LT4793B - The strain candida lipolytica c. 6.1-5capable of oxydising oil and oil products - Google Patents

Abstract

The invention belongs to the field of ecology and microbiology, namely, to microorganism strains that oxidise oil and its products. The proposed strain can be used in biological cleaning of ground and water polluted by oil and oil products. The strain Candida lipolytica C.6.1-5 (deposited in the Institute of Botany, Biodestructor Research Laboratory (LT), collection number IBL: Y-12) has the especially prominent property of destroying carbohydrates.

Description

The present invention relates to the fields of ecology and microbiology, namely strains of microorganisms oxidizing petroleum and oil products. The proposed strain can be used for biological treatment of soil, water contaminated with petroleum or petroleum products.

The known strain of the yeast Candida guillermondii ΒΚΠΜ Y-241 described in patent RU 2 007 372, C'02F 3/34, E02B 15/04 (deposited on Ι4ΜΠΜ BHHH Sintezbelok). This strain is used to purify water and soil contaminated with oil or petroleum products. The contaminated environment is treated by suspension of Yeast Candida guillermondii ΒΚΓΙ.Μ Y-241 cells in mineral nitrogen and phosphorus fertilizer medium.

The disadvantage of this strain is that it is not effective enough to bio-purify heavy oil fractions such as fuel oil.

Known method for microbiological cleaning of oil-contaminated soil and water as described in patent RU 2 014 286, C02F 3/34, E02B 15/04, wherein oil-contaminated water or soil is purified by an association of oil-oxidizing microorganisms: Candida [ropicalis.V / ycoccocw .y or Micococcus lactis + Micococcus lactis, or Candida guillermondii + Acinetobacter oleovorum subsp. paraffinium.

The disadvantage of said microorganism composition is the inadequate disintegration of different composition of oil pollutants and other hydrocarbons. In addition, the association of microorganisms is not stable in the natural environment. It is difficult to determine the quantitative composition of an association of microorganisms that will allow complete and rapid degradation of contaminants.

A known association of microorganisms called "Devouroil" is described in the specification of patent RU 2 023 686, C02F 3/34, E02B 15/04. This association includes Rhodococcus sp., Rhodococcus maris, Rhodococcus erythropolis, Pseudomonas stutzeri and Candida sp .. This microorganism kit is intended for the purification of water surface and soil contaminated by petroleum products.

The main disadvantage of this microorganism association is that it is more suitable for cleaning seawater or saline soil. In addition, aromatic hydrocarbons and naphthenes are poorly oxidized by this association.

The object of the present invention is to isolate a yeast strain which oxidizes petroleum and oil products which can be used to intensify the cleaning of contaminated soil and water.

The proposed strain of Candida lipolytica C. 6.1-5 was isolated from the soil of the Oil Base of Vilnius City. The method of isolation comprises preparing an aqueous soil suspension, seeding the prepared soil suspension on agarized synthetic glucoseatnonium medium in the following composition (g / l): glucose - 20; (NHjjiSOj - 5; KH2PO4 - 0.85; K.2HPO4 0.15: \ lgSO4 - 0.5; NaCl - 0.1; CaCl · - 0.1; levomycetin - 0.1; agar - 20; water - 1000 ml. Yeast culture was grown for 4 days in a thermostat at 26 ° C. ± 2 ° C. Identification of the yeast was carried out on the basis of morphological, physiological and biochemical features using the NJW Kreger - van Rij (1984) classification system.

Candida lipolytica C. 6.1-5 was deposited in the Microorganism Collection of the Biodestructors Research Laboratory of the Botanical Institute in 1998. September. Dec. 6, Collector Number IBL: Y - 12.

The proposed strain 6.1-5 of Candida lipolytica C. has the following properties.

Morphological properties. After culturing for 3 days in liquid malt extract medium at 25 ° C, the cells are oval or elongated, (2-4.5) x (4-22) pm. Buds on a narrow, sometimes wide base. Forms a film and a precipitate in a liquid medium. Observe pseudomycel or true micelle.

After 1 month of cultivation, the agarized malt medium is cream-colored, moist, soft, slightly wrinkled or pleated. Microscopy observed pseudomycel and branched, septal true micelle. Occasionally forms arthrospores. Blastospores are connected in small chains.

Physiological properties. Does not perform the bitter process. As the only source of carbon it can assimilate: glucose. L - sorbose, ethanol, glycerol, erythritol, D - mannitol, lactic, succinic and citric acids, n - hexadecane. n-decane, naphthalene, petroleum, fuel oil. Does not assimilate: galactose, D - ribose, L rhamnose, sucrose, maltose, cellobiose, tregahose, lactose, melibiosis, raffinose, melecytosis, inulin. starch, D - xylose, L and D - arabinose, adonitol (ribitol), salicyl, inositol. There is no hydrolysis of arbutine. Produces acids. It grows in Vitamin Free Yeast Base DIFCO (USA). Growth is stimulated by thiamine. Optimum growth temperature 27 ° C. The maximum growth temperature is 33-37 ° C. Optimal pH 5.0 - 5.5.

Enzymatic activity. Qualitative screening methods have shown that the strain has urease and lipase enzymatic activity.

Strain storage. The strain is stored in agarized glucose-ammonium medium in tubes. Regularly transplanted. Petroleum hydrocarbons are added to the culture medium of the protected strain. The strains are stored at ± 4 ° C.

