SU1687608A1 - Consortium of bacterial strains pseudomonas sp.and methylobacillus methanolovorus, decomposing methylacetate - Google Patents

Description

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(21) 4759597/13 (22) 08.28.89 (46) 10.30.91. Bul ISfc40 (71) Institute of Biochemistry and Physiology of Microorganisms of the USSR Academy of Sciences and Institute of Microbiology and Virology of the Academy of Sciences of the Kazakh SSR (72) D.Yu. Rakov, N.V. Doronina, Yu.A. Trotsenko, and R.M. Aliyeva (53) 663.15 (088.8)

(56) Buhler N., Pearson D. Organic Synthesis. Part 2. M .: Mir, 1973.

Weigand-Hilgetach Experimental methods in organic chemistry, M .: Chemistry, 1968.

(54) CONSORTIUM OF STRAINS OF BACTERIA PSEUDOMONAS SP. AND METHYLOBACILLUS METHANOLOVORUS, DECOMPOSE METHYL ACETATE

(57) The invention relates to biotechnology and concerns a new consortium of bacterial strains that can be used in the biological treatment of wastewater from acetaldehyde production. The purpose of the invention is a consortium of bacterial strains completely decomposing methyl acetate. The bacterial consortium completely decomposes methyl acetate, using its hydrolysis products — methanol and acetate — as sources of carbon and energy, at concentrations up to 5 g / l for 15–36 hours. The optional methylotrophic bacteria strain Pseudomonas sp BKM B-1735D, possessing carboxylesterase, together with Methylobaclllus methanolovorus BKM B-1447D strain, obligately using methanol. Strain Pseudomonas sp. isolated from wastewater acetaldehyde production in the Kazakh SSR. The strain Methylobaclllus methanolovorus was isolated from the active bark of the treatment facilities of the pulp and paper mill of the Estonian SSR. The consortium is used at a ratio of 2: 1 strains. 1 tab.

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This invention relates to biotechnology and concerns a new consortium of bacterial strains that can be used in the biological treatment of wastewater from acetaldehyde production.

Methyl acetate (acetic acid methyl ester) is a volatile, highly toxic synthetic pollutant that is chemically stable under normal conditions. In the patent and scientific literature there is no information about the microbial degradation of this compound.

The purpose of the invention is a new consortium of bacterial strains completely decomposing methyl acetate.

The invention consists in the use of a consortium consisting of a strain of faku tati in no-methyrophic bacteria Pseudomonas sp. As a destructor of methyl acetate. 24 RAs with carboxylesterase and Methylobaclllus methanolovorus BKM B-1447D strain. obligatorily using methanol.

A bacterial consortium completely decomposes methyl acetate using its products.

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hydrolysis (mgm nol and acetate) in clusters of carbon and energy sources

The strain Pseudomonas sp 24 RA was isolated from the icy waters of acetaldehyde arbitrarily hundred in the Kazakh SSR.

The strain has the following characteristics

Morphological and cultural signs. The cells are colorless mobile rods 0.5 0.7 x 1.3-1.7 μm with one polar flagellum, gram-negative, non-porous, not having complex intracytoplasmic membranes like methanotrophic bacteria. The stack is multiplied by dividing in half, forming polarly spaced saws.

Colonies on MPA are up to 5 mm in diameter, rounded, opaque, with a smooth edge, white, the surface is smooth, the profile is convex, the structure is uniform, the texture is viscous.

Physiological and biochemical signs. Aerobe, grows at 10-39 ° С, optimum is 28-32 ° С. The optimum pH is 6.8-7.2, at pH 3.6 it does not grow.

Fluorescent pigments, pyocionine, carotenoids do not form. It has oxidase, catalase, arginine dihydrolase. Nitrates restores to nitrites. Levan from sucrose, acetoin, indole, acetylmethyl carbinol does not form. Gelatin, cellulose, poly- / -oxybutyrate does not decompose, but hydrolyzes starch and tween-80. Poly-α-oxybutyrate does not accumulate intracellularly. DL- / -oxybutyrate does not use as a carbon source in a nitrogen-deficient medium.

