RU2534819C2 - Vibrio aquamarinus strain, method of determining sample toxicity using same and testing culture for determining sample toxicity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology and can be used for biotesting of toxicity of environmental media. The invention discloses a Vibrio aquamarinus bacterial strain, a method of determining toxicity of samples using said strain and use of the strain as a testing culture for determining chemical toxicity of samples. The method comprises measuring luminescence of the Vibrio aquamarinus VKPM V-11245 strain in the presence of at least one chemical toxicant of a sample, said toxicant being ZnSO₄ or CuSO₄ or K₂Cr₂O₇ or oil or diesel fuel or bilge water or phenol or a heavy metal. Luminescence of said strain is measured without a chemical toxicant and the measured luminescence levels are compared. Change in luminescence indicates toxicity of the analysed sample.

EFFECT: invention improves reliability of detecting toxic substances in the environment.

9 cl, 4 tbl, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to biotechnology, namely, bacterial strains for biotesting the toxicity of environmental objects and can be used in environmental-toxic studies, while monitoring aquatic ecosystems.

State of the art

A known strain of bacteria Ph. phosphoreum (Cohn) Ford, which is recommended for biotesting water in Ukraine (I.Yu. Malygina, A.M. Katsev. Luminous bacteria of the Black and Azov Seas, KND 211.1.4.060-97) (1, 2). The known strain during testing is used in lyophilized form and requires the restoration of its active form. In addition, the sensitivity of this strain to toxic substances is not high enough, in particular, the known strain allows you to determine the presence of toxic substances in concentrations exceeding the MPC.

The known bacterial test "Ekolyum", developed in Russia (Moscow State University, Moscow) (3). The Ekolyum biosensor is a lyophilized culture of a genetically engineered strain of luminescent bacteria contained in an inert gas medium in special glass bottles. The culture was obtained through genetic engineering introduction of a lux operon into a specially selected strain of E. coli. It is made according to TU 6-09-20-236-93. (3) Used to determine the toxicity of water (4), soil (5), chemical compounds, polymers, materials and products (6).

A disadvantage of the known strain is not sufficiently high sensitivity to toxic substances, in particular to their presence in concentrations at the MPC level and below. For testing, a lyophilized culture is used, which is in a biologically inactive form and it takes time for it to go through several cell divisions to go to an active physiological state. The disadvantage is the high cost of biosensor production due to the use of the expensive lyophilization process. In addition, the biosensor preparation is supplied only subject to the initial acquisition of the Biotoke-10M environmental monitoring device.

The known strains of Vibrio fischeri VKPM B-9580 and Vibrio fischeri VKPM B-9579 isolated from the Black Sea water can be used to test environmental toxicity (7) (8).

As a prototype of the selected strain of Vibrio fischeri VKPM B-9579 (8). Biotesting is based on determining the change in the intensity of bioluminescence of the strain Vibrio fischeri VKPM B-9579 when exposed to toxic substances present in the analyzed sample, compared with the control.

The sensitivity of this strain to toxic substances is not high enough, in particular, the known strain does not reliably determine the presence of toxic substances in concentrations at the MPC level and below.

Disclosure of invention

The aim of this invention is to obtain a new, affordable and cheap strain of bacteria characterized by high sensitivity of bioluminescence to the action of toxic substances of a wide variety of chemical nature.

To achieve this goal, the proposed strain of bacteria Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054), isolated from the Black Sea, selected in the region of Abrau-Durso.

One of the embodiments of the present invention is a strain of bacteria Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) to determine the toxicity of the samples.

In another embodiment, the present invention relates to a method for determining the toxicity of samples, comprising measuring the luminescence of the bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) in the presence of at least one toxicant sample, measuring the luminescence of said strain in the absence of a toxicant, and comparing the measured luminescence levels .

In another embodiment of the present invention, the toxicity of environmental samples is determined by the above method.

In yet another embodiment of the present invention, the toxicant is ZnSO ₄ , CuSO ₄ or K ₂ Cr ₂ O ₇ . The toxicant may also be a petroleum product, polychlorinated biphenyl or a heavy metal.

Also, the present invention in one embodiment is the use of the bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) as a test culture for determining toxicity of samples.

The implementation of the invention

To isolate a strain of bioluminescent bacteria from seawater, the membrane filter method was used. Bacteria were concentrated from the analyzed water onto a membrane filter and then grown on selective media for luminous bacteria. Bioluminescent bacteria were isolated by visually analyzing the presence of bioluminescence. When luminescence was detected, a pure bacterial culture was isolated by standard methods.

