RU2292392C2 - BACTERIUM Pseudomonas alcaligence STRAIN USEFUL IN PURIFICATION OF SOILS, GROUND WATER AND SURFACE WATER FROM TRINITROTOLUENE - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to production of biopreparation for purification of soils, ground water and surface water from trinitrotoluene. Bacterium Pseudomonas alcaligenes BS300 strain was isolated from soil. Said strain utilizes trinitrotoluene, is stable to heavy metal ions and is useful in purification of soil and water from combined contamination by trinitrotoluene and heavy metal ions. Pseudomonas alcaligenes BS300 strain produces biological surfactant accelerating trinitrotoluene degradation in water medium and soil.

EFFECT: improved agent for effective purification of soil and water.

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Description

The invention relates to the field of microbiology and is a new bacterial strain that can be used to clean soil, groundwater and surface water when Trinitrotoluene (TNT) enters the environment.

2,4,6-Trinitrotoluene (TNT) has been produced by nitration of toluene on an industrial scale since the end of the 19th century in many countries. It is used as an explosive for military and industrial purposes. TNT was found in wastewater, surface and soil waters, in soil and sediments near enterprises producing it. Environmental pollution TNT poses a serious threat to public health and nature. TNT is slightly soluble in water, is well adsorbed on clay minerals and humic substances of the soil and very slowly passes into the aqueous phase, where it is metabolized by microorganisms.

Known strains of microorganisms: Penicillium sp. [1], Phanerochaete chrysosporium [2], Pseudomonas fluorescens I-C [3], Clostridium thermoaceticum [4], Pseudomonas savastanoi [5], Rhodococcus (opacus) erythropolis HL PM-I [6], Desulfovibrio sp. (B stram) [7], Enterobacter cloacae PB2 [8], Anabena sp. [9], Pseudomonas putida [10], which can decompose TNT in soil and water. The closest to the proposed strain is a bacterial strain Pseudomonas putida [10], which has high characteristics for biodegradation of TNT. However, the disadvantage of this strain is that to enhance the biodegradation of TNT, it is necessary to add surfactants.

The objective of the invention is to obtain a new strain of microorganisms with high utilizing ability against TNT, producing extracellular biological surfactants (biosurfactants), which can be used to clean soils and water bodies contaminated with TNT and salts of heavy metals.

The proposed strain Pseudomonas alcaligenes BS300 isolated from soil contaminated with TNT, and selected by reseeding of individual colonies of bacteria on plates with minimal agar A, which contains (g / DM ³ ) Na ₂ HPO ₄ · N ₂ O - 6.0; KH ₂ PO ₄ - 3.0; NaCl - 0.5; NH ₄ Cl - 1.0; Mg ₂ SO ₄ · 7H ₂ O - 0,3; CaCl ₂ · 2H ₂ O - 0.01; Agar-agar - 15.0; sodium pyruvate - 5 g, TNT - 100 mg. Distilled water - up to 1 dm ³ ; pH 7.2.

The strain Pseudomonas alcaligenes BS300 was identified in accordance with Berg's determinant [12] and deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number VKPM B-8810.

The proposed strain is characterized by the following morphological and physiological and biochemical characteristics. Gram-negative movable sticks of 2-3 · 0.6-0.8 microns in size, does not form a spore. On the agar medium, from the acid hydrolyzate of fish meal, flat, dull colonies with a diameter of 2-3 mm are formed on the second day. In a broth made from acid hydrolyzate, fishmeal grows in the form of a film and uniform turbidity.

The strain is an aerobic, has oxidase and catalase activity, grows in the temperature range from 8 to 41 ° C, the optimum is from 26 to 28 ° C. As a source of carbon, it consumes acetate, citrate, succinate, alanine, arginine. It has arginine dihydrolase activity, restores nitrates, and denitrification activity is absent. Prototroph, does not need additional growth factors. Hydrolyzes gelatin, has lecithinase and urease activity. Does not hydrolyze starch and poly-β-hydroxybutyrate.

The strain transforms TNT cometabolic in the presence of citrate, succinate.

The strain is not pathogenic (non-virulent, non-toxic, non-toxic).

A test for pathogenicity was performed on white mice and white rats. The strain issued a safety opinion.

Genetic features. The culture is resistant to ampicillin antibiotics. The strain is resistant to heavy metal ions: Pb, Zn, Fe, Cr, Co - 50 mg / DM ³ .

The strain produces biological surfactants (biosurfactants). The strain grows well on rich nutrient media based on meat and peptone broth and enzymatic hydrolyzate of fish meal, on synthetic nutrient media, where sucrose, pyruvate, molasses are used as a carbon source.

Storage conditions: in the lyophilized state in ampoules at 4 ° C. The strain can be maintained by regular reseeding (1 time in 2 weeks) on an agarized growth medium, (g / dm ³ ): Na ₂ HPO ₄ · N ₂ O - 6.0; KH ₂ PO ₄ - 3.0; NaCl - 0.5; Mg ₂ SO ₄ · 7H ₂ O - 0.3; CaCl ₂ · 2H ₂ O - 0.01; (NH ₄ ) ₂ SO ₄ 0.25; agar-agar - 15.0; sucrose - 5 g; TNT - 50 mg; distilled water - up to 1 dm ³ ; pH 7.2.