The sources of carbon for growth are formate, methanol or ethanol at low concentrations (0.05-0.1%), methyl acetate, ethyl acetate, ethyl formate, butyl acetate, isopropanol, isobutanol, α -arabinose, fructose, and α-rhamnose, D -ribose, D-xylose, glucose, sucrose, D-serine, acetate, betaine, sarcosine, glycine, fumarate, succinate "-ketoglutarate.

The medium with glucose is acidified, gas formation is absent. Glucose does not ferment anaerobically. Formaldehyde, methylamine, trimethylamine, ethylamine, trehalose, l-alanine, L-hporin, phenylalanine, DL-arginine, glyoxylate, p-hydroxybenzoate, M-hydroxybenzoate, meso-inositol do not provide growth.

Does not grow autotrophic in an atmosphere of CO2 f H2 + 02. As the nitrogen source uses ammonium salts, nitrates and some amino acids. Growth factors are not needed.

I | D mt ,,% DNA (,, „) G1 (Ilmm Moihylobacilliis moihnnolovonjs VMK B 144 7D dust from the active bark of the wastewater treatment plant of the Estonian SSR and has the following characteristics

Morphological and cultural signs. Colorless moving sticks 0.5 x x2 µm with one polar flagellum, literate. Spores and capsules do not form, do not have complex intracytoplasmic membrane structures, multiply by dividing in half.

The colonies on the mineral agar medium with methanol as a source of carbon and energy are round, convex, colorless, smooth, up to 1 mm in diameter, the edge is even, the structure is homogeneous, viscous consistency.

0 Physiological and biochemical characteristics.

Obligate aerobic, optimal temperature for growth is 35 ° C, optimum pH is 6.8-7.4. Methylotropha Obligate, uses methanol as a carbon source and

5 energy, not capable of growing on media with polycarbon and other one-carbon substrates. Sources of nitrogen are ammonium salts or nitrates. No additional growth factors are needed.

0In the presence of 0.5% methanol in

the medium is hydrolyzed starch, does not destroy cellulose, hydrogen sulfide, indole, acetoin does not form, does not thin gelatin. Catalog-, ureazo-, oxidatively. Nitrates

5 restores to nitrite. Uses methanol through the 2-keto-3-deoxy-6-phosphoglu-gluconate variant of the ribulose monophosphate formaldehyde fixation cycle. Hz mol% DNA (TL) - 52.2.

0Type of interaction between strains in

consortium using symbiotic methyl acetate. Strain Pseudomonas sp. BKM B-1735D has a carboxyl esterase catalyzing the hydrolysis of ester

5 Methyl acetate to form methanol and acetate. Acetate is assimilated by this strain through a tricarboxylic acid cycle and a glyoxylate shunt (isocitrate lyase and malate synthase). Methanol is assimilated

0 Pseudomonas sp. BKM B-1735D through the serine Ci-metabolism pathway, however, the use of methanol is limited by the low activity of alcohol oxidase. Methanol dehydrogenesis (FMS, NAD) is absent in this strain. As a result, the culture of Pseudomonas sp. BKM B-1735D with growth on a medium with a high concentration of methyl acetate (more than 2 g / l) actively consumes one of its degradation products — acetate, and methanol gradually accumulates in front of it.

and begins to inhibit further cleavage of the megylate

Methylobacillus methanolovorus BKM B-1447D is an obligate methylotroph with methanol dehydrogenase (FMS) and actively assimilating methanol. Methylobaclllus methanolovorus BKM B-1447D is not inhibited by methyl acetate, but does not have carboxylesterase and cannot use methyl acetate as a source of carbon and energy.

When co-cultivated, Pseudomonas sp. BKM B-1735D and M. methanolovorus BKM B-1447D is achieved by complete utilization of methyl acetate.

Getting a consortium.

A consortium may be composed of a suspension of Pseudomonas sp. Cells. BKM B- 1735D grown on D medium with 2 g / l methyl acetate and Methylobacillus methanolovorus BKM B-1447D suspension grown on K medium with 5 g / l methanol in a ratio at equal density of suspensions, respectively, 2: 1. When this proportion changes (within 0.5: 1-4: 1) at the beginning of the consortium cultivation on methyl acetate, the strains gradually grow to an optimal ratio of cells (2: 1) during growth. This is due to the fact that the growth of obligate methylotroph Methylobacillus methanolovorus BKM B-1447D is limited by methanol, which is formed during the decomposition of methyl acetate by the strain Pseudomonas sp. VMK V-1735D.

