RU2346035C1 - Vibrio fischeri BACTERIA STRAIN, USED AS TEST-CULTURE FOR DETERMINING TOXIC LEVEL OF ENVIRONMENTAL MEDIA - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: proposed invention pertains to biotechnology and can be used for carrying out ecological toxic tests when monitoring water ecosystems. The Vibrio fischeri "ВКПМ" B-9580 strain, with high sensitivity to effect of toxic substances with very diverse chemical nature, can be used in testing toxic levels of sea-floor sediments.

EFFECT: increased sensitivity of the test-culture to effect of toxic substances of very diverse chemical nature.

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Description

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.

A known strain of bacteria Ph. phosphoreum (Cohn) Ford, which is recommended for biotesting water in Ukraine (1) (2). The disadvantage of the strain is that during testing it is used in a lyophilized form and restoration of its active form is required. 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.

Known, selected as a prototype, the 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 produced according to TU 6-09-20-236-93. (3) It is used to determine the toxicity of water, soils (4), soils (5), chemical compounds, polymers, materials and products (6).

Biotesting is based on determining the change in the intensity of the bioluminescence of the Ekolyum biosensor when exposed to toxic substances present in the analyzed sample, compared with the control.

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 switch 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 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 fischeri VKPM B-9580, isolated from the Black Sea, selected in the area of metro B. Utrish (certificate of deposit attached)

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 identification of the strain was carried out on the basis of a study of its cultural, morphological and physiological-biochemical characteristics in accordance with the description given in the definition of the Bergey bacteria (Determinant of the Bergey bacteria. T.1 / Edited by J. Holet, N. Krig, P. Snit, S. Williams. - M.: Mir, 1997. - 432 p .; Brief identifier of Bergi bacteria. Edited by J. Hoult. - M .: Mir, 1980. - 496 p.). 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. According to the results of the analysis, the test strain of the microorganism belongs to the species Vibrio fischeri (99% homology).

This strain is characterized by the following cultural and morphological characteristics: the culture is a mobile, gram-negative rod-shaped cells. Cell size in the range of (0.5-0.8) × (1.4-2.6) microns.

The strain grows on many natural and synthetic environments. On nutrient agar, after 1 day of growth at 20 ° C, the culture forms small translucent colonies of a round shape.

On a nutrient broth at 20 ° C after 1 day, a turbidity of the medium is observed, the formation of a thin wall ring, a weak film on the surface of the medium are noticeable. In semi-liquid agar, a homogeneous culture growth is observed.

Physiological and biochemical characteristics: Optional anaerobic. The oxidase test is positive. Indole does not form. Catabolizes glucose to form acid. Fermentation of maltose, D-mannose. Urease-negative. Arginine dehydrolase is absent. Ornithine decarboxylase is absent. Acetylmethylcarbinol does not form. Hydrogen sulfide does not form. Citrate does not dispose. The optimum growth is 20-25 ° C, pH 7.2-7.4. The optimal concentration of Nad is 1.7-3%. The strain does not have toxic and pathogenic properties. It emits light visually detected in the dark.

The strain is stored on nutrient agar with 1.7% 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% Nad 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 1.7% 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 1.7% 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 Vibrio fischeri VKPM B-9580 bacterial strain as a test culture for determining the toxicity of environmental factors of chemical origin was carried out using toxicants ZnSO ₄ , CuSO ₄ , K ₂ Cr ₂ O ₇ , sodium dodecyl sulfate (SDS) and phenol.

The strain was grown on nutrient agar with 1.7% NaCl at a temperature of 20 ° C for 18 hours. Then a suspension of a night culture of luminous bacteria was prepared, the optical density of which was 0.1 units. at a wavelength of 590 nm. Next, the resulting suspension was diluted a hundred times. A bacterial suspension was prepared so that the final concentration of NaCl was 1.7%; glucose - 0.01% in 0.001 M Tris - HCl buffer (pH 7.4).

Aliquots of this culture (900 μl) were transferred to sterile tubes and the studied substances were added in a volume of 100 μl. The contents of the tubes were thoroughly mixed. The tubes were left at room temperature (20 ° C) for 30 minutes. During the incubation, the tubes were shaken several times. The bioluminescence intensity of cultures containing the analyzed compounds, as well as control cultures, was measured using an LT-01 luminometer. 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. A 1.7% NaCl solution was used as a control.

For a comparative assessment of sensitivity, we used the characteristic EU ₅₀ (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 the strain of luminous bacteria to all the studied toxicants are presented in table 1. The table also provides data on the sensitivity to the same toxicants of the lux strain of E. coli.

