate - 10 g / l, pH 7-7.2, temperature + 28 ± 1 ° С, aeration 50-70% of saturation. The inoculum is prepared as described in Example 1 and used after 36 hours of fasting.

A suspension of cells for introduction is obtained as described in Example 4 and introduced into the soil at the rate of 1 liter per 1 m ² , the cell titer is (1 ± 0.5) × 10 ⁷ cells / g of absolutely dry soil.

In the control variant, soil contaminated with glyphosate as part of the Grass Bio herbicide is used without introducing microorganisms.

Control over the bioremediation process is carried out by changing the amount of residual glyphosate in the soil, integrated toxicity and phytotoxicity, as described in example 4. The duration of the experiment is 28 days.

The results shown in table 6 show that in the process of bioremediation of the soil in a field experiment using the proposed bacterial strain Achromobacter sp. VKM B-2534 D degradation undergoes 75% of glyphosate in the soil compared to 40% in the control.

After bioremediation, water extracts from the soil do not have integrated toxicity to Daphnia magna daphnia and phytotoxicity according to the gels of the oat test culture.

It was shown that the bacterial strain Achromobacter sp. VKM B-2534 D reduces the toxicity of contaminated soil to indicators of unpolluted soil. The results of microbiological studies show that the titer of introduced microorganisms during the observation period is reduced by two orders of magnitude.

Thus, it was shown that the proposed bacterial strain Achromobacter sp. BKM B-2534 D is capable of degrading a number of organophosphonates, in particular glyphosate, methylphosphonate, aminomethylphosphonate, phosphonoacetate, 2-aminoethylphosphonate, N- (phosphonomethyl) iminodiacetate. It utilizes glyphosate in high concentrations (up to 10 g / l) in a liquid mineral medium, which is 3 times higher than the concentration described for the known strain of Pseudomonas sp. Lbr.

The bacterial strain Achromobacter sp.BKM B-2534 D has a high destruction efficiency in environmental conditions, is not pathogen and can be used to clean the soil and liquid media from contamination with glyphosate and other organophosphonates.

The proposed method using a strain of bacteria Achromobacter sp. BKM B-2534 D provides the possibility of bioremediation of soils contaminated with organophosphonates.

Information sources

1. Rueppel M., Brightwell B., Schaefer J., Marcel J. "Metabolism and degradation of glyphosate in soil and water." - J. Agric. Food Chem., 1977, vol. 25, p. 517-528.

2. Strange-Hansen R., Holm P. E, Jacobsen O.S., Jacobsen C.S. "Sorption, mineralization and mobility of N- (phosphonomethyl) glycine (glyphosate) in five different types of gravel." - Pest Manag Sci., 2004, vol.60, No.6, p. 570-578.

3. Jacob G.S., Garbow J.R., Hallas L.E., Kimak N.M., Kishore G.M., Schaefer J. "Metabolism of Glyphosate in Pseudomonas sp. Strain LBr." - Appl. Environ. Microbiol, 1988, vol. 54, No.12, p. 2953-2958.

4. Balthazor T.M. and Hallas L.E. "Glyphosate-Degrading Microorganisms from Industrial Activated Sludge." - Appl. Environ. Microbiol., 1986. vol. 51 No.2, p. 432-434.

5. Pipke R., Amrhein N., Jacob G.S., Schaefer J., Kishore G.M. "Metabolism of glyphosate by an Arthrobacter sp.GLP-1." - Eur. J. Biochem., 1987. vol. 165, p. 267-273.

6. Ermakova I.T., Shushkova T.V., Leontievsky A.A. "Microbial destruction of organophosphonates by soil bacteria." - Microbiology, 2008, T.77, No. 5, S.689-695.

7. EP 0465452 "Degradation of N-phosphonomethylglycine (glyphosate) in an aqueous system and the microorganisms used", 1993-08-26.

8. Getenga M., Kengara F.O. "Mineralization of glyphosate in compost-amended soil under controlled condition." - Bull. Environ. Contam. Toxycol., 2004, vol. 72, p. 266-275.

9. Moore J.K., Braymer H. D., Larson A. D. "Isolation of a Pseudomonas sp. Which utilizes the phosphate herbicide glyphosate." - Appl. Environ. Microbiol., 1983, vol. 46, p. 316-320.

10. Guidelines for biotesting of soil and water, approved. Mosoblkompriroda, 1994.

11. O.A. Berestetskiy. Methods for determining soil toxicity. Microbiological and biochemical studies of soils, Kiev: Harvest, 1971, p. 28-31.

Table 1 Comparison of the destructive activity of strains of destructors with respect to glyphosate. Options Ochrobactrum sp. GPK 3 [6] Pseudomonas sp.PG2982 [9] Achromobacter sp. VKM B-2534 D (the claimed strain) The concentration of glyphosate in the medium, g / l 0.5 0.15 0.5 10.0 Decrease, mg / l 220 150 75 500 The destruction efficiency, mg glyphosate / g biomass 78 55 56 345

table 2 Change in the efficiency of glyphosate destruction depending on the duration of inoculum starvation on phosphorus in the bacterial strain Achromobacter sp. VKM B-2534 D (glyphosate concentration 0.5 g / l) Defined parameter Inoculum starvation time, h 0 36 48 72 Glyphosate degradation efficiency, mg / g biomass 40 56 37 39

Table 3 The efficiency of glyphosate destruction by the bacterial strain Achromobacter sp. VKM V-2534 D in a medium with different concentrations Options Glyphosate concentration, g / l 0.05 0.25 0.5 1,0 5,0 10.0 Glyphosate degradation efficiency, mg / g biomass 37 41 56 107 320 345

Table 4 Biodegradation of various organophosphonates by the bacterial strain Achromobacter sp.BKM B-2534 D under conditions of periodic cultivation 2-amino-ethylphosphonate Aminomethylphosphonate N- (Phosphonomethyl) iminodiacetate Phosphonoacetate Fosfoni
Qing Methyl Phosphonate The concentration of organophosphonate, mg / l 400 350 700 450 500 300 Biomass, units of OP ₅₆₀ 2.9 2,8 2,8 2.2 2.1 4.2 Decrease, mg organophosphonate / l 71 49 36 22 35 59 Efficiency, mg organophosphonate / g biomass 49 35 26 twenty 33 28

Table 5 The effectiveness of the use of the bacterial strain Achromobacter sp. BKM B-2534 D for cleaning the soil of glyphosate in a laboratory experiment Experience Options Glyphosate destruction Integral toxicity,% daphnia death Phytotoxicity after 28 days,% mg / kg % ref. 28 days Soil + glyphosate (control) eighteen 24 37 23 40 Soil + glyphosate + strain 48 57 37 5 24

Table 6 The effectiveness of the use of the strain of bacteria Achromobacter sp. VKM V-2534 D for soil bioremediation Experience Options Glyphosate destruction Integral toxicity,% daphnia death Phytotoxicity after 28 days,% mg / kg % ref. 28 days Soil + glyphosate (control) 24 40 47 23 7 Soil + glyphosate + strain 45 75 47 0 0

Claims (2)

1. The bacterial strain Achromobacter sp.BKM B-2534 D is a destructor of organophosphonates. 2. The method of bioremediation of soils contaminated with organophosphonates, including the introduction of bacteria-destructors in the soil, characterized in that the bacterial strain Achromobacter sp.BKM B-2534 D. is introduced into the soil.