The determination was carried out according to the analysis of sequencing of variable regions of 16S rDNA. According to the results of the analysis, the test strain of the microorganism belongs to the genus Vibrio (homology 98%).

The new bioluminescent strain was phenotypically and genotypically characterized. The strain was identified based on the study of its cultural, morphological, physiological, and biochemical characteristics in accordance with the description given in the Bergey bacterial identifier (9).

For the final determination of the strain type, genetic identification was performed at the Federal State Unitary Enterprise “GosNIIGenetika”, VKPM. The determination was carried out according to the analysis of sequencing of variable regions of 16S rDNA.

Phylogenetic analysis based on a comparison of 16S rRNA sequences showed that this strain belongs to a new unknown species of the genus Vibrio. Based on all these data, the strain was assigned to a new species, which was called Vibrio aquamarinus VKPM B-11245 {Vibrio aquamarinus DSM 26054).

This strain is characterized by the following cultural and morphological characteristics: cells with a diameter of about 1-1.5 μm and a length of 2-3 μm, straight, slightly curved rods, motile due to a single polar flagellum. The strain grows on many natural and synthetic environments. On TCBS (Thiosulfate Citrate Bile Salts Sucrose Agar) green colonies, 3-5 mm in diameter. On LB medium supplemented with 1.7% NaCl, it grows in the form of luminescent translucent colonies of a round shape, with a smooth edge, yellowish in color. For growth, they need the presence in the medium from 0.5 to 5% (optimum 1-4%) NaCl (w / v), but not at 8 or 10%; and temperatures of 10-35 ° C (optimum 20-25 ° C).

Physiological and biochemical signs: optional anaerobic. Gram-negative, oxide-positive, catalase-positive. The formation of indole is positive. Hydrogen sulfide does not form. Gelatin does not hydrolyze. Starch hydrolyzes. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative.

Positive for the following enzymatic activity: Ala-Phe-Pro-arylamidase, β-glucosidase, L-prolinarylamidase, amylase, nitrate reductase.

Negative for the following enzymatic activity: L-pyrrolidonarylamidase, D-cellobiose, β-galactosidase, β-N-acetylglucosaminidase, glutamylarylamidase pNA, γ-glutamyltransferase, β-xylosidase, β-alaninarylamidase pNAnase, lipase, pNA, lipase, glucosidase, β-N-acetylgalactosaminidase, α-galactosidase, phosphatase, glycinarylamidase, ornithine decarboxylase, lysine decarboxylase, β-glucuronidase, Glu-Gly-Arg arylamidase, phenylalanine deaminase, arginine dehydrogen.

It catabolizes carbohydrates such as D-glucose, D-maltose. It does not utilize D-cellobiose, arabinose, D-mannose, rhamnose, D-tagatose, D-trehalose, sucrose, raffinose, lactose. Adonitol, L-arabitol, D-mannitol, D-sorbitol, inositol, dulcite, and the following salts do not utilize alcohols: malonate, 3-keto-B-gluconate, L-lactate, succinate, L-malate, citrate (sodium) .

It is not resistant to O / 129 (2,4-diamino-6,7-diisopropylpteridine) vibriostatic agent.

The strain does not have toxic and pathogenic properties. It emits light visually detected in the dark. The luminescence spectrum of Vibrio aquamarinus is in the blue region. The maximum of the bioluminescence spectrum is 478 nm.

The strain is stored on nutrient agar with 3% NaCl, pH 7.2-7.4.

The method, conditions and composition of the media for long-term storage of the strain:

- storage of bacteria in the freezer of the refrigerator-refrigerator at a temperature of -18 ° C in a nutrient broth with 1.7% NaCl containing sterile glycerin as a cryoprotectant;

- storage of bacteria in a low-temperature refrigerator at a temperature of -60 ° C in a nutrient broth with 3.0% NaCl containing sterile glycerin as a cryoprotectant;

- freezing cells in liquid nitrogen in a nutrient broth with 1.7% NaCl containing sterile glycerin as a cryoprotectant;

- in hermetically sealed ampoules in a freeze-dried state.

Cultivation is carried out by reseeding on nutrient agar with 3.0% NaCl.

The practical application of this strain is determined by its physiological and biochemical characteristics. The strain is intended for bioassay of toxicity, as it is characterized by high sensitivity of bioluminescence to the action of a wide range of toxicants.