Table 1 Strain EU 50 mg / l ZnSO ₄ CuSO ₄ SDS K ₂ Cr ₂ O ₇ Phenol Vibrio fischeri VKPM B-9580 4-5 2 200 10-50 125-150 E.coli 2-3 5-7.5 300 150 > 300

Comparison of the sensitivity of the strains to the studied toxic substances showed that the Vibrio fischeri VKPM B-9580 strain is more sensitive than the E. coli lux strain (according to average EC ₅₀ values).

The EC ₅₀ for ZnSO ₄ · 7H ₂ O for the Vibrio fischeri strain VKPM B-9580 lies in the range from 4 to 5 mg / L (zinc concentration is from 0.904 to 1.13 mg / L), and the minimum EC ₅₀ for CuSO ₄ · 5H ₂ O is 2 mg / L (respectively, the copper concentration is 0.512 mg / L).

For potassium dichromate, the EC ₅₀ value of the Vibrio fischeri VKPM B-9580 strain is in the concentration range from 10 to 50 mg / L.

For phenol, the EC ₅₀ value of the Vibrio fischeri VKPM B-9580 strain was 125-150 mg.

Comparison of the sensitivity of the bioluminescence of the strain Vibrio fischeri VKPM B-9580 to toxic substances with the sensitivity of the lux strain E.coli showed that the strain Vibrio fischeri VKPM B-9580 is more sensitive to CuSO ₄ (about 3 times), chipboard (1.5 times ), K ₂ Cr ₂ O ₇ (approximately 5 times), as well as phenol (approximately 2 times).

This indicates the promise of using the strain Vibrio fischeri VKPM B-9580 to determine the toxicity of aqueous media.

Example 2

Of great interest is the reaction of luminous bacteria to low concentrations of pollutants, comparable with the MPC level and below, which allows the use of this strain as a test culture in determining very 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 , K ₂ Cr ₂ O ₇ , particleboard and phenol.

Toxicity testing was performed as described in example 1.

Table 2 presents data on the sensitivity of the isolated strains of luminous bacteria to all the studied toxicants in the concentration range from 0.00001 mg / L to 1.0 mg / L.

As can be seen from table 2, copper in a concentration of 0.001-0.005 mg / l (MAC of copper - 0.005 mg / l for sea water) increases the luminescence of the strain within 12%. At copper concentrations starting from 1 mg and above, a noticeable quenching of bioluminescence is observed.

Zinc at a concentration of 0.001-0.01 mg / l (maximum concentration of zinc - 0.05 mg / l for sea water) increases the intensity of bioluminescence of the strain Vibrio fischeri VKPM B-9580 within 20-24%.

The intensity of the bioluminescence of the strain Vibrio fischeri VKPM B-9580 under the influence of potassium dichromate (MPC 0.05 mg / l), in the concentration range of 0.00001-1.0 mg / l, changed slightly - to the maximum within 20%.

Sodium dodecyl sulfate (MPC 0.5 mg / L), at a concentration of 0.00001-1.0 mg / L, increased the luminescence intensity of the Vibrio fischeri VKPM B-9580 strain by an average of 25% (the maximum was noted at a concentration of 0.01 mg / L - by 40%).

As follows from table 2, phenol in concentrations of 0.00001-1.0 mg / l in General had a similar effect with phenol and potassium dichromate on the intensity of the glow of the strain. At a phenol concentration of 0.001–0.01 mg / L (MPC of phenol 0.001 mg / L), fluorescence of the Vibrio fischeri VKPM B-9580 strain was induced by 25%. At a concentration of 1 mg / l, the glow increased by 38%.

Thus, low concentrations of toxicants cause stable induction of bioluminescence in bacteria. It should be noted that a noticeable induction of the luminescence of the strain Vibrio fischeri VKPM B-9580 (by 20-40%) was observed at the MPC level of toxicants (7), or significantly lower than the MPC (5-10 times). Higher concentrations of pollutants caused inhibition of bioluminescence.

The data obtained indicate a high sensitivity of the strain Vibrio fischeri VKPM B-9580 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 fischeri VKPM B-9580 as a test culture for the toxicity of petroleum products.

To determine the sensitivity of the Vibrio fischeri VKPM V-9580 strain to petroleum products, oil, diesel fuel, and sub-bilge (bilge) water from the engine room of the vessel were tested in concentrations from 0.001 mg / l to 1.0 g / l.