The composition of the medium D, g / l of distilled water:

(NI-M) H2PCM1,0

K2NRSm1,0

NaCI5.0

MgS04 7 NaO0,2

FeSCM 7 H200,004

A medium of 100 ml is sterilized in 750 ml flasks at 1 atm for 30 minutes. After seeding, 30 ml of the suspension are added with 2 g / l methyl acetate and sealed with rubber stoppers to prevent the methyl acetate from evaporating.

Culture Pseudomonas sp. BKM B-1735D grown on a rocking chair at 180 rpm at 30 ° C for 30 hours

The composition of the medium of water: KN2RSm (NN4) 2 SCM MgSCM 7 NaO FeSCM 7 Н20

K, g / l distilled2, 0 2.0 0.175 0.002

pH 7.2 is adjusted with NaOH solution. The medium is sterilized at 1 atm. 30 min.

Meihylobacillii culture; mrtb -.in BKM B-1447D grown to tnnh-i-. 750 ml tui with 200 ml of medium and 5 g / l of methanol on a shaker at 180 rpm at 30 ° С R turning 5 24 h.

Storage conditions of the consortium and its constituent strains.

The consortium is stored in a liquid medium D at 4 ° C and undergoes reseeding after every two weeks, while the culture retains a high biochemical activity.

In laboratory conditions, the culture of Pseudomonas sp. BKM B-1735D is maintained on spit with MPA and transplanted after two weeks, the culture of Methylobacillus methanolovorus BKM B-1447D is maintained on agar (2%) spit with medium K and 0.5% (v / v) of methanol and re-0 is set once per month.

Strains of Pseudomonas sp. BKM B-1735D and Methylobaclllus methanolovorus BKM B-1447D are stored in a lyophilized state, the protective medium is milk, and the properties of the strains are stable, Example. 30 ml of a suspension of 109 cells / ml grown on medium with methyl acetate of two-day association of cultures of Pseudomonas sp. BKM B-1735D and Methylobaclllus 0 methanolovorus BKM B-1447D are sown in flasks with medium D. 5 g / l methyl acetate is introduced as a carbon source and energy. The cultivation is carried out on a shaker at 180 rpm at 30 ° C. A quantitative gas chromatographic analysis of the culture liquid for the content of methyl acetate, methanol and acetate is carried out every 3 hours for 36 hours of cultivation.

The control over the process of methyl acetate degradation is carried out by gas chromatography on a Rue Unlearn 104 chromatograph by the loss of methyl acetate and the formation of methanol. The column (70 cm, 02 mm) 5 is filled with Porapak Q. The H2 rate is 30 ml / min, the air is 300 ml / min, and the N2 is 30 ml / min. The temperature of the column 110 ° C.

Acetate is determined on a Chrom 4 gas chromatograph. Column (1.8 m, 04 mm) 0 is filled with Porapak QS. The rate of H2 is 35 ml / min, air - 300 ml / min, N2 - 35 ml / min. The temperature of the column 160 ° C.

By 15 h of cultivation, decomposes

65% methyl acetate, methanol and acetate in the medium

5 are missing. K 36 h methyl acetate and products

its degradation (acetate and methanol) in the environment

not detected.

The experimental data are presented in the table.

As follows from the data table, the resulting microorganism consortium completely decomposes methyl acetate. One of the consortium strains (Pseudomonas sp. VKM B-1735D) is able to completely decompose methyl acetate at low concentrations (0.1-0.5 g / l). With a higher content of methyl acetate in the environment, its degradation and strain growth are inhibited by accumulating toxic methanol.

Thus, the consortium of bacteria Pseudomonas sp. VKM B-1735D and

Methylobaclllus methanolovorus VKM B-1447D has the ability to completely decompose methyl acetate at a concentration of up to 5 g / l for 15-36 hours, which makes it possible to use it in industrial wastewater treatment of acetaldehyde production.

Claims (1)

Claims of the Invention Consortium of Bacterial Strains Pseudomonas sp. VKM B-1735D and Methylobaclllus methanolovorus VKM B-1447D decomposing methyl acetate.