Example 1

The use of the bacterial strain Vibrio aquamarinus VKPM B-11245 as a test culture for determining the toxicity of environmental factors of chemical origin was carried out on toxicants ZnSO ₄ , CuSO ₄ , KiCr ₂ O ₇ . sodium dodecyl sulfate (SDS) and phenol.

The strain Vibrio aquamarinus VKPM B-11245 was grown on LB agar with 3.0% NaCl at 25 ° C overnight.

Then a suspension of an overnight culture of luminous bacteria was prepared. For this, the overnight culture was bred using a DEN-1 densitometer (BioSan) to a turbidity of 1 McFarland unit (concentration of 3 × 10 ⁸ cells / ml). Then the suspension was diluted 100 times. A bacterial suspension was prepared so that the final concentration of NaCl was 3%; glucose - 0.01% in 0.001 M Tris-HCl buffer (pH 7.4).

Aliquots of the culture of 180 μl were transferred into the wells, some of them served as a control (20 μl of distilled water was added to them). 20 μl of toxicant solutions were added to other wells.

The luminescence was measured on a LM-01T microplate luminometer (Immunotech). The measurement was carried out for 35 minutes with an interval between measurements of 5 minutes at a temperature of 20-25 ° C.

The results of measurements of the intensity of bioluminescence were presented in arbitrary units of luminescence (ues).

Assessment of the toxicity of the sample was carried out by the relative difference in the intensity of bioluminescence of the control and experimental samples. For a comparative assessment of sensitivity, we used the characteristic EC50 (effective concentration) - the concentration of the substance, causing a 50% decrease in the bioluminescence of the bacterial suspension.

The obtained data on the sensitivity of strains of luminous bacteria (average values of EC ₅₀ ) to all the studied toxicants are presented in table 1. The table also contains data on the sensitivity to the same toxicants of the lux strain Vibrio jischeri VKPM B-9579.

A comparison of the sensitivity of the bioluminescence of the strains to the studied toxic substances showed that the Vibrio aquamarinus VKPM B-11245 strain is more sensitive than the Vibrio fischeri VKPM B-9579 strain (according to the average values of EC ₅₀ ).

Table 1 EU 50 mg / l Strain ZnSO ₄ CuSO ₄ SDS K ₂ Cr ₂ O ₇ Phenol Vibrio aquamarinus VKPM B-11245 0.3 0.4 10 1-10 225-275 Vibrio fischeri 2-3 1-1.5 150 10-50 125-150 VKPM B-9579

The EC ₅₀ value for ZnSO ₄ · 7H ₂ O in the strain Vibrio aquamarinus VKPM B-11245 is 0.3 mg / L (zinc concentration is 0.068 mg / L), and the EC ₅₀ concentration for CuSO ₄ · 3H ₂ O is 0.4 mg / l (respectively, the concentration of copper is 0.102 mg / l).

The EC ₅₀ value of the strain Vibrio aquamarinus VKPM B-11245 for potassium dichromate is in the concentration range from 1 to 10 mg / L.

For phenol, the EC ₅₀ value for the Vibrio aquamarinus VKPM B-11245 strain was 225-275 mg.

Compared with the biosensor strain Vibrio fischeri VKPM B-9579 (at the lower limit of its sensitivity), the strain Vibrio aquamarinus VKPM B-11245 is more sensitive to ZnSO ₄ (7 times), CuSO ₄ (2.5 times), SDS (15 time). The strain of Vibrio aquamarinus VKPM B-11245 is more sensitive to potassium dichromate (10 times). Vibrio aquamarinus is slightly less sensitive to phenol.

The high sensitivity of Vibrio aquamarinus VKPM B-11245 indicates the promise of using this strain to determine the toxicity of aqueous media.

Example 2

Of undoubted interest is the reaction of luminous bacteria to low concentrations of pollutants, comparable with the MPC level and below, which allows using this strain as a test culture in determining low concentrations of pollutants.

The bioluminescence sensitivity of the isolated strains to the action of toxic factors of chemical origin in very low concentrations was measured. The following toxicants were used: ZnSO ₄ , CuSO ₄ , K2Cr2O7.

Toxicity testing was performed as described in example 1.

Table 2 presents data on the sensitivity of Vibrio aquamarinus VKPM B-11245 bioluminescence to the above toxicants in the concentration range from 0.00001 mg / L to 1.0 mg / L.

As can be seen from table 2, low concentrations of toxicants cause stable induction of bioluminescence in Vibrio aquamarinus VKPM B-11245.