Toxicity testing was performed as described in example 1.

The obtained data on the bioluminescence sensitivity of the Vibrio fischeri VKPM B-9580 strain to all studied petroleum products are presented in Table 3. The table also presents data on the bioluminescence sensitivity of the E. coli lux strain to the same toxicants.

Oil at a concentration of 0.01 mg / L, which is significantly lower than the MPC of oil (0.05 mg / L) stimulated the luminescence of Vibrio fischeri VKPM B-9580 by 53%.

table 2 Toxicant Name Con
troll Toxicant concentration, mg / l 0.00001 0.0001 0.001 0.005 0.01 0.05 0.1 one The intensity of bioluminescence Vibrio fischeri VKPMV-9580, ues Copper 2.68 3.02 3.27 3.4 3.41 3.28 2.92 2.67 1,04 Zinc 2,780 3,350 3,400 3,430 3,450 3,330 3,170 3,150 1,364 K ₂ Cr ₂ O ₇ 3.20 3.27 3.42 3,54 3.8 3.53 3.52 3.42 2.82 Sodium dodecyl sulfate 3.0 3.18 3,51 3.60 3.74 4.2 3.72 3,58 3,54 Phenol 2.63 3.18 3.26 3.27 3.26 3.28 3.31 3,51 3.63

Table 3 The concentration of oil, mg / l Name of oil product Con
troll 0.001 0.01 0.1 1,0 10 one hundred 250 500 1000 The intensity of the bioluminescence of the strain Vibrio fischeri VKPMV-9580, ues Oil 0.53 0.67 0.81 0.62 0.59 0.49 0.47 0.45 0.42 0.40 Diz. fuel 0.53 0.73 0.82 0.72 0.66 0.63 0.51 0.46 0.43 0.40 Bilge water 0.55 0.71 0.72 0.64 0.61 0.58 0.52 0.47 0.44 0.41 The intensity of bioluminescence strain E.coli.yec Oil 0,057 0,048 0,053 0,039 0,031 0,026 0.014 Diz. fuel 0.058 0,053 0,060 0,043 0,040 0,039 0,029 Bilge water 0,061 0,050 0,048 0,042 0,038 0,037 0,035

Bilge water, diesel fuel also increased the glow of this strain at a concentration of 0.01 mg / l (30-55%, respectively). Luminescence induction was observed in the range of tested concentrations of petroleum products from 0.001 to 0.1 mg / L.

A comparison of the sensitivity of the strain Vibrio fischeri VKPM B-9580 and the lux strain of E. coli indicates a higher sensitivity of the strain Vibrio fischeri VKPM B-9580 and the prospects of its use for determining the toxicity of the components of the environment of water bodies.

Example 4

To determine the toxicity of natural samples using the strain Vibrio fischeri VKPM B-9580, samples of bottom sediments of marine water bodies were taken. One sample with a high content of oil products and heavy metals was taken in an area with a high anthropogenic load in the waters of one of the ports of the Sea of Azov. Another soil for biotesting with a similar particle size distribution was selected in a conditionally clean sea estuary isolated from the sea by a natural pebble scythe (5 km from B. Utrish metro station).

For testing, soil extracts were prepared on water.

Toxicity testing was performed as described in example 1.

Table 4 presents the results of the toxicity bioassay of averaged soil samples taken in the sea estuary and in the port.

Table 4 Sediment sampling area The intensity of the bioluminescence Vibrio fischeri VKPM B-9580, ues The control Experience Deviation from the control,% Estuary 0.645 0.640 -0.78 Sea port 0.413 0.650 57.4

As can be seen from the data presented in table 4, the luminescence intensity of the aqueous extract of bottom sediments from the estuary did not practically differ from the control, and the intensity of bioluminescence of the sample with soil extract from the seaport waters exceeded the control level by 57.4%. The data obtained indicate the sensitivity of the bioluminescence of the strain Vibrio fischeri VKPM B-9580 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 fischeri VKPM B-9580 as a test culture.

Literature

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. Recognized toxicity in the bacteria Photobacterium phosphoreum (Cohn) Ford. - 05/21/97.

3. TU 6-09-20-236-93 (prototype).

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

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

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

7. The list of fishery standards: maximum permissible concentrations (MPC) and tentatively safe exposure levels (SEC) of harmful substances for water, water bodies of fishery value - M.: VNIRO, 1999. - 304 C.

Claims (1)

The bacterial strain Vibrio fischeri VKPM B-9580, used as a test culture for determining the toxicity of environmental objects.