The invention is illustrated by the following examples.

Example 1. The bacterial strain Pseudomonas alcaligenes BS300 is grown on a mineral medium of the following composition, g / l: Na ₂ HPO ₄ · H ₂ O - 6.0; KH ₂ PO ₄ - 3.0; NaCl - 0.5; Mg ₂ SO ₄ · 7H ₂ O - 0.3; CaCl ₂ · 2H ₂ O - 0.01; (NH ₄ ) ₂ SO ₄ 0.25; agar-agar - 15.0; sucrose (or molasses) - 5; distilled water - up to 1 dm ³ ; pH 7.2. 30 ml of nutrient medium and 0.1% xenobiotic (TNT) are added to each 100 ml cone separately. Flasks are inoculated with cells of the proposed strain at a concentration of 1.0 · 10 ⁷ mcl / cm ³ . As a control, put the same flask with medium, TNT and without bacteria to determine the total (natural) losses. The experiment is carried out in five replicates. The flasks were cultured on a shaker at 200 rpm for 2.5 days. Cometabolic biodegradation of TNT is determined using high pressure liquid chromatography. Experimental data show that the proposed strain utilizes 88% of TNT after 48 hours, whereas TNT did not establish biodegradation as a carbon source in a medium without sucrose or molasses.

Example 2. In flasks per 100 cm ³ contribute 30 cm ^{3 of} synthetic medium (the composition of the medium is shown in example 1), TNT at the rate of 100 mg / l, 20 mg / l of salts of Pb, Zn, Fe, Cr, Co - 40 mg / dm ³ , Hg - 86 mg / dm ³ and cells of the strain Pseudomonas alcaligenes BS300 at a concentration of 1.0 · 10 mcl / cm ³ . The control is a seeded flask with TNT. The flasks under investigation were cultured in five replicates on a shaker at 28 ° C and 200 rpm for 2 days. The experimental results show that the TNT degradation efficiency of the proposed strain in variants with and without metal salts does not differ (table 1).

Table 1 Biodegradation of TNT in the presence of heavy metals by the strain Pseudomonas alcaligenes BS 300,% (M ± m) Substance Biodegradation With metals Metal free TNT 80.0 ± 5.6 78.2 ± 5.9 Note: M is the average of four replicates; m - confidence interval with a probability of 95%

Example 3. The culture fluid of the strain Pseudomonas alcaligenes BS300, grown as in example 1, is separated from microbial cells by centrifugation at 5000 rpm./min for 10 minutes. As a biosurfactant-containing liquid in the experiment, a culture liquid diluted with distilled water by a factor of 10 is used. The surface tension of this fluid is determined using a ring tensiometer. The control is distilled water. The results of determining the surface tension show (table 2) that the addition of the culture medium to distilled water leads to a decrease in the surface tension of distilled water from 53.1 dyne / cm to 26.4 dyne / cm. The addition of an inoculated culture medium in the same ratio of 1:10 does not affect the surface tension of distilled water. Thus, the culture fluid of the proposed strain contains biological surfactants.

table 2 The effect of biosurfactant-forming culture fluid of the strain Pseudomonas aicaligenes BS300 on the surface tension of distilled water, dyne / cm (M ± m) Test fluid Surface tension Distilled water 56.1 ± 4.2 Biosurfactant-containing fluid 28.4 ± 3.3 Distilled water + culture medium 53.1 ± 2.6 Note: M is the average of four replicates; m - confidence interval with a probability of 95%

Example 4. In flasks per 100 cm ³ contribute 27 cm ^{3 of the} mineral medium (the composition of the medium is shown in example 1) and TNT at the rate of 100 mg / l To the flasks add 3 cm ³ biosurfactant-containing culture medium as in example 3. The flask is seeded with a culture of Pseudomonas alcaligenes BS300 to a concentration of 1.0 · 10 CFU / cm ³ . As controls use flasks with TNT. Cultivation is carried out on a rocking chair at 200 rpm./min, a temperature of 20 ° C for 3 days. The effectiveness of TNT biodegradation is determined by high pressure liquid chromatography. The experimental results show (table 3) that the addition of a biosurfactant increases the efficiency of TNT biodegradation. So, for 2 days in the experimental variants with the addition of a biosurfactant, TNT biodegradation was 100%.

Table 3 Biodegradation of trinitrotoluene by the strain Pseudomonas alcaligenes BS300 with the addition of a biosurfactant,% (M ± m) Substance Biodegradation,% Biosurfactant added No biosurfactant TNT one hundred% 62.2 ± 5.8 Note: M is the average of four replicates; m - confidence interval with a probability of 95%

Example 5. In a desiccator with a volume of 3 dm ³ make 2 kg of sod-podzolic soil contaminated with 0.1% by weight of TNT, and mix thoroughly.