Copper in a concentration of 0.001-0.005 mg / l (MAC of copper - 0.005 mg / l for sea water) increases the luminescence intensity in the range of 35-32%.

Zinc in a concentration of 0.001-0.005 mg / l (MAC of zinc - 0.05 mg / l for sea water) increases the intensity of the bioluminescence of the strain in the range of 19-31%.

Potassium dichromate (MPC 0.05 mg / L), at a concentration of 0.001-0.005 mg / L, increases the intensity of the bioluminescence of the strain by about 38-35%.

It should be noted that a noticeable induction of luminescence of the strain Vibrio aquamarinus VKPM B-11245 was observed at the MPC level of toxicants (10) for copper and potassium dichromate, as well as below the MPC for copper, zinc and potassium dichromate.

According to the data given in the patent (8), for Vibrio fischeri VKPM B-9579 the following indicators were recorded:

Copper in a concentration of 0.001-0.005 mg / l increased luminescence in the range of 36-42%. Zinc in a concentration of 0.001-0.01 mg / l increased the intensity of the bioluminescence of the strain in the range of 18-26%. Potassium dichromate in a concentration of 0.001-0.01 mg / l increased the intensity of the bioluminescence of the strain by about 30%.

Compared with the biosensor strain Vibrio jischeri VKPM B-9579, Vibrio aquamarinus VKPM B-11245 is more sensitive to toxicant concentrations at the MPC level and lower - the magnitude of the fluorescence induction of the strain often reaches a higher value and is recorded at lower concentrations of substances.

The data obtained indicate a high sensitivity of the strain Vibrio aquamarinus VKPM B-11245 and the prospects of its use for determining the toxicity of components of the aquatic environment.

Example 3

Experiments were conducted on the use of the bacterial strain Vibrio aquamarinus VKPM B-11245 as a test culture for the toxicity of petroleum products.

Oil, diesel fuel, and sub-base (bilge) water from the engine room of the vessel were tested at concentrations from 0.00001 mg / l to 5.0 g / l.

Toxicity testing was performed as described in example 1.

The obtained data on the sensitivity of the bioluminescence of the strain Vibrio aquamarinus VKPM B-11245 to all studied petroleum products are presented in table 3.

Oil, starting from concentrations below the MPC of oil (0.05 mg / l), suppressed the glow of Vibrio aquamarinus VKPM B-11245. At a concentration of 0.05 mg / L (MPC), a decrease in the intensity of luminescence reached 13%.

Bilge water and diesel fuel also suppressed the glow of this strain, starting from concentrations below the MAC. At the MPC level (0.05 mg / L), the suppression of luminescence was 36% and 20%, respectively.

The value of the EU ₅₀ for oil, diesel fuel and bilge water was 100 mg / l.

The value of EC ₅₀ for oil, diesel fuel and bilge water for the strain Vibrio fischeri VKPM B-9579, apparently, is more than 1000 mg / l.

A comparison of the sensitivity of the strain Vibrio aquamarinus VKPM B-11245 and Vibrio jlscheri VKPM B-9579 (8) indicates a higher sensitivity of the strain Vibrio aquamarinus VKPM B-11245 and the prospects of its use for determining the toxicity of the components of the environment of marine water bodies.

table 2 Toxicant Name The control Toxicant concentration, mg / l 0.00001 0.0001 0.001 0.005 0.01 0.05 0.1 one The intensity of bioluminescence Vibrio aquamarinus VKPM B-11245, ues Copper 349 359 394 473 461 356.5 221.5 188.5 38.5 Zinc 349 351 361 417 456 398 309 294.5 97 K ₂ Cr ₇ O ₇ 349 365.5 451 481 473 460 397 346.5 181.5 The intensity of bioluminescence Vibrio fischeri VKPM B-9579, ues Copper 1,62 2.15 2.22 2.3 * 2.2 2.08 1.83 1,69 0.803 Zinc 0.444 0.495 0.519 0.526 0.540 0.559 0,500 0.520 0.385 K ₂ Cr ₂ O ₇ 0.430 0.512 0.545 0.562 0.575 0.557 0.553 0.544 0.552

Table 3 Name of oil product The concentration of oil, mg / l The control 0.01 0.05 0.1 0.5 one 5 10 fifty one hundred 1000 5000 The intensity of the bioluminescence of the strain Vibrio aquamarinus, yec oil 661 617.5 577 546.5 542 551.5 503 486.5 498.5 361.5 284.5 138 diesel fuel 661 438 421 416 407 400 399, 396 382.5 350.5 336.5 197.5 bilge water 661 538.5 531 531 498 451.5 426.5 446.5 432.5 343.5 309 214 The intensity of the bioluminescence of the strain Vibrio fischeri VKPM B-9579, yec oil 0.30 0.49 0.62 0.64 0.56 0.56 0.36 diesel fuel 0.34 0.52 0.52 0.63 0.55 0.54 0.36 bilge water 0.31 0.45 0.46 0.48 0.53 0.54 0.30

Example 4

To determine the toxicity of natural samples using the bacterial strain Vibrio aquamarinus VKPM B-11245, samples of bottom sediments of marine water bodies were taken.

