SU1377290A1 - Strain of bacteria alcaligenes faccalis used for cleaning waste water of 2-chloro-cis- muconic acid - Google Patents

Abstract

This invention relates to the microbiological industry and relates to a new bacterial strain used to treat persistent and toxic compounds in wastewater. The aim of the invention is to obtain a new strain of bacteria capable of utilizing 2-chloro-cononic acid, with the result that this compound is completely decomposed with 100% dechlorination of the organic molecule. The bacterial strain Alcali genes faecalis VKPM B-3636 (NIMI-. -KZ-5) was isolated from garden soil by the accumulative culture method and is capable of destroying completely 0.4-0.7 g / l of 2-chloro-cis in 120-160 hours , cis-muconic acid. 3 tab. (L

Description

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The invention relates to the microbiological industry and concerns a new bacterial strain used to treat wastewater from persistent and toxic compounds.

The purpose of the invention is to obtain a bacterial strain capable of utilizing 2-chloroconic acid, with the result that this compound is completely decomposed with 100% dechlorination of the organic molecule.

The bacterial strain Alcaligenes faecalis VKPM B-3636 (INMI-KZ-5) was isolated from garden soil by the method of accumulative culture on a mineral medium obtained in the following way.

Flasks, 0.5 liter in volume, containing 100 ml cpe; joi of the following composition, g / l: KjHPO ЗП-гО 4.0 NaH5.P04 2Н20 0.4 $ (NH 4) 50, 0.5; MgS04-7H20 0.15-, Ca (NOj) 5-4H20-0.005; Na MoO 2H-iO 0.004; 3-chlorobenzoic acid 0.5, inoculated with Acinetobacter calcoaceticus bacteria INMI-KZ-3i and cultured until the substrate disappeared. Simultaneously with the disappearance of 3-hl6rbenzoic acid, 2-chloro-dismuconic acid accumulates in the medium. After the growth was completed, the culture fluid was separated from the cells by centrifugation (20 min at 10 kb / min) and then sterilized by filtration (Zeitd filter). A sterile solution of yeast extract (final concentration 10 mg / l) was added to the medium thus obtained and poured 2 ml into test tubes.

In preparing an agarized agar, the culture liquid obtained as described above was concentrated under vacuum 2 times, sterilized by filtration, and a sterile solution of yeast extract was added. The concentrated culture liquid was added to 3% molten aqueous agar in a 1: 1 ratio and the medium was poured into cups. The liquid and dense medium obtained by this method contained 0.25 g / l of 2-chloro-cis, cis-muconic acid.

When isolating the strain, 2 g of garden soil was introduced into a 250 ml flask.

containing 50 ml of medium of the following composition, g / l: KiHP04-3HiG 4.0; 0.4i (NH), S04 0.5 j MgS04.-7HjO 0.15; Ca (NOi), - 4HaO 0.005; 2H jO 0.004; 3-chlorobenzoic acid

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0,25, and kept at 28 ° C. After 20 days of incubation, 5 ml of the cumulative culture was introduced into a flask with fresh medium and cultured under the same conditions. After 10 days, the flask was observed complete disappearance of 3-chlorobenzoic acid. From this flask we made seeding in 20 tubes containing

2 ml of mineral medium with 2-chloro-cis, cis-muconic acid. After 10 days in the test tubes, the complete disappearance of 2-chloro-cis, cis-mucous, acid was observed. In order to obtain the most active strain utilizing 2-chloro-cis, cis-mukonovuga acid, the enrichment culture was enriched with bacteria utilizing this compound. To do this, from the test tube, in which the process of utilization of 2-chloro-cis, cis-muconic acid went most quickly, seeding was done in 10 test tubes containing the mineral medium with 2-chloro-cis, cis-muconic acid. One of the test tubes was selected again, in which the process of xenobiotic disposal went faster. This operation was repeated 2 times. A pure bacterial culture was isolated from a separate colony obtained by sieving enriched cumulative culture on plates with agar mineral medium with 2-chloro-cis, cis-muconic acid. A pure bacterial culture was replanted weekly for

3 months on a liquid mineral medium containing 0.25 g / l of 2-chloro-cis, cis-cononic acid. The final strain of Alcaligenes faecalis, INMI-KZ-5, was obtained by isolating the fastest growing colony on a mineral agar medium with 0.5 g / l of 2-chloro-cis, cis-muconic acid.

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Strain retains viability

for 6 months when stored on beveled micro-peptone agar and for 2 years in a lyophilized state.

The strain Alcaligenes faecalis INMI-KZ-5 has the following culture-morphological and physico-biochemical characteristics.