A bacterial suspension of the strain Pseudomonas alcaligenes BS300 is diluted with a phosphate buffer solution of pH 7.2 and introduced into the soil contaminated with xenobiotic at the rate of 1.0 · 10 ⁷ CFU per 1 g of soil. The soil is thoroughly mixed, moistened to 60% of the total moisture capacity and exposed at 20 ° C for 2 months. For analysis, soil samples are taken at the beginning of the experiment and after 2 months. The effectiveness of TNT biodegradation of the proposed strain in the soil is evaluated by high pressure liquid chromatography. The research results show that the proposed strain for 2 months at a temperature of 20 ° C carries out the degradation of 65.6% of TNT.

Table 4 Biodegradation of TNT in soil at a temperature of 20 ° C by strain Pseudomonas alcaligenes BS300 for 2 months,% (M ± m) Substance Biodegradation,% TNT 72.2 ± 6.4 Note: M is the average of four replicates; m - confidence interval with a probability of 95%

Example 6. In desiccators with a volume of 3 dm ³ with 2 kg of soil contaminated with TNT (100 mg / kg), add 15 mg / kg of soil of salts of Pb, Zn, Fe, Co and Cr - 50 μg / kg and cells of the strain Pseudomonas putida BS300 to concentrations of 1.0 · 10 ⁷ CFU / g of soil. The soil is thoroughly mixed, moistened to 60% of the total moisture capacity and exposed at 20 ° C for 2 months. For analysis, soil samples are taken at the beginning of the experiment and after 2 months. The effectiveness of the biodegradation of oil by the proposed strain in the soil is evaluated by high pressure liquid chromatography. The control is soil contaminated with TNT and introduced microorganisms. The repetition of the experiment is fivefold. The experimental results show that the TNT degradation efficiency of the proposed strain in variants with and without metal salts does not differ (table 5).

Table 5 Biodegradation of TNT in the soil in the presence of heavy metals by the strain Pseudomonas alcaligenes BS300,% (M ± m) Substance Biodegradation With metals Metal free TNT 88.7 ± 7.2 84.5 ± 5.9 Note: M is the average of four replicates; m - confidence interval with a probability of 95%

Thus, the advantage of the proposed strain is that at a temperature of 20 ° C it utilizes TNT in soil and water. The proposed strain produces biological surfactants, which accelerates the degradation of TNT in the aquatic environment and soil. The resistance of the strain to heavy metal ions extends the range of its application in the treatment of soil and water from combined pollution of TNT and metals.

LITERATURE

1. Patent of Russia №2001111823, 2003.05.10. Microbiological method for removing nitroaromatic compounds present in solution or soil.

2. Jalal Hawari, Annamaria Halasz, Sylvie Beaudet, Louise Paquet, Guy Ampleman, and Sonia Thiboutot. Biotransformation of 2,4,6-Trinitrotoluene with Phanerochaete chrysosporium in Agitated Cultures at pH 4.5. Applied and Environmental Microbiology, 1999, Vol.65, No.7, p.2977-2986.

3. Jeong W. Pak, Kyle L. Knoke, Daniel R. Noguera, Brian G. Fox, and Glenn H. Chambliss. Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C. Applied and Environmental Microbiology, 2000, 66 (11): 4742-4750.

4. Huang S., Lindahl P.A., Wang C., Bennett G.N., Rudolph F.B., Hughes J.B. 2,4,6-trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum. Applied and Environmental Microbiology, 2000, 66 (4): 1474-1478.

5. Martin J.L., Comfort S.D., Shea P.J., Kokjohn T.A., Drijber R.A. Denitration of 2,4,6-trinitrotoluene by Pseudomonas savastanoi. Can J Microbiol, 1997, 43 (5): 447-55.

6. Heiss, G., Hofmann, K. W., Trachtmann, N., Walters, D.M., Rouviere, P., Knackmuss, H.-J. (2002). Npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation. Microbiology, 148: 799-806.

7. Boopathy R., Kulpa C., Wilson M. Metabolism of 2,4,6-trinitrotoluene by Desulfovibrio sp. (B strain). Appl. Microbiol. Biotechnol. 1993, v.393, pp. 270-275.

8. French C.E., Nicklin S., Bruce N.C. Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase. Applied and Environmental Microbiology, 1998, 64 (8): 2864-22868.

9. Pavlostathis S.G., Jackson G.H. Biotransformation of 2,4,6- trinitrotoluene in Anabena sp. Cultures Environmental Toxicology and Chemistry, 1999, 18 (3): 412-419.

10. Chul Hwan Park, Tak-Hyun Kim, Sangyong Kim, Seung-Wook Kim, Jinwon Lee, and Sun-Hwan Kim. Optimization for Biodegradation of 2,4,6-Trinitrotoluene (TNT) by Pseudomonas putida, Journal of Bioscience and Bioengineering, 2003.

11. Bergey′s Manual of Determinative Bacteriology. Ninth Edition. Baltimore, Maryland: Williams & Wilkins, 1994 .-- 787 p.

Claims (1)

The bacterial strain Pseudomonas alcaligenes VKPM B-8810 used to clean the soil, groundwater and surface water from trinitrotoluene in situ.