A sample with a high content of heavy metals and polychlorinated biphenyls was taken in an area with a high anthropogenic load in the Black Sea at the port of Tuapse.

Another soil for biotesting was selected in a relatively clean area of the Black Sea near the village of Arkhipo-Osipovka.

Toxicity testing was performed as described in example 1.

Table 4 presents the results of the toxicity bioassay of averaged soil samples taken in a relatively clean area of the sea and in the port. As a control, a 3.0% NaCl solution was used.

Table 4 Sediment sampling area The intensity of bioluminescence Vibrio aquamarinus VKPM B-11245, ues the control experience deviation from control,% Water area near the village of Arkhipo-Osipovka 1023 812 20.6 Port of Tuapse 1798 851 52.7

From table 3 it follows that the intensity of bioluminescence of the aqueous extract of bottom sediments taken in a relatively clean region of the Black Sea exceeded the control level by 20.6%, and the intensity of the luminescence of the sample with soil extract from the water area of the seaport - by 52.7%. The data obtained indicate the sensitivity of the bioluminescence of the strain Vibrio aquamarinus VKPM B-11245 to the action of toxicants present in the bottom sediments of marine water bodies.

Thus, testing the toxicity of one of the main components of the medium - bottom sediments, demonstrated the possibility of using the proposed strain of Vibrio aquamarinus VKPM B-11245 as a test culture.

Information sources

1. I.Yu. Malygina, A.M. Katsev. Luminous bacteria of the Black and Azov Seas. Ecology of the sea. 2003. Issue 64.

2. KND 211.1.4.060-97. Identified toxicity of bacteria in Photobacterium phosphoreum (Cohn) Ford bacteria. - 05/21/97.

3. TU 6-09-20-236-93.

4. MP No. 11-1 / 133-09. Methodology for the rapid determination of water toxicity using the luminescent bacterial test "Ekolyum".

5. MP No. 11-1 / 134-09. Determination of total soil toxicity by the intensity of bacterial bioluminescence.

6. MP No. 11-1 / 131-09. Determination of toxicity of chemical compounds, polymers, materials and products using a luminescent bacterial test.

7. Patent RU 2346035, IPC C12N 1/20, C12R 1/01.

8. Patent RU 2342434, IPC C12Q 1/02, C12R 1/63 (prototype).

9. The determinant of bacteria Bergey. T.1. / Ed. J.Houlta, N.Krieg, P. Snit, S. Williams. - M .: Mir, 1997 .-- 432 p .; A brief guide to Bergi bacteria. Ed. J.Hoult. - M .: Mir, 1980 .-- 496 p.

10. Water quality standards for water bodies of fishery importance, including standards for maximum permissible concentrations of harmful substances in the waters of water bodies of fishery value. "- Approved by the Order of the Federal Agency for Fishery on January 18, 2010. No. 20. - 214 p.

Claims (9)

1. The bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) to determine the chemical toxicity of the samples. 2. A method for determining the chemical toxicity of samples, including measuring the luminescence of the strain according to claim 1 in the presence of at least one chemical toxicant of the sample, measuring the luminescence of the strain according to claim 1 in the absence of the specified toxicant and comparing the measured levels of luminescence, while the measurement of luminescence indicates toxicity samples. 3. The method according to claim 2, in which the toxicity of environmental samples is determined. 4. The method according to claim 2 or 3, in which the toxicant is ZnSO ₄ , CuSO ₄ or K ₂ Cr ₂ O ₇ . 5. The method according to claim 2 or 3, in which the toxicant is an oil product. 6. The method according to claim 5, in which the oil product is oil, diesel fuel or bilge water. 7. The method according to claim 2 or 3, in which the toxicant is phenol. 8. The method according to claim 2 or claim 3, in which the toxicant is a heavy metal. 9. The use of the strain according to claim 1 as a test culture for determining the chemical toxicity of samples.