Preliminary screening of the proposed strain was performed using qualitative methods. Selection was performed on agarized medium with the following composition (g / 1): fuel oil - 10; (NFLhSCL - 5; KH2PO4 - 0.85; K2HPO4 - 00:15; MgSO ₄ - 0.5; NaCl - 0.1; CaCl ₂ - 0.1; agar - 20; water to 1000 ml. Of the strain was grown for 5 days in a thermostat. 26 ± 2 ° C. PH of the medium 5. The activity of the strain oxidizing the oil and its products was evaluated by the formation of clear zones around the microorganism.

The influence of various sources of phosphorus (bone meal, superphosphate, Kemira Horti-2 (NPK 6-i2-24)) on the oil-oxidizing yeast Candida lipolytica C. 6.15 was evaluated according to the medium content optimization method in the modified Chapeck medium based on the absolute dry biomass (ASB) content. for growth. Study data are presented in Table 1.

table. Effect of various sources of phosphorus on the growth of Candida lipolytica C. strain 6.1-5

; Name of the source of the phosphorus i to Phosphorus content, g / l Absolutely dry biomass weight, g / 1 Control (KH2PO4) 0.23 8.2 ± 0.1 Bone meal 0.115 11.1 = 0.1 0.23 13.6 = 0.2 0.345 12.4 = 0.1 Kemira Horti -2 0.115 6.2 = 0.1 i (NPK 6-12-24) 0.23 7.5 = 0.2 0.345 5.1 = 0.1 Superphosphate 0.115 2.36 ± 0.04 0.23 2.90 ± 0.03 0.345 5.70 ± 0.10

fs presented data show that different sources of phosphorus affect the growth of the selected strain in different ways. It was found that the highest amount of ASB was obtained by culturing the selected strain with ₄ LT 4793 B bone meal. When cultured with superphosphate and Kemira Horti - 2 fertilizers, ASB content was lower than in control (Table 1).

The ability of the proposed yeast strain to degrade petroleum and petroleum products and some aromatic hydrocarbons is shown in the examples.

example

The proposed strain of yeast Candida lipolytica C. 6.1-5 is cultivated in a modified Chapeck medium by deep-water shaking. Oil is the sole source of carbon. Medium composition (g / 1): bone meal - 1.75; Mg SO ₄ - 0.5; Fe SO ₄ - 0.001; NH ₄ NO ₃ - 1.5; KCl = 0.5; water - 1000 ml. Medium pH 5.0 - 5.5. Cultivation temperature 26 ± 2 ° C. Quantitative oil analysis after 7 and 20 days of yeast cultivation was performed by spectrometry using an oil analyzer ANVP -79.

It was found that after 7 days of cultivation the oil content decreased by 82.18% and after 20 days by 90.67%.

example

The selected yeast strain was cultured in a medium containing only fuel oil as the sole carbon source. Composition of medium (g / 1): fuel oil - 10; (NH ₄ ) ₂ SO ₄ - 5; KH ₂ PO ₄ - 0.85; K ₂ HPO ₄ - 0.15; MgSO ₄ - 0.5; NaCl - 0.1; CaCl ₂ - 0.1; water - 1000 ml. Medium pH 5.0-6.5. The strain was cultivated in depth, in shakes. Growth temperature 26 ± 2 ° C. Fuel oil was quantitated after 7 and 20 days of culturing Candida lipolytica C. Strain 6.1-5. It was found that after 7 days of cultivation the fuel oil content decreased by 51.89% and after 20 days by 81.68%.

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A test was performed which, by its parameters, corresponds to the test described in Example 1. The strain selected was cultured in the same composition as in Example 1. The difference is that aromatic hydrocarbon - naphthalene 10 g / l was chosen as the sole carbon source. Quantitative analysis of naphthalene after culturing Candida lipolytica C. 6.1-5 was performed by gas chromatography mass spectrometry using a Satum 3 gas chromatograph.

It was found that after 7 days of cultivation, naphthalene content decreased by 64.91%, and after 20 days by 85.01%.

example

Oil-contaminated soil remediation. The treatment site contains 100 tonnes of contaminated soil with a pH of 5.0 to 5.5. 35 g / kg of petroleum products. The soil is spread in a layer 0.5 m thick. Prepare a suspension of strain 6.1-5 of Candida lipolytica C. and mix with an aqueous saline solution containing 30 mg / l nitrogen (ammonium sulphate used) and 25 mg / l phosphorus (bone meal). The cell titer in solution is about 3 x 10 'cells / ml. The prepared solution is applied to the soil to be cleaned by spraying at a rate of 3 1 / m ² . The soil is cleaned and fertilized with NPK mineral fertilizers and then cultivated on a weekly basis. Under natural conditions with an air temperature of at least 14 ° C, the soil remediation process takes 1.5-2.5 months. Pollutant reduction is 91.02%.

Claims (3)

1. Strain Candida lipolytica C. 6.1-5 (deposited at the Institute of Botany, Biodestructor Research Laboratory (LT), Collector Number IBL: Y - 12) Oxidising petroleum and its products with a pronounced hydrocarbon destructive activity. 2. Strain Candida lipolytica C. 6.1-5 for the purification of oil-contaminated water and soil. 3. A process for the preparation of a strain of Candida lipolytica C. 6.1-5 according to claim 1, characterized in that the strain biomass is grown under laboratory conditions using different nutrient media and maintained at a temperature of 14 to 29 ° C and a pH of 5.0 to 6.5, prepare a suspension mixed with an aqueous saline solution containing 30 mg / l nitrogen and 25 mg / l phosphorus with a cell titer in the solution of 3 × 10 ⁷ cells / ml under natural conditions suitable for the growth of yeast.