Morphological and cultural properties.

Coccoid sticks, O, 5x0.5-1.3 microns in size, mobile, have peritrichally arranged flagella, gram-negative, non-spongiferous.

Growth on dense environments. M co-peptone agar, 28 ° C, 7 days (Table 1).

Table 1 5

Hatch

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The colony

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Colorless, flat, smooth, glossy with smooth edges, dough-like, pigment does not penetrate the medium

Potato agar, 28 ° C, 7 days (Table 2).

Table 2

Hatch

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The colony

d, testos even

Colorless, flat, smooth, glossy with smooth edges, dough-like, pigment does not penetrate the medium

Mineral agar medium with 2-chloro-cis, cis-muconic acid (0.7 g / l), 28 ° C, 7 days (Table 3). 35

Table 3

Hatch

The colony

Colorless, flat, smooth, glossy with smooth edges, dough-like, pigment is not released on Wednesday.

Growth on liquid media.

M co-peptone broth, 28 C, 5 days.

Character of growth - a turbidity is formed, a dense precipitate, which is easily stirred up when agitating. No pigment is released on Wednesday.

Liquid mineral medium with 2-chloro-cis, cis-muconic acid (0.7 g / l) 28 ° C, 7 days.

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Character of growth - a turbidity is formed, a dense precipitate, which is easily stirred up when agitating.

Physical and biochemical properties.

Aerob, capable of anaerobic respiration in the presence of nitrate. Gives a positive catalase and oxidase reactions. Gelatin does not thin, starch does not hydrolyze. Does not fix gaseous nitrogen. Grows at 37 C, does not grow at 38 ° C. The optimum temperature for growth is 28-30 ° C. Sensitive to 50 units. penicillin.

Sources of carbon.

The strain grows well on m-co-peptone broth, mac-peptone agar, potato agar and mineral agar medium of the following composition, g / l: K, HP04-ZN, 0 4, OiNaH, 2H., 0 0, 4j (NH c), S0 / -0.5; MgS04-7HjO 0.15, Ca (NO-i) 0.005, nicotinic acid 0.005, carbon source 0.5, pH 7.0-7.3. The following sources of carbon are used: organic acids — acetate, citrate, succinate, malonate, levulinate, 2-chloro-cis, cis-muconic acid, amino acids — proline, serine, norleucine.

Does not absorb carbohydrates: D-glucose, D-galactose, D-fructose, D-rhamnose, D-xylose, cellobiose, sucrose, lactose, sugar alcohols: inositol, mannitol, sorbitol, adonite, organic acids: 3-hydroxybenzoate, 4-hydroxybenzoate , 3,4-α-dioxybenzoate, maleate, adipate, amino acids: valine, ornithine, glycine, arginine, lysine, leucine, tryptophan.

Growth factors - nicotinic acid (0.0005 g / l).

Sources of nitrogen - absorbs ammonium and nitrate nitrogen.

Example 1. Decomposition of 2-chloro-cis, cis-muconic acid in mineral medium. Culture A.faecalis INMI-KZ-5 grown on potato or meat-peptone agar for 5 days. The grown culture is inoculated with 250 ml flasks containing 50 ml of medium. The composition of the medium, g / l: К2НР04 ЗН О 4.0i NaH, jP04-2H,; 0 0.4 (№14) 7304 0.5; MgSO 4 7H20 0.15 ;, nicotinic acid 0.0005-, 2-chloro-cis, cis-muconic acid 0.7, tap water. The cultivation is carried out on a rocking chair for 7 days. A prepared seed in the amount of 10 ml is added to

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750 ml flasks containing 100 ml of medium of the same composition. Under these conditions, 2-chloro muconic acid disappears in 120 hours, the release of chlorine atoms is 100%,

Example 2. Decomposition of 2-chloro-cis, cis-muconic acid in waste water. . .

The strain was grown on wastewater containing the following components, g / l

Reducing substances 1,2

Monocarboxylic acids

(in terms of acetic acid) Volatile acids 2-chloro-cis, cis-muonic acid

2.0

0.3 0.4

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Nitrogen 0.12

Phosphorus 0.07

Wastewater was seeded with 10% seed.

Cultivation was carried out in 500 ml flasks containing 100 ml of waste water on a rocking chair at 180 rpm at 28 ° C. Under these conditions, AO faecal is strain INMI-KZ-5 completely decomposes 0.4 g / l of 2-chloro-cis, cis-muconic acid in 160 hours,

Claims (1)

Invention Formula The bacterial strain Alcaligenes faecalis VKGM B-3636 used for wastewater treatment from 2-chloro-cis, cis-